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Post-translational modifications influence transcription factor activity: A view from the ETS superfamily
Abstract
Transcription factors provide nodes of information integration by serving as nuclear effectors of multiple signaling cascades, and thus elaborate layers of regulation, often involving post-translational modifications, modulating and coordinate activities. Such modifications can rapidly and reversibly regulate virtually all transcription factor functions, including subcellular localization, stability, interactions with cofactors, other post-translational modifications and transcriptional activities. Aside from analyses of the effects of serine/threonine phosphorylation, studies on post-translational modifications of transcription factors are only in the initial stages. In particular, the regulatory possibilities afforded by combinatorial usage of and competition between distinct modifications on an individual protein are immense, and with respect to large families of closely related transcription factors, offer the potential of conferring critical specificity. Here we will review the post-translational modifications known to regulate ETS transcriptional effectors and will discuss specific examples of how such modifications influence their activities to highlight emerging paradigms in transcriptional regulation.
... The sea urchin genome contains eleven genes encoding ETS transcription factors and some of them are presumed to be transcriptional repressors [20,21]. We focused on the gene encoding the ETS transcription factor Yan/Tel since this gene is known to encode a prototypical repressor [22][23][24][25][26] (reviewed in [27]). ...
... This result is consistent with previous studies showing that the activity of Drosophila Yan or of vertebrate Tel is not regulated at the transcriptional level but at the post-transcriptional level by phosphorylation by MAP kinases. MAPK dependent phosphorylation of Yan and Tel is the central mechanism that regulates the activity and stability of these transcriptional repressors [22][23][24][25][26] (reviewed in [27]). Specific phosphorylation events triggered by either ERK, JNK or p38 downregulate the transcriptional repressor function of Drosophila Yan or of vertebrate Tel, leading to their export out of the nucleus and to their degradation [22][23][24][25][26] (reviewed in [27]). ...
... MAPK dependent phosphorylation of Yan and Tel is the central mechanism that regulates the activity and stability of these transcriptional repressors [22][23][24][25][26] (reviewed in [27]). Specific phosphorylation events triggered by either ERK, JNK or p38 downregulate the transcriptional repressor function of Drosophila Yan or of vertebrate Tel, leading to their export out of the nucleus and to their degradation [22][23][24][25][26] (reviewed in [27]). Mutations that convert the serine and threonines residues normally phosphorylated by MAPK into non-phosphorylatable residues transform these factors into constitutively active repressors while mutations that convert them into phospho-mimetic residues promote degradation of these factors. ...
In the sea urchin embryo, specification of the dorsal-ventral axis critically relies on the spatially restricted expression of nodal in the presumptive ventral ectoderm. The ventral restriction of nodal expression requires the activity of the maternal TGF-beta ligand Panda but the mechanism by which Panda restricts nodal expression is unknown. Similarly, what initiates expression of nodal in the ectoderm and what are the mechanisms that link patterning along the primary and secondary axes is not well understood. We report that in Paracentrotus lividus, the activity of the maternally expressed ETS-domain transcription factor Yan/Tel is essential for the spatial restriction of nodal. Inhibiting translation of maternal yan/tel mRNA disrupted dorsal-ventral patterning in all germ layers by causing a massive ectopic expression of nodal starting from cleavage stages, mimicking the phenotype caused by inactivation of the maternal Nodal antagonist Panda. We show that like in the fly or in vertebrates, the activity of sea urchin Yan/Tel is regulated by phosphorylation by MAP kinases. However, unlike in the fly or in vertebrates, phosphorylation by GSK3 plays a central role in the regulation Yan/Tel stability in the sea urchin. We show that GSK3 phosphorylates Yan/Tel in vitro at two different sites including a β-TRCP ubiquitin ligase degradation motif and a C-terminal Ser/Thr rich cluster and that phosphorylation of Yan/Tel by GSK3 triggers its degradation by a β-TRCP/proteasome pathway. Finally, we show that, Yan is epistatic to Panda and that the activity of Yan/Tel is required downstream of Panda to restrict nodal expression. Our results identify Yan/Tel as a central regulator of the spatial expression of nodal in Paracentrotus lividus and uncover a key interaction between the gene regulatory networks responsible for patterning the embryo along the dorsal-ventral and animal-vegetal axes.
... For instance, a complex of transcription factors and an RNA polymerase might stabilize a promoter-enhancer loop; upon transcription termination, the complex could dissociate and the loop disappear [2,3]. Alternatively, phosphorylation, or other post-translational modifications of transcription factors [27], may affect their affinity for chromatin, as might a conformational change in a protein or the reversible addition of a sub-unit to a protein complex, which might be driven by ATP hydrolysis. ...
... To conclude, we have shown that active post-translational protein modication (e.g., phosphorylation, methylation, acetylation [27], or any other non-equilibrium reaction where a protein switches between binding and non-binding states) has a profound and generic effect on the behaviour of a chromatin-protein mixture. The interplay between protein bridging and protein modification is therefore an important principle underlying nuclear organization within eukaryotes. ...
Fluorescence microscopy reveals that the contents of many (membrane-free) nuclear “bodies” exchange rapidly with the soluble pool whilst the underlying structure persists; such observations await a satisfactory biophysical explanation. To shed light on this, we perform large-scale Brownian dynamics simulations of a chromatin fiber interacting with an ensemble of (multivalent) DNA-binding proteins; these proteins switch between two states – active (binding) and inactive (non-binding). This system provides a model for any DNA-binding protein that can be modified post-translationally to change its affinity for DNA (e.g., like the phosphorylation of a transcription factor). Due to this out-of-equilibrium process, proteins spontaneously assemble into clusters of self-limiting size, as individual proteins in a cluster exchange with the soluble pool with kinetics like those seen in photo-bleaching experiments. This behavior contrasts sharply with that exhibited by “equilibrium”, or non-switching, proteins that exist only in the binding state; when these bind to DNA non-specifically, they form clusters that grow indefinitely in size. Our results point to post-translational modification of chromatin-bridging proteins as a generic mechanism driving the self-assembly of highly dynamic, non-equilibrium, protein clusters with the properties of nuclear bodies. Such active modification also reshapes intra-chromatin contacts to give networks resembling those seen in topologically-associating domains, as switching markedly favors local (short-range) contacts over distant ones.
... Members of the ETS (E twenty six),a transcription factor super family, operating as a dynamic and reversible sensor of upstream signaling events, may provide a way to modulate all facets of transcription factor function. 37 In metazoans transcription factors ETS are conserved and, during development, play a major role, which function as own stream effectors of signal transduction cascades to control the broad spectrum of cell processes. ETS play a vital role in regulation of cell differentiation, proliferation, apoptosis, migration, and epithelial-mesenchymal interactions during normal development; whereas mis-regulated ETS proteins, by a variety of mechanisms, lead to both the initiation and progression of many human cancers. ...
... ETS play a vital role in regulation of cell differentiation, proliferation, apoptosis, migration, and epithelial-mesenchymal interactions during normal development; whereas mis-regulated ETS proteins, by a variety of mechanisms, lead to both the initiation and progression of many human cancers. 37 ETS transcription factors are formed of a vastly conserved, 85 amino acid motif called the ETS domain, which belongs to the super family of helix-turn helix (HTH) DNA binding domains and, by a recognition sequence GGAA/T, is referred to as the ETS binding site (EBS). There are variable sequences for EBS, which subsidize to the individual specificity of individual ETS transcription factors, which are approximately 8 in drosophila and 30 in mammals. ...
... Pathway analysis of the microarray data revealed that perturbation of Ets-1 activity is another likely contributor to the phenotypes exhibited by Gam1 embryos [91][92][93][94]. The regulation of Ets-1 during development is complex and occurs through the integration of multiple types of post-translational modification: phosphorylation, acetylation, ubiquitination, and SUMOylation [92,94,95]. ...
... Pathway analysis of the microarray data revealed that perturbation of Ets-1 activity is another likely contributor to the phenotypes exhibited by Gam1 embryos [91][92][93][94]. The regulation of Ets-1 during development is complex and occurs through the integration of multiple types of post-translational modification: phosphorylation, acetylation, ubiquitination, and SUMOylation [92,94,95]. Furthermore, Ets factors can either enhance or repress transcription [96]. ...
Background
Adenovirus protein, Gam1, triggers the proteolytic destruction of the E1 SUMO-activating enzyme. Microinjection of an empirically determined amount of Gam1 mRNA into one-cell Xenopus embryos can reduce SUMOylation activity to undetectable, but nonlethal, levels, enabling an examination of the role of this post-translational modification during early vertebrate development.
Results
We find that SUMOylation-deficient embryos consistently exhibit defects in neural tube and heart development. We have measured differences in gene expression between control and embryos injected with Gam1 mRNA at three developmental stages: early gastrula (immediately following the initiation of zygotic transcription), late gastrula (completion of the formation of the three primary germ layers), and early neurula (appearance of the neural plate). Although changes in gene expression are widespread and can be linked to many biological processes, three pathways, non-canonical Wnt/PCP, snail/twist, and Ets-1, are especially sensitive to the loss of SUMOylation activity and can largely account for the predominant phenotypes of Gam1 embryos. SUMOylation appears to generate different pools of a given transcription factor having different specificities with this post-translational modification involved in the regulation of more complex, as opposed to housekeeping, processes.
Conclusions
We have identified changes in gene expression that underlie the neural tube and heart phenotypes resulting from depressed SUMOylation activity. Notably, these developmental defects correspond to the two most frequently occurring congenital birth defects in humans, strongly suggesting that perturbation of SUMOylation, either globally or of a specific protein, may frequently be the origin of these pathologies.
Electronic supplementary material
The online version of this article (10.1186/s12864-019-5773-3) contains supplementary material, which is available to authorized users.
... HEK293) (9,10). Consequently, these approaches do not assay the impact of cell-specific post-translational modifications (PTMs), which are known to have diverse effects on TF binding and function (11,12), and do not account for the impact of cell-specific cofactors that can bind cooperatively with TFs. ...
... HT throughput methods for characterizing TF-DNA binding provide critical biophysical data for genomic analyses of gene regulation (1-3). Cell-specific PTMs (11,12) and cofactors (1,44) can affect TF binding, but are not implicitly accounted for in current HT methods. Here we describe the nextPBM methodology for the characterization of protein-DNA binding that uses nuclear extracts to account for the impact of cell-specific PTMs and cofactors. ...
High-throughput (HT) in vitro methods for measuring protein-DNA binding have become invaluable for characterizing transcription factor (TF) complexes and modeling gene regulation. However, current methods do not utilize endogenous proteins and, therefore, do not quantify the impact of cell-specific post-translational modifications (PTMs) and cooperative cofactors. We introduce the HT nextPBM (nuclear extract protein-binding microarray) approach to study DNA binding of native cellular TFs that accounts for PTMs and cell-specific cofactors. We integrate immune-depletion and phosphatase treatment steps into our nextPBM pipeline to characterize the impact of cofactors and phosphorylation on TF binding. We analyze binding of PU.1/SPI1 and IRF8 from human monocytes, delineate DNA-sequence determinants for their cooperativity, and show how PU.1 affinity correlates with enhancer status and the presence of cooperative and collaborative cofactors. We describe how nextPBMs, and our accompanying computational framework, can be used to discover cell-specific cofactors, screen for synthetic cooperative DNA elements, and characterize TF cooperativity.
... Current HT methods use either purified or in vitro produced protein (Badis et al., 2009;Berger et al., 2006a;Slattery et al., 2011), or tagged protein overexpressed in cells (e.g., HEK293) (Fang et al., 2012;Jolma et al., 2013). Consequently, these approaches do not assay the impact of cell-specific post-translational modifications (PTMs), which are known to have diverse effects on TF binding and function (Filtz et al., 2014;Tootle and Rebay, 2005). Furthermore, these approaches do not implicitly account for the impact of cell-specific cofactors that can bind cooperatively with a target TF to affect its DNA binding. ...
... Relating biophysical principles of TF binding to in vivo occupancy is critical to a mechanistic view of gene regulation. Cell-specific PTMs (Filtz et al., 2014;Tootle and Rebay, 2005) and cofactors (Garvie and Wolberger, 2001;Siggers and Gordân, 2013) can affect TF binding, but are not implicitly accounted for in current HT methods for analyzing protein-DNA binding. Here we describe the nextPBM methodology for the HT characterization of protein-DNA binding that accounts for cell-specific PTMs and cofactors. ...
Determining the biophysical principles that shape transcription factor (TF) binding in a cell-specific manner is key to quantitative models of gene expression. High-throughput (HT) in vitro methods measuring protein-DNA binding are invaluable for relating TF binding affinity to genome-wide binding; however, the impact of cell-specific post-translational modifications (PTMs) and cofactors are not routinely assessed. To address these limitations, we describe a new HT approach, called nextPBMs (nuclear extract protein-binding microarrays), to characterize TF binding that accounts for PTMs and endogenous cofactors. We use nextPBMs to examine the DNA binding of the lineage factor PU.1/Spi1 and IRF8 in human monocytes. We identify two binding modes for PU.1 in monocytes - autonomous binding unaffected by PTMs and cooperative binding with IRF8, and identify a single cooperative mode for IRF8. We characterize the DNA binding of PU.1:IRF8 complexes, and show how nextPBMs can be used to discover cell-specific cofactors and characterize TF cooperativity at single-nucleotide resolution. We show that chromatin state and cofactors both influence the affinity requirements for PU.1 binding sites. Furthermore, we find that the influences of cooperative (IRF8) and collaborative (C/EBPα) cofactors on PU.1-binding site affinity are independent and additive.
... To enable the identification of single DR3 variants exhibiting improved TL1A binding affinity, we sub-cloned the enriched library from the yeast surface display-based screening into a mammalian vector fused to the sequences encoding an N-terminal leader peptide and a C-terminal human IgG1 Fc. It was previously shown that the extracellular domain of many receptors is heavily glycosylated and that glycosylation can contribute to receptor binding to the target ligand [31]. Thus, the mammalian-based expression of soluble DR3 receptor offers the advantage of subjecting the native protein to post-translational modifications, including glycosylation. ...
... As demonstrated before, the ECD of many receptors is highly glycosylated and such glycosylation can significantly contribute to receptor conformation and binding to the target ligand [31]. Thus, an important component of our screening strategy involved the expression of the DR3 mutants in mammalian cells so as to maintain receptor glycosylation. ...
TNF-like 1A (TL1A) is a cytokine belonging to the TNF superfamily that promotes inflammation in autoimmune diseases. Inhibiting the interaction of TL1A with the endogenous death-domain receptor 3 (DR3) offers a therapeutic approach for treating TL1A-induced autoimmune diseases. Here, we generated improved DR3 variants showing increased TL1A binding affinity and stability using a directed evolution approach. Given the high cysteine content and post-translational modification of DR3, we employed yeast surface display and expression in mammalian cell lines for screening, expression and characterization of improved DR3 variants. A cell-based assay performed with the human TF-1 cell line and CD4⁺ T cells showed that two improved DR3 mutants efficiently inhibited TL1A-induced cell death and secretion of IFN-γ, respectively. These DR3 mutants can be used as drug candidates for the treatment of inflammatory bowel diseases and for other autoimmune diseases, including rheumatic arthritis and asthma.
... In some cases, the formation of the composition of the metabolome can be an event that determines ontogeny and the implementation of the genetic program of the organism. Thus, the reduction of the vernalization period or its complete absence turns winter wheat into "grass by grass" [87,90]. ...
The result of the genotype/environment (G/E) interaction affects the success of the implementation of the genetic program of a plant biological system of any level, from a cell population to a multicellular organism. During this interaction, the plant system absorbs trophic and energy resources, processes and assimilates them. Under normal conditions, signal perception and transduction occurs against the background of homeostasis regulated by the genome. Genetic control is exercised at all stages of growth and development of plant systems via differential gene expression. The activity of metabolism is coordinated by the cooparated action of the ionome, proteome, metabolome, and transcriptome. Direct and cross connections between these aspects of life activity are established and developed constantly and manifest themselves in the form of dynamic phenotypic effects from structural formations and enzyme chains. Disturbanses within the individual stages of metabolism and the disconnection between them reveal differences between stable, sensitive and unstable forms. The obtained information is the basis for experiments to obtain forms with improved characteristics. A range of tasks has been outlined in this direction, and there have already been significant developments. Comparison of the dynamics of the functioning of creative variants of plant systems of any level showed their significant differences from the original forms. Changes in creative systems are determined by the interactions of transgenes with endogenous genes and can manifest themselves in the form of positive/negative/combined characteristics of the new system. Comparative studies of the dynamics of vital activity will provide information about the coordinated process of communication both within the cell and between the tissues of a multicellular organism. The use of various combinations of “omic tools” will facilitate the discovery of new promising candidates among structural and regulatory genes, as well as among promoters. On the other hand, the obtained biological information will be a stimulus for improving the methods and directions of research.
... Sixteen out of the 24 significantly enriched transcription factors identified by TRANSFAC analysis were within the ETS family, indicating enrichment for ETS-factors, such as GABP, above expected levels (chi-square, p < 8.52 × 10 −30 ). Though the ETS transcription factors were not differentially expressed themselves, they are known to be regulated by post-translational modifications [33]. Although there are fewer C250T cell lines in the DepMap dataset, analysis of C250T vs TPM-cells also demonstrated that ETS-factors are important mediators of DEG in C250T cell lines (PPI p < 0.00007, chi-square, p = 1.44 × 10 −16 , Fig. 5d-f). ...
Pooled genetic screens represent a powerful approach to identify vulnerabilities in cancer. Here we used pooled CRISPR/Cas9-based approaches to identify vulnerabilities associated with telomerase reverse transcriptase (TERT) promoter mutations (TPMs) found in >80% of glioblastomas. We first developed a platform to detect perturbations that cause long-term growth defects in a TPM-mutated glioblastoma cell line. However, we could not detect dependencies on either TERT itself or on an E-twenty six transcription (ETS) factor known to activate TPMs. To explore this finding, we cataloged TPM status for 441 cell lines and correlated this with genome-wide screening data. We found that TPM status was not associated with differential dependency on TERT, but that E-twenty six (ETS) transcription factors represent key dependencies in both TPM+ and TPM- lines. Further, we found that TPMs are associated with expression of gene programs regulated by a wide array of ETS-factors in both cell lines and primary glioblastoma tissues. This work contributes a unique TPM cell line reagent, establishes TPM status for many deeply-profiled cell lines, and catalogs TPM-associated vulnerabilities. The results highlight challenges in executing genetic screens to detect TPM-specific vulnerabilities, and suggest redundancy in the genetic network that regulates TPM function with therapeutic implications.
... These nucleoside triphosphatases (NTPases) hydrolyze nucleoside triphosphates (NTP) such as ATP, thereby switching to an NDP-bound state, and restore their NTP-bound conformation by nucleotide exchange (28). For example, specialized proteins actively modify DNA-bound histones and transcription factors via nonequilibrium reactions (23,29,30). In the context of eukaryotes, active processes are enriched in transcriptionally active euchromatin compared to inactive heterochromatin. ...
From proteins to chromosomes, polymers fold into specific conformations that control their biological function. Polymer folding has long been studied with equilibrium thermodynamics, yet intracellular organization and regulation involve energy-consuming, active processes. Signatures of activity have been measured in the context of chromatin motion, which shows spatial correlations and enhanced subdiffusion only in the presence of adenosine triphosphate. Moreover, chromatin motion varies with genomic coordinate, pointing toward a heterogeneous pattern of active processes along the sequence. How do such patterns of activity affect the conformation of a polymer such as chromatin? We address this question by combining analytical theory and simulations to study a polymer subjected to sequence-dependent correlated active forces. Our analysis shows that a local increase in activity (larger active forces) can cause the polymer backbone to bend and expand, while less active segments straighten out and condense. Our simulations further predict that modest activity differences can drive compartmentalization of the polymer consistent with the patterns observed in chromosome conformation capture experiments. Moreover, segments of the polymer that show correlated active (sub)diffusion attract each other through effective long-ranged harmonic interactions, whereas anticorrelations lead to effective repulsions. Thus, our theory offers nonequilibrium mechanisms for forming genomic compartments, which cannot be distinguished from affinity-based folding using structural data alone. As a first step toward exploring whether active mechanisms contribute to shaping genome conformations, we discuss a data-driven approach.
... Likely the necessity to maintain a manageable genome size led to the evolution of other strategies consenting cells to re-use the same TF in multiple ways. One strategy consists in post-translationally modifying TFs to modulate their stability, localization as well as affinity for the DNA, co-activators and/or the GTFs (13,14). In the past decade, another cellular strategy emerged as an effective way of achieving multiplexing: controlling TF dynamics, that is, the time-resolved activity of the TF (10,15,16). ...
In response to different stimuli many transcription factors (TFs) display different activation dynamics that trigger the expression of specific sets of target genes, suggesting that promoters have a way to decode dynamics. Here, we use optogenetics to directly manipulate the nuclear localization of a synthetic TF in mammalian cells without affecting other processes. We generate pulsatile or sustained TF dynamics and employ live cell microscopy and mathematical modelling to analyse the behaviour of a library of reporter constructs. We find decoding of TF dynamics occurs only when the coupling between TF binding and transcription pre-initiation complex formation is inefficient and that the ability of a promoter to decode TF dynamics gets amplified by inefficient translation initiation. Using the knowledge acquired, we build a synthetic circuit that allows obtaining two gene expression programs depending solely on TF dynamics. Finally, we show that some of the promoter features identified in our study can be used to distinguish natural promoters that have previously been experimentally characterized as responsive to either sustained or pulsatile p53 and NF-κB signals. These results help elucidate how gene expression is regulated in mammalian cells and open up the possibility to build complex synthetic circuits steered by TF dynamics.
... Combinatorial actions of cell-type specific and ubiquitous transcription factors (TFs) and chromatin modifiers at distinct genomic loci generate temporally and spatially restricted gene expression profiles (6). The activity of TFs can be modulated by divergent signaling pathways that mediate the impact of extracellular cues and/or the endogenous microenvironment via post-translational modifications (PTMs) (7)(8)(9). PTMs offer a rapid, effective and flexible means to integrate diverse cellular responses by altering the function of TFs, with phosphorylation representing a key regulatory switch in signal transduction (10,11). Misregulation of transcriptional machinery can lead a plethora of diseases including cancer, diabetes and developmental disorders (12,13). ...
Maf-family basic motif leucine zipper protein NRL specifies rod photoreceptor cell fate during retinal development and, in concert with homeodomain protein CRX and other regulatory factors, controls the expression of most rod-expressed genes including the visual pigment gene Rhodospin (Rho). Transcriptional regulatory activity of NRL is modulated by post-translational modifications (PTMs), especially phosphorylation, and mutations at specific phosphosites can lead to retinal degeneration. During our studies to elucidate NRL-mediated transcriptional regulation, we identified Protein Kinase CK2 in NRL-enriched complexes bound to Rho promoter-enhancer regions and in NRL-enriched high molecular mass fractions from the bovine retina. The presence of CK2 in NRL complexes was confirmed by co-immunoprecipitation from developing and adult mouse retinal extracts. In vitro kinase assay and bioinformatic analysis indicated phosphorylation of NRL at Ser117 residue by CK2. Co-transfection of Csnk2a1 cDNA encoding murine CK2 with human NRL and CRX reduced the bovine Rho promoter-driven luciferase expression in HEK293 cells and mutagenesis of NRL-Ser117 residue to Ala restored the reporter gene activity. In concordance, over-expression of CK2 in the mouse retina in vivo by electroporation resulted in reduction of Rho promoter-driven DsRed reporter expression as well as the transcript level of many phototransduction genes. Thus, our studies demonstrate that CK2 can phosphorylate Ser117 of NRL. Modulation of NRL activity by CK2 suggests intricate inter-dependence of transcriptional and signaling pathways in maintaining rod homeostasis.
... Among them, the varieties with shorter culture periods to form fruiting bodies had strong expression of the 14-kDa protein in the primordial or fruiting body stage. Previous findings demonstrated that the modification of a transcription factor changed its activity (Klenova et al. 1997;Tootle and Rebay 2005). Therefore, it is conceivable that the 14-kDa protein may be produced from a 56-kDa protein through protease digestion. ...
The cDNA library prepared from Lentinula edodes , Hokken 600 (H600), primordia was screened using cDNA expressed specifically in Dictyostelium discoideum prestalk as a probe. Twenty-one clones, Le-Dd1 ~ 21, were isolated from the L. edodes primordia cDNA library. Functional analysis of each gene was carried out by transformation into protoplast cells from L. edodes Mori 252 (M252) mycelia with the overexpression vector pLG-RasF1 of each gene because M252 protoplast cells were transformed with an 11-fold higher efficiency than H600 cells. Transformants with the overexpression vector of Le-Dd10 formed a fruiting body at almost the same time as H600, a positive control, although M252, a negative control, did not form a fruiting body under culture conditions. This suggested that Le-Dd10 is involved in the formation of fruiting bodies. Single-strand conformation polymorphism analysis revealed that Le-Dd10 is located on No. 4 linkage group of L. edodes . The properties of Le-Dd10 products were investigated by Western blotting analysis using polyclonal antibodies against GST:Le-Dd10 fusion proteins. As a result, 56-kDa, 27-kDa, and 14-kDa protein bands appeared in primordial and fruiting body stages, although the expected molecular weight of the Le-Dd10 product was 50 kDa.
... Functions of TFs are usually modulated by their regulatory elements that bind to cofactors and metabolite molecules (Xie et al., 2006;Puga et al., 2014;Le et al., 2016;Qi et al., 2017;Zhu et al., 2019;Wang et al., 2020) or by post-translational modifications (Gill, 2003;Tootle and Rebay, 2005;Schütze et al., 2008). Therefore, we hypothesized that the underlying mechanism related to nitrogen status-dependent CmMYB1 function could be revealed by the identification of the CmMYB1 regulatory element. ...
Nitrogen assimilation is an essential process that controls plant growth and development. Plant cells adjust the transcription of nitrogen assimilation genes through transcription factors (TFs) to acclimatize to changing nitrogen levels in nature. However, the regulatory mechanisms of these TFs under nitrogen-repleted (+N) conditions in plant lineages remain largely unknown. Here, we identified a negative domain (ND) of CmMYB1, the nitrogen-depleted (−N)-activated TF, in a unicellular red alga Cyanidioschyzon merolae. The ND deletion changed the localization of CmMYB1 from the cytoplasm to the nucleus, enhanced the binding efficiency of CmMYB1 to promoters of nitrate assimilation genes, and increased the transcripts of nitrate assimilation genes under +N condition. A pull-down assay using an ND-overexpressing strain combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis helped us to screen and identify an unknown-function protein, the CmNDB1. Yeast two-hybrid analysis demonstrated that CmNDB1 interacts with ND. Similar to ND deletion, CmNDB1 deletion also led to the nucleus localization of CmMYB1, enhanced the promoter-binding ratio of CmMYB1 to the promoter regions of nitrate assimilation genes, and increased transcript levels of nitrate assimilation genes under +N condition. Thus, these presented results indicated that ND and CmNDB1 negatively regulate CmMYB1 functions under the +N condition in C. merolae.
... Among them, the varieties with shorter culture periods to form fruiting bodies had strong expression of the 14-kDa protein in the primordial or fruiting body stage. There is evidence that a modi cation of a transcription factor changes its function (Klenova et al. 1997;Tootle and Rebay 2005). It is conceivable that the 14-kDa protein may be produced from a 56-kDa protein through protease digestion. ...
The cDNA library prepared from Lentinula edodes , Hokken 600 (H600), primordia was screened by using cDNA expressed specifically in Dictyostelium discoideum prestalk as a probe. Twenty-one clones, Le-Dd 1~21, were isolated from the L. edodes primordia cDNA library. Functional analysis of each gene was carried out by transformation into protoplast cells from L. edodes Mori 252 (M252) mycelia with the overexpression vector pLG-RasF1 of each gene because M252 protoplast cells were transformed with 11-fold higher efficiency than H600 cells. Transformants with the overexpression vector of Le-Dd10 formed a fruiting body at almost the same time as H600, a positive control, although M252, a negative control, did not form a fruiting body under culture conditions. This suggested that Le-Dd10 is involved in the formation of fruiting bodies. Single-strand conformation polymorphism analysis revealed that Le-Dd10 is located on No. 4 linkage group of L. edodes . The properties of Le-Dd10 products were investigated by Western blotting analysis using polyclonal antibodies against GST:Le-Dd10 fusion proteins. As a result, 56-kDa, 27-kDa, and 14-kDa protein bands appeared in primordial and fruiting body stages, although the expected molecular weight of the Le-Dd10 product was 50 kDa.
... The various codes described by Trifonov is compatible with other approaches and his conclusion may be generally valid. Using Trifonov's definition, we can define at least 8 sequence codes in genomes of living creatures, along with the classic triplet code [307] • The translation pausing code • The Histone Code [309][310][311][312] • The Adhesive Code [313,314] • A Nuclear Receptors Combinatorial Code [315] • A Regulatory Code in mammalian organogenesis [316] • The RNA Codes [317] • The Error-Correcting Codes [318,319] • The Neural Transcriptional Codes [320,321] • The Signal Transduction Codes [322] • A Code of Post Translational Modifications [323] • The Musical Code [324] • The Sugar Code [325,326] • An Acetylation Code [327] • The Codes of Language [328] • A Neural Code for written words [329] • The Metabolic Codes [330] • A Transcription Factors Code [331] • The Immune Self Code [332] • The Modular Code of the Cytoskeleton [333] • An Estrogen Receptor Code [334] • A Lipid-based Code in nuclear signaling [335] These discoveries have shown how complex life is. Genetic codes prove that cells are semiotic systems and organic codes bring to light insights into evolutionary pathways. ...
... Protein phosphorylation is a well-known regulatory event that plays a key role in the control of multiple functions of viral and cellular proteins [41][42][43]. Modulation of protein functions can affect multiple mechanisms including regulation of subcellular localization, protein-protein interactions, protein stability, and protein-nucleic acid binding, as previously reported for viruses such as varicella-zoster virus [44], human papillomavirus [45], rotavirus [46], rabies virus [47], and hepatitis C virus [48,49], among several others [43]. For the Hepadnaviridae family members, protein phosphorylation is a central event during the replication of the DNA genome. ...
Hepatitis B virus (HBV) is a circular, and partially double-stranded DNA virus. Upon infection, the viral genome is translocated into the cell nucleus, generating the covalently closed circular DNA (cccDNA) intermediate, and forming a mini chromosome. HBV HBx is a small protein displaying multiple roles in HBV-infected cells, and in different subcellular locations. In the nucleus, the HBx protein is required to initiate and maintain viral transcription from the viral mini chromosome. In contrast, HBx also functions in the cytoplasm, where it is able to alter multiple cellular functions such as mitochondria metabolism, apoptosis and signal transduction pathways. It has been reported that in cultured cells, at low expression levels, the HBx protein is localized in the nucleus, whereas at high expression levels, it accumulates in the cytoplasm. This dynamic subcellular distribution of HBx might be essential to exert its multiple roles during viral infection. However, the mechanism that regulates different subcellular localizations of the HBx protein is unknown. We have previously taken a bioinformatics approach to investigate whether HBx might be regulated via post-translational modification, and we have proposed that the multiple nucleocytoplasmic functions of HBx might be regulated by an evolutionarily conserved mechanism via phosphorylation. In the current study, phylogenetically conserved amino acids of HBx with a high potential of phosphorylation were targeted for site-directed mutagenesis. Two conserved serine (Ser25 and Ser41), and one conserved threonine (Thr81) amino acids were replaced by either alanine or aspartic acid residues to simulate an unphosphorylated or phosphorylated state, respectively. Human hepatoma cells were transfected with increasing amounts of the HBx DNA constructs, and the cells were analyzed by fluorescence microscopy. Together, our results show that the nucleocytoplasmic distribution of the HBx protein could be regulated by phosphorylation since some of the modified proteins were mainly confined to distinct subcellular compartments. Remarkably, both HBx Ser41A, and HBx Thr81D proteins were predominantly localized within the nuclear compartment throughout the different expression levels of HBx mutants.
... Phosphorylation is a common reversible regulatory event with a central role in controlling trans-acting/transcription (23). Earlier studies have demonstrated that Tax is a phosphoprotein (24) and that phosphorylation can regulate Tax activation of the HTLV-1 LTR (25,26). ...
The human T-cell leukemia virus type 1 oncoprotein Tax is a phosphoprotein with a predominately nuclear subcellular localization that accomplishes multiple functions via protein-protein interactions. It has been proposed that regulation of this protein’s pleiotropic functions may be accomplished through phosphorylation of specific amino acid residues. We have conducted a phosphoryl mapping of mammalian-expressed Tax protein using a combination of affinity purification, liquid chromatography tandem mass spectrometry, and site-directed substitution mutational analysis. We achieved physical coverage of 77% of the Tax sequence and identified four novel sites of phosphorylation at Thr-48, Thr-184, Thr-215, and Ser-336. Previously identified potential serine phosphorylation sites at Ser-10, Ser-77, and Ser-274 could not be confirmed by mass spectrometry. The functional significance of these novel phosphorylation events was evaluated by mutational analysis and subsequent evaluation for activity via both CREB and NF-κB-responsive promoters. Our results demonstrate that phosphorylation at Thr-215 is associated with loss of both Tax functions, phosphorylation at Thr-48 was specifically deficient for activation via NF-κB, and phosphorylation at Thr-184 and Ser-336 had no effect on these Tax functions. Semiquantitation of phosphopeptides revealed that the majority of Tax was phosphorylated at Thr-48, Thr-184, Thr-215, and Ser-336, whereas only a minor population of Tax was phosphorylated at either Ser-300 or Ser-301. These results suggest that both positive and negative phosphorylation signals result in the maintenance of a subfraction of Tax as “active” protein.
... As transcription factors have the capability of reconfiguring cellular physiology and function, their regulation through molecular modification is essential to proper function. Post-translational modifications (PTMs) play a large role in modulating protein stability, protein-protein interaction, DNA-binding, subcellular localization and so on (174). Many of these PTMs occur as individual, isolated events to dictate some aspect of transcription factor function, though some PTMs are sequentially linked, enabling (or inhibiting) one another (175). ...
Over the past couple of decades, lymphatics research has accelerated and gained a much-needed recognition in pathophysiology. As the lymphatic system plays heavy roles in interstitial fluid drainage, immune surveillance and lipid absorption, the ablation or excessive growth of this vasculature could be associated with many complications, from lymphedema to metastasis. Despite their growing importance in cancer, few anti-lymphangiogenic therapies exist today, as they have yet to pass phase 3 clinical trials and acquire FDA approval. As such, many studies are being done to better define the signaling pathways that govern lymphangiogenesis, in hopes of developing new therapeutic approaches to inhibit or stimulate this process. This review will cover our current understanding of the Ras signaling pathways and their interactions with Prox1, the master transcriptional switch involved in specifying lymphatic endothelial cell fate and lymphangiogenesis, in hopes of providing insights to lymphangiogenesis-based therapies.
... There are multiple ways in which we can interfere with the functionality of TFs, including altering the absolute abundance of a given TF, either by regulating how much of the protein is being produced or by regulating proteolytic degradation. Another approach is to alter the relative abundance of TFs in the nucleus (where a TF is active) by modulating post-translational modifications, such as sumoylation and phosphorylation [16][17][18], that affect nuclear shuttling. However, these strategies do not physically target TFs per se and are therefore subject to the limitation of drugging conventional enzyme targets in upstream cell signalling. ...
In the post-genome era, pathologies become associated with specific gene expression profiles and defined molecular lesions can be identified. The traditional therapeutic strategy is to block the identified aberrant biochemical activity. However, an attractive alternative could aim at antagonizing key transcriptional events underlying the pathogenesis, thereby blocking the consequences of a disorder, irrespective of the original biochemical nature. This approach, called transcription therapy, is now rendered possible by major advances in biophysical technologies. In the last two decades, techniques have evolved to become key components of drug discovery platforms, within pharmaceutical companies as well as academic laboratories. This review outlines the current biophysical strategies for transcription manipulation and provides examples of successful applications. It also provides insights into the future development of biophysical methods in drug discovery and personalized medicine.
... In breast cancer, several lineage TFs have been identified as mediators of tumor progression. Although the expression of TFs may be a surrogate for their function, there are many epigenetic processes that ultimately affect their activity [81,82]. Chromatin accessibility has been successfully used to identify the functional effects of pioneering TFs [83,84]. ...
Background:
Few somatic mutations have been linked to breast cancer metastasis, whereas transcriptomic differences among primary tumors correlate with incidence of metastasis, especially to the lungs and brain. However, the epigenomic alterations and transcription factors (TFs) which underlie these alterations remain unclear.
Methods:
To identify these, we performed RNA-seq, Chromatin Immunoprecipitation and sequencing (ChIP-seq) and Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) of the MDA-MB-231 cell line and its brain (BrM2) and lung (LM2) metastatic sub-populations. We incorporated ATAC-seq data from TCGA to assess metastatic open chromatin signatures, and gene expression data from human metastatic datasets to nominate transcription factor biomarkers.
Results:
Our integrated epigenomic analyses found that lung and brain metastatic cells exhibit both shared and distinctive signatures of active chromatin. Notably, metastatic sub-populations exhibit increased activation of both promoters and enhancers. We also integrated these data with chromosome conformation capture coupled with ChIP-seq (HiChIP) derived enhancer-promoter interactions to predict enhancer-controlled pathway alterations. We found that enhancer changes are associated with endothelial cell migration in LM2, and negative regulation of epithelial cell proliferation in BrM2. Promoter changes are associated with vasculature development in LM2 and homophilic cell adhesion in BrM2. Using ATAC-seq, we identified a metastasis open-chromatin signature that is elevated in basal-like and HER2-enriched breast cancer subtypes and associates with worse prognosis in human samples. We further uncovered TFs associated with the open chromatin landscapes of metastatic cells and whose expression correlates with risk for metastasis. While some of these TFs are associated with primary breast tumor subtypes, others more specifically correlate with lung or brain metastasis.
Conclusions:
We identify distinctive epigenomic properties of breast cancer cells that metastasize to the lung and brain. We also demonstrate that signatures of active chromatin sites are partially linked to human breast cancer subtypes with poor prognosis, and that specific TFs can independently distinguish lung and brain relapse.
... In aop, one MAPK phosphorylation sites, S127, is required for RAS/ERK pathway responsiveness, phosphorylation at the other sites appears important for amplifying and modulating the response (Rebay & Rubin, 1995). Studies on pnt P2 by mutagenesis have indicated that phosphorylation of Thr151 is critically required for the in vivo function (Tootle & Rebay, 2005;Lau et al., 2012). To our knowledge no study has searched for edl phosphorylation, thus, this potential edl modification type deserves further investigations. ...
Drosophila melanogaster is a main model in biology, notably immunity and evolution. Although the Drosophila innate immune processes to fight bacteria and fungi have largely been explored, less is known of the defence against endoparasitoid wasps whose successful development inside the insect host leads to its death. One of the most studied Drosophila – parasitoid wasp interaction involves Leptopilina boulardi that lays eggs inside host larvae and develop at their expense. Once the parasitoid egg has been recognized as a foreign invader, the Drosophila larva can mount a successful immune response, the encapsulation: the egg is surrounded by several layers of hemocytes and there is an increase of a specific types of hemocytes, the lamellocytes. The so-formed capsule is melanised and there is formation of reactive oxygen species, which kills the parasitoid. Alternatively, the immune response can be circumvented thanks to the venom components injected by the female wasp together with the egg. Using two Drosophila strains, resistant and susceptible to L. boulardi, which differ only in a region of chromosome 2R containing a major resistance gene. The resistance was found to be monogenic, with two alleles, the resistance allele being dominant (Rlb+>Rlb). The team previously identified edl/mae (R and S alleles) as a candidate gene. Mae (Modulator of the Activity of ETS) or edl (ETS-domain lacking) was described as a mediator of specific transcription factors of the ETS (E26 transformation-specific) family in Drosophila. Mae interacts with transcription factors trough a SAM (Steril Alpha Domain), a protein – protein interaction domain. Mae is known to regulate yan and pnt P2 transcription factors during the eye development and yan and pnt P2 appear to have a role during haematopoiesis. The objectives of my thesis were to decipher the possible role of edl/mae and identify the molecular and cellular events leading to success or failure of encapsulation. I used various approaches from fly genetics to flow cytometry. The involvement of edl/mae in Drosophila resistance was confirmed by using overexpression and interference of mae expression. The overexpression of the resistant allele in a susceptible background leads to a resistant phenotype. The interference of the expression of the susceptible allele results in an increased rate of parasitoid encapsulation. At the cellular level, an increased in the number of hemocytes after parasitism occurred earlier in the resistant strain than in the susceptible strain. It was also observed that the hematopoietic lymph gland of the resistant larvae busted before the one of the susceptible larvae. At the molecular level, potential interactants of mae were identified in silico and 2 were tested using interference of their expression which led to observing an increase of encapsulation. Overall, a key player in the resistance mechanism of the Drosophila to the parasitic wasp have been identify during this work and it lays the path for future work on regulation mechanism of the response at the molecular level.
... Specifically, TF activity is regulated via post-translational modifications (PTMs) by such modification enzymes as a response to cellular stimuli (38). Modification enzymes directly interact with TFs and modify specific residues of the TF protein and alter subcellular localization, stability, interaction with more cofactors and other transcriptional activities (39). Some of the modifications those enzymes undertake are phosphorylation, acetylation, methylation and glycosylation (38). ...
... Pathway analysis identified deregulation of not only those consistent with previously established ELF3-related signaling events-for example, IL-1β, NFκB, p38, and JNK signaling in inflammation [30][31][32][33] , ETS transcription factors in MAPK signaling 34,35 , and NOTCH and WNT in cancer stem cells and colorectal cancer 14,27,36 -but other pathways that agreed with phenotypes established in our isogenic systems and pointed to previously undescribed functions. These included cell cycle, apoptosis, adhesion, and motility functions (organization, junction, adherens, cadherin), as well as AR signaling (Supplementary Fig. 12 and Supplementary Data 1). ...
Gene function in cancer is often cell type-specific. The epithelial cell-specific transcription factor ELF3 is a documented tumor suppressor in many epithelial tumors yet displays oncogenic properties in others. Here, we show that ELF3 is an oncogene in the adenocarcinoma subtype of lung cancer (LUAD), providing genetic, functional, and clinical evidence of subtype specificity. We discover a region of focal amplification at chromosome 1q32.1 encompassing the ELF3 locus in LUAD which is absent in the squamous subtype. Gene dosage and promoter hypomethylation affect the locus in up to 80% of LUAD analyzed. ELF3 expression was required for tumor growth and a pan-cancer expression network analysis supports its subtype and tissue specificity. We further show that ELF3 displays strong prognostic value in LUAD but not LUSC. We conclude that, contrary to many other tumors of epithelial origin, ELF3 is an oncogene and putative therapeutic target in LUAD. Tissue context can dictate why a gene can have seemingly opposing functions in different settings. ELF3 is tumor suppressive in many cancers of epithelial origin but in lung cancer, the authors describe an oncogenic role in the adenocarcinoma histology of non-small cell lung cancer.
... Drosophila Yan and vertebrate Tel which demonstrated that regulation is not at the transcriptional level but instead is post-transcriptionally controlled by MAP kinase phosphorylation. MAPK dependent phosphorylation of Yan and Tel is the central mechanism that regulates the activity and stability of these transcriptional repressors (Lai & Rubin, 1992;Maki et al., 2004;O'Neill, Rebay, Tjian, & Rubin, 1994;Poirel et al., 1997;Rebay & Rubin, 1995) (reviewed in (Tootle & Rebay, 2005)). Specific phosphorylation events triggered by either ERK, JNK or p38 downregulate the transcriptional repressor function of Drosophila Yan or of vertebrate Tel, leading to their nuclear export, promoting their degradation, and/or reducing DNA binding interactions. ...
Specification of the main axes of polarity of the embryo is an essential process during embryonic development. In many species, this process is achieved by the localization of maternal factors into discrete regions of the egg. However, in other animals, like in amniotes and in echinoderms, the considerable plasticity of the early blastomeres seems to preclude the existence of maternal determinants and the mechanisms by which the radial symmetry of the egg is broken remain largely enigmatic. In this chapter, we review recent progress on the identification of maternal components involved in symmetry breaking and dorsal-ventral (D/V) axis formation of the sea urchin embryo. We will first review some key experiments on D/V axis formation from classical embryologists that provided evidence for a weak maternal D/V prepattern. We will then detail more recent molecular analyses that established the critical role played by Nodal signaling in allocating cell fates along the secondary axis and led to the discovery that maternal transcription factors such as the Sry-related HMG box B1 (SoxB1), the Octamer binding factor1/2 (Oct1/2), the T-cell factor/Lymphoid enhancer-binding factor (TCF/LEF) and the Erythroblastosis virus E26 Oncogene Homolog (ETS) domain transcriptional repressor Translocation-Ets-Leukemia virus protein (Yan/Tel) as well as maternal signaling molecules like Univin are essential for the initiation of nodal expression. Finally, we will describe recent advances that uncovered a role in symmetry breaking and dorsal-ventral axis orientation for the transforming growth factor beta (TGF-beta)-like factor Panda, which appears to be both necessary and sufficient for D/V axis orientation. Therefore, even in the highly regulative sea urchin embryo, the activity of localized maternal factors provides the embryo with a blueprint of the D/V axis.
... Post-translational modifications of DNA-binding proteins can also be included by modifying their affinity to DNA [54,61]. Moreover, the bridging proteins can switch between active and inactive states at a pre-specified rate, which has been found to regulate the micro-phase separation of DNA-protein clusters [54], see Fig. 3. ...
The molecular machinery of life is largely created via self-organisation of individual molecules into functional assemblies. Minimal coarse-grained models, in which a whole macromolecule is represented by a small number of particles, can be of great value in identifying the main driving forces behind self-organisation in cell biology. Such models can incorporate data from both molecular and continuum scales, and their results can be directly compared to experiments. Here we review the state of the art of models for studying the formation and biological function of macromolecular assemblies in living organisms. We outline the key ingredients of each model and their main findings. We illustrate the contribution of this class of simulations to identifying the physical mechanisms behind life and diseases, and discuss their future developments.
... This approach overcomes several limitations of the previous TFBS prediction method such as the cell type specificities (i.e., predictions are tailored toward the underlying expression profile) and the TF coverage (i.e., regulons can be inferred for any TF whose expression varies sufficiently in the corresponding gene expression data set). Still, the approach may fail to infer TF-target interactions for TFs regulated at a molecular level other than transcription (such as posttranslational modification and protein-protein interactions) (Tootle and Rebay 2005), and their power to distinguish direct and indirect regulation is controversial (Margolin and Califano 2007;Marbach et al. 2010Marbach et al. , 2012. Taken together, currently there is no universal strategy to identify all bona fide targets of the full collection of TFs across all possible cell conditions. ...
The prediction of transcription factor (TF) activities from the gene expression of their targets (i.e., TF regulon) is becoming a widely used approach to characterize the functional status of transcriptional regulatory circuits. Several strategies and data sets have been proposed to link the target genes likely regulated by a TF, each one providing a different level of evidence. The most established ones are (1) manually curated repositories, (2) interactions derived from ChIP-seq binding data, (3) in silico prediction of TF binding on gene promoters, and (4) reverse-engineered regulons from large gene expression data sets. However, it is not known how these different sources of regulons affect the TF activity estimations and, thereby, downstream analysis and interpretation. Here we compared the accuracy and biases of these strategies to define human TF regulons by means of their ability to predict changes in TF activities in three reference benchmark data sets. We assembled a collection of TF-target interactions for 1541 human TFs and evaluated how different molecular and regulatory properties of the TFs, such as the DNA-binding domain, specificities, or mode of interaction with the chromatin, affect the predictions of TF activity. We assessed their coverage and found little overlap on the regulons derived from each strategy and better performance by literature-curated information followed by ChIP-seq data. We provide an integrated resource of all TF-target interactions derived through these strategies, with confidence scores, as a resource for enhanced prediction of TF activities.
... Upon a distinct stimulus, suitable transcription factors are posttranslationally modified and translocate to the nucleus where they can bind to their DNA target sites. Posttranslational modifications are also able to control the binding strength of TFs to the DNA and the activity of their activation/silencing domain 66,67 . ...
The expression of genetic information into proteins is a key aspect of life. The efficient and exact regulation of this process is essential for the cell to produce the correct amounts of these effector molecules to a given situation. For this purpose, eukaryotic cells have developed many different levels of transcriptional and posttranscriptional gene regulation. These mechanisms themselves heavily rely on interactions of proteins with associated nucleic acids. In the case of posttranscriptional gene regulation an orchestrated interplay between RNA-binding proteins, messenger RNAs (mRNA), and non-coding RNAs is compulsory to achieve this important function. A pivotal factor hereby are RNA secondary structures. One of the most stable and diverse representatives is the G-quadruplex structure (G4) implicated in many cellular mechanisms, such as mRNA processing and translation. In protein biosynthesis, G4s often act as obstacles but can also assist in this process. However, their presence has to be tightly regulated, a task which is often fulfilled by helicases. One of the best characterized G4-resolving factors is the DEAH-box protein DHX36. The in vitro function of this helicase is extensively described and individual reports aimed to address diverse cellular functions as well. Nevertheless, a comprehensive and systems-wide study on the function of this specific helicase was missing, so far. The here-presented doctoral thesis provides a detailed view on the global cellular function of DHX36. The binding sites of this helicase were defined in a transcriptome-wide manner, a consensus binding motif was deviated, and RNA targets as well as the effect this helicase exerts on them were examined. In human embryonic kidney cells, DHX36 is a mainly cytoplasmic protein preferentially binding to G-rich and G4-forming sequence motifs on more than 4,500 mRNAs. Loss of DHX36 leads to increased target mRNA levels whereas ribosome occupancy on and protein output of these transcripts are reduced. Furthermore, DHX36 knockout leads to higher RNA G4 levels and concomitant stress reactions in the cell. I hypothesize that, upon loss of this helicase, translationally-incompetent structured DHX36 target mRNAs, prone to localize in stress granules, accumulate in the cell. The cell reacts with basal stress to avoid cytotoxic effects produced by these mis-regulated and structured transcripts.
... Post-translational modifications of DNA-binding proteins can also be included by modifying their affinity to DNA [54,61]. Moreover, the bridging proteins can switch between active and inactive states at a pre-specified rate, which has been found to regulate the micro-phase separation of DNA-protein clusters [54], see Fig. 3. ...
The molecular machinery of life is largely created via self-organisation of individual molecules into functional assemblies. Minimal coarse-grained models, where a whole macromolecule is represented by a small number of particles, can be of great value in identifying the main driving forces behind self-organisation in cell biology. Such models can incorporate data from both molecular and continuum scales, and their results can be directly compared to experiments. Here we review the state of the art of models for studying the formation and biological function of macromolecular assemblies in cells. We outline the key ingredients of each model and their main findings. We illustrate the contribution of this class of simulations to identifying the physical mechanisms behind life and diseases, and discuss their future developments.
... Transcription factors (TFs) play major roles in the regulation of genes involved in development and tolerance to biotic and abiotic stresses (Dang et al. 2012), by serving as nuclear effectors of multiple signaling cascades and often involved in posttranslational modifications (Tootle and Rebay 2005). The analyses of the promoter are crucial for improving the basic understanding of the expressions of adaptive/stressresponsive genes and will promote their application in plant breeding. ...
Key message:
SNP alleles on chromosomes 4BL and 6AL are associated with sensitivity to salt tolerance in wheat and upon validation can be exploited in the development of salt-tolerant wheat varieties. The dissection of the genetic and molecular components of salt stress response offers strong opportunities toward understanding and improving salt tolerance in crops. In this study, GWAS was employed to identify a total of 106 SNP loci (R2 = 0.12-63.44%) linked to salt stress response in wheat using leaf chlorophyll fluorescence, grain quality and shoot ionic (Na+ and K+ ions) attributes. Among them, 14 SNP loci individually conferred pleiotropic effects on multiple independent salinity tolerance traits including loci at 99.04 cM (R2 ≥ 14.7%) and 68.45 cM (R2 ≥ 4.10%) on chromosomes 6AL and 4BL, respectively, that influenced shoot Na+-uptake, shoot K+/Na+ ratio, and specific energy fluxes for absorption (ABS/RC) and dissipation (DIo/RC). Analysis of the open reading frame (ORF) containing the SNP markers revealed that they are orthologous to genes involved in photosynthesis and plant stress (salt) response. Further transcript abundance and qRT-PCR analyses indicated that the genes are mostly up-regulated in salt-tolerant and down-regulated in salt-sensitive wheat genotypes including NRAMP-2 and OPAQUE1 genes on 4BL and 6AL, respectively. Both genes showed highest differential expression between contrasting genotypes when expressions of all the genes within their genetic intervals were analyzed. Possible cis-acting regulatory elements and coding sequence variation that may be involved in salt stress response were also identified in both genes. This study identified genetic and molecular components of salt stress response that are associated with Na+-uptake, shoot Na+/K+ ratio, ABS/RC, DIo/RC, and grain quality traits and upon functional validation would facilitate the development of gene-specific markers that could be deployed to improve salinity tolerance in wheat.
... All proteins were purified by affinity column chromatography and gel filtration as soluble proteins (Supplementary Figure 3A), yielding highly pure (95%-98%) N-TIMP2, V3, and HD N-TIMP2,V3 (Supplementary Figure 3B). It was previously shown that the extracellular domain of many receptors is highly glycosylated, and that such glycosylation can significantly contribute to the conformation of the receptor and its binding to the target ligand [47]. Indeed, using SDS-PAGE, we found that the mobility of both the bi-specific heterodimer HD N-TIMP2,V3 and V3 reflects proteins with a much higher molecular weight than predicted based on the amino acid sequence, namely, ~120 kDa instead of 76 kDa for HD N-TIMP2,V3 and ~100 kDa instead of 60 kDa for V3 (Supplementary www.oncotarget.com ...
The cytokine IL-17A is associated with the progression of various cancers, but little is known about the molecular cross-talk between IL-17A and other tumor-promoting factors. Previous studies have shown that the IL-17A-mediated invasion of breast cancer cells can be inhibited by selective antagonists of the matrix metalloproteinase 9 (MMP-9), suggesting that the cross-talk between IL-17A and MMP-9 may promote cancer invasiveness and metastasis. Here, we present a novel strategy for developing cancer therapeutics, based on the simultaneous binding and inhibition of both IL-17A and MMP-9. To this end, we use a bi-specific heterodimeric fusion protein, comprising a natural inhibitor of MMPs (N-TIMP2) fused with an engineered extracellular domain (V3) of the IL-17A receptor. We show that, as compared with the mono-specific inhibitors of IL-17A (V3) and MMP-9 (N-TIMP2), the engineered bi-specific fusion protein inhibits both MMP-9 activation and IL-17A-induced cytokine secretion from fibroblasts and exhibits a synergistic inhibition of both the migration and invasion of breast cancer cells. Our findings demonstrate, for the first time, that dual targeting of inflammatory (IL-17A) and extracellular matrix remodeling (MMP) pathways can potentially be used as a novel therapeutic approach against cancer. Moreover, the platform developed here for generating the bi-specific IL-17A/MMP-9 inhibitor can be utilized for generating bi-specific inhibitors for other cytokines and MMPs.
... regulons can be inferred for any TF whose expression varies sufficiently in the corresponding gene expression dataset). Still, the approach may fail to infer TF-target interactions for TFs regulated at a molecular level other than transcription (such as post-translational modification and protein-protein interactions) 32 and their power to distinguish direct and indirect regulation is controversial [33][34][35] . Taken together, currently there is no universal strategy to identify all bona fide targets of the full collection of TFs across all possible cell conditions. ...
Prediction of transcription factor (TF) activities from the gene expression of their targets (i.e. TF regulon) is becoming a widely-used approach to characterize the functional status of transcriptional regulatory circuits. Several strategies and datasets have been proposed to link the target genes likely regulated by a TF, each one providing a different level of evidence. The most established ones are: (i) manually curated repositories, (ii) interactions derived from ChIP-seq binding data, (iii) in silico prediction of TF binding on gene promoters, and (iv) reverse-engineered regulons from large gene expression datasets. However, it is not known how these different sources of regulons affect the TF activity estimations, and thereby downstream analysis and interpretation. Here we compared the accuracy and biases of these strategies to define human TF regulons by means of their ability to predict changes in TF activities in three reference benchmark datasets. We assembled a collection of TF-target interactions among 1,541 TFs, and evaluated how the different molecular and regulatory properties of the TFs, such as the DNA-binding domain, specificities or mode of interaction with the chromatin, affect the predictions of TF activity changes. We assessed their coverage and found little overlap on the regulons derived from each strategy and better performance by literature-curated information followed by ChIP-seq data. We provide an integrated resource of all TF-target interactions derived through these strategies with a confidence score, as a resource for enhanced prediction of TF activities.
... The change in conformation induced by phosphorylation can be involved in transcriptional regulation because it can cause a change in the nature of the binding sites affecting protein-protein and protein-DNA interactions [4]. It can also interfere with cellular localisation, activity and stability of the TF [5]. Many TFs such as members of the Ets (E26 transformation-specific) family [6], NF-IL6 and STAT3 [7] have been shown to be regulated by phosphorylation in this way. ...
FOXP2 is a transcription factor expressed in multiple tissues during embryonic development. FOXP2 regulates transcription by binding to DNA at its DNA binding domain, the forkhead domain (FHD) through the recognition helix. Ser557 is a residue located within the recognition helix that has the potential to become phosphorylated posttranslationally. In this study we investigated whether phosphorylation of Ser557 can influence the structure and DNA binding of the FOXP2 FHD. We did this by constructing S557E, a phosphomimetic mutant, and comparing its behaviour to the wild type. The mutation did not affect the secondary or tertiary structure of the protein although it did decrease the propensity of the FOXP2 FHD to form dimers. Most notably, the mutation showed significantly reduced DNA binding compared to the wild type as detected using electrophoretic mobility shift assays. Molecular docking was also performed in which the wild type, phosphomimetic mutant and phosphorylated wild-type were docked to DNA and their interactions with DNA were compared. These results indicated that the wild type forms more interactions with the DNA and that the phosphomimetic mutant as well as the phosphorylated wild type did not associate as favourably with the DNA. This indicates that phosphorylation of Ser557 could disrupt DNA binding likely due to electrostatic and steric hindrance. This suggests that phosphorylation of Ser557 in the FOXP2 FHD could act as a control mechanism for FOXP2 and ultimately could be involved in regulation of transcription.
... Protein kinase signaling pathways transduce extracellular signals that are integrated at gene promotors, transcription factors, co-regulators and chromatin proteins [1]. Therefore, transcription factors are critical for signal transduction pathways that relay information from the cell surface to the nucleus [2]. Tyrosine phosphorylation is an important mechanism for modulating biological processes such as differentiation and growth [3]. ...
Yin Yang 1 (YY1) is a multifunctional transcription factor that can activate or repress transcription depending on the promotor and/or the co-factors recruited. YY1 is phosphorylated in various signaling pathways and is critical for different biological functions including embryogenesis, apoptosis, proliferation, cell-cycle regulation and tumorigenesis. Here we report that YY1 is a substrate for c-Abl kinase phosphorylation at conserved residue Y254 in the spacer region. Pharmacological inhibition of c-Abl kinase by imatinib, nilotinib and GZD824, knock-down of c-Abl using siRNA, and the use of c-Abl kinase-dead drastically reduces tyrosine phosphorylation of YY1. Both radioactive and non-radioactive in vitro kinase assays, as well as co-immunoprecipitation in different cell lines, show that the target of c-Abl phosphorylation is tyrosine residue 254. c-Abl phosphorylation has little effect on YY1 DNA binding ability or cellular localization in asynchronous cells. However, functional studies reveal that c-Abl mediated phosphorylation of YY1 regulates YY1's transcriptional ability in vivo. In conclusion, we demonstrate the novel role of c-Abl kinase in regulation of YY1's transcriptional activity, linking YY1 regulation with c-Abl tyrosine kinase signaling pathways.
... Extensive evidence indicates that AS and PTMs of TFs commonly alter DNA-binding affinity/specificity or their interactions with cofactors in cell-or tissue-specific manner [28,[102][103][104][105]. Here we present data for one important subfamily--the nuclear factors of activated T-cells (NFATs), to show how specific combinations of IDR-localized AS and PTMs affect the detailed functions of NFATs. ...
... Runx1 plays a major role in silencing CD4 expression by binding to the intronic silencer element ( Sun et al, 1995;Hayashi et al, 2001;Taniuchi et al, 2002a). Other TFs with transactivating properties, such as Ets family members that are generally activators, can also recruit co-repressors depending on post-translational modifications (Cowley and Graves 2000;Tootle and Rebay 2005). One such example is the case of Erg-mediated repression (Yuan et al, 2009). ...
Correct gene expression is achieved by controlling the balance of transcriptional activation and repression, the latter including direct silencer-, Polycomb- and, more recently identified lncRNA- mediated repression. Here, we describe the known and possible cross-talk mechanisms within and between these modes of repression. We discuss these mechanisms within the framework of recruitment to as well as organization of the chromatin and propose possible research orientations that would aim to further resolve the links within certain of these modes of repression, notably between Polycomb-, lncRNA- and silencer-mediated silencing
... Additionally, in silico analysis of the full putative mouse Nt5e promoter reveals the presence of potential binding sites for Ets-related Elk1 and ERG transcription factors. Second, Ets-related transcription factors can be regulated by post-translational modifications such as phosphorylation [45] and glycosylation [46] that modulate their DNA-binding ability and function(s) [35]. For instance, a recent study has shown that transgenic mice overexpressing a mutant form of Ets2 transcription factor that cannot be site-specific phosphorylated are protected against experimental lung fibrosis when compared to wild-type animals [47]. ...
Hepatic fibrosis represents a pathological wound healing and tissue repair process triggered in response to chronic liver injury. A heterogeneous population of activated non-parenchymal liver cells, known as liver myofibroblasts, functions as the effector cells in hepatic fibrosis. Upon activation, liver myofibroblasts become fibrogenic, acquiring contractile properties and increasing collagen production capacity, while developing enhanced sensitivity to endogenous molecules and factors released in the local microenvironment. Hepatic extracellular adenosine is a bioactive small molecule, increasingly recognized as an important regulator of liver myofibroblast functions, and an important mediator in the pathogenesis of liver fibrosis overall. Remarkably, ecto-5'-nucleotidase/Nt5e/Cd73 enzyme, which accounts for the dominant adenosine-generating activity in the extracellular medium, is expressed by activated liver myofibroblasts. However, the molecular signals regulating Nt5e gene expression in liver myofibroblasts remain poorly understood. Here, we show that activated mouse liver myofibroblasts express Nt5e gene products and characterize the putative Nt5e minimal promoter in the mouse species. We describe the existence of an enhancer sequence upstream of the mouse Nt5e minimal promoter and establish that the mouse Nt5e minimal promoter transcriptional activity is negatively regulated by an Elf2-like Ets-related transcription factor in activated mouse liver myofibroblasts.
... Post-translational modifications such as phosphorylation have been shown with a variety of other proteins to provide a fine-tuned level of control over activity and expression (Lomelí and Vázquez, 2011;Talamillo et al., 2008;Tootle and Rebay, 2005). We have shown that phosphorylation plays important and different roles in regulating the activity of Vg during larval wing and embryonic muscle development. ...
The Drosophila vestigial gene is required for proliferation and differentiation of the adult wing and for differentiation of larval and adult muscle identity. Vestigial is part of a multi-protein transcription factor complex, which includes Scalloped, a TEAD-class DNA binding protein. Binding Scalloped is necessary for translocation of Vestigial into the nucleus. We show that Vestigial is extensively post-translationally modified and at least one of these modifications is required for proper function during development. We have shown that there is p38-dependent phosphorylation of Serine 215 in the carboxyl-terminal region of Vestigial. Phosphorylation of Serine 215 occurs in the nucleus and requires the presence of Scalloped. Comparison of a phosphomimetic and non-phosphorylatable mutant forms of Vestigial shows differences in the ability to rescue the wing and muscle phenotypes associated with a null vestigial allele.
With decades of research seeking to generalize sterile alpha motif (SAM) biology, many outstanding questions remain regarding this multi-tool protein module. Recent data from structural and molecular/cell biology has begun to reveal new SAM modes of action in cell signaling cascades and biomolecular condensation. SAM-dependent mechanisms underlie blood-related (hematologic) diseases, including myelodysplastic syndromes and leukemias, prompting our focus on hematopoiesis for this review. With the increasing coverage of SAM-dependent interactomes, a hypothesis emerges that SAM interaction partners and binding affinities work to fine tune cell signaling cascades in developmental and disease contexts, including hematopoiesis and hematologic disease. This review discusses what is known and remains unknown about the standard mechanisms and neoplastic properties of SAM domains and what the future might hold for developing SAM-targeted therapies.
Post-translational modification (PTM) is crucial for many biological events, such as the modulation of bone metabolism. Phosphorylation and O-GlcNAcylation are two examples of PTMs that can occur at the same site in the protein: serine and threonine residues. This phenomenon may cause crosstalk and possible interactions between the molecules involved. Protein phosphatase 2 A (PP2A) is widely expressed throughout the body and plays a major role in dephosphorylation. At the same location where PP2A acts, O-GlcNAc transferase (OGT) can introduce uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) molecules and mediates O-GlcNAc modifications. To examine the effects of PP2A inhibition on OGT localization and expression, osteoblastic MC3T3-E1 cells were treated with Okadaic Acid (OA), a potent PP2A inhibitor. In the control cells, OGT was strictly localized in the nucleus. However, OGT was observed diffusely in the cytoplasm of the OA-treated cells. This change in localization from the nucleus to the cytoplasm resulted from an increase in mitochondrial OGT expression and translocation of the nucleocytoplasmic isoform. Furthermore, knockdown of PP2A catalytic subunit α isoform (PP2A Cα) significantly affected OGT expression (p < 0.05), and there was a correlation between PP2A Cα and OGT expression (r = 0.93). These results suggested a possible interaction between PP2A and OGT, which strengthens the notion of an interaction between phosphorylation and O-GlcNAcylation.
From proteins to chromosomes, polymers fold into specific conformations that control their biological function. Polymer folding has long been studied with equilibrium thermodynamics, yet intracellular organization and regulation involve energy-consuming, active processes. Signatures of activity have been measured in the context of chromatin motion, which shows spatial correlations and enhanced subdiffusion only in the presence of adenosine triphosphate (ATP). Moreover, chromatin motion varies with genomic coordinate, pointing towards a heterogeneous pattern of active processes along the sequence. How do such patterns of activity affect the conformation of a polymer such as chromatin? We address this question by combining analytical theory and simulations to study a polymer subjected to sequence-dependent correlated active forces. Our analysis shows that a local increase in activity (larger active forces) can cause the polymer backbone to bend and expand, while less active segments straighten out and condense. Our simulations further predict that modest activity differences can drive compartmentalization of the polymer consistent with the patterns observed in chromosome conformation capture experiments. Moreover, segments of the polymer that show correlated active (sub)diffusion attract each other through effective long-ranged harmonic interactions, whereas anticorrelations lead to effective repulsions. Thus, our theory offers non-equilibrium mechanisms for forming genomic compartments, which cannot be distinguished from affinity-based folding using structural data alone. As a first step toward disentangling active and passive mechanisms of folding, we discuss a data-driven approach to discern if and how active processes affect genome organization.
In cancer biology, ETS transcription factors promote tumorigenesis by mediating transcriptional regulation of numerous genes via the conserved ETS DNA-binding domain. MicroRNAs (miRNAs) act as posttranscriptional regulators to regulate various tumor-promoting or tumor-suppressing factors. Interactions between ETS factors and miRNAs regulate complex tumor-promoting and tumor-suppressing networks. This review discusses the progress of ETS factors and miRNAs in cancer research in detail. We focused on characterizing the interaction of the miRNA/ETS axis with competing endogenous RNAs (ceRNAs) and its regulation in posttranslational modifications (PTMs) and the tumor microenvironment (TME). Finally, we explore the prospect of ETS factors and miRNAs in therapeutic intervention. Generally, interactions between ETS factors and miRNAs provide fresh perspectives into tumorigenesis and development and novel therapeutic approaches for malignant tumors.
Transcription factors (TFs) regulate gene expression by binding to DNA sequences and modulating transcriptional activity through their effector domains. Despite the central role of effector domains in TF function, there is a current lack of a comprehensive resource and characterization of effector domains. Here, we provide a catalog of 924 effector domains across 594 human TFs. Using this catalog, we characterized the amino acid composition of effector domains, their conservation across species and across the human population, and their roles in human diseases. Furthermore, we provide a classification system for effector domains that constitutes a valuable resource and a blueprint for future experimental studies of TF effector domain function.
In response to different stimuli many transcription factors (TFs) display different activation dynamics that trigger the expression of specific sets of target genes, suggesting that promoters have a way to decode them. Combining optogenetics, deep learning-based image analysis and mathematical modeling, we find that decoding of TF dynamics occurs only when the coupling between TF binding and transcription pre-initiation complex formation is inefficient and that the ability of a promoter to decode TF dynamics gets amplified by inefficient translation initiation. Furthermore, we propose a theoretical mechanism based on phase separation that would allow a promoter to be activated better by pulsatile than sustained TF signals. These results provide an understanding on how TF dynamics are decoded in mammalian cells, which is important to develop optimal strategies to counteract disease conditions, and suggest ways to achieve multiplexing in synthetic pathways.
Transcription factors (TFs) are key regulators of intrinsic cellular processes, such as differentiation and development, and of the cellular response to external perturbation through signaling pathways. In this review we focus on the role of TFs as a link between signaling pathways and gene regulation. Cell signaling tends to result in the modulation of a set of TFs that then lead to changes in the cell's transcriptional programme. We highlight the molecular layers at which TF activity can be measured and the associated technical and conceptual challenges. These layers include post‐translational modifications of the TF, regulation of TF binding to DNA through chromatin accessibility and epigenetics, and expression of target genes. We highlight that a large number of TFs are understudied in both signaling and gene regulation studies, and that our knowledge about known TF targets has a strong literature bias. We argue that TFs serve as a perfect bridge between the fields of gene regulation and signaling, and that separating these fields hinders our understanding of cell functions. Multi‐omics approaches that measure multiple dimensions of TF activity are ideally suited to study the interplay of cell signaling and gene regulation using TFs as the anchor to link the two fields. This article is protected by copyright. All rights reserved
The increase in the number of Web-based resources on posttranslational modification sites (PTMSs) in proteins is accelerating. This chapter presents a set of computational protocols describing how to work with the Internet resources when dealing with PTMSs. The protocols are intended for querying in PTMS-related databases, search of the PTMSs in the protein sequences and structures, and calculating the pI and molecular mass of the PTM isoforms. Thus, the modern bioinformatics prediction tools make it feasible to express protein modification in broader quantitative terms.
That genes are indispensable is indisputable but that they are the source of information for protein synthesis-to the extent reflected by statements such as "genes are blueprints for proteins" or "genomes constitute developmental programs"-is challenged by discoveries such as post-translational modification of protein and alternative splicing. © 2018 National Association of Biology Teachers. All rights reserved.
The third subunit of the COP9 signalosome (COPS3) is associated with cell proliferation and tumorigenesis process in cancer. The present study showed that the expression level of COPS3 was upregulated in malignant cell lines and COPS3 overexpression was related with clinical stage, T stage, historical grade. Kaplan-Meier survival curves showed that COPS3 may function as a prognostic factor for overall survival. CCK-8 and colony formation assays revealed that knockdown of COPS3 in ACHN and 786-O significantly impacted proliferation in vitro. In addition, flow cytometry showed that inhibition of COPS3 induced G0/G1 arrest and promoted apoptosis. COPS3 may promote kidney cancer progression by altering Phospho-AKT(Thr308), Cyclin D1 and Caspase-3 expression. Collectively, Our findings suggest that COPS3 may be a new potential target of ccRCC.
TEL is a transcriptional repressor that is a frequent target of chromosomal translocations in a large number of hematalogical malignancies. These rearrangements fuse a potent oligomerization module, the SAM domain of TEL, to a variety of tyrosine kinases or transcriptional regulatory proteins. The self-associating property of TEL–SAM is essential for cell transformation in many, if not all of these diseases. Here we show that the TEL–SAM domain forms a helical, head-to-tail polymeric structure held together by strong intermolecular contacts, providing the first clear demonstration that SAM domains can polymerize. Our results also suggest a mechanism by which SAM domains could mediate the spreading of transcriptional repression complexes along the chromosome.
The Ets family of transcription factors includes nuclear phosphoproteins that are involved in cell proliferation, differentiation and oncogenic transformation. The family is defined by a conserved DNA-binding domain (the ETS-DBD), which forms a highly conserved, winged, helix-turn-helix structural motif. As targets of the Ras–MAPK signaling pathway, Ets proteins function as critical nuclear integrators of ubiquitous signaling cascades. To direct signals to specific target genes, Ets proteins interact with (other) transcription factors that promote the binding of Ets proteins to composite Ras-responsive elements.
T cell activation leads via multiple intracellular signaling pathways to rapid induction of interleukin-2 (IL-2) expression, which can be mimicked by costimulation with 12-O-tetradecanoylphorbol-13-acetate (TPA) and ionomycin. We have identified a distal IL-2 enhancer regulated by the Raf-MEK-ERK signaling pathway, which can be induced by TPA/ionomycin treatment. It contains a dyad symmetry element (DSE) controlled by the Ets-like transcription factor GA-binding protein (GABP), a target of activated ERK. TPA/ionomycin treatment of T cells stimulates both mitogen-activated ERK, as well as the stress-activated mitogen-activated protein kinase family members JNK/SAPK and p38. In this study, we investigated the contribution of the stress-activated pathways to the induction of the distal IL-2 enhancer. We show that JNK- but not p38-activating pathways regulate the DSE activity. Furthermore, the JNK/SAPK signaling pathway cooperates with the Raf-MEK-ERK cascade in TPA/ionomycin-induced DSE activity. In T cells, overexpression of SPRK/MLK3, an activator of JNK/SAPK, strongly induces DSE-dependent transcription and dominant negative kinases of SEK and SAPK impair TPA/ionomycin-induced DSE activity. Blocking both ERK and JNK/SAPK pathways abolishes the DSE induction. The inducibility of the DSE is strongly dependent on the Ets-core motifs, which are bound by GABP. Both subunits of GABP are phosphorylated upon JNK activation in vivo and three different isoforms of JNK/SAPK, but not p38, in vitro. Our data suggest that GABP is targeted by signaling events from both ERK and JNK/SAPK pathways. GABP therefore is a candidate for signal integration and regulation of IL-2 transcription in T lymphocytes.
TEL is a novel member of the ETS family of transcriptional regulators which is frequently involved in human leukemias as the result of specific chromosomal translocations. We show here by co-immunoprecipitation and GST chromatography analyses that TEL and TEL-derived fusion proteins form homotypic oligomers in vitro and in vivo. Deletion mutagenesis identifies the TEL oligomerization domain as a 65 amino acid region which is conserved in a subset of the ETS proteins including ETS-1, ETS-2, FLI-1, ERG-2 and GABP alpha in vertebrates and PNTP2, YAN and ELG in Drosophila. TEL-induced oligomerization is shown to be essential for the constitutive activation of the protein kinase activity and mitogenic properties of TEL-platelet derived growth factor receptor beta (PDGFR beta), a fusion oncoprotein characteristic of the leukemic cells of chronic myelomonocytic leukemia harboring a t(5;12) chromosomal translocation. Swapping experiments in which the TEL oligomerization domain was exchanged by the homologous domains of representative vertebrate ETS proteins including ETS-1, ERG-2 and GABP alpha show that oligomerization is a specific property of the TEL amino-terminal conserved domain. These results indicate that the amino-terminal domain conserved in a subset of the ETS proteins has evolved to generate a specialized protein-protein interaction interface which is likely to be an important determinant of their specificity as transcriptional regulators.
The recent definition of a consensus DNA binding sequence for the Ets family of transcription factors has allowed the identification of potential Ets binding sites in the promoters and enhancers of many inducible T-cell genes. In the studies described in this report, we have identified two potential Ets binding sites, EBS1 and EBS2, which are conserved in both the human and murine interleukin-2 enhancers. Within the human enhancer, these two sites are located within the previously defined DNase I footprints, NFAT-1 and NFIL-2B, respectively. Electrophoretic mobility shift and methylation interference analyses demonstrated that EBS1 and EBS2 are essential for the formation of the NFAT-1 and NFIL-2B nuclear protein complexes. Furthermore, in vitro mutagenesis experiments demonstrated that inducible interleukin-2 enhancer function requires the presence of either EBS1 or EBS2. Two well-characterized Ets family members, Ets-1 and Ets-2, are reciprocally expressed during T-cell activation. Surprisingly, however, neither of these proteins bound in vitro to EBS1 or EBS2. We therefore screened a T-cell cDNA library under low-stringency conditions with a probe from the DNA binding domain of Ets-1 and isolated a novel Ets family member, Elf-1. Elf-1 contains a DNA binding domain that is nearly identical to that of E74, the ecdysone-inducible Drosophila transcription factor required for metamorphosis (hence the name Elf-1, for E74-like factor 1). Elf-1 bound specifically to both EBS1 and EBS2 in electrophoretic mobility shift assays. It also bound to the purine-rich CD3R element from the human immunodeficiency virus type 2 long terminal repeat, which is required for inducible virus expression in response to signalling through the T-cell receptor. Taken together, these results demonstrate that multiple Ets family members with apparently distinct DNA binding specificities regulate differential gene expression in resting and activated T cells.
The human ets-2 gene is a homolog of the v-ets oncogene of the E26 virus and codes for a 56-kilodalton nuclear protein. The ets-2 protein is phosphorylated and has a rapid turnover, with a half-life of 20 min. When human lymphocytic CEM cells were treated with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA), the level of the ets-2 protein was quickly elevated 5- to 20-fold. This effect of TPA was mimicked by a synthetic diacylglycerol, 1-oleoyl-2-acetyl glycerol, and was blocked by the protein kinase C inhibitor H7, indicating that protein kinase C is involved in the induction. The increase in the ets-2 protein was due to stabilization of the protein, because the protein had a half-life of more than 2 h in the presence of TPA and the ets-2 mRNA level did not increase significantly upon TPA treatment. The protein synthesis inhibitor cycloheximide enhanced the effect of TPA on the ets-2 protein and could itself slow turnover of the protein. Properties of the ets-2 protein, such as nuclear localization, phosphorylation, rapid turnover, and response to protein kinase C, indicate that this protein belongs to a group of oncogene proteins which are generally thought to have regulatory functions in the nucleus (e.g., myc, fos, myb, and p53). Our results suggest that protein kinase C, either directly or indirectly, regulates the level of the ets-2 protein by posttranslational mechanisms.
ERF (ETS2 Repressor Factor) is a novel member of the ets family of genes, which was isolated by virtue of its interaction with the ets binding site (EBS) within the ETS2 promoter. The 2.7 kb ubiquitously expressed ERF mRNA encodes a 548 amino acid phosphoprotein that exhibits strong transcriptional repressor activity on promoters that contain an EBS. The localization of the DNA-binding domain of the protein at the N-terminus and th repression domain at the C-terminus is reminiscent of the organization of ELK1-like members of the ets family; however, there is no significant homology between ERF and ELK1 or any other ets member outside the DNA-binding domain. The repressor activity of ERF can antagonize the activity of other ets genes that are known transcriptional activators. Furthermore, ERF can suppress the ets-dependent transforming activity of the gag-myb-ets fusion oncogene of ME26 virus. Although ERF protein levels remain constant throughout the cell cycle, the phosphorylation level of the protein is altered as a function of the cell cycle and after mitogenic stimulation. The ERF protein is also hyperphosphorylated in cells transformed by the activated Ha-ras and v-src genes and the transcription repressor activity of ERF is decreased after co-transfection with activated Ha-ras or the kinase domain of the c-Raf-1 gene, indicating that ERF activity is probably regulated by the ras/MAPK pathway. Consistent with the in vivo phosphorylation and inactivation by ras, ERF is efficiently phosphorylated in vitro by Erk2 and cdc2/cyclin B kinases, at sites similar to those detected in vivo. Furthermore, a single mutation at position 526 results in the loss of a specific phosphopeptide both in in vivo and in vitro (by Erk2) labeling. Substitution of Thr526 for glutamic acid also decreases the repression ability of ERF. Our data suggest a model in which modulation of ERF activity is involved in the transcriptional regulation of genes activated during entry into G1 phase. Obstruction of the ERF repressor function by the transactivating members of the ets family of genes (i.e.gag-myb-ets) may be essential for the control of genes involved in cell proliferation and may also underlie their tumorigenic effects.
The ets-1 protein has been primarily studied as a sequence-specific transcriptional regulator that is predominately expressed
in lymphoid cells. In this report, we show that ets-1 is also expressed in astrocytes and astrocytoma cells and is regulated
during both signal transduction and differentiation. Both isoforms of ets-1, p51 and p42, were found in astrocytes and astrocytoma
cells, but whereas expression of p51 was strong, p42, the alternate splice product previously shown to lack the phosphorylation
domain, was difficult to detect and was present at a level 10- to 40-fold lower than that of p51. This differed by roughly
an order of magnitude from the ratio generally observable in T cells and thymocytes. In two astrocytoma lines of human origin,
CCF and 1321N1, ets-1 phosphorylation was stimulated by bradykinin and carbachol, respectively. Glutamate, norepinephrine,
and bradykinin elicited phosphorylation of p51 in cultures of primary rat type 1 astrocytes. ets-1 phosphorylation was dramatically
blocked by KT5926, an inhibitor of myosin light-chain kinase, suggesting that this kinase may be involved in phosphorylation
of ets-1 in vivo. Investigations of retinoic acid-induced differentiation in P19 cells provided further support for a strong
correlation of ets-1 with the pathway for astrocyte differentiation.
Induction of the human c-fos proto-oncogene by mitogens depends on the formation of a ternary complex by p62TCF with the serum response factor (SRF) and the serum response element (SRE). We demonstrate that Elk-1, a protein closely related to p62TCF in function, is a nuclear target of two members of the MAP kinase family, ERK1 and ERK2. Phosphorylation of Elk-1 increases the yield of ternary complex in vitro. At least five residues in the C-terminal domain of Elk-1 are phosphorylated upon growth factor stimulation of NIH3T3 cells. These residues are also phosphorylated by purified ERK1 in vitro, as determined by a combination of phosphopeptide sequencing and 2-D peptide mapping. Conversion of two of these phospho-acceptor sites to alanine impairs the formation of ternary complexes by the resulting Elk-1 proteins. Removal of these serine residues also drastically diminishes activation of the c-fos promoter in epidermal growth factor-treated cells. Analogous mutations at other sites impair activation to a lesser extent without affecting ternary complex formation in vitro. Our results indicate that phosphorylation regulates ternary complex formation by Elk-1, which is a prerequisite for the manifestation of its transactivation potential at the c-fos SRE.
Ras signaling appears to be mediated in part by transcription factors that belong to the ets gene family. To identify downstream targets for the Ras signal transduction pathway, we have used Ras-transformed mouse fibroblasts to isolate a new member of the ets gene family, net. Net has sequence similarity in three regions with the ets factors Elk1 and SAP1, which have been implicated in the serum response of the fos promoter. Net shares various properties with these proteins, including the ability to bind to ets DNA motifs through the Ets domain of the protein and form ternary complexes with the serum response factor SRF on the fos serum response element, SRE. However, Net differs from Elk1 and SAP1 in a number of ways. The pattern of net RNA expression in adult mouse tissues is different. Net has negative effects on transcription in a number of assays, unlike Elk1. Strikingly, Ras, Src, and Mos expression switch Net activity to positive. The study of Net should help in understanding the interplay between Net and other members of the Elk subfamily and their contribution to signal transduction through Ras to the nucleus.
The Ras oncogene products regulate the expression of genes in transformed cells, and members of the Ets family of transcription factors have been implicated in this process. To determine which Ets factors are the targets of Ras signaling pathways, the abilities of several Ets factors to activate Ras-responsive enhancer (RRE) reporters in the presence of oncogenic Ras were examined. In transient transfection assay, reporters containing RREs composed of Ets-AP-1 binding sites could be activated 30-fold in NIH 3T3 fibroblasts and 80-fold in the macrophage-like line RAW264 by the combination of Ets1 or Ets2 and Ras but not by several other Ets factors that were tested in the assay. Ets2 and Ras also superactivated an RRE composed of Ets-Ets binding sites, but the Ets-responsive promoter of the c-fms gene was not superactivated. Mutation of a threonine residue to alanine in the conserved amino-terminal regions of Ets1 and Ets2 (threonine 38 and threonine 72, respectively) abrogated the ability of each of these proteins to superactivate reporter gene expression. Phosphoamino acid analysis of radiolabeled Ets2 revealed that Ras induced normally absent threonine-specific phosphorylation of the protein. The Ras-dependent increase in threonine phosphorylation was not observed in Ets2 proteins that had the conserved threonine 72 residue mutated to alanine or serine. These data indicate that Ets1 and Ets2 are specific nuclear targets of Ras signaling events and that phosphorylation of a conserved threonine residue is a necessary molecular component of Ras-mediated activation of these transcription factors.
An allele of the yan locus was isolated as an enhancer of the Ellipse mutation of the Drosophila epidermal growth factor receptor (Egfr) gene. This yan allele is an embryonic lethal and also fails to complement the lethality of anterior open (aop) mutations. Phenotypic and complementation analysis revealed that aop is allelic to yan and genetically the lethal alleles act as null mutations for the yan gene. Analysis of the lethal alleles in the embryo and in mitotic clones showed that loss of yan function causes cells to overproliferate in the dorsal neuroectoderm of the embryo and in the developing eye disc. Our studies suggest that the role of yan is defined by the developmental context of the cells in which it functions. An important role of this gene is in allowing a cell to choose between cell division and differentiation. The relationship of the Egfr and Notch pathways to this developmental role of yan is discussed.
Ets-1 is the prototypic member of the ets family of transcription factors. This family is characterized by the conserved ETS domain that mediates specific DNA binding. Using NMR methods, we have determined the structure of a fragment of murine Ets-1 composed of the 85 residue ETS domain and a 25 amino acid extension that ends at its native C-terminus. The ETS domain folds into a helix-turn-helix motif on a four-stranded anti-parallel beta-sheet scaffold. This structure places Ets-1 in the winged helix-turn-helix (wHTH) family of DNA binding proteins and provides a model for interpreting the sequence conservation of the ETS domain and the specific interaction of Ets-1 with DNA. The C-terminal sequence of Ets-1, which is mutated in the v-Ets oncoprotein, forms an alpha-helix that packs anti-parallel to the N-terminal helix of the ETS domain. In this position, the C-terminal helix is poised to interact directly with an N-terminal inhibitory region in Ets-1 as well as the wHTH motif. This explains structurally the concerted role of residues flanking the ETS domain in the intramolecular inhibition of Ets-1 DNA binding.
The Ets family of transcription factors, of which there are now about 35 members regulate gene expression during growth and development. They share a conserved domain of around 85 amino acids which binds as a monomer to the DNA sequence 5'-C/AGGAA/T-3'. We have determined the crystal structure of an ETS domain complexed with DNA, at 2.3-A resolution. The domain is similar to alpha + beta (winged) 'helix-turn-helix' proteins and interacts with a ten-base-pair region of duplex DNA which takes up a uniform curve of 8 degrees. The domain contacts the DNA by a novel loop-helix-loop architecture. Four of amino acids that directly interact with the DNA are highly conserved: two arginines from the recognition helix lying in the major groove, one lysine from the 'wing' that binds upstream of the core GGAA sequence, and another lysine, from the 'turn' of the 'helix-turn-helix' motif, which binds downstream and on the opposite strand.
The insulin-response element from the prolactin gene is identical to the Ets-binding site, and dominant-negative Ets protein inhibits insulin-increased prolactin gene expression. Immunoblotting identified the Ets-related transcription factor GABP in nuclear extracts from GH cells. Expression of GABP alpha and GABP beta 1 squelches insulin-increased prolactin gene expression. GABP alpha and GABP beta 1 bind the insulin-response element of the prolactin promoter, and anti-GABP alpha and anti-GABP beta 1 antibodies supershift a species seen with nuclear extracts from GH cells. GABP alpha immunoprecipitated from insulin-treated, 32P-labeled GH cells was phosphorylated 3-fold more than GABP alpha from control cells. There was no increase in phosphorylation of GABP beta in response to insulin. Mitogen-activated protein (MAP) kinase activity is increased 10-fold in insulin-treated GH4 cells. MAP kinase immunoprecipitated from control cells does not phosphorylate GABP alpha while MAP kinase immunoprecipitated from insulin-treated cells shows substantial phosphorylation of GABP alpha. These studies suggest that GABP mediates insulin-increased transcription of the prolactin gene. GABP may be regulated by MAP kinase phosphorylation.
Mitogenic and stres signals results in the activation of extracellular signal-regulated kinases (ERKs) and stress-activated protein kinase/c-Jun N-terminal kinases (SAPK/JNKs), respectively, which are two subgroups of the mitogen-activated protein kinases. A nuclear target of mitogen-activated protein (MAP) kinases is the ternary complex factor Elk-1, which underlies its involvement in the regulation of c-fos gene expression by mitogenic and stress signals. A second ternary complex factor, Sap1a, is coexpressed with Elk-1 in several cell types and shares attributes of Elk-1, the significance of which is not clear. Here we show that Sap1a is phosphorylated efficiently by ERKs but not by SAPK/JNKs. Serum response factor-dependent ternary complex formation by Sap1a is stimulated by ERK phosphorylation but not by SAPK/JNKs. Moreover, Sap1a-mediated transcription is activated by mitogenic signals but not by cell stress. These results suggest that Sap1a and Elk-1 have distinct physiological functions.
The three ternary complex factors (TCFs), Net (ERP/ SAP-2), ELK-1 and SAP-1, are highly related ets oncogene family members that participate in the response of the cell to Ras and growth signals. Understanding the different roles of these factors will provide insights into how the signals result in coordinate regulation of the cell. We show that Net inhibits transcription under basal conditions, in which SAP-1a is inactive and ELK-1 stimulates. Repression is mediated by the NID, the Net Inhibitory Domain of about 50 amino acids, which autoregulates the Net protein and also inhibits when it is isolated in a heterologous fusion protein. Net is particularly sensitive to Ras activation. Ras activates Net through the C-domain, which is conserved between the three TCFs, and the NID is an efficient inhibitor of Ras activation. The NID, as well as more C-terminal sequences, inhibit DNA binding. Net is more refractory to DNA binding than the other TCFs, possibly due to the presence of multiple inhibitory elements. The NID may adopt a helix-loop-helix (HLH) structure, as evidenced by homology to other HLH motifs, structure predictions, model building and mutagenesis of critical residues. The sequence resemblance with myogenic factors suggested that Net may form complexes with the same partners. Indeed, we found that Net can interact in vivo with the basic HLH factor, E47. We propose that Net is regulated at the level of its latent DNA-binding activity by protein interactions and/or phosphorylation. Net may form complexes with HLH proteins as well as SRF on specific promotor sequences. The identification of the novel inhibitory domain provides a new inroad into exploring the different roles of the ternary complex factors in growth control and transformation.
The ETS family of proteins is a large group of transcription factors implicated in many aspects of normal hematopoietic development, as well as oncogenesis. For example, the TEL1/ETV6 (TEL1) gene is required for normal yolk sac angiogenesis, adult bone marrow hematopoiesis, and is rearranged or deleted in numerous leukemias. This report describes the cloning and characterization of a novelETS gene that is highly related to TEL1 and is therefore called TEL2. The TEL2 gene consists of 8 exons spanning approximately 21 kilobases (kb) in human chromosome 6p21. Unlike the ubiquitously expressed TEL1 gene, however,TEL2 appears to be expressed predominantly in hematopoietic tissues. Antibodies raised against the C-terminus of the TEL2 protein were used to show that TEL2 localizes to the nucleus. All ETS proteins can bind DNA via the highly conserved ETS domain, which recognizes a purine-rich DNA sequence with a GGAA core motif. DNA binding assays show that TEL2 can bind the same consensus DNA binding sequence recognized by TEL1/ETV6. Additionally, the TEL2 protein is capable of associating with itself and with TEL1 in doubly transfected Hela cells, and this interaction is mediated through the pointed (PNT) domain of TEL1. The striking similarities ofTEL2 to the oncogenic TEL1, its expression in hematopoietic tissues, and its ability to associate withTEL1 suggest that TEL2 may be an important hematopoietic regulatory protein.
ETS domain transcription factor Elk-1 serves as an integration point for different mitogen-activated protein (MAP) kinase pathways. Phosphorylation of Elk-1 by MAP kinases triggers its activation. However, while the activation process is well understood, its downregulation-inactivation is less well characterized. The ETS DNA-binding domain plays a role in the downregulation of Elk-dependent promoter activity following mitogenic activation by recruiting the mSin3A-HDAC complex. Here we have identified a novel evolutionarily conserved repression domain in Elk-1, termed the R motif, which serves to reduce the basal transcriptional activity of Elk-1 and dampen its response to mitogenic signals. This domain is highly potent and portable and can repress transcription in trans. The R motif is related to the CRD1 repression domain in p300 and can functionally replace this domain and confer p21(waf1/cip1) inducibility on p300. However, the R motif acts in a context-dependent manner and is not p21(waf1/cip1) responsive in Elk-1. Thus, the Elk-1 R motif and the p300 CRD1 motif represent a new class of repression domains that are regulated in a context-dependent manner.
Glial-neuronal cell interactions at the ventral midline are necessary for the proper elaboration of commissures in the embryonic CNS of Drosophila. In particular, migrating midline glial cells are required for the separation of segmental commissures. During this process the glial cells recognize specific neuronal cells at the midline, they migrate posteriorly along their cell processes and thereby separate the segmental commissures. The gene pointed (pnt) is required for this glial-neuronal cell interaction, as loss of function mutations lead to a change in the migration behavior of the midline glial cells. As a consequence, anterior and posterior commissures do not become separated and appear fused. Molecular analysis of pointed has revealed two differently spliced types of transcripts, which are encoded in a region extending over 55 kb of genomic sequence. In the CNS both transcript classes are expressed in cells of the midline, including the midline glial cells. Sequence analysis of cDNA clones corresponding to both transcript types reveals two different pointed proteins which share an ETS domain common to a number of transcription factors related to the vertebrate ets oncogene. Furthermore, one pointed protein form contains an additional domain of homology of approx. 80 amino acids in length, which is shared by only a subset of the ETS protein family.
Many eukaryotic proteins contain O-linked N-acetylglucosamine (O-GlcNAc) on their serine and threonine side chain hydroxyls. In contrast to classical cell surface glycosylation, O-GlcNAc occurs on resident nuclear and cytoplasmic proteins. O-GlcNAc exists as a single monosaccharide residue, showing no evidence of further elongation. Like phosphorylation, O-GlcNAc is highly dynamic, transiently modifying proteins. These post-translational modifications give rise to functionally distinct subsets of a given protein. Furthermore, all known O-GlcNAc proteins are also phosphoproteins that reversibly form multimeric complexes that are sensitive to the state of phosphorylation. This observation implies that O-GlcNAc may work in concert with phosphorylation to mediate regulated protein interactions. The proteins that bear the O-GlcNAc modification are very diverse, including RNA polymerase II and many of its transcription factors, numerous chromatin-associated proteins, nuclear pore proteins, proto-oncogenes, tumor suppressors and proteins involved in translation. Here, we discuss the functional implications of O-GlcNAc-modifications of proteins involved in various aspects of gene expression, beginning with proteins involved in transcription and ending with proteins involved in regulating protein translation.
Elf-1, a member of the Ets transcription factor family with an estimated molecular mass of 68 kDa, is involved in the transcriptional regulation of several hematopoietic cell genes. It is shown that following O-GlcNAc glycosylation and phosphorylation by PKC ϑ, the cytoplasm-located, 80-kDa Elf-1 translocates to the nucleus as a 98-kDa protein. In the nucleus, Elf-1 binds to the promoter of the TCRζ gene and promotes its transcription in Jurkat and fresh human T cells. It is also shown that in the majority of patients with systemic lupus erythematosus (SLE), who are known to express decreased levels of T cell receptor (TCR) ζ chain and mRNA, the 80-kDa Elf-1 protein does not undergo proper post-transcriptional modification, which results in low levels of the 98-kDa protein, lack of Elf-binding to the TCR ζ promoter, and decreased gene transcription. Therefore, a novel activation pathway for a member of the Ets family of transcription factors, which is defective in patients with systemic autoimmunity, has been revealed.
The molecular complexity that defines different cell types and their biological responses occurs at the level of the cell’s proteome. The recent increase in availability of genomic sequence information is a valuable tool for the field of proteomics. While most proteomic studies focus on differential expression levels, post-translational modifications such as phosphorylation, glycosylation, and acetylation, provide additional levels of functional complexity to the cell’s proteome. The reversible post-translational modification O-linked β-N-acetylglucosamine (O-GlcNAc) is found on serines and threonines of nuclear and cytoplasmic proteins. It appears to be as widespread as phosphorylation. While phosphorylation is recognized as a fundamental mechanism for controlling protein function, less is known about the specific roles of O-GlcNAc modification. However, evidence is building that O-GlcNAc may compete with phosphate at some sites of attachment. Aberrant O-GlcNAc modification has been linked to several disease states, including diabetes and Alzheimer’s disease. Regulated enzymes catalyzing the addition (O-GlcNAc transferase, OGT) and removal (O-GlcNAcase) of the modification have been cloned and OGT is required for life at the single cell level. Here we review the properties of O-GlcNAc that suggest it is a regulatory modification analogous to phosphorylation. We also discuss the use of comparative functional proteomics to elucidate functions for this ubiquitous intracellular carbohydrate modification.
Loss-of-function mutations in the yan gene result in the differentiation of supernumerary photoreceptors in the Drosophila eye. The yan gene encodes a protein with an ETS DNA-binding domain that accumulates in the nuclei of undifferentiated cells during the early stages of eye development. Our data suggest that yan functions as a cell-autonomous negative regulator of photoreceptor development; in the presumptive R7 and cone cells, yan appears to act antagonistically to the proneural signal mediated by sevenless and Ras1.
The Ets-1 proto-oncogene is a member of the Ets family of eukaryotic transcription factors. Members of this family play important roles in regulating gene expression in response to multiple developmental and mitogenic signals. Ets-1 is preferentially expressed at high levels in B and T cells of adult mice and is regulated during both thymocyte development and T-cell activation. To study the role of Ets-1 in T-cell development and function we have used the RAG-2-/- complementation system and murine embryonic stem (ES) cells containing homozygous deletions in the Ets-1 gene (Ets-1-/-). Ets-1-/(-)-RAG-2-/- chimaeric mice displayed markedly decreased numbers of mature thymocytes and peripheral T cells. Ets-1-/- T cells expressed normal levels of CD3 and T-cell antigen receptor (TCR)-alpha/beta. However, they displayed a severe proliferative defect in response to multiple activational signals and demonstrated increased rates of spontaneous apoptosis in vitro. These findings demonstrate that Ets-1 is required for the normal survival and activation of murine T cells.
The Ets-1 proto-oncogene is a member of a transcription factor family characterized by homology to the v-ets oncogene. In adult mice, Ets-1 is expressed predominantly in lymphoid cells where it has been implicated in regulating transcription of lymphocyte-specific genes. Following T-cell activation, the specific DNA binding activity of Ets-1 is inactivated by transient phosphorylation, suggesting a function in the transition from the resting to activated state. Ets-1 has also been suggested to cooperate with the AP-1 transcription factor complex to mediate cellular growth factor responses. Here we show, by using RAG-2-deficient blastocyst complementation, that Ets-1 deficiency has dramatic, but different, effects on development and function of T- and B-lineage cells. Ets-1-deficient T cells were present in reduced numbers and were highly susceptible to cell death in vitro. In contrast, Ets-1-deficient B cells were present in normal numbers but a large proportion were IgM plasma cells. Our data demonstrate that Ets-1 is essential for maintenance of the normal pool of resting T- and B-lineage cells.
The ternary complex factor (TCF) subfamily of ETS-domain transcription factors bind with serum response factor (SRF) to the serum response element (SRE) and mediate increased gene expression. The TCF protein Elk-1 is phosphorylated by the JNK and ERK groups of mitogen-activated protein (MAP) kinases causing increased DNA binding, ternary complex formation, and transcriptional activation. Activated SRE-dependent gene expression is induced by JNK in cells treated with interleukin-1 and by ERK after treatment with phorbol ester. The Elk-1 transcription factor therefore integrates MAP kinase signaling pathways in vivo to coordinate biological responses to different extracellular stimuli.
Drosophila yan has been postulated to act as an antagonist of the proneural signal mediated by the sevenless/Ras1/MAPK pathway. We have mutagenized the eight MAPK phosphorylation consensus sites of yan and examined the effects of overexpressing the mutant protein in transgenic flies and transfected S2 cultured cells. Our results suggest that phosphorylation by MAPK affects the stability and subcellular localization of yan, resulting in rapid down-regulation of yan activity. Furthermore, MAPK-mediated down-regulation of yan function appears to be critical for the proper differentiation of both neuronal and nonneuronal tissues throughout development, suggesting that yan is an essential component of a general timing mechanism controlling the competence of a cell to respond to inductive signals.
We show that the activities of two Ets-related transcription factors required for normal eye development in Drosophila, pointed and yan, are regulated by the Ras1/MAPK pathway. The pointed gene codes for two related proteins, and we show that one form is a constitutive activator of transcription, while the activity of the other form is stimulated by the Ras1/MAPK pathway. Mutation of the single consensus MAPK phosphorylation site in the second form abrogates this responsiveness. yan is a negative regulator of photoreceptor determination, and genetic data suggest that it acts as an antagonist of Ras1. We demonstrate that yan can repress transcription and that this repression activity is negatively regulated by the Ras1/MAPK signal, most likely through direct phosphorylation of yan by MAPK.
The fate of the R7 photoreceptor cell in the developing eye of Drosophila is controlled by the Sevenless (Sev) receptor tyrosine kinase. Sev activates a highly conserved signal transduction cascade involving the proteins Ras1 and Raf and the Rolled/mitogen-activated protein (Rl/MAP) kinase. Here we show that the ETS domain protein encoded by the P2 transcript of the pointed (pnt) gene is a nuclear target of this signalling cascade which acts downstream of Rl/MAP kinase. The PntP2 protein is phosphorylated by Rl/MAP kinase in vitro at a single site and this site is required for its function in vivo. Furthermore, we present genetic and biochemical data suggesting that MAP kinase controls neural development through phosphorylation of two antagonizing transcription factors of the ETS family, Yan and PntP2.
Chronic myelomonocytic leukemia (CMML) is a myelodysplastic syndrome characterized by abnormal clonal myeloid proliferation and by progression to acute myelogenous leukemia (AML). CMML thus offers an opportunity to study early genetic events in the transition to AML. A recently recognized subgroup of CMML has a t(5;12)(q33;p13) balanced translocation. We report that the consequence of the t(5;12) translocation is expression of a fusion transcript in which the tyrosine kinase domain of the platelet-derived growth factor receptor beta (PDGFR beta) on chromosome 5 is coupled to a novel ets-like gene, tel, on chromosome 12. The tel-PDGFR beta fusion demonstrates the oncogenic potential of PDGFR beta and may provide a paradigm for early events in the pathogenesis of AML.
Glial-neuronal cell interactions at the ventral midline are necessary for the proper elaboration of commissures in the embryonic CNS of Drosophila. In particular, migrating midline glial cells are required for the separation of segmental commissures. During this process the glial cells recognize specific neuronal cells at the midline, they migrate posteriorly along their cell processes and thereby separate the segmental commissures. The gene pointed (pnt) is required for this glial-neuronal cell interaction, as loss of function mutations lead to a change in the migration behavior of the midline glial cells. As a consequence, anterior and posterior commissures do not become separated and appear fused. Molecular analysis of pointed has revealed two differently spliced types of transcripts, which are encoded in a region extending over 55 kb of genomic sequence. In the CNS both transcript classes are expressed in cells of the midline, including the midline glial cells. Sequence analysis of cDNA clones corresponding to both transcript types reveals two different pointed proteins which share an ETS domain common to a number of transcription factors related to the vertebrate ets oncogene. Furthermore, one pointed protein form contains an additional domain of homology of approx. 80 amino acids in length, which is shared by only a subset of the ETS protein family.
Ternary complex factors (TCFs), one of which is Elk-1, have been implicated in mediation of c-fos induction. They have been shown to be phosphorylated by mitogen-activated protein kinases (MAPKs) in vitro. We demonstrate that recombinant Elk-1 is hyperphosphorylated in vivo upon joint overexpression of MAPKs and constitutively activated Raf-1 kinase, the latter serving as an indirect in vivo activator of MAPKs. This phosphorylation is accompanied by a conformational change and results in an elevated transactivation potential of Elk-1. Mutation of mapped in vivo phosphorylation sites, which are potential targets for MAPKs, reduced Elk-1-mediated transcription. Thus, MAPKs are very probably controlling Elk-1 activity by direct phosphorylation in vivo. Furthermore, Elk-1 was shown to stimulate transcription from both the c-fos serum response element and also from an Ets binding site. While binding of TCFs to the c-fos promoter is dependent on the serum response factor, TCFs can autonomously interact with Ets binding sites. This indicates that TCFs may participate in the transcriptional regulation of two different sets of genes.
The Elk-1 and SRF transcription factors form a ternary complex at the c-fos serum response element (SRE). Growth factor stimulation rapidly induces a reversible change in the electrophoretic mobility of the ternary complex, accompanied by increased phosphorylation of the Elk-1 C-terminal region and by the activation of a 42 kd cellular Elk-1 kinase. Phosphorylation of Elk-1 in vitro by partially purified p42/p44 MAP kinase induces a similar reduction in ternary complex mobility but has little effect on the efficiency of its formation. In vitro, MAP kinase phosphorylates the Elk-1 C-terminal region at multiple sites, which are also phosphorylated following growth factor stimulation in vivo. The Elk-1 C-terminal region functions as a regulated transcriptional activation domain whose activity in vivo is dependent on the integrity of the MAP kinase sites. These findings directly link transcriptional activation by the SRE to the growth factor-regulated phosphorylation of an SRE-binding protein.
EGF-induction of human astrocytoma and A431 cells leads to c-fos transcriptional activation and then repression. This could be correlated with changes in the DNA binding characteristics of the c-fos regulatory protein ternary complex factor (TCF) present in nuclear extracts from these cells. Band shifts showed the appearance of induction-related slowly migrating protein-DNA complexes, detected as ternary complexes on the c-fos SRE using a truncated SRF molecule and by direct binding to the Drosophila E74 Ets-protein recognition sequence. By several criteria both types of complexes represented TCF. The appearance of the slow ternary and direct complexes correlated with c-fos transcriptional activation, and their disappearance coincided with the ensuing c-fos shut-off. Blocking c-fos transcriptional repression with the phosphatase inhibitor okadaic acid led to their continued presence. They were sensitive to protein phosphatase 2A but not 1 alpha, and similar slow complexes were formed by partially purified p62TCF phosphorylated by a copurifying kinase activity. Thus the phosphorylation state of TCF correlated strongly with c-fos promoter activity. Since ternary complex formation mediated by full-sized SRF was only slightly affected under comparable conditions, we propose a model for c-fos regulation involving modification of constitutively bound TCF.
PU.1 recruits the binding of a second B cell-restricted nuclear factor, NF-EM5, to a DNA site in the immunoglobulin kappa
3' enhancer. DNA binding by NF-EM5 requires a protein-protein interaction with PU.1 and specific DNA contacts. Dephosphorylated
PU.1 bound to DNA but did not interact with NF-EM5. Analysis of serine-to-alanine mutations in PU.1 indicated that serine
148 (Ser148) is required for protein-protein interaction. PU.1 produced in bacteria did not interact with NF-EM5. Phosphorylation
of bacterially produced PU.1 by purified casein kinase II modified it to a form that interacted with NF-EM5 and that recruited
NF-EM5 to bind to DNA. Phosphopeptide analysis of bacterially produced PU.1 suggested that Ser148 is phosphorylated by casein
kinase II. This site is also phosphorylated in vivo. Expression of wild-type PU.1 increased expression of a reporter construct
containing the PU.1 and NF-EM5 binding sites nearly sixfold, whereas the Ser148 mutant form only weakly activated transcription.
These results demonstrate that phosphorylation of PU.1 at Ser148 is necessary for interaction with NF-EM5 and suggest that
this phosphorylation can regulate transcriptional activity.
The retinoblastoma gene product (Rb) is a nuclear phosphoprotein that regulates cell cycle progression. Elf-1 is a lymphoid-specific
Ets transcription factor that regulates inducible gene expression during T cell activation. In this report, it is demonstrated
that Elf-1 contains a sequence motif that is highly related to the Rb binding sites of several viral oncoproteins and binds
to the pocket region of Rb both in vitro and in vivo. Elf-1 binds exclusively to the underphosphorylated form of Rb and fails
to bind to Rb mutants derived from patients with retinoblastoma. Co-immunoprecipitation experiments demonstrated an association
between Elf-1 and Rb in resting normal human T cells. After T cell activation, the phosphorylation of Rb results in the release
of Elf-1, which is correlated temporally with the activation of Elf-1-mediated transcription. Overexpression of a phosphorylation-defective
form of Rb inhibited Elf-1-dependent transcription during T cell activation. These results demonstrate that Rb interacts specifically
with a lineage-restricted Ets transcription factor. This regulated interaction may be important for the coordination of lineage-specific
effector functions such as lymphokine production with cell cycle progression in activated T cells.
We describe the identification of the ERG-2 gene products using an antibody raised against recombinant human ERG-2 protein. ERG-2 is a nuclear phosphoprotein and binds to purine-rich sequences (C/G)(C/a)GG-AA(G/a)T. ERG-2 protein, with a half-life of 21 h, is considerably more stable than the short-lived ETS-1 or ETS-2 proteins. Its phosphorylation is stimulated by phorbol myristate acetate (PMA), but not by Ca2+ ionophore treatment. ETS-1 protein is phosphorylated by Ca(2+)-dependent events, whereas ERG-2 protein is phosphorylated by activation of protein kinase C, suggesting their involvement in distinct signal transduction mechanisms. The expression of ERG-2 protein is restricted to few cell types and is high in early myeloid cells, indicating that it may function at an early stage of hematopoietic lineage determination. The DNA-binding sequence for ERG-2 protein is identified by using a random oligonucleotide selection procedure. The selected sequence is very similar to the binding sequence determined for human ETS-1 using the same method. Like other ets proteins, ERG-2 is a sequence-specific DNA-binding protein and is expressed at higher levels in early myeloid cells than in mature lymphoid cells. These results suggest that it may act as a regulator of genes required for maintenance and/or differentiation of early hematopoietic cells.
Background:
The mammalian response to stress results in the activation of stress-activated protein kinases (also known as cJun N-terminal kinases; SAPKs or JNKs), which are a sub-group of the mitogen-activated protein (MAP) kinase family. The SAPKs are involved in the upregulation of activity of the transcription factor AP-1 by post-translational modification of two of its components, cJun and ATF2. AP-1 activity can also be elevated by increased expression of the Fos protein, a further AP-1 component. Elk-1 (also called p62TCF), a transcription factor involved in the induction of the expression from the c-fos promoter through the promoter's serum response element, is known to be activated as a result of phosphorylation by the MAP kinases ERK1 and ERK2. However, induction of c-fos expression in response to noxious agents takes place in the absence of ERK activation. We therefore investigated whether SAPKs similarly upregulate c-fos expression by phosphorylating Elk-1.
Results:
Elk-1 is activated in response to stimuli other than mitogenic signals. Both p46SAPK and p54SAPK interact physically with, and phosphorylate, Elk-1. The capacity of Elk-1 to form a ternary complex with serum response factor in vitro is thereby elevated. In vivo, selective activation of SAPKs stimulates formation of the ternary complex containing Elk-1, serum response factor and the serum response element, and enhances Elk-1-dependent transcription. Expression of the SAPK upstream-activator kinase, MEKK1, induces SAPK activation and c-fos transcription in the absence of ERK activity. Phosphopeptide mapping of Elk-1 phosphorylated with p46SAPK or p54SAPK reveals Ser383, a residue critical for ternary complex formation and transcriptional activation, to be the major phosphorylation site.
Conclusion:
Elk-1 responds to stress-induced, as well as mitogenic, signals by stimulating c-fos transcription through the serum response element. Phosphorylation of Elk-1 by SAPKs and the ensuing expression of Fos protein thus constitutes an additional mechanism by which cells can upregulate AP-1 activity in response to stress.
Spi-1/PU-1 and Spi-B are hematopoietic transcription factors, which, in vitro, display similar affinities for DNA target sequences containing the consensus binding site 5'-GGAA-3'. While the role of Spi-1 in the transcriptional regulation of B cell and myeloid specific genes has been largely demonstrated, the biological function of Spi-B still remains to be elucidated. Since Spi-B and Spi-1 are very divergent in their transactivator domain, these domains might acquire functional specificity in vivo by interacting with different co-factors and/or by undergoing different phosphorylations. First, we observed that casein kinase II phosphorylates Spi-B as well as Spi-1, in vitro. Then, by affinity chromatographies and in vitro kinase assays with fusion proteins between glutathione-S-transferase and the transactivator domain of Spi-B, two kinases were identified on their ability to interact and phosphorylate this domain; the MAP kinase ERK1 and the stress activated protein kinase JNK1. The Threonine 56 was defined as the ERK1 phosphorylation site by using phosphoamino-acid analyses and a Spi-B mutant version with the substitution T56 to A56. Strikingly, ERK1 failed to phosphorylate Spi-1, in vitro, whereas JNK1, like CK II, phosphorylated Spi-B and Spi-1. In addition, other purified Spi-B-kinase activities, unidentified as yet, display similar specificity than ERK1 for Spi-B versus Spi-1. Furthermore, the evident interaction of pRb protein with the transactivator domain of Spi-B in an unphosphorylated state disappeared when this domain was first phosphorylated in vitro either by ERK1 or by the purified Spi-B-kinase activities. Our data revealed multiple phosphorylation sites within Spi-B whose some of them appeared specific for Spi-B versus Spi-1 and which may account for differential regulation of their activities.
A plethora of extracellular signals leads to the stimulation of Ras, which triggers intracellular protein kinase cascades,
resulting in activation of transcription factors and thus in enhanced gene activity. In this report, it is demonstrated that
the ETS transcription factor ER81, which appears to be localized within the cell nucleus by virtue of its DNA binding domain,
is transcriptionally activated by oncogenic Ras. Since this activation was dependent on the presence of Raf-1 and ERK-1, ER81
is a target of the Ras/Raf/MEK/ERK signaling cascade. Consistently, activated ERK-1 is capable to phosphorylate ER81. However,
the carboxy-terminal region of ER81, which contains no potential ERK phosphorylation sites, is also transcriptionally activated
by ERK-1, suggesting that an ERK-stimulated protein kinase phosphorylates and thus stimulates ER81 activity. Two acidic stretches
of amino acids, which are conserved in the related PEA3 and ERM proteins, are localized within the amino-and carboxy-terminal
transactivation domains of ER81. In addition, an inhibitory domain may dampen the activation function of these two domains.
In conclusion, ER81 is a target of Ras-dependent signaling cascades and may thus contribute to the nuclear response upon stimulation
of cells and also to cellular transformation due to oncogenic Ras.
The Ets family of transcription factors has been implicated in the etiology of several types of cancer. We cloned and characterized the gene encoding the murine homologue of one Ets family member, Elf-1 (mElf-1), in order to gain insight into its cellular physiology. We examined mElf-1 mRNA expression in normal mouse tissues and in several murine and human cell lines. Expression of mElf-1, although highest in lymphocytes, was observed in a number of hematopoietic cell lineages, including the myeloid, macrophages and erythroid lineages, and was lowest in the murine fibroblast cell line, NIH3T3. Analysis of human and fetal tissue mRNAs confirmed that mElf-1 is expressed in a variety of cell lineages, principally in hematopoietic cells. Western blot analysis using antiserum generated to a synthetic C-terminal peptide of mElf-1, and extracts prepared from cell lines that expressed the mElf-1 mRNA identified multiple mElf-1 species that migrated near the 97-kDa molecular weight marker. Site selection analysis indicated that the binding site preferred by mElf-1 is very similar to that of the Drosophila melanogaster homologue, E74, and to that of Fli-1, another Ets family member. We conclude that the expression of mElf-1 is not restricted to the lymphoid lineage, and suggest that Elf-1 may regulate the transcription of a broad spectrum of genes.
Chromosomal translocations have provided valuable insights into the molecular mechanisms of leukemogenesis in humans. Examples of genes implicated in pathogenesis of hematologic malignancy by virtue of involvement in translocation breakpoints include the PML-RARa fusion associated with t(15;17) acute promyelocytic leukemia (APML, refs. 1–3), the AML1-ETO fusion associated with t(8;21) acute myeloid leukemia (AML, ref.4), the CBFß-MYHII fusion associated with inv(16) acute myelomonocytic leukemia and eosinophilia (5), and MLL fusions at 11q23 breakpoints with various fusion proteins associated with acute myeloid leukemias (6).
PEA3, a member of the Ets family of transcriptional regulatory proteins, binds to the PEA3 promoter element and stimulates transcription through this site. The activity of the PEA3 element is regulated by mitogens, activated receptor tyrosine kinases, and oncogenic members of the Ras signal transduction pathway. However, it is not clear whether PEA3 mediates transcriptional regulation by these agents because a number of different Ets proteins can functionally interact with the PEA3 element. To specifically learn whether the activity of PEA3 is regulated, we investigated the ability of constitutively-activated Ras (Ha-RasV12) and signaling proteins downstream of Ras to alter PEA3-dependent reporter gene expression in COS cells. Ha-RasV12 and activated proteins in both the extra-cellular regulated kinase (ERK) and the stress-activated protein kinase (SAPK) or Jun N-terminal kinase (JNK) cascades independently stimulated PEA3-mediated gene expression. Ha-RasV12 stimulation of PEA3 activity was reduced by dominant-negative mutants in each of these protein kinase cascades, suggesting that Ras activates PEA3 through both pathways. Furthermore, the ability of unique activators of each kinase cascade to stimulate PEA3-dependent gene expression was selectively reduced by dominant-negative mutants within the homologous but not the heterologous pathway. Hence two distinct mitogen-activated protein kinase (MAPK) cascades regulate PEA3 activity. PEA3 was phosphorylated in vivo at serine residues consistent with the possibility that it may be a direct target of MAPKs.
Recent studies support a model for signal transduction from activated receptor tyrosine kinases to Ras which, in turn, activates the pathway of the mitogen-activated protein kinase (MAPK). Although some members of the Ets transcription factor family have been shown to be activated by this signaling pathway, no data are available on the activation of the PEA3 group of Ets proteins. This group is composed of three members -- PEA3, ER81 and ERM -- which are very similar in the DNA-binding domain, the ETS domain, in the 32 residue amino-terminal acidic domain and in the 61 residue carboxy-terminal domain. First of all we demonstrated that ERM-transfected cells contain a positive labeling in the nucleus, and we concluded that a nuclear localization signal might be situated in the ETS domain. We then showed that of four putative reporter plasmids, ERM activated the artificial 3 x TORU plasmid which contains an Ets binding site contiguous to an AP1 one. This transactivation enhancement requires the presence of the ERM amino-terminal domain. In contrast, although the lack of the carboxy-terminal domain induced a decrease in transactivation, this latter domain is not crucial. By using the E74-reporter plasmid system which is not basically activated by ERM, we showed that the activation of the Ras/Raf-1/MAPK pathway significantly enhanced ERM-mediated transactivation. The deletion of the amino-terminal transactivation domain abolished the capacity of stimulated MAPK to activate ERM. We also demonstrated that ERM can also be activated through the protein kinase A (PKA), another signaling pathway. Nevertheless, the MAPK and PKA activation of ERM are not synergistic. Finally, we showed that this Ets transcription factor is in vitro phosphorylated by both activated ERK-2 and activated PKA. ERM has thus been identified as a transcription factor which is a target for two different signaling pathways and might therefore be involved in the mitogenic response of cells.
A plethora of signals induce the c-fos proto-oncogene via phosphorylation of the transcription factor Elk-1 by MAP kinase. We show that the coactivator CBP cooperates with Elk-1 to stimulate c-fos. Elk-1 physically interacts with CBP, which is dependent on the transactivation domain of Elk-1 but is independent of MAP kinase phosphorylation. However, functional cooperation between Elk-1 and CBP requires phosphorylation of Elk-1. Importantly, a carboxy-terminal transactivation domain of CBP itself is phosphorylated by MAP kinase, whereby the transactivation potential of CBP is enhanced. Thus, MAP kinase may not solely activate specific transcription factors but also the coactivator CBP, identifying a novel aspect of MAP kinase function. Thereby MAP kinase stimulation can pleiotropically affect activation of genes regulated by different transcription factors interacting with the same coactivator CBP.