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The Level of C/EBP Protein Is Critical for Cell Migration during Drosophila Oogenesis and Is Tightly Controlled by Regulated Degradation
Abstract
The C/EBP transcription factor, Slbo, is required for migration of border cells during Drosophila oogenesis. Unexpectedly, we find that neither increase nor decrease of Slbo activity is tolerated in border cells. Correct protein level is in part ensured by cell type-specific regulated turnover of Slbo protein. Through genetic screening, we identify two genes that are involved in this regulation. The Ubp64 ubiquitin hydrolase acts as a stabilizer of Slbo protein. A novel gene, tribbles, is a negative regulator of slbo in vivo. Tribbles acts by specifically targeting Slbo for rapid degradation via ubiquitination.
... Functional studies in multiple organisms have shown that these pseudokinases play diverse roles in innate immunity, cell signalling, energy homeostasis and cell division [2,4]. The Drosophila tribbles gene is required for cell proliferation and migration in embryogenesis and oogenesis [5][6][7][8]. The mammalian Tribbles family includes three genes, Trib1, Trib2 and Trib3, each of which plays unique roles in signalling networks regulating adipose tissue, metabolic homeostasis and the immune system [2,4]. ...
... Mammalian Tribbles can interact with components of the mitogen-activated protein (MAP) kinase pathway and act as adaptor proteins to modulate the strength and output of kinase signalling cascades [10][11][12]. Like fly Tribbles [7,13], mammalian Tribbles bind to the basic leucine zipper (bZIP) transcription factors. Trib1 and Trib2 can induce degradation of several CCAAT/enhancer-binding protein (C/EBP) members in a context-dependent manner [1,14,15]. ...
... In further analyses using the yeast two-hybrid assay, we found that the N-terminal region (amino acids 1-115) of CEBP-1 was sufficient for binding NIPI-3 (Additional file 2: Figure S2). This interaction is reminiscent of those observed for fly and vertebrate Tribbles proteins, which bind and degrade C/EBP family proteins [1,7,[13][14][15]29], with human Trib1 binding the N-terminus of C/EBPα [1]. Thus, the ability of Tribbles and C/EBP proteins to interact directly is likely conserved from C. elegans to humans. ...
Background
Tribbles proteins are conserved pseudokinases that function to control kinase signalling and transcription in diverse biological processes. Abnormal function in human Tribbles has been implicated in a number of diseases including leukaemia, metabolic syndromes and cardiovascular diseases. Caenorhabditis elegans Tribbles NIPI-3 was previously shown to activate host defense upon infection by promoting the conserved PMK-1/p38 mitogen-activated protein kinase (MAPK) signalling pathway. Despite the prominent role of Tribbles proteins in many species, our knowledge of their mechanism of action is fragmented, and the in vivo functional relevance of their interactions with other proteins remains largely unknown. ResultsHere, by characterizing nipi-3 null mutants, we show that nipi-3 is essential for larval development and viability. Through analyses of genetic suppressors of nipi-3 null mutant lethality, we show that NIPI-3 negatively controls PMK-1/p38 signalling via transcriptional repression of the C/EBP transcription factor CEBP-1. We identified CEBP-1’s transcriptional targets by ChIP-seq analyses and found them to be enriched in genes involved in development and stress responses. Unlike its cell-autonomous role in innate immunity, NIPI-3 is required in multiple tissues to control organismal development. Conclusions
Together, our data uncover an unprecedented crosstalk involving multiple tissues, in which NIPI-3 acts as a master regulator to inhibit CEBP-1 and the PMK-1/p38 MAPK pathway. In doing so, it keeps innate immunity in check and ensures proper organismal development.
... The first tribbles protein was identified in Drosophila as a protein required for oogenesis [1]. By comparison, higher eukaryotes express three distinct tribbles, TRIB1, 2 and 3 which are characterized by a relatively well conserved kinase-like domain (40-70% identity) and variable N and C-terminal regions [2]. ...
... A prominent hypothesis to account for the role of TRIB1 in disease involves the degradation of C/EBP proteins by TRIB1 [5,[14][15][16]; this process is conserved in Drosophila where degradation of the C/EBP ortholog (slbo) is stimulated by tribbles [1]. TRIB1 could serve as a scaffold for the assembly of C/EBPA and/or C/EBPB and COP1, an E3 ligase, thereby promoting the degradation of these C/EBPs via the Ubiquitin Proteasome System (UPS) [17]. ...
... By contrast, no NLS, mono-or bipartite is predicted for TRIB2, in line with reports indicating that TRIB2 does not show preferential localization to the nucleus, and might actually be excluded in some systems [40,41,45]. A similar analysis of Drosophila tribbles fails to identify classical NLSs; tribbles, like TRIB2, has been reported to be present in the nucleus as well as the cytoplasm [1]. Thus the appearance of a bipartite NLS seems to be a recent addition that may have taken advantage of the poorly conserved and loosely structured N-terminal region to tailor certain tribbles for exclusive nuclear functions. ...
The TRIB1 gene has been associated with multiple malignancies, plasma triglycerides and coronary artery disease (CAD). Despite the clinical significance of this pseudo-kinase, there is little information on the regulation of TRIB1. Previous studies reported TRIB1 mRNA to be unstable, hinting that TRIB1 might be subject to post-transcriptional regulation. This work explores TRIB1 regulation, focusing on its post-transcriptional aspects. In 3 distinct model systems (HEK293T, HeLa and arterial smooth muscle cells) TRIB1 was undetectable as assessed by western blot. Using recombinant TRIB1 as a proxy, we demonstrate TRIB1 to be highly unstable at the protein and RNA levels. By contrast, recombinant TRIB1 was stable in cellular extracts. Blocking proteasome function led to increased protein steady state levels but failed to rescue protein instability, demonstrating that the 2 processes are uncoupled. Unlike as shown for TRIB2, CUL1 and TRCPβ did not play a role in mediating TRIB1 instability although TRCPβ suppression increased TRIB1 expression. Lastly, we demonstrate that protein instability is independent of TRIB1 subcellular localization. Following the identification of TRIB1 nuclear localization signal, a cytosolic form was engineered. Despite being confined to the cytosol, TRIB1 remained unstable, suggesting that instability occurs at a stage that precedes its nuclear translocation and downstream nuclear function. These results uncover possible avenues of intervention to regulate TRIB1 function by identifying two distinct regulatory axes that control TRIB1 at the post-transcriptional level.
... They have been implicated in embryonic development, as well as in various human diseases, including cancers, atherosclerosis, and insulin resistance [79]. Drosophila tribbles is a key regulator of cell cycle progression in morphogenesis [80,81,82] as well as cell migration during drosophila oogenesis [83]. Drosophila tribbles inhibits the activity of mitotic activator String/Cdc25 by inducing its proteolytic degradation via the proteasome [81,80]. ...
... Drosophila tribbles inhibits the activity of mitotic activator String/Cdc25 by inducing its proteolytic degradation via the proteasome [81,80]. Further, slbo, a homolog of the mammalian C/EBP transcription factor, is required for border cell migration during drosophila oogenesis [83]. Tribbles negatively regulates Slbo expression by specifically targeting it for proteasomal degradation [83]. ...
... Further, slbo, a homolog of the mammalian C/EBP transcription factor, is required for border cell migration during drosophila oogenesis [83]. Tribbles negatively regulates Slbo expression by specifically targeting it for proteasomal degradation [83]. ...
... To facilitate the degradation of C/EBPα, TRIB1 scaffolds an interaction between C/EBPα and the E3 ubiquitin ligase, COP1 (Figure 3), leading to the ubiquitination of C/EBPα [12,14,26]. This mechanism of C/EBPα degradation is evolutionarily conserved from Drosophila where Trbl regulates the C/EBPα homolog Slbo through a similar ubiquitin-dependent mechanism [12,14,27,58]. The structural investigation of COP1-TRIB1-C/EBPα interaction was discussed earlier in this review. ...
... To facilitate the degradation of C/EBPα, TRIB1 scaffolds an interaction between C/EBPα and the E3 ubiquitin ligase, COP1 ( Figure 3), leading to the ubiquitination of C/EBPα [12,14,26]. This mechanism of C/EBPα degradation is evolutionarily conserved from Drosophila where Trbl regulates the C/EBPα homolog Slbo through a similar ubiquitin-dependent mechanism [12,14,27,58]. The structural investigation of COP1-TRIB1-C/EBPα interaction was discussed earlier in this review. ...
Simple Summary
Tribbles proteins possess the structure of protein kinases but function by forming protein complexes rather than phosphorylating substrates. Here we review the structure–function relationship of TRIB1 in cancers. Of the Tribbles proteins, TRIB1 is currently the most well characterised structurally. TRIB1 has different states that could potentially be targeted by small-molecule inhibitors and well-established relevance in acute myeloid leukaemia through degradation of transcription factors. Less is understood about the role of TRIB1 in solid tumours. Further research is required to fully realise the potential of TRIB1 as either a direct target of small-molecule drugs or a biomarker of treatment response across diverse cancer types.
Abstract
The Tribbles family of proteins—comprising TRIB1, TRIB2, TRIB3 and more distantly related STK40—play important, but distinct, roles in differentiation, development and oncogenesis. Of the four Tribbles proteins, TRIB1 has been most well characterised structurally and plays roles in diverse cancer types. The most well-understood role of TRIB1 is in acute myeloid leukaemia, where it can regulate C/EBP transcription factors and kinase pathways. Structure–function studies have uncovered conformational switching of TRIB1 from an inactive to an active state when it binds to C/EBPα. This conformational switching is centred on the active site of TRIB1, which appears to be accessible to small-molecule inhibitors in spite of its inability to bind ATP. Beyond myeloid neoplasms, TRIB1 plays diverse roles in signalling pathways with well-established roles in tumour progression. Thus, TRIB1 can affect both development and chemoresistance in leukaemia; glioma; and breast, lung and prostate cancers. The pervasive roles of TRIB1 and other Tribbles proteins across breast, prostate, lung and other cancer types, combined with small-molecule susceptibility shown by mechanistic studies, suggests an exciting potential for Tribbles as direct targets of small molecules or biomarkers to predict treatment response.
... During Drosophila gastrulation, increased Tribbles levels in the ventral mesoderm temporarily block mitosis during invagination and promote migration of these cells between the overlying ectoderm and internal endoderm [1][2][3][4]. Subsequently, Tribbles expression decreases in these mesodermal precursors as they resume proliferation to form the muscle, heart, and peripheral nervous system. Recently, binding sites for the transcriptional repressor Tramtrack (Ttk) have been identified in a mesoderm-specific cis regulatory motif upstream of the trbl gene, which together with evidence that Ttk is expressed in the mesoderm upon migration, suggest a mechanism by which trbl gene expression is down-regulated after gastrulation [61,62]. ...
... Loss of Trib function promotes undifferentiated tumor formation in mammals and this tumor suppressor effect is explained in part by the potent ability of Trib family members to block cell growth and proliferation as well as cell differentiation during normal development [74]. The effect of Trbl on cell differentiation was recognized in a misexpression genetic screen conducted in the year 2000 that showed increased Trbl levels degrade the C/EBP homolog Slbo (slow border cells) to block differentiation of the border cells in the ovary [2,75]. Subsequent work showed that mammalian Trib1 and 2 bind and degrade C/EBP to promote T cell maturation, revealing the deep conservation of the interaction between Tribs and C/EBPs [10,76]. ...
Simple Summary
Tribbles pseudokinases represent a sub-branch of the CAMK (Ca²⁺/calmodulin-dependent protein kinase) subfamily and are associated with disease-associated signaling pathways associated with various cancers, including melanoma, lung, liver, and acute leukemia. The ability of this class of molecules to regulate cell proliferation was first recognized in the model organism Drosophila and the fruit fly genetic model and continues to provide insight into the molecular mechanism by which this family of adapter molecules regulates both normal development and disease associated with corruption of their proper regulation and function.
Abstract
The Tribbles (Trib) family of pseudokinase proteins regulate cell growth, proliferation, and differentiation during normal development and in response to environmental stress. Mutations in human Trib isoforms (Trib1, 2, and 3) have been associated with metabolic disease and linked to leukemia and the formation of solid tumors, including melanomas, hepatomas, and lung cancers. Drosophila Tribbles (Trbl) was the first identified member of this sub-family of pseudokinases and shares a conserved structure and similar functions to bind and direct the degradation of key mediators of cell growth and proliferation. Common Trib targets include Akt kinase (also known as protein kinase B), C/EBP (CAAT/enhancer binding protein) transcription factors, and Cdc25 phosphatases, leading to the notion that Trib family members stand athwart multiple pathways modulating their growth-promoting activities. Recent work using the Drosophila model has provided important insights into novel facets of conserved Tribbles functions in stem cell quiescence, tissue regeneration, metabolism connected to insulin signaling, and tumor formation linked to the Hippo signaling pathway. Here we highlight some of these recent studies and discuss their implications for understanding the complex roles Tribs play in cancers and disease pathologies.
... Several unrelated screens for Drosophila genes required for cell division and cell migration conducted in the year 2000 converged on the gene Tribbles (Trbl), an adaptor protein that facilitates the proteosomal turnover of key regulators of cell differentiation (Grosshans and Wieschaus, 2000;Rorth et al., 2000;Mata et al., 2000;Seher and Leptin, 2000). Subsequent unbiased fly genetic screens using both misexpression and loss-offunction approaches have uncovered diverse roles for Trbl in (1) neural differentiation during bristle patterning (Abdelilah-Seyfried et al., 2000), (2) stem cell proliferation in the germline (Schulz et al., 2004), (3) JAK/STAT signaling to modulate eye growth (Mukherjee et al., 2006), and (4) memory formation in the adult brain (LaFerriere et al., 2008). ...
... To test if these wing phenotypes were sensitive to interactions with known Trbl targets, we first examined the C/EBP encoded by the slow border cells (slbo) gene (Montell et al., 1992;Rorth et al., 2000). EnGAL4 misexpressing UAS-slbo was lethal, likely due to its potent transcription factor activity (Fig. 2B). ...
Drosophila Tribbles (Trbl) is the founding member of a family of pseudokinases with conserved roles in antagonizing cell division, tissue growth and cell differentiation. In humans, three Tribbles isoforms serve as adaptor proteins, binding targets such as Cdc25 phosphatase, Akt kinase or the transcription factor C/EBP to block their activity or direct their proteosomal degradation. Mutations in Tribbles family members are associated with susceptibility to diabetes and cancer, notably Notch-induced tumor growth. Trbl misexpression in the fly wing disk leads to a block in mitosis associated with decreased levels of String/Cdc25 and increased levels of Cyclin B leading to reduced overall wing size and reduced trichome density. We show these Trbl growth-restricting phenotypes can be suppressed by manipulating levels of known Trbl targets, and use this sensitized wing system to screen a collection of growth regulating open reading frames (ORFs) to search for enhancers and suppressors affecting cell and tissue size. By precisely measuring morphometric changes in wing phenotypes using a computer-based tool, we detected synthetic interactions with several E3 ubiquitin ligases, and focused our analysis on the Notch pathway components Neuralized (Neur) and Mindbomb1 (Mib1). In the wing, notum and egg chamber epithelia, Trbl misexpression suppressed Neur and Mib1 activities and stabilized the accumulation of both proteins. To understand these interactions, we used yeast two-hybrid assays to show Trbl physically bound to both Neur and Mib1. Our data are consistent with published reports that mammalian Tribbles3 modulates Notch responses by binding and stabilizing Mindbomb and indicate that a wing misexpression approach is useful to identify novel components in a conserved Tribbles signaling pathway.
AUTHOR SUMMARY
Tribbles pseudokinases are adaptor molecules, binding diverse targets regulating cell differentiation, growth and proliferation and directing their proteasomal degradation. To search for novel targets of Drosophila Tribbles, we adopted a wing co-misexpression scheme and measured changes in cell/tissue size to identify enhancers and suppressors of the Tribbles phenotype. We show the Notch pathway components Neuralized and Mindbomb1 E3 ligases act as Tribbles suppressors and demonstrate that Tribbles modulates their levels and activites. Recent demonstration that mammalian Tribbles 3 binds the E3 ligase Mindbomb to promote ligand-mediated Notch activation implies a conserved role for Tribbles family members in Notch activation.
... Deficiency in C/EBP expression is reported to be strongly linked to AML development (section 1.2.3.2) (Pabst and Mueller, 2007), albeit through an undefined mechanism. Interestingly, a conserved C-terminal-binding site for E3 ubiquitin ligases in Trib proteins facilitates the degradation of Trib targets such as C/EBP in several biological systems Keeshan et al., 2006;Mata et al., 2000;Qi et al., 2006;Rorth et al., 2000) ( Fig. 1.9). Therefore, it is suggested that ...
... Moreover, Drosophila Trib protein negatively regulates the level of Drosophila Slbo, which controls border cell migration and is a fly orthologue of the mammalian C/EBP transcription factor . In order to investigate its function, (Bowers et al., 2003;Rorth et al., 2000). E3 ...
Tribbles (Trib) protein was first described in Drosophila as a regulator of
proliferation, later being implicated as a G2/M modulator. In mammalian systems,
three Trib gene family members have been identified, which share a conserved motif
similar to the catalytic domain of serine/threonine kinases. However, they lack
several conserved residues in the ATP-binding pocket and the core motif of the
catalytic domain necessary for catalytic function. Tribbles 1 (Trib1) is involved in
inflammation through its ability to regulate MAPK, NF-κB and the CCAAT
Enhancer Binding Protein (C/EBP). Moreover, Trib1 is associated with human
disease, such as atherosclerosis and acute myeloid leukaemia. In this thesis, I
investigated the functional role of Trib1 in Toll-like Receptor (TLR)-induced
inflammatory responses together with pro- or anti-inflammatory cytokines. The
RAW264.7 myeloid cell line was stimulated with TLR2/9 ligands in the presence or
absence of IFN-γ or IL-10. I observed a high level of Trib1 expression in the
presence of IFN-γ and TLR2 ligands, but weak Trib1 expression following treatment
with IL-10 and TLR9 ligands. In gene knock-down experiments using small
interfering RNAs (siRNA) to reduce Trib1 expression, C/EBPβ was up-regulated in
both stimulated (by IFN-γ and TLR2 ligands) and resting macrophage populations.
TNF-α production was increased following Trib1 knockdown after treatment with
IFN-γ and/or TLR2 ligands but IL-6 secretion remained unchanged. Furthermore,
ERK1/2 expression was reduced in Trib1 siRNA-treated cells and failed to induce
chemokinesis in macrophages. Finally, Trib1 was demonstrated to act as a modulator
of cell cycle (G2/M) transition and displays a delayed apoptotic phenotype. The work in this thesis demonstrates that mammalian Trib1 contributes to the
pro-inflammatory response and functions as a regulator of the ERK1/2 and C/EBPβ
pathways following TLR ligand-mediated activation. Its novel functions include
acting as a modulator of G2/M arrest and suppressing macrophage migration.
... Trib function was first characterized in the Drosophila embryo, where Tribbles (Trbl) binds and degrades String/Twine phosphatase to block cell division both early during the midblastula transition and later in the invaginating mesoderm during gastrulation [3][4][5][6]. In the fly ovary, Trbl binds and degrades the C/EBP homolog Slow Border Cells (Slbo) to modulate cell migration [7]. Trib family members share three conserved motifs: (1) a divergent kinase-like domain notably lacking key residues required for catalytic activity, (2) a ubiquitin ligase COP1 binding domain and (3) a site for binding the MAP kinase kinase MEK1 [8,9]. ...
... Previously, Trbl has been shown to regulate activities of the C/ EBP Slbo during cell migration and in separate work, Trbl has been shown to regulate activity of String/Twine during cell proliferation [4,5,7], in both cases by binding the respective proteins and directing their proteasome-dependent degradation. Here we show that Drosophila Trbl binds Akt to block its phosphorylation-dependent activation and does so without affecting Akt levels (model in Fig. 7D). ...
Drosophila Tribbles (Trbl) is the founding member of the Trib family of kinase-like docking proteins that modulate cell signaling during proliferation, migration and growth. In a wing misexpression screen for Trbl interacting proteins, we identified the Ser/Thr protein kinase Akt1. Given the central role of Akt1 in insulin signaling, we tested the function of Trbl in larval fat body, a tissue where rapid increases in size are exquisitely sensitive to insulin/insulin-like growth factor levels. Consistent with a role in antagonizing insulin-mediated growth, trbl RNAi knockdown in the fat body increased cell size, advanced the timing of pupation and increased levels of circulating triglyceride. Complementarily, overexpression of Trbl reduced fat body cell size, decreased overall larval size, delayed maturation and lowered levels of triglycerides, while circulating glucose levels increased. The conserved Trbl kinase domain is required for function in vivo and for interaction with Akt in a yeast two-hybrid assay. Consistent with direct regulation of Akt, overexpression of Trbl in the fat body decreased levels of activated Akt (pSer505-Akt) while misexpression of trbl RNAi increased phospho-Akt levels, and neither treatment affected total Akt levels. Trbl misexpression effectively suppressed Akt-mediated wing and muscle cell size increases and reduced phosphorylation of the Akt target FoxO (pSer256-FoxO). Taken together, these data show that Drosophila Trbl has a conserved role to bind Akt and block Akt-mediated insulin signaling, and implicate Trib proteins as novel sites of signaling pathway integration that link nutrient availability with cell growth and proliferation.
... Slbo, a Drosophila homolog of C/EBP, is regulated at the protein level by Tribbles-mediated degradation in oogenesis [11]. In mammals, degradation of C/EBPb is reduced by Trib1 (tribbles homolog 1) ...
Cop1 encodes a ubiquitin E3 ligase that has been well preserved during evolution in both plants and metazoans. In metazoans, the C/EBP family transcription factors are targets for degradation by Cop1, and this process is regulated by the Tribbles pseudokinase family. Over-expression of Tribbles homolog 1 ( Trib1 ) induces acute myeloid leukemia (AML) via Cop1-dependent degradation of the C/EBPa p42 isoform. Here, we induced rapid growth arrest and granulocytic differentiation of Trib1 -expressing AML cells using a Cop1 conditional knockout (KO), which is associated with a transient increase in the C/EBPa p42 isoform. The growth-suppressive effect of Cop1 KO was canceled by silencing of Cebpa and reinforced by exogenous expression of the p42 isoform. Moreover, Cop1 KO improved the survival of recipients transplanted with Trib1 -expressing AML cells. We further identified a marked increase in Trib1 protein expression in Cop1 KO, indicating that Trib1 is self-degraded by the Cop1 degradosome. COP1 downregulation also inhibits the proliferation of human AML cells in a TRIB1 -dependent manner. Taken together, our results provide new insights into the role of Trib1/Cop1 machinery in the C/EBPa p42-dependent leukemogenic activity, and a novel idea to develop new therapeutics.
... Tribbles was determined to act by negatively regulating the cell cycle regulator String/Cdc25, supporting its suppression of expression, thus allowing the gastrulation phase to take place and delaying the initiation of mitosis [3,4]. These early studies highlighted essential hallmarks of TRIB protein family members, with specific roles of TRIBs in specific developmental stages [2,3], in modulating specific protein targets [3,5], and having cell-specific roles [6]. Research advances in TRIBs have established the consensus that this protein family plays roles in diverse biological conditions and regulates intracellular signaling networks and multiple human diseases, as reviewed in [7]. ...
Simple Summary
TRIB1 is at the center of major cell signaling pathways. In this review, we describe its role in immune cells and highlight TRIB1 interacting partners which suggests cell-specific functions and that TRIB1 is involved in cellular homeostasis and also in different cancers and immune-related disorders.
Abstract
Research advances in Tribbles homolog (TRIB) genes have established the consensus that this protein family plays roles in diverse biological conditions and regulates intracellular signaling networks and several human diseases. In this review, we focus on one member of the family, TRIB1, and its role at the crossroads of immune signaling. TRIB1 directly interacts with transcription factors such as FOXP3 and C/EBPα, with several signaling molecules such as MEK1 and MALT1 and directly acts on key cell signaling pathways such as the MAPK and NF-κB pathways. Altogether, these interactions emphasize that TRIB1 is at the center of major cell signaling pathways while TRIB1 has cell-specific roles, potentially depending on the expressing cells and binding partners. In this review, we describe its roles in immune cells and highlight the interacting partners explaining these functions which suggests TRIB1 as a precise mediator of cellular homeostasis as well as in different cancers and immune-related disorders.
... One of the best understood molecular functions for TRIB1 is its role in mediating the ubiquitination and degradation of the transcription factor C/EBPa by bringing it into proximity of the COP1 E3 ubiquitin ligase. Tribbles-mediated regulation of C/EBPa protein levels has been shown to be an important function in several models, including as a causal mechanism for the hepatic lipogenesis phenotype in Trib1 liver-specific knockout mice [13], for myeloid cell proliferation in the context of leukemia [31], and in oogenesis in drosophila [58]. We report here that Trib1_ASKO adipocytes also exhibit increased C/EBPa protein levels in the absence of any change in gene expression, and that C/EBPa protein levels are normalized between control and Trib1_ASKO SVF-derived adipocytes under conditions of proteasomal inhibition. ...
Objective
Multiple GWAS have identified SNPs in the 8q24 locus near TRIB1 that significantly associate with plasma lipids and other markers of cardiometabolic health, and prior studies have uncovered roles for hepatic and myeloid Trib1 in plasma lipid regulation and atherosclerosis. The same 8q24 SNPs additionally associate with plasma adiponectin levels in humans, implicating TRIB1 in adipocyte biology. Here, we hypothesize that TRIB1 in adipose tissue regulates plasma adiponectin, lipids, and metabolic health.
Methods
We investigate the metabolic phenotype of adipocyte-specific Trib1 knockout mice (Trib1_ASKO) on chow and high fat diet. Through secretomics of adipose tissue explants and RNA-seq of adipocytes and livers from these mice, we further investigate the mechanism of TRIB1 in adipose tissue.
Results
Trib1_ASKO mice have an improved metabolic phenotype with increased plasma adiponectin levels, improved glucose tolerance, and decreased plasma lipids. Trib1_ASKO adipocytes have increased adiponectin production and secretion independent of the known TRIB1 function of regulating proteasomal degradation. RNA-seq analysis of adipocytes and livers from Trib1_ASKO mice suggests that alterations in adipocyte function underlie the observed plasma lipid changes. Adipose tissue explant secretomics further reveals that Trib1_ASKO adipose tissue has decreased ANGPTL4 production, and we demonstrate an accompanying increase in LPL activity that likely underlies the triglyceride phenotype.
Conclusion
Adipocyte Trib1 regulates multiple aspects of metabolic health, confirming previously observed genetic associations in humans and shedding light on further mechanisms by which TRIB1 regulates plasma lipids and metabolic health.
... TRB3 is highly conserved through the evolutionary process and has numerous functions, including cell cycle regulation, apoptosis, differentiation, and metabolism [5e13]. TRB3 interacts with its target proteins, regulating their activities or functions [5,7,14,15]. In liver, TRB3 inhibits Akt through direct binding and therefore results in impaired insulin signaling, decreased glycogen content, and increased hepatic glucose output [9,10]. ...
Brown adipose tissue (BAT) and stimulating adaptive thermogenesis have been implicated as anti-obese and anti-diabetic tissues due to their ability to dissipate energy as heat by the expression of UCP1. We have recently demonstrated that TRB3 impairs differentiation of brown preadipocytes via inhibiting insulin signaling. However, the roles of the protein in BAT function and thermogenesis in vivo have not yet been established. For this study we tested the hypothesis that TRB3 mediates obesity- and diabetes-induced impairments in BAT differentiation and function, and that inhibition of TRB3 improves BAT function. TRB3 expression was increased in BAT from high-fat fed mice and ob/ob mice, which was associated with decreased UCP1 expression. Incubation of brown adipocytes with palmitate increased TRB3 expression and decreased UCP1. Knockout of TRB3 in mice displayed higher UCP1 expression in BAT and cold resistance. Incubation of brown adipocytes with ER stressors increased TRB3 but decreased UCP1 and ER stress markers were elevated in BAT from high-fat fed mice and ob/ob mice. Finally, high-fat feeding in TRB3KO mice were protected from obesity-induced glucose intolerance and displayed cold resistance and higher expression of BAT-specific markers. These data demonstrate that high-fat feeding and obesity increase TRB3 in BAT, resulting in impaired tissue function.
... [IFL]...[IL]"), given the tribbles-mediated degradation of a CEBP family transcription factor is still conserved in drosophila melanogaster (Rørth et al., 2000). The multiple sequence alignment was generated by Clustal Omega using default parameters (https://www.ebi.ac.uk/Tools/msa/clustalo) (Sievers et al., 2011), and visualized using Jalview (Waterhouse et al., 2009). ...
Despite remarkable clinical efficacies of immune checkpoint blockade (ICB) in cancer treatment, ICB benefits in triple-negative breast cancer (TNBC) remain limited. Through pooled in vivo CRISPR knockout (KO) screens in syngeneic TNBC mouse models, we found that inhibition of the E3 ubiquitin ligase Cop1 in cancer cells decreases the secretion of macrophage-associated chemokines, reduces tumor macrophage infiltration, and shows synergy in anti-tumor immunity with ICB. Transcriptomics, epigenomics, and proteomics analyses revealed Cop1 functions through proteasomal degradation of the C/ebpδ protein. Cop1 substrate Trib2 functions as a scaffold linking Cop1 and C/ebpδ , which leads to polyubiquitination of C/ebpδ . Cop1 inhibition stabilizes C/ebpδ to suppress the expression of macrophage chemoattractant genes. Our integrated approach implicates Cop1 as a target for improving cancer immunotherapy efficacy by regulating chemokine secretion and macrophage levels in the TNBC tumor microenvironment.
Highlights
Large-scale in vivo CRISPR screens identify new immune targets regulating the tumor microenvironment
Cop1 knockout in cancer cells enhances anti-tumor immunity
Cop1 modulates chemokine secretion and macrophage infiltration into tumors
Cop1 targets C/ebp δ degradation via Trib2 and influences ICB response
... To check this, we overexpressed exogenous copy of the slbo coding sequence (UAS-slbo) in BCs using the slbo-Gal4 (Novo16) driver. In a wild-type background, this led to about 10.5-fold increase in slbo mRNA level and resulted only in a minimal disruption of the collective cell migration process (Figure 4), which is principally consistent with earlier observations [25]. In Trl 362 /Trl R85 mutants, slbo-Gal4 (Novo16) -driven slbo overexpression increased the expression level of the gene only about 2.0-fold ( Figure 4A). ...
Collective cell migration is a complex process that happens during normal development of many multicellular organisms, as well as during oncological transformations. In Drosophila oogenesis, a small set of follicle cells originally located at the anterior tip of each egg chamber become motile and migrate as a cluster through nurse cells toward the oocyte. These specialized cells are referred to as border cells (BCs) and provide a simple and convenient model system to study collective cell migration. The process is known to be complexly regulated at different levels and the product of the slow border cells (slbo) gene, the C/EBP transcription factor, is one of the key elements in this process. However, little is known about the regulation of slbo expression. On the other hand, the ubiquitously expressed transcription factor GAGA, which is encoded by the Trithorax-like (Trl) gene was previously demonstrated to be important for Drosophila oogenesis. Here, we found that Trl mutations cause substantial defects in BC migration. Partially, these defects are explained by the reduced level of slbo expression in BCs. Additionally, a strong genetic interaction between Trl and slbo mutants, along with the presence of putative GAGA binding sites within the slbo promoter and enhancer, suggests the direct regulation of this gene by GAGA. This idea is supported by the reduction in the slbo-Gal4-driven GFP expression within BC clusters in Trl mutant background. However, the inability of slbo overexpression to compensate defects in BC migration caused by Trl mutations suggests that there are other GAGA target genes contributing to this process. Taken together, the results define GAGA as another important regulator of BC migration in Drosophila oogenesis.
... 12,13 Tribbles proteins were first described in Drosophila when multiple genetic screens revealed that trbl regulates the proteins string and slbo, the latter being the Drosophila homolog of the C/EBPα basic region-leucine zipper transcription factor in humans. [14][15][16] Subsequent work established that TRIB1 and TRIB2 can regulate the proteasomal degradation of C/ EBPα and β by binding the E3 ubiquitin ligase COP1, which in turn ubiquitinates the target C/EBP proteins and promotes their proteasomal degradation. 17,18 A recently resolved crystal structure of this complex revealed an elegant system in which binding of C/EBPα to the catalytic pocket of TRIB1 induces a conformation change allowing for better recruitment of COP1 to the C terminus of TRIB1. ...
Genome-wide association studies have identified hundreds of genomic loci in humans that are significantly associated with plasma cholesterol, triglycerides, and coronary artery disease. Although some loci contain genes with known regulatory roles in lipid metabolism and atherosclerosis, the majority were being implicated for the first time. The 8q24 locus, containing the gene Tribbles-1 (TRIB1), is the only novel genome-wide association studies locus that associates with all 4 plasma lipid traits and coronary artery disease, an observation that has spurred immense interest in this locus. Subsequent in vivo loss and gain of function studies confirmed that Trib1 plays a role in hepatic lipid metabolism, validating the initial genetic observation. Yet, many challenges remain in discerning the nature of the association between the TRIB1 locus and cardiometabolic phenotypes. Is TRIB1 the causal gene at the 8q24 locus and what is the functional consequence of the associated noncoding variation? Is the relationship between TRIB1 and the transcription factor C/EBPα the primary molecular mechanism governing the genetic association or could it be an as yet unknown function for this interesting pseudokinase? Is hepatic TRIB1 the sole regulator of lipid metabolism or could extrahepatic TRIB1 play a role as well? The following review summarizes key findings related to these questions and highlights both the challenges and excitement in pursuing translational research of a novel gene in the post-genome-wide association studies era.
... Tribbles proteins primarily act as adaptors to promote protein degradation and/or sequestration. [5][6][7] There are 3 mammalian tribbles homologs (Trib1-3) that are defined by a central serine/threonine kinase-like domain and C-terminal sequences that bind the E3 ubiquitin ligase COP1. [8][9][10][11][12] Mice with germline deletion of Trib1 lack "M2" macrophages and eosinophils, and have more neutrophils, a phenotype that is influenced by the failure of Trib1 to promote CCAAT/enhancer binding protein a (C/EBPa) protein degradation. ...
Eosinophils and neutrophils are critical for host defense, yet gaps in understanding how granulocytes differentiate from hematopoietic stem cells (HSCs) into mature effectors remain. The pseudokinase tribbles homolog 1 (Trib1) is an important regulator of granulocytes; knockout mice lack eosinophils and have increased neutrophils. However, how Trib1 regulates cellular identity and function during eosinophilopoiesis is not understood. Trib1 expression markedly increases with eosinophil-lineage commitment in eosinophil progenitors (EoPs), downstream of the granulocyte/macrophage progenitor (GMP). Using hematopoietic- and eosinophil-lineage-specific Trib1 deletion, we found that Trib1 regulates both granulocyte precursor lineage commitment and mature eosinophil identity. Conditional Trib1 deletion in HSCs reduced the size of the EoP pool and increased neutrophils, whereas deletion following eosinophil lineage commitment blunted the decrease in EoPs without increasing neutrophils. In both modes of deletion, Trib1-deficient mice expanded a stable population of Ly6G+ eosinophils with neutrophilic characteristics and functions, and had increased CCAAT/enhancer binding protein α (C/EBPα) p42. Using an ex vivo differentiation assay, we found that interleukin 5 (IL-5) supports the generation of Ly6G+ eosinophils from Trib1-deficient cells, but is not sufficient to restore normal eosinophil differentiation and development. Furthermore, we demonstrated that Trib1 loss blunted eosinophil migration and altered chemokine receptor expression, both in vivo and ex vivo. Finally, we showed that Trib1 controls eosinophil identity by modulating C/EBPα. Together, our findings provide new insights into early events in myelopoiesis, whereby Trib1 functions at 2 distinct stages to guide eosinophil lineage commitment from the GMP and suppress the neutrophil program, promoting eosinophil terminal identity and maintaining lineage fidelity.
... The Tribbles family of proteins occupies a dedicated branch of the kinome composed of four members-TRIB1, TRIB2, TRIB3, and the more distantly related STK40 (also known as SgK495) (4). The family derives its name from the Drosophila tribbles protein (5,6) and shares a common domain architecture: They have a variable N-terminal extension, a pseudokinase domain, and a C-terminal extension that binds to the ubiquitin E3 ligase COP1 (Fig. 1A). The pseudokinase and COP1-binding motif are key for the function of Tribbles proteins-by binding to substrates through their pseudokinase domain and to COP1 through their C terminus, they act as substrate adaptors to facilitate ubiquitylation by COP1. ...
The Tribbles family of pseudokinases recruits substrates to the ubiquitin ligase COP1 to facilitate ubiquitylation. CCAAT/enhancer-binding protein (C/EBP) family transcription factors are crucial Tribbles substrates in adipocyte and myeloid cell development. We found that the TRIB1 pseudokinase was able to recruit various C/EBP family members and that the binding of C/EBPβ was attenuated by phosphorylation. To explain the mechanism of C/EBP recruitment, we solved the crystal structure of TRIB1 in complex with C/EBPα, which revealed that TRIB1 underwent a substantial conformational change relative to its substrate-free structure and bound C/EBPα in a pseudosubstrate-like manner. Crystallographic analysis and molecular dynamics and subsequent biochemical assays showed that C/EBP binding triggered allosteric changes that link substrate recruitment to COP1 binding. These findings offer a view of pseudokinase regulation with striking parallels to bona fide kinase regulation—by means of the activation loop and αC helix—and raise the possibility of small molecules targeting either the activation “loop-in” or “loop-out” conformations of Tribbles pseudokinases.
... Similar to that in hepatic Trib3 overexpression, we also observed reduced AKT phosphorylation in skeletal muscle tissues of Trib3TG mice; however, this primarily contributed from the decreased total AKT protein level, particularly AKT2, rather than blocking the phosphorylation of AKT. Trib3 regulates protein activity or stability by enhancing ubiquitination and proteasome-dependent degradation of its binding substrates [4,20,32]. Similarly, we revealed that Trib3, without altering AKT2 mRNA level, dose-dependently induced AKT2 ubiquitination. In regulating AKT activity by the ubiquitination process, Suizu et al. showed that TTC3 bound and preferentially ubiquitinated phosphorylated, or activated AKT [33]. ...
Background/aims:
Obesity is a serious health risk factor strongly associated with insulin resistance and type 2 diabetes; however, the underlying mechanisms associating obesity with insulin resistance remain unknown. In this study, we explored the physiological role of Trib3 in regulating glucose metabolism in skeletal muscle tissues in a Trib3 transgenic mice model.
Methods:
Glucose metabolism in transgenic mice overexpressing Trib3 specifically in the skeletal muscle was examined by glucose/insulin tolerance test, metabolic cage studies, and glucose uptake assay. The effect of Trib3 overexpression on AKT phosphorylation and AKT protein turnover were assessed by RT-PCR and immunoblot analysis. Subcellular distribution of Trib3 and AKT1/2 was determined by microscopic analysis, co-immunoprecipitation experiments, and limited-detergent extraction of subcellular organelles. Ubiquitin assay was performed and ATG7 deficient cell line was employed to address the mechanisms of Trib3-dependent AKT protein homeostasis.
Results:
We found that Trib3 expression in skeletal muscle is elevated in obese conditions, and transgenic mice that overexpressed Trib3, specifically in skeletal muscle tissues, displayed impaired glucose homeostasis by suppressing insulin-stimulated glucose uptake. Disruption of insulin signaling in skeletal muscle Trib3 transgenic mice may occur due to the specific downregulation of AKT2 but not AKT1. Autophagy regulated AKT2 protein turnover, and Trib3 overexpression stimulated autophagic degradation of AKT2 by promoting AKT2 ubiquitination.
Conclusion:
Because diet-induced obesity upregulates Trib3 and downregulates AKT2 in skeletal muscle tissues, Trib3 may play a key role in establishing an association between obesity and insulin resistance by regulating AKT2 protein homeostasis.
... Group 3 (Cell migration) is a fundamental process in the development and maintenance of multicellular organisms. Several reproductive events depend on this process, as embryo formation (Jovanović et al., 2010), immunological responses (Sánchez-Madrid and del Pozo, 1999), spermatogenesis , and oogenesis (Rørth et al., 2000). Genes clustered in this group were associated with daughter pregnancy rate, cow conception rate (Ortega et al., 2016), development of nervous system (Kapur et al., 1991;Kanaani et al., 2005;Jessberger et al., 2009), embryo lethality (Nourizadeh-Lillabadi et al., 2010), postnatal mortality and growth retardation (Pallares and Gonzalez-Bulnes, 2010), immunity (Bogdan, 2015), embryo differentiation (Rosa and Brivanlou, 2011), and perinatal mortality (Brites et al., 2003). ...
Multi-trait meta-analyses are a strategy to produce more accurate genome-wide association studies (GWAS), especially for complex phenotypes. We carried out a meta-analysis study for traits related to sexual precocity in tropical beef cattle (Nellore and Brahman) aiming to identify important genomic regions affecting these traits. The traits included in the analyses were age at first calving (AFC), early pregnancy (EP), age at first corpus luteum (AGECL), first postpartum anoestrus interval (PPAI) and scrotal circumference (SC). The traits AFC, EP and SCN were measured in Nellore cattle, while AGECL, PPAI and SCB were measured in Brahman cattle. Meta-analysis resulted in 108 significant single nucleotide polymorphisms (SNP), at an empirical threshold P-value of 1.39x10⁻⁵ (FDR < 0.05). Within 0.5 Mb of the significant SNP, candidate genes were annotated and analyzed for functional enrichment. Most of the closest genes to the SNP with higher significance in each chromosome have been associated with important roles in reproductive function, these are TSC22D2, KLF7, ARHGAP29, 7SK, MAP3K5, TLE3, WDR5, TAF3, TMEM68, PPP1R15B, NR2F2, GALR1, SUFU and KCNU1. We did not observe any significant SNP in BTA5, BTA12, BTA17, BTA18, BTA19, BTA20, BTA22, BTA23, BTA25 and BTA28. Although the majority of significant SNP are in BTA14, it was identified significant associations in multiple chromosomes (19 out of 29 autosomes), which is consistent with the postulation that reproductive traits are complex polygenic phenotypes. Five proposed association regions harbor the majority of the significant SNP (76 %) and were distributed over four chromosomes (P < 1.39x10⁻⁵, FDR < 0.05): BTA2 (5.55%) from 95 to 96 Mb, BTA4 (5.55%) from 94.1 to 94.8 Mb, BTA14 (59.26%) from 24 to 25 Mb and 29 to 30 Mb, and BTA21 (5.55%) from 6.7 Mb to 11.4 Mb. These regions harbored key genes related to reproductive function. Moreover, these genes were enriched for functional groups associated with immune response, maternal-fetal tolerance, pregnancy maintenance, embryo development, fertility and response to stress. Further studies including other breeds and precocity traits could confirm the importance of these regions and identify new candidate regions for sexual precocity in beef cattle.
... In addition, multiple transcription factors are involved in this process. For example, the C/EBP transcription factor encoded by the slow border cells (slbo) gene (Saadin and Starz-Gaiano 2016) is specifically expressed in BCs starting from stage 8, and required for their migration (Montell et al. 1992;Rørth et al. 2000). Actin-binding proteins such as Rac, Filamin, Cortactin, Cofilin/ADF, Profilin, and Twinfilin, also play important roles during BC migration (Chen et al. 2001;Stossel et al. 2001;Weed and Parsons 2001;Sokol and Cooley 2003;Weaver et al. 2003;Burridge and Wennerberg 2004;Raftopoulou and Hall 2004;Somogyi and Rørth 2004). ...
Border cell (BC) migration during Drosophila oogenesis is an excellent model for the analysis of the migratory and invasive cell behavior. Most studies on BC migration have exploited a slbo-Gal4 driver to regulate gene expression in these cells or to mark them. Here, we report that the slbo-Gal4 transgene present in the line #6458 from the Bloomington Stock Center is inserted within chickadee (chic), a gene encoding the actin-binding protein Profilin, which promotes actin polymerization and is known to be involved in cell migration. The chic6458 mutation caused by the transgene insertion behaves as a null chic allele and is homozygous lethal. To evaluate possible effects of chic6458 on the assessment of BC behavior, we generated new lines bearing the slbo-Gal4 transgene inserted into different second chromosome loci that do not appear to be involved in cell migration. Using these new lines and the slbo-Gal4-chic6458 line, we defined the functional relationships between the twinfilin (twf) and chic in BC migration. Migration of BCs is substantially reduced by mutations in twf, which encodes an actin-binding protein that inhibits actin filament assembly. The defects caused by twf mutations are significantly suppressed when the slbo-Gal4-chic6458, but not the new slbo-Gal4 drivers were used. These findings indicate twf and chic interact and function antagonistically during BC migration in Drosophila oogenesis.
... The Tribbles family of proteins occupy a dedicated branch of the kinome comprised of four members-TRIB1, TRIB2, TRIB3, and the more distantly related STK40 (also known as SgK495) (4). The family derives its name from the Drosophila Tribbles protein (5,6), and share a common domain architecture: they have a variable N-terminal extension; a pseudokinase domain; and a C-terminal extension that binds to the COP1 ubiquitin E3 ligase (Fig. 1A). The pseudokinase and COP1-binding motif are key for the function of Tribbles proteins-by binding to substrates through their pseudokinase domain and to COP1 through their C-terminus they act as substrate adaptors for ubiquitination by COP1. ...
One Sentence Summary
Substrate binding to Tribbles-homolog 1 (TRIB1) pseudokinase induces allosteric changes that allow formation of a complex with the COP1 ubiquitin ligase.
Abstract
The Tribbles family of pseudokinases recruit substrates to the COP1 ubiquitin ligase for ubiquitination. CCAAT-enhancer binding protein (C/EBP) family transcription factors are crucial Tribbles substrates in adipocyte and myeloid development. Here we show that the TRIB1 pseudokinase can recruit various C/EBP family members, with binding of C/EBPβ attenuated by phosphorylation. To explain the mechanism of substrate recruitment, we solved the crystal structure of TRIB1 in complex with C/EBPα. TRIB1 undergoes a significant conformational change relative to its substrate-free structure, to bind C/EBPα in a pseudo-substrate-like manner. Crucially, substrate binding triggers allosteric changes that link substrate recruitment to COP1 binding, which is consistent with molecular dynamics and biochemical studies. These findings offer a view of pseudokinase regulation with striking parallels to bona fide kinase regulation— via the activation loop and αC-helix—and raise the possibility of small molecules targeting either the activation loop-in, or loop-out, conformations of Tribbles pseudokinases.
... Tribbles promotes the proteasome-dependent degradation of slbo, the Drosophila homolog of the C/EBP family, which is critical for cell migration during oogenesis. 6 Mammals have 3 homologs of tribbles, Trib1, Trib2, and Trib3, which are characterized by a pseudokinase domain and C-terminal COP1-binding motif. 7,8 Trib1 and Trib2, but not Trib3, downregulate C/EBPa and induce AML in mouse models. ...
C/EBPα is a key transcription factor regulating myeloid differentiation and leukemogenesis. The Trib1-COP1 complex is an E3 ubiquitin ligase that targets C/EBPα for degradation, and its overexpression specifically induces acute myeloid leukemia (AML). Here we show that myeloid leukemia factor 1 (MLF1) stabilizes C/EBPα protein levels by inhibiting the ligase activity of the Trib1-COP1 complex. MLF1 directly interacts with COP1 in the nucleus and interferes with the formation of the Trib1-COP1 complex, thereby blocking its ability to polyubiquitinate C/EBPα for degradation. MLF1 overexpression suppressed the Trib1-induced growth advantage in a murine bone marrow (BM) culture and Trib1-induced AML development in BM-transplanted mouse models. MLF1 was expressed in hematopoietic stem cells and myeloid progenitors (common myeloid progenitors and granulocyte-macrophage progenitors) in normal hematopoiesis, which is consistent with the distribution of C/EBPα. An MLF1 deficiency conferred a more immature phenotype on Trib1-induced AML development. A higher expression ratio of Trib1 to MLF1 was a key determinant for AML development in mouse models, which was also confirmed in human patient samples with acute leukemia. These results indicate that MLF1 is a positive regulator that is critical for C/EBPα stability in the early phases of hematopoiesis and leukemogenesis.
... The link of zygotic transcription and the switch-like decrease in the half-life of Twine suggests that zygotic factors may be involved. One of these factors is the pseudokinase Tribbles [101][102][103], as RNAimediated depletion of tribbles accelerates Twine degradation [97]. ...
The activation of the zygotic genome and onset of transcription in blastula embryos is linked to changes in cell behavior and remodeling of the cell cycle and constitutes a transition from exclusive maternal to zygotic control of development. This step in development is referred to as mid-blastula transition and has served as a paradigm for the link between developmental program and cell behavior and morphology. Here, we discuss the mechanism and functional relationships between the zygotic genome activation and cell cycle control during mid-blastula transition with a focus on Drosophila embryos.
... The Tribbles protein regulates cell proliferation, migration, and morphogenesis during Drosophila embryo development 21,22 , but the functions of the mammalian Tribbles family in vivo remain largely unknown. To investigate the effects of Trib2 on mammalian physiology, we generated Trib2-deficient mice using a conventional gene targeting approach. ...
Tribbles homolog 2 (Trib2) is a member of Tribbles protein pseudokinases and involves in apoptosis, autoimmunity, cancer, leukemia and erythropoiesis, however, the physiological function of Trib2 in hematopoietic system remains to be elucidated. Here, we report that Trib2 knockout (KO) mice manifest macrocytic anemia and increase of T lymphocytes. Although Trib2 deficient RBCs have similar half-life as the control RBCs, Trib2 KO mice are highly vulnerable to oxidant-induced hemolysis. Endogenous Trib2 mRNA is expressed in early hematopoietic progenitors, erythroid precursors, and lymphoid lineages, but not in mature RBCs, myeloid progenitors and granulocytes. Consistently, flow cytometric analysis and in vitro colony forming assay revealed that deletion of Trib2 mainly affected erythroid lineage development, and had no effect on either granulocyte or megakaryocyte lineages in bone marrow. Furthermore, a genetic approach using double knockout of Trib2 and C/ebpα genes in mice suggested that Trib2 promotes erythropoiesis independent of C/ebpα proteins in vivo. Finally, ectopic expression of human Trib2 in zebrafish embryos resulted in increased expression of erythropoiesis-related genes and of hemoglobin. Taking all data together, our results suggest that Trib2 positively promotes early erythrocyte differentiation and is essential for tolerance to hemolysis.
... USP47 is a deubiquitinase (DUB) of the ubiquitin specific protease (USP) family ( Fig 1A) [9]. It has previously been associated with the regulation of the transcription factors TTK (tramtrack; FBgn0003870) and SLBO (FBgn0005638) in Drosophila [10,11]. Its human orthologue, USP47 interacts with the beta-TRCP E3 ligase complex [12] and has been found to regulate base-excision repair (BER) by controlling the levels of Polymerase β [13,14]. ...
Author
The RAS-MAPK pathway plays a central role in the development of multicellular organisms, predominantly by regulating cell proliferation and differentiation. At its core, the pathway includes the RAS GTPase and three kinases (RAF, MEK, and MAPK) that transmit RAS signals through a phosphorylation cascade. Several factors have been discovered over the years that modulate signal transmission by altering the kinetics of phosphorylation/dephosphorylation of the core pathway components. In contrast, scant information is available on the mechanisms governing their expression and steady-state levels. Here, we report the characterization of a deubiquitinase (DUB) known as USP47, which stabilizes MAPK protein levels by opposing the activity of the proteasome, which is a major protein degradation machinery in eukaryotic cells. A search for enzymes that opposed USP47 activity identified several components of the N-end rule pathway centered on the poorly characterized POE/UBR4 E3 ligase. These components appear to work together to target MAPK for proteasome-mediated protein degradation and thereby reduce MAPK protein half-life. Together, this work identifies a new means by which RAS-MAPK signaling output is controlled in Drosophila. As the role of the N-end rule pathway remains largely uncharacterized in metazoans, this work opens new opportunities for studying its function in higher organisms.
... A recent study demonstrated that Trbl also regulates Twine degradation, a homologue of String, in the Drosophila blastoderm during the midblastula transition [4]. Trbl was also found to promote the degradation of Slbo, the Drosophila orthologue of the important CCAAT/enhancer binding protein (C/EBP) family of transcription factors, which are critical for transcriptional programmes associated with cell migration during oogenesis [5]. Recently, the proto-oncogene AKT was identified as a third Trbl interacting protein in flies. ...
Tribbles homolog 2 (TRIB2) is a member of the mammalian Tribbles family of serine/threonine pseudokinases (TRIB1-3). Studies of TRIB2 indicate that many of the molecular interactions between the single Drosophila Tribbles (Trbl) protein and interacting partners are evolutionary conserved. In this study, we examined the relationship between TRIB2 and cell division cycle 25 (CDC25) family of dual-specificity protein phosphatases (mammalian homologues of Drosophila String), which are key physiological cell cycle regulators. Using co-immunoprecipitation we demonstrate that TRIB2 interacts with CDC25B and CDC25C selectively. Forced overexpression of TRIB2 caused a marked decrease in total CDC25C protein levels. Following inhibition of the proteasome, CDC25C was stabilized in the nuclear compartment. This implicates TRIB2 as a regulator of nuclear CDC25C turnover. In complementary ubiquitination assays, we show that TRIB2-mediated degradation of CDC25C is associated with lysine-48-linked CDC25C polyubiquitination driven by the TRIB2 kinase-like domain. A cell cycle associated role for TRIB2 is further supported by the cell cycle regulated expression of TRIB2 protein levels. Our findings reveal mitotic CDC25C as a new target of TRIB2 that is degraded via the ubiquitin proteasome system. Inappropriate CDC25C regulation could mechanistically underlie TRIB2 mediated regulation of cellular proliferation in neoplastic cells.
... One recent study showed that significantly associated SNPs in the TRIB1 locus alter the expression of a long noncoding RNA named TRIBAL (TRIB1 associated locus), although the role of TRIBAL in any disease pathology is currently unclear. 146 The TRIB1 gene encodes a protein known as Tribbles-1, which was originally identified in a drosophila mutagenesis screen which revealed that the protein Trbl (the drosophila homolog of TRIB1) participates in oogenesis via promoting the proteasomal degradation of String, Twine, and Slbo, the latter of which is the Drosophila homolog of the human transcription factor C/EBPa. [147][148][149][150] Subsequent work in the myeloblast 32D cell line showed that human Tribbles-1 can induce the proteasomal degradation of C/EBPa and C/EBPb by promoting their ubiquitination by the E3 ligase COP1 (constitutive photomorphogenesis protein 1 homolog, RFWD2) through direct binding to both targets and the ligase. 151,152 This function is critical for Tribbles-mediated leukemogenesis 153,154 and also coordinates Tribbles regulation of macrophage polarization and differentiation. ...
Genome-wide association studies have provided a rich collection of ≈58 coronary artery disease (CAD) loci that suggest the existence of previously unsuspected new biology relevant to atherosclerosis. However, these studies only identify genomic loci associated with CAD, and many questions remain even after a genomic locus is definitively implicated, including the nature of the causal variant(s) and the causal gene(s), as well as the directionality of effect. There are several tools that can be used for investigation of the functional genomics of these loci, and progress has been made on a limited number of novel CAD loci. New biology regarding atherosclerosis and CAD will be learned through the functional genomics of these loci, and the hope is that at least some of these new pathways relevant to CAD pathogenesis will yield new therapeutic targets for the prevention and treatment of CAD. (Circ Res. 2016;118:586-606.
... Since Cdc25 activates the major mitotic kinase Cdk1, its degradation decreases mitosis and growth of mammalian cells. Tribbles was also found to increase the ubiquitination and proteasome-mediated degradation of the Drosophila protein, Slbo (slow border cells) [74], later identified as a homolog of the mammalian transcription factor C/EBP (CAAT enhancer binding protein) [75]. Indeed, multiple C/EBP transcription factors, e.g. ...
Metabolic diseases like obesity, atherosclerosis and diabetes are frequently associated with increased risk of aggressive cancers. Although metabolic dysfunctions in normal cells are manifested due to defective signaling networks that control cellular homeostasis, malignant cells utilize these signaling networks for their increased survival, growth and metastasis. Despite decades of research, a common mechanistic link between these chronic pathologies is still not well delineated. Evidences show that the unfolded protein response (UPR) and the endoplasmic reticulum stress (ERS) pathways are often dysregulated in both metabolic diseases and cancer. The UPR also triggers coordinated signaling with both PI3K/AKT/mTOR and Autophagy pathways in order to promote stress-adaptive mechanisms. Whereas, uncontrolled UPR and the resultant ERS escalates cells towards metabolic dysfunctions and ultimately cell death. In this review, we will discuss findings that implicate a crucial role for the multifunctional ERS-induced protein, TRIB3. The 'pseudokinase' function of TRIB3 facilitates the inactivation of multiple transcription factors and signaling proteins. The MEK1 binding domain of TRIB3 enables it to deactivate multiple MAP-kinases. In addition, the COP1 motif of TRIB3 assists ubiquitination and proteasomal degradation of numerous TRIB3 associated proteins. The most well studied action of TRIB3 has been on the PI3K/AKT/mTOR pathway, where TRIB3-mediated inhibition of AKT phosphorylation decreases insulin signaling and cell survival. Conversely, cancer cells can either upregulate the AKT survival pathway by suppressing TRIB3 expression or alter TRIB3 localization to degrade differentiation inducing nuclear transcription factors such as C/EBPα and PPARγ. The gain-of-function Q84R polymorphism in TRIB3 is associated with increased risk of diabetes and atherosclerosis. TRIB3 acts as a crucial 'stress adjusting switch' that links homeostasis, metabolic disease and cancer; and is being actively investigated as a disease biomarker and therapeutic target.
... C/EBPα protein abundance in liver is regulated by TRIB1, and thus the TRIB1-C/EBPα axis may be an underappreciated moderator of human hepatic lipogenesis. The tribbles protein was originally identified in Drosophila as a regulator of cell migration and mitosis during oogenesis (9)(10)(11), with its role in oogenesis determined to be regulating the proteasomal degradation of Slbo, the Drosophila homolog of the human gene C/EBPα (12). There are 3 mammalian tribbles genes (tribbles1-3), all of which are known as pseudokinases; they have a protein domain homologous to a kinase domain yet are lack-tal Table 1). ...
Variants near the gene TRIB1 are significantly associated with several plasma lipid traits, circulating liver enzymes, and the development of coronary artery disease in humans; however, it is not clear how its protein product tribbles-1 regulates lipid metabolism. Here, we evaluated mice harboring a liver-specific deletion of Trib1 (Trib1_LSKO) to elucidate the role of tribbles-1 in mammalian hepatic lipid metabolism. These mice exhibited increased hepatic triglyceride (TG) content, lipogenic gene transcription, and de novo lipogenesis. Microarray analysis revealed altered transcription of genes that are downstream of the transcription factor C/EBPα, and Trib1_LSKO mice had increased hepatic C/EBPα protein. Hepatic overexpression of C/EBPα in WT mice phenocopied Trib1_LSKO livers, and hepatic knockout of Cebpa in Trib1_LSKO mice revealed that C/EBPα is required for the increased lipogenesis. Using ChIP-Seq, we found that Trib1_LSKO mice had increased DNA-bound C/EBPα near lipogenic genes and the Trib1 gene, which itself was transcriptionally upregulated by C/EBPα overexpression. Together, our results reveal that tribbles-1 regulates hepatic lipogenesis through posttranscriptional regulation of C/EBPα, which in turn transcriptionally upregulates Trib1. These data suggest an important role for C/EBPα in mediating the lipogenic effects of hepatic Trib1 deletion and provide insight into the association between TRIB1 and plasma lipids, and liver traits in humans.
... 7,8) TRB1 is a mammalian ortholog of Tribbles, which controls cell division and cell migration during embryonic Drosophila development. [9][10][11] TRB1 and other Tribbles family members (TRB2 and TRB3) contain the classic substrate-binding domain of a protein kinase, but not the ATP-binding and kinase-activating domains; therefore, these proteins do not have kinase activity and are classified as pseudokinases. 12) Recent reports suggest that Tribbles family members function as adaptor or scaffold proteins to facilitate degradation of target proteins and to regulate activation of various signaling pathways. ...
Tribbles 1 (TRB1), a member of the Tribbles family, is a pseudokinase that is conserved among species and implicated in various human diseases including leukemia, cardiovascular diseases, and metabolic disorders. However, the role of TRB1 in the immune response is not understood. To evaluate this role, we examined regulation of TRB1 expression and the function of TRB1 in interleukin-2 (IL-2) induction in Jurkat cells, a human acute T cell leukemia cell line. We found that TRB1 was strongly induced by phorbol 12-myristate 13-acetate (PMA) and ionomycin in these cells. IL-2 expression was induced in Jurkat cells activated by PMA and ionomycin; however, knockdown of TRB1 resulted in decreased induction of IL-2. TRB1 null Jurkat cells established using the CRISPR/Cas9 system also showed reduction of IL-2 expression on PMA/ionomycin stimulation. TRB1 knockdown also markedly inhibited IL-2 promoter activation. To determine the mechanism of the stimulatory effect on IL-2 induction, we focused on histone deacetylases (HDACs), and found that HDAC1 preferentially interacts with TRB1. TRB1 suppressed the interaction of HDAC1 with nuclear factor of activated T cells 2 (NFAT2), which is a crucial transcription factor for IL-2 induction. These results indicate that TRB1 is a positive regulator of IL-2 induction in activated T cells.
... Collective migration modes have also been uncovered in other systems using similar experimental approaches. For example, zebrafish lateral line cells lacking the Cxcl12a chemokine receptor, or border cells of the fly egg chamber lacking the slbo transcription factor, can migrate only in the presence of wild type migrationcompetent cells (Haas and Gilmour, 2006;Rorth et al., 2000). There are interesting parallels between collective migration and the "domineering non-cell autonomy" of PCP signaling described in the fly, in which mutant clones influence the behavior of adjacent In immature FBMNs born in r4, Pk1b translocates Rest into the nucleus to repress its target genes. ...
The vertebrate brain arises from the complex organization of millions of neurons. Neurogenesis encompasses not only cell fate specification from neural stem cells, but also the terminal molecular and morphological maturation of neurons at correct positions within the brain. RE1-silencing transcription factor (Rest) is expressed in non-neural tissues and neuronal progenitors where it inhibits the terminal maturation of neurons by repressing hundreds of neuron-specific genes. Here we show that Rest repression of maturation is intimately linked with the migratory capability of zebrafish facial branchiomotor neurons (FBMNs), which undergo a characteristic tangential migration from hindbrain rhombomere (r) 4 to r6/r7 during development. We establish that FBMN migration is increasingly disrupted as Rest is depleted in zebrafish rest mutant embryos, such that around two-thirds of FBMNs fail to complete migration in mutants depleted of both maternal and zygotic Rest. Although Rest is broadly expressed, we show that de-repression or activation of Rest target genes only within FBMNs is sufficient to disrupt their migration. We demonstrate that this migration defect is due to precocious maturation of FBMNs, based on both morphological and molecular criteria. We further show that the Rest target gene and alternative splicing factor srrm4 is a key downstream regulator of maturation; Srrm4 knockdown partially restores the ability of FBMNs to migrate in rest mutants while preventing their precocious morphological maturation. Rest must localize to the nucleus to repress its targets, and its subcellular localization is highly regulated: we show that targeting Rest specifically to FBMN nuclei rescues FBMN migration in Rest-deficient embryos. We conclude that Rest functions in FBMN nuclei to inhibit maturation until the neurons complete their migration.
Copyright © 2015. Published by Elsevier Inc.
... In flies, Tribbles (the founding Drosophila homologue from which the name was derived) regulates the proteasomal turnover of the C/EBP homologue Slbo, which is required for the migration of border cells within the Drosophila ovary. Tribbles-mediated degradation of Slbo blocks this migration [88,89]. ...
Protein phosphorylation lies at the heart of cell signalling, and somatic mutation(s) in kinases drives and sustains a multitude of human diseases, including cancer. The human protein kinase superfamily (the kinome) encodes approximately 50 'pseudokinases', which were initially predicted to be incapable of dynamic cell signalling when compared with canonical enzymatically active kinases. This assumption was supported by bioinformatics, which showed that amino acid changes at one or more key loci, making up the nucleotide-binding site or phosphotransferase machinery, were conserved in multiple vertebrate and non-vertebrate pseudokinase homologues. Protein kinases are highly attractive targets for drug discovery, as evidenced by the approval of almost 30 kinase inhibitors in oncology, and the successful development of the dual JAK1/2 (Janus kinase 1/2) inhibitor ruxolitinib for inflammatory indications. However, for such a large (>550) protein family, a remarkable number have still not been analysed at the molecular level, and only a surprisingly small percentage of kinases have been successfully targeted clinically. This is despite evidence that many are potential candidates for the development of new therapeutics. Indeed, several recent reports confirm that disease-associated pseudokinases can bind to nucleotide co-factors at concentrations achievable in the cell. Together, these findings suggest that drug targeting using either ATP-site or unbiased ligand-discovery approaches should now be attempted using the validation technology currently employed to evaluate their classic protein kinase counterparts. In the present review, we discuss members of the human pseudokinome repertoire, and catalogue somatic amino acid pseudokinase mutations that are emerging as the depth and clinical coverage of the human cancer pseudokinome expand.
... Nonstop scheint also direkt oder indirekt an der Progression der Gliazellen von der glialen Anlage aus in die Laminavorläuferregion beteiligt zu sein. Interessanterweise existiert in Drosophila eine Ubiquitin spezifische Protease, Ubp-64, welche indirekt an der Zellmigration während der Oogenese beteiligt ist; in diesem Fall stabilisiert das Enzym den Transkriptionsfaktor C/EBP (Rorth et al., 2000). Es läßt sich eine gewisse Parallele zu den (Huang et al., 1995;Wu et al., 1999). ...
The pseudokinase Tribbles Homolog 1 ( TRIB1 ) is a known driver of tumorigenesis in acute myeloid leukemia and is encoded upstream of the oncogene MYC at the 8q24 locus. We observed that TRIB1/MYC co-amplification is associated with decreased relapse-free and overall survival in breast cancer patients, but the role of TRIB1 in this disease has not been well characterized. TRIB1 knockdown in multiple breast cancer cell lines inhibited cell proliferation and suppressed MYC expression, implicating TRIB1 in breast cancer cell proliferation. Transcriptomic and cell cycle analysis revealed cell cycle regulation as the likely mechanism through which TRIB1 influences breast cancer cell proliferation. TRIB1 knockdown also resulted in significant changes in both estrogen receptor (ER) and β-catenin associated transcription. Interrogating the TRIB1 interactome in breast cancer cells by qPLEX-RIME reinforced the known association between TRIB1 and ubiquitination, while revealing a range of previously undescribed TRIB1 associated factors. Further analysis of the association between TRIB1, β-catenin and FERMT2 suggests TRIB1 may regulate β-catenin activity by controlling the levels of both β-catenin, and its co-factor FERMT2. Together, these results suggest that coregulation of β-catenin and ER-driven transcription by TRIB1, facilitates regulation of MYC expression and breast cancer cell proliferation.
Significance
The pseudokinase TRIB1 is frequently co-amplified in breast cancers with the potent oncogene MYC , although the functional consequences of this event are not well understood. This study demonstrates TRIB1 is a regulator of cell cycle progression and MYC expression in breast cancer cells. It also profiles TRIB1 -associated proteins in breast cancer cells, demonstrating conservation of TRIB1’s canonical interaction with COP1 and reveals associations with members of the wider ubiquitination machinery, a range of transcriptional regulators and chromatin remodelers. The data presented provide insight into the function of TRIB1 in breast cancer and the role of TRIB1 in transcriptional regulation.
Wound response programs are often activated during neoplastic growth in tumors. In both wound repair and tumor growth, cells respond to acute stress and balance the activation of multiple programs including apoptosis, proliferation, and cell migration. Central to those responses are the activation of the JNK/MAPK and JAK/STAT signaling pathways. Yet, to what extent these signaling cascades interact at the cis-regulatory level, and how they orchestrate different regulatory and phenotypic responses is still unclear. Here, we aim to characterize the regulatory states that emerge and cooperate in the wound response, using the Drosophila melanogaster wing disc as a model system, and compare these with cancer cell states induced by rasV12scrib-/- in the eye disc. We used single-cell multiome profiling to derive enhancer Gene Regulatory Networks (eGRNs) by integrating chromatin accessibility and gene expression signals. We identify a 'proliferative' eGRN, active in the majority of wounded cells and controlled by AP-1 and STAT. In a smaller, but distinct population of wound cells, a 'senescent' eGRN is activated and driven by C/EBP-like transcription factors (Irbp18, Xrp1, Slow border, and Vrille) and Scalloped. These two eGRN signatures are found to be active in tumor cells, at both gene expression and chromatin accessibility levels. Our single-cell multiome and eGRNs resource offers an in-depth characterisation of the senescence markers, together with a new perspective on the shared gene regulatory programs acting during wound response and oncogenesis.
Delaminating cells undergo complex, precisely regulated changes in cell–cell adhesion, motility, polarity, invasiveness, and other cellular properties. Delamination occurs during development and in pathogenic conditions such as cancer metastasis. We analyzed the requirements for epithelial delamination in Drosophila ovary border cells, which detach from the structured epithelial layer and begin to migrate collectively. We used live imaging to examine cellular dynamics, particularly epithelial cells’ acquisition of motility and invasiveness, in delamination-defective mutants during the time period in which delamination occurs in the wild-type ovary. We found that border cells in slow border cells (slbo), a delamination-defective mutant, lacked invasive cellular protrusions but acquired basic cellular motility, while JAK/STAT-inhibited border cells lost both invasiveness and motility. Our results indicate that invasiveness and motility, which are cooperatively required for delamination, are regulated independently. Our reconstruction experiments also showed that motility is not a prerequisite for acquiring invasiveness.
The formation of a functional organ such as the eye requires specification of the correct cell types and their terminal differentiation into cells with the appropriate morphologies and functions. Here we show that the zinc-finger transcription factor Blimp-1 acts in secondary and tertiary pigment cells in the Drosophila retina to promote the formation of a bi-convex corneal lens with normal refractive power, and in cone cells to enable complete extension of the photoreceptor rhabdomeres. Blimp-1 expression depends on the hormone ecdysone, and loss of ecdysone signaling causes similar differentiation defects. Timely termination of Blimp-1 expression is also important, as its overexpression in the eye has deleterious effects. Our transcriptomic analysis revealed that Blimp-1 regulates the expression of many structural and secreted proteins in the retina. Blimp-1 may function in part by repressing another transcription factor; Slow border cells is highly upregulated in the absence of Blimp-1, and its overexpression reproduces many of the effects of removing Blimp-1. This work provides insight into the transcriptional networks and cellular interactions that produce the structures necessary for visual function.
Despite remarkable clinical efficacy of immune checkpoint blockade (ICB) in cancer treatment, ICB benefits for triple-negative breast cancer (TNBC) remain limited. Through pooled in vivo CRISPR knockout (KO) screens in syngeneic TNBC mouse models, we found that deletion of the E3 ubiquitin ligase Cop1 in cancer cells decreases secretion of macrophage-associated chemokines, reduces tumor macrophage infiltration, enhances anti-tumor immunity, and strengthens ICB response. Transcriptomics, epigenomics, and proteomics analyses revealed that Cop1 functions through proteasomal degradation of the C/ebpδ protein. The Cop1 substrate Trib2 functions as a scaffold linking Cop1 and C/ebpδ, which leads to polyubiquitination of C/ebpδ. In addition, deletion of the E3 ubiquitin ligase Cop1 in cancer cells stabilizes C/ebpδ to suppress expression of macrophage chemoattractant genes. Our integrated approach implicates Cop1 as a target for improving cancer immunotherapy efficacy in TNBC by regulating chemokine secretion and macrophage infiltration in the tumor microenvironment.
Cell migration is a key component in development, homeostasis, immune function, and pathology. It is important to understand the molecular activity that allows some cells to migrate. Drosophila melanogaster is a useful model system because its genes are largely conserved with humans and it is straightforward to study biologically. The well-conserved transcriptional regulator Signal Transducer and Activator of Transcription (STAT) promotes cell migration, but its signaling is modulated by downstream targets Apontic (APT) and Slow Border Cells (SLBO). Inhibition of STAT activity by APT and cross-repression of APT and SLBO determines whether an epithelial cell in the Drosophila egg chamber becomes motile or remains stationary. Through mathematical modeling and analysis, we examine how the interaction of STAT, APT, and SLBO creates bistability in the Janus Kinase (JAK)/STAT signaling pathway. In this paper, we update and analyze earlier models to represent mechanistically the processes of the JAK/STAT pathway. We utilize parameter, bifurcation, and phase portrait analyses, and make reductions to the system to produce a minimal three-variable quantitative model. We analyze the manifold between migratory and stationary steady states in this minimal model and show that when the initial conditions of our model are near this manifold, cell migration can be delayed.
Purpose of review:
Lineage commitment is governed by instructive and stochastic signals, which drive both active induction of the lineage program and repression of alternative fates. Eosinophil lineage commitment is driven by the ordered interaction of transcription factors, supported by cytokine signals. This review summarizes key findings in the study of eosinophil lineage commitment and examines new data investigating the factors that regulate this process.
Recent findings:
Recent and past studies highlight how intrinsic and extrinsic signals modulate transcription factor network and lineage decisions. Early action of the transcription factors C/EBPα and GATA binding protein-1 along with C/EBPε supports lineage commitment and eosinophil differentiation. This process is regulated and enforced by the pseudokinase Trib1, a regulator of C/EBPα levels. The cytokines interleukin (IL)-5 and IL-33 also support early eosinophil development. However, current studies suggest that these cytokines are not specifically required for lineage commitment.
Summary:
Together, recent evidence suggests a model where early transcription factor activity drives expression of key eosinophil genes and cytokine receptors to prime lineage commitment. Understanding the factors and signals that control eosinophil lineage commitment may guide therapeutic development for eosinophil-mediated diseases and provide examples for fate choices in other lineages.
The recently developed single-cell CRISPR screening techniques, independently termed Perturb-Seq, CRISP-seq, or CROP-seq, combine pooled CRISPR screening with single-cell RNA-seq to investigate functional CRISPR screening in a single-cell granularity. Here, we present MUSIC, an integrated pipeline for model-based understanding of single-cell CRISPR screening data. Comprehensive tests applied to all the publicly available data revealed that MUSIC accurately quantifies and prioritizes the individual gene perturbation effect on cell phenotypes with tolerance for the substantial noise that exists in such data analysis. MUSIC facilitates the single-cell CRISPR screening from three perspectives, i.e., prioritizing the gene perturbation effect as an overall perturbation effect, in a functional topic-specific way, and quantifying the relationships between different perturbations. In summary, MUSIC provides an effective and applicable solution to elucidate perturbation function and biologic circuits by a model-based quantitative analysis of single-cell-based CRISPR screening data.
Members of the Tribbles family of proteins are conserved pseudokinases with diverse roles in cell growth and proliferation. Both Drosophila Tribbles (Trbl) and vertebrate Trib3 proteins bind to Akt kinase to block its phosphorylation-activation and reduce downstream insulin-stimulated anabolism. A single nucleotide polymorphism (SNP) variant in human Trib3, which results in a glutamine (Q) to arginine (R) missense mutation in a conserved motif at position 84, confers stronger Akt binding resulting in reduced Akt phosphorylation and is associated with a predisposition to Type II diabetes, cardiovascular disease, diabetic nephropathy, chronic kidney disease and leukemogenesis. Here we used a Drosophila model to understand the importance of the conserved R residue in several Trbl functions. In the fly fat body, misexpression of a site-directed Q mutation at position R141 resulted in weakened binding to dAkt, leading to increased levels of phospho-dAkt, increased cell and tissue size and increases in levels of stored glycogen and triglycerides. Consistent with the functional conservation of this arginine in modulating Akt activity, mouse Trib3R84 misexpressed in the fly fat body blocked dAkt phosphorylation with a strength similar to wild type (WT) Trbl. Limited mutational analysis shows that the R141 site dictates the strength of Akt binding but does not affect other Trbl-dependent developmental processes, suggesting a specificity that may serve as drug target for metabolic diseases.
The tribbles (trbl) pseudokinases play important roles in signaling and physiology in multiple contexts, ranging from innate immunity to cancer, suggesting fundamental cellular functions for the trbls’ gene products. Despite expression of the trbl pseudokinases in the nervous systems of invertebrate and vertebrate animals, and evidence that they have a function within mouse and human dopamine neurons, there is no clear case for a function of a Trbl protein that influences behavior. Indeed, the first and only evidence for this type of function comes from Drosophila melanogaster, where a mutation of the single trbl gene was identified in a genetic screen for short-term memory mutant flies. The current study tested flies containing multiple trbl mutant alleles and potential transgenic rescue in both operant place memory and classical olfactory memory paradigms. Genetic complementation tests and transgenic rescue of memory phenotypes in both paradigms show that the D. melanogaster trbl pseudokinase is essential for proper memory formation. Expression analysis with a polyclonal antiserum against Trbl shows that the protein is expressed widely in the fly brain, with higher expression in the cellular rind than the neuropil. Rescue of the behavioral phenotype with transgenic expression indicates the trbl function can be localized to a subset of the nervous system. Thus, we provide the first compelling case for the function of a trbl pseudokinase in the regulation of behavior.
Drosophila border cells undergo a straightforward and stereotypical collective migration during egg development. However, a complex genetic program underlies this process. A variety of approaches, including biochemical, genetic, and imaging strategies have identified many regulatory components, revealing layers of control. This complexity suggests that the active processes of evaluating the environment, remodeling the cytoskeleton, and coordinating movements among cells, demand rapid systems for modulating cell behaviors. Multiple signaling inputs, nodes of integration, and feedback loops act as molecular rheostats to fine-tune gene expression levels and physical responses. Since key genetic regulators of border cell migration have been shown to be required in other types of cell migration, this model system continues to provide an important avenue for genetic discovery.
The protein tribbles-1, encoded by the gene TRIB1, is increasingly recognized as a major regulator of multiple cellular and physiological processes in humans. Recent human genetic studies, as well as molecular biological approaches, have implicated this intriguing protein in the aetiology of multiple human diseases, including myeloid leukaemia, Crohn's disease, non-alcoholic fatty liver disease (NAFLD), dyslipidaemia and coronary artery disease (CAD). Genome-wide association studies (GWAS) have repeatedly identified variants at the genomic TRIB1 locus as being significantly associated with multiple plasma lipid traits and cardiovascular disease (CVD) in humans. The involvement of TRIB1 in hepatic lipid metabolism has been validated through viral-mediated hepatic overexpression of the gene in mice; increasing levels of TRIB1 decreased plasma lipids in a dose-dependent manner. Additional studies have implicated TRIB1 in the regulation of hepatic lipogenesis and NAFLD. The exact mechanisms of TRIB1 regulation of both plasma lipids and hepatic lipogenesis remain undetermined, although multiple signalling pathways and transcription factors have been implicated in tribbles-1 function. Recent reports have been aimed at developing TRIB1- based lipid therapeutics. In summary, tribbles-1 is an important modulator of human energy metabolism and metabolic syndromes and worthy of future studies aimed at investigating its potential as a therapeutic target.
The tribbles protein family, an evolutionarily conserved group of pseudokinases, have been shown to regulate multiple cellular events including those involved in normal and malignant haematopoiesis. The three mammalian Tribbles homologues, Trib1, Trib2 and Trib3 are characterized by conserved motifs, including a pseudokinase domain and a C-terminal E3 ligase-binding domain. In this review, we focus on the role of Trib (mammalian Tribbles homologues) proteins in mammalian haematopoiesis and leukaemia. The Trib proteins show divergent expression in haematopoietic cells, probably indicating cell-specific functions. The roles of the Trib proteins in oncogenesis are also varied and appear to be tissue-specific. Finally, we discuss the potential mechanisms by which the Trib proteins preferentially regulate these processes in multiple cell types.
CCAAT-enhancer binding proteins (C/EBPs) are transcription factors that play a central role in the differentiation of myeloid cells and adipocytes. Tribbles pseudokinases govern levels of C/EBPs by recruiting them to the COP1 ubiquitin ligase for ubiquitination. Here, we present the first crystal structure of a Tribbles protein, which reveals a catalytically inactive TRIB1 pseudokinase domain with a unique adaptation in the αC helix. A second crystal structure and biophysical studies of TRIB1 with its C-terminal extension, which includes the COP1-binding motif, show that the C-terminal extension is sequestered at a site formed by the modified TRIB1 αC helix. In addition, we have identified and characterized the TRIB1 substrate-recognition sequence within C/EBPα, which is evolutionarily conserved in C/EBP transcription factors. Binding studies indicate that C/EBPα recruitment is weaker in the presence of the C-terminal COP1-binding motif, but the magnitude of this effect suggests that the two bind distinct rather directly overlapping binding sites.
Cell movements are essential for animal development and homeostasis but also contribute to disease. Moving cells typically extend protrusions towards a chemoattractant, adhere to the substrate, contract and detach at the rear. It is less clear how cells that migrate in interconnected groups in vivo coordinate their behaviour and navigate through natural environments. The border cells of the Drosophila melanogaster ovary have emerged as an excellent model for the study of collective cell movement, aided by innovative genetic, live imaging, and photomanipulation techniques. Here we provide an overview of the molecular choreography of border cells and its more general implications.
This report describes the identification and purification of a nuclear protein from rat liver that binds selectively to DNA sequences associated with several animal virus enhancers. The binding activity was tracked by direct DNase I footprinting through four steps of biochemical fractionation. These procedures led to the identification of a polypeptide species exhibiting an apparent molecular weight of 20 kD that accounts for enhancer binding activity. DNase I and dimethyl sulfate footprinting assays were used to examine the manner in which the purified protein binds to enhancer elements associated with SV40, murine sarcoma virus, and polyoma virus. The results of these assays indicate that the initial interaction established between the 20-kD protein and each viral enhancer occurs via a common DNA sequence known as the enhancer core homology.
The autonomous selector capacity of the homeotic proboscipedia (pb) gnee of the Drosophila Antennapedia Complex was tested. We introduced into the germline a P element containing a transcriptional fusion of a mini-gene for pb (normally required for formation of the labial and maxillary palps of the mouthparts) and the Hsp70 promoter. Uninduced expression of this Hsp70:pb element (HSPB) directs a novel, fully penetrant dominant transformation of antennae toward maxillary palps. Gene dosage experiments varying the number of HSPB elements indicate that the extent of the dominant transformation depends on the level of PB protein. At the same time, expression from the transgene also rescues recessive pb mutations. Finally, HSPB function may override the dominant antennal transformations caused by Antennapedia (Antp) mutations in a dose-sensitive manner, directing a switch of the antennal disc-derived appendage from ectopic leg to ectopic maxillary palp. This switch correlated with strikingly reduced ANTP protein accumulation when PB concentrations exceeded a genetically defined threshold level. These observations support a crucial role for quantitative aspects of pb function in determining segmental identity, including cross-regulatory events involved in this determination.
A dominant insertional P-element mutation enhances position-effect variegation in Drosophila melanogaster. The mutation is
homozygous, viable, and fertile and maps at 64E on the third chromosome. The corresponding gene was cloned by transposon tagging.
Insertion of the transposon upstream of the open reading frame correlates with a strong reduction of transcript level. A transgene
was constructed with the cDNA and found to have the effect opposite from that of the mutation, namely, to suppress variegation.
Sequencing of the cDNA reveals a large open reading frame encoding a putative ubiquitin-specific protease (Ubp). Ubiquitin
marks various proteins, frequently for proteasome-dependent degradation. Ubps can cleave the ubiquitin part from these proteins.
We discuss the link established here between a deubiquitinating enzyme and epigenetic silencing processes.
The Drosophila fat facets (faf) gene encodes a deubiquitination enzyme with a putative function in proteasomal protein degradation. Mutants lacking zygotic faf function develop to adulthood, but have rough eyes caused by the presence of one to two ectopic outer photoreceptors per ommatidium. Here we show that faf interacts genetically with the receptor tyrosine kinase (RTK)/Ras pathway, which induces photoreceptor differentiation in the developing eye. The results indicate that RTK/Ras signalling is increased in faf mutants, causing normally non-neuronal cells to adopt photoreceptor fate. Consistently, the protein level of at least one component of the Ras signal transduction pathway, the transcription factor D-Jun, is elevated in faf eye discs at the time when the ectopic photoreceptors are induced. We propose that defective ubiquitin-dependent proteolysis leads to increased and prolonged D-Jun expression, which together with other factors contributes to the induction of ectopic photoreceptors in faf mutants. These studies demonstrate the relevance of ubiquitin-dependent protein degradation in the regulation of RTK/Ras signal transduction in an intact organism.
A modular misexpression system was used to carry out systematic gain-of-function genetic screens in Drosophila. The system is based on inducible expression of genes tagged by insertion of a P-element vector carrying a GAL4-regulated promoter oriented to transcribe flanking genomic sequences. To identify genes involved in eye and wing development, the 2300 independent lines were screened for dominant phenotypes. Among many novel genes, the screen identified known genes, including hedgehog and decapentaplegic, implicated in these processes. A genetic interaction screen for suppressors of a cell migration defect in a hypomorphic slow border cells mutant identified known genes with likely roles in tyrosine kinase signaling and control of actin cytoskeleton, among many novel genes. These studies demonstrate the ability of the modular misexpression system to identify developmentally important genes and suggest that it will be generally useful for genetic interaction screens.
Signaling by Notch family receptors is involved in many cell-fate decisions during development. Several modifiers of Notch activity have been identified, suggesting that regulation of Notch signaling is complex. In a genetic screen for modifiers of Notch activity, we identified a gene encoding a novel WD40-repeat protein. The gene is called Notchless, because loss-of-function mutant alleles dominantly suppress the wing notching caused by certain Notch alleles. Reducing Notchless activity increases Notch activity. Overexpression of Notchless in Xenopus or Drosophila appears to have a dominant-negative effect in that it also increases Notch activity. Biochemical studies show that Notchless binds to the cytoplasmic domain of Notch, suggesting that it serves as a direct regulator of Notch signaling activity.
Cadherins are involved in a variety of morphogenetic movements during animal development. However, it has been difficult to pinpoint the precise function of cadherins in morphogenetic processes due to the multifunctional nature of cadherin requirement. The data presented here indicate that homophilic adhesion promoted by Drosophila E-cadherin (DE-cadherin) mediates two cell migration events during Drosophila oogenesis. In Drosophila follicles, two groups of follicle cells, the border cells and the centripetal cells migrate on the surface of germline cells. We show that the border cells migrate as an epithelial patch in which two centrally located cells retain epithelial polarity and peripheral cells are partially depolarized. Both follicle cells and germline cells express DE-cadherin, and border cells and centripetal cells strongly upregulate the expression of DE-cadherin shortly before and during their migration. Removing DE-cadherin from either the follicle cells or the germline cells blocks migration of border cells and centripetal cells on the surface of germline cells. The function of DE-cadherin in border cells appears to be specific for migration as the formation of the border cell cluster and the adhesion between border cells are not disrupted in the absence of DE-cadherin. The speed of migration depends on the level of DE-cadherin expression, as border cells migrate more slowly when DE-cadherin activity is reduced. Finally, we show that the upregulation of DE-cadherin expression in border cells depends on the activity of the Drosophila C/EBP transcription factor that is essential for border cell migration.
even-skipped represses wingless and transforms cells that would normally secrete naked cuticle into denticle secreting cells. The GAL4 system can thus be used to study regulatory interactions during embryonic devel- opment. In adults, targeted expression can be used to generate dominant phenotypes for use in genetic screens. We have directed expression of an activated form of the Dras2 protein, resulting in dominant eye and wing defects that can be used in screens to identify other members of the Dras2 signal transduction path- way. SUMMARY
During Drosophila oogenesis six to ten follicle cells, the border cells, undergo a dramatic and stereotypic migration through the developing egg chamber. We identified four independent P element insertion mutations that specifically blocked border cell migration. They defined a single, novel locus that was named slow border cells (slbo), because hypomorphic alleles caused delayed onset of the migration. Laser ablation of the border cells, or failure of their migration, caused improper morphogenesis of the micropyle, the egg-shell structure through which the sperm enters at fertilization. The slbo locus was found to encode a product homologous to the CCAAT/enhancer-binding protein (C/EBP), a basic region-leucine zipper transcription factor. Drosophila C/EBP may be required for the expression of gene products mediating border cell migration.
Recently, we reported the cloning of the Drosophila melanogaster homolog of the vertebrate CCAAT/enhancer-binding protein (C/EBP). Here, we describe studies of the DNA-binding and dimerization properties of Drosophila C/EBP (DmC/EBP), as well as its tissue distribution, developmental regulation, and essential role in embryonic development and conclude that it bears functional as well as structural similarity to mammalian C/EBP. DmC/EBP contains a basic region/leucine zipper (bZIP) DNA-binding domain very similar to that of mammalian C/EBP and the purified C/EBPs bound to DNA with the same sequence specificity. Among the DNA sequences that DmC/EBP bound with high affinity was a conserved site within the promoter of the DmC/EBP gene itself. In vitro, DmC/EBP and mammalian C/EBP specifically formed functional heterodimers; however, as we found no evidence for a family of DmC/EBPs, DmC/EBP may function as a homodimer in vivo. The DmC/EBP protein was expressed predominantly during late embryogenesis in the nuclei of a restricted set of differentiating cell types, such as the lining of the gut and epidermis, similar to the mammalian tissues that express C/EBP. We have characterized mutations in the DmC/EBP gene and found that deleting the gene caused late embryonic lethality. Embryos that lack C/EBP die just before or just upon hatching. The lethal phenotype of C/EBP mutants can be rescued with the cloned C/EBP gene introduced by P-element-mediated germ-line transformation. The strict requirement for C/EBP during Drosophila embryogenesis, coupled with its structural and functional similarities to mammalian C/EBP, provides a useful genetic system in which to study the role of C/EBP in development.
Most of the thoracic and abdominal segments of Drosophila are specified early in embryogenesis by the overlapping activities of the hunchback (hb), Krüppel, knirps, and giant gap genes. The orderly expression of these genes depends on two maternal determinants: bicoid, which activates hb transcription anteriorly, and nanos, which blocks translation of hb transcripts posteriorly. Here we provide evidence that the resulting gradient of hb protein dictates where the Krüppel, knirps, and giant genes are expressed by providing a series of concentration thresholds that regulate each gene independently. Thus, hb protein functions as a classical morphogen, triggering several distinct responses as a function of its graded distribution.
In an effort to identify protein factors that play a regulatory role in the differentiation of adipocytes, we have isolated two genes that encode polypeptides related to CCAAT/enhancer-binding protein (C/EBP; hereafter termed C/EBP alpha). The proteins encoded by these C/EBP-related genes, termed C/EBP beta and C/EBP delta, exhibit similar DNA-binding specificities and affinities compared with C/EBP alpha. Furthermore, C/EBP beta and C/EBP delta readily form heterodimers with one another as well as with C/EBP alpha. The transcriptional activating capacity of these two newly identified C/EBP isoforms was demonstrated by transient transfection experiments in which expression vectors encoding C/EBP beta and C/EBP delta were observed to induce transcription from the promoter of the serum albumin gene in cultured hepatoma cells. The mRNAs encoding C/EBP beta and C/EBP delta were detected in a number of tissues, most of which corresponded to sites of expression of C/EBP alpha. The expression pattern of C/EBP beta and C/EBP delta during adipose conversion of 3T3-L1 cells was examined by Western and Northern blotting assays. In contrast to the expression profile of the gene encoding C/EBP alpha, whose product is not detectable until the late phase of adipocyte differentiation, the c/ebp beta and c/ebp delta genes were actively expressed very early during adipocyte differentiation. Moreover, transcription of the c/ebp beta and c/ebp delta genes was observed to be induced directly by adipogenic hormones. The accumulation of C/EBP beta and C/EBP delta reached a maximal level during the first 2 days of differentiation and declined sharply before the onset of C/EBP alpha accumulation. The temporal pattern of expression of these three C/EBP isoforms during adipocyte differentiation may reflect the underpinnings of a regulatory cascade that controls the process of terminal cell differentiation.
The promoter from the metallothiortein gene may be a useful conditional promoter for the construction of chimeric genes to
be expressed in Drosophila cells in culture. To explore this possibility the responses of the endogenous metallothionein gene and an in vitro constructed
chimeric gene containing the metallothiortein promoter were examined. Copper and cadmium, when added to the growth medium
of Drosophila Schneider's line 2 cells, can produce a 30–100 fold induction of metallothionein mRNA levels. The level of induction depends
on the amount of copper or cadmium added to the medium and these mRNA levels remain high for at least four days. Copper is
less toxic than cadmium and does not induce a typical heat-shock response in the cells. Finally, a chimeric gene containing
the metallothionein promoter shows a similar induction when transformed into the cells.
The bicoid (bcd) protein in a Drosophila embryo is derived from an anteriorly localized mRNA and comes to be distributed in an exponential concentration gradient along the anteroposterior axis. To determine whether the levels of bcd protein are directly related to certain cell fates, we manipulated the density and distribution of bcd mRNA by genetic means, measured the resultant alterations in height and shape of the bcd protein gradient, and correlated the gradient with the fate map of the respective embryos. Increases or decreases in bcd protein levels in a given region of the embryo cause a corresponding posterior or anterior shift of anterior anlagen in the embryo. The bcd protein thus has the properties of a morphogen that autonomously determines positions in the anterior half of the embryo.
The biologically relevant interactions of a transcription factor are those that are important for function in the organism. Here, a transgenic rescue assay was used to determine which molecular functions of Drosophila CCAAT/enhancer binding protein (C/EBP), a basic region-leucine zipper transcription factor, are required for it to fulfill its essential role during development. Chimeric proteins that contain the Drosophila C/EBP (DmC/EBP) basic region, a heterologous zipper, and a heterologous activation domain could functionally substitute for DmC/EBP. Mammalian C/EBPs were also functional in Drosophila. In contrast, 9 of 25 single amino acid substitutions in the basic region disrupted biological function. Thus, the conserved basic region specifies DmC/EBP activity in the organism.
The role of the ubiquitin-dependent proteolysis system in c-Jun breakdown was investigated. Using in vitro experiments and a novel in vivo assay that utilizes molecularly-tagged ubiquitin and c-Jun proteins, it was shown that c-Jun, but not its transforming counterpart, retroviral v-Jun, can be efficiently multiubiquitinated. Consistently, v-Jun has a longer half-life than c-Jun. Mutagenesis experiments indicate that the reason for the escape of v-Jun from multiubiquitination and its resulting stabilization is the deletion of the delta domain, a stretch of 27 amino acids that is present in c-Jun but not in v-Jun. c-Jun sequences containing the delta domain, when transferred to the bacterial beta-galactosidase protein, function as a cis-acting ubiquitination and degradation signal. The correlation between transforming ability and the escape from ubiquitin-dependent degradation described here suggests a novel route to oncogenesis.
We have constructed a series of strains to facilitate the generation and analysis of clones of genetically distinct cells in developing and adult tissues of Drosophila. Each of these strains carries an FRT element, the target for the yeast FLP recombinase, near the base of a major chromosome arm, as well as a gratuitous cell-autonomous marker. Novel markers that carry epitope tags and that are localized to either the cell nucleus or cell membrane have been generated. As a demonstration of how these strains can be used to study a particular gene, we have analyzed the developmental role of the Drosophila EGF receptor homolog. Moreover, we have shown that these strains can be utilized to identify new mutations in mosaic animals in an efficient and unbiased way, thereby providing an unprecedented opportunity to perform systematic genetic screens for mutations affecting many biological processes.
Throughout their lives, adult Drosophila females continuously produce oocytes, each surrounded by an epithelial monolayer of follicle cells. To characterize the somatic stem cells that give rise to ovarian follicle cells, we marked dividing cells using FLP-catalyzed mitotic recombination and analyzed the resulting clones. Each ovariole in young females contains, on average, two somatic stem cells located near the border of germarium regions 2a and 2b. The somatic stem cells do not coordinate their divisions either with each other or with the germline stem cells. As females age, initially mosaic ovarioles become monoclonal, indicating that functional somatic stem cells have a finite life span. Analysis of agametic flies revealed that somatic cells continue to divide in the absence of a germline. Under these conditions, the somatic stem cells develop near the tip of the ovariole (the normal site of the germline stem cells), and a subpopulation of somatic cells that normally separates the germline and somatic stem cells is missing.
The proteasome and the small protein ubiquitin are key elements in the intracellular pathway of general protein degradation. Recent evidence shows that the proteasome and other less well defined cytoplasmic proteases can participate in specific events which control inducible gene expression. A number of eukaryotic transcriptional regulators, including NF-kappa B/l kappa B, p53, c-Jun, Notch, sterol regulated element binding proteins and MAT2 alpha, have recently been shown to be regulated by proteolytic events, a regulation which results in the activation or inactivation of gene expression.
The proto-oncogene-encoded transcription factor c-Jun activates genes in response to a number of inducers that act through
mitogen-activated protein kinase (MAPK) signal transduction pathways. The activation of c-Jun after phosphorylation by MAPK
is accompanied by a reduction in c-Jun ubiquitination and consequent stabilization of the protein. These results illustrate
the relevance of regulated protein degradation in the signal-dependent control of gene expression.
We show that Tramtrack (TTK88) expression represses neuronal fate determination in the developing Drosophila eye. Phyllopod (PHYL) acts to antagonize this repression by a mechanism that requires Seven In Absentia (SINA) and is associated with decreased TTK88 protein levels, but not reduced ttk88 gene transcription or mRNA stability. We present evidence that SINA, PHYL, and TTK88 physically interact and that SINA interacts genetically and physically with UBCD1, a component of the ubiquitin-dependent protein degradation pathway. Our results suggest a model in which activation of the Sevenless receptor tyrosine kinase induces PHYL expression, which then acts with SINA to target the transcriptional repressor TTK88 for degradation, thereby promoting R7 cell fate specification.
We have isolated cDNA clones encoding the dog and human forms of a novel protein whose function is still unknown. Sequence analysis indicates that dog clone c5fw protein contains 343 amino acid residues. several potential phosphorylation sites. and two of the 12 conserved subdomains (VIII and IX) that fold into a common catalytic core structure of the large family of protein kinases. Human clone c5fw shares 95% amino acid identity with its dog counterpart. We have also isolated another human-related clone c5fw sharing 70% amino acid identity with the dog sequence. We transiently expressed c-myc epitope-tagged clone c5fw protein in COS-7 cells and infected thyrocytes in primary culture with a recombinant adenovirus containing clone c5fw cDNA (adenovirus c5fw). In both experiments, a 46-kDa protein was detected and subsequently more extensively characterized. By two-dimensional gel electrophoresis and V8 protease digestion, we showed that this overexpressed protein is phosphorylated on different sites. Moreover, cells stimulated with thyrotropin or epidermal growth factor, thyrotropin and fetal calf serum increased the level of clone c5fw protein produced after infection by adenovirus containing clone c5fw. The disappearance of this 46-kDa protein after 1 h of puromycin treatment indicates that it is a labile protein. Immunofluorescence and subcellular fractionation analysis have revealed that c-myc-tagged clone c5fw was insoluble and localized mainly in the cytoplasm, in the form of granules.
We show that the intracellular concentration of transcriptional activator proteins is regulated by the proteasome-mediated protein degradation pathway. The rate of degradation of activators by proteasomes correlates with activation domain potency in vivo. Mutations either in the activation domain residues involved in target protein interaction or in the DNA-binding domain residues essential for DNA binding abolish the transcriptional activation function in vivo and render the activator resistant to degradation by proteasomes. Finally, using a rapamycin-regulated gene expression system, we show that recruiting activation domains to DNA-bound receptor proteins greatly enhanced the rate of degradation of reconstituted activators. These observations suggest that in mammalian cells efficient recruitment of activator-target protein complexes to the promoter means that they are subjected to rapid degradation by proteasomes. We propose that proteasome-mediated control of the intracellular levels of transcriptional activators could play an important role in the regulation of gene expression.
Although it is known that the establishment of polarity during Drosophila oogenesis is initiated by signalling from the oocyte to the overlying follicle cells, much less is understood about the role of specific follicular subpopulations. One powerful approach for addressing this question, toxigenic cell ablation of specific subpopulations, has not previously been applicable to studying follicular subpopulations because many of the genes and Gal4 enhancer trap insertions that are expressed in the ovary are also expressed at earlier times in development. To overcome this problem, we have utilized a fusion protein between Gal4 and the human estrogen receptor to achieve hormone-dependent, tissue-specific gene expression of UAS-linked transgenes in flies. We used this system to study the role of the polar subpopulations of follicle cells during oogenesis by expressing within them a modified form of diphtheria toxin that causes cell death. Our results confirmed previous functions ascribed to these cells, and also demonstrated a previously undescribed role for the border cells in facilitating the migration of the anterior Fasciclin III-expressing polar pair cells to the edge of the oocyte.
Morphogenesis and cell differentiation in multicellular organisms often require accurate control of cell divisions. We show that a novel cell cycle regulator, tribbles, is critical for this control during Drosophila development. During oogenesis, the level of tribbles affects the number of germ cell divisions as well as oocyte determination. The mesoderm anlage enters mitosis prematurely in tribbles mutant embryos, leading to gastrulation defects. We show that Tribbles acts by specifically inducing degradation of the CDC25 mitotic activators String and Twine via the proteosome pathway. By regulating CDC25, Tribbles serves to coordinate entry into mitosis with morphogenesis and cell fate determination.
Levels of homeotic protein function can determine developmental identity: evidence from low-level expression of the Analysis of genetic mosaics in develila homeotic gene proboscipedia under Hsp70 control
- T Drosoph-Xu
(1995). Levels of homeotic protein function can determine developmental identity: evidence from low-level expression of the Drosoph-
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oping and adult Drosophila tissues. Development 117, 1223–1237.
767–778.
PHYL Development 118, 401–415. acts to down-regulate TTK88, a transcriptional repressor of neuronal Characterizacell fates, by a SINA-dependent mechanism
- A H Tang
- T P Neufeld
- E Kwan
means of altering cell fates and generating dominant phenotypes.
Tang, A.H., Neufeld, T.P., Kwan, E., and Rubin, G.M. (1997). PHYL
Development 118, 401–415.
acts to down-regulate TTK88, a transcriptional repressor of neuronal
Bunch, T.A., Grinblat, Y., and Goldstein, L.S.B. (1988). Characterizacell fates, by a SINA-dependent mechanism. Cell 90, 459–467.
Ubiquitin- Drosophila melanogaster cells
- M Staszewski
- L M Bohmann
tion and use of the Drosophila metallothionein promoter in cultured
Treier, M., Staszewski, L.M., and Bohmann, D. (1994). Ubiquitin-
Drosophila melanogaster cells. Nucleic Acids Res. 16, 1043–1061.
dependent c-Jun degradation in vivo is mediated by the delta