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Rapid histone H3 phosphorylation in response to growth factors, phorbol esters, okadaic acid, and protein synthesis inhibitors
Abstract
When quiescent cells are stimulated with growth factors, phorbol esters, okadaic acid, or protein synthesis inhibitors, the early-response genes, which include c-fos and c-jun, are rapidly induced. The earliest growth factor- and phorbol ester-stimulated nuclear signaling events concomitant with proto-oncogene induction are the rapid phosphorylation of two chromatin-associated proteins, pp33 and pp15. We show here that the tumor promoter okadaic acid, which inhibits protein phosphatases 1 and 2A, and the protein synthesis inhibitors anisomycin and cycloheximide also stimulate pp33 and pp15 phosphorylation. Using transcriptional inhibitors, we show that this response is not a consequence of early gene induction. By peptide mapping and microsequencing, chromatin-associated pp15 is identified as histone H3. Upon stimulation, histone H3 is rapidly phosphorylated on serine residues within its highly charged, basic N-terminal domain. Thus, these diverse agents elicit a common early nuclear signal modulating nucleosomal structure or function, potentially contributing to conformational regulation of proto-oncogene induction.
... When quiescent fibroblasts are stimulated with growth factors or phorbol esters, a complex series of intracellular signalling and immediate-early (IE) gene induction responses is rapidly initiated (reviewed in references 1, 2, 4, 9, 18, 24, 26, and 29). Concomitant with c-fos/c-jun induction, early mitogen-stimulated responses in chromatin include phosphorylation of histone H3, a high-mobility-group (HMG)-like protein (identified by protein microsequencing studies as HMG-14 [3a]), and a nucleolar fraction of S6 (3,21,31,43). The hallmark of these early signalling and genetic responses is their transience; through concerted phosphatase action, key epidermal growth factor (EGF)-activated protein kinases are switched off within minutes and their substrates are dephosphorylated, producing a phosphorylation response that is rapidly terminated (29,51). ...
... Mailing address: Nuclear Signalling Laboratory, Developmental Biology Research Centre, The Randall Institute, King's College London, 26-29 Drury Ln., London WC2B 5RL, United Kingdom. induced by EGF or tetradecanoyl phorbol acetate (TPA) (3,18,31,42,43). The striking observation is that whereas growth factor-elicited responses are transient, those elicited by anisomycin are extremely sustained (18). ...
... Further, they remain demonstrable at subinhibitory anisomycin concentrations and are not a consequence of translational arrest (42). Finally, anisomycin stimulates an extremely restricted subset of the complex cytoplasmic signals elicited by EGF or TPA (reviewed in references 18 and 31) and will not, for instance, activate protein kinase C (43). However, it elicits chromatin-associated protein phosphorylation and gene induction responses virtually identical to those induced by EGF or TPA (3,18,31,42,43), suggesting its possible use to define an area of signalling from which the nuclear responses arise (31). ...
Independent of its ability to block translation, anisomycin intrinsically initiates intracellular signals and immediate-early gene induction [L. C. Mahadevan and D. R. Edwards, Nature (London) 349:747-749, 1991]. Here, we characterize further its action as a potent, selective signalling agonist. In-gel kinase assays show that epidermal growth factor (EGF) transiently activates five kinases: the mitogen-activated protein (MAP) kinases ERK-1 and -2, and three others, p45, p55, and p80. Anisomycin, at inhibitory and subinhibitory concentrations, does not activate ERK-1 and -2 but elicits strong sustained activation of p45 and p55, which are unique in being serine kinases whose detection is enhanced with poly-Glu/Tyr or poly-Glu/Phe copolymerized in these gels. Translational arrest using emetine or puromycin does not activate p45 and p55 but does prolong EGF-stimulated ERK-1 and -2 activation. Rapamycin, which blocks anisomycin-stimulated p70/85S6k activation without affecting nuclear responses, has no effect on p45 or p55 kinase. p45 and p55 are activable by okadaic acid or UV irradiation, and both kinases phosphorylate the c-Jun NH2-terminal peptide 1-79, putatively placing them within c-Jun NH2-terminal kinase/stress-activated protein kinase (JNK/SAPK) subfamily of MAP kinases. Thus, the EGF- and anisomycin-activated kinases p45 and p55 are strongly implicated in signalling to c-fos and c-jun, whereas the MAP kinases ERK-1 and -2 are not essential for this process.
... Phosphorylation of serine 10 on histone H3 (H3S10ph) is an unique histone modification as it is involved in two structurally opposed processes: transcriptional activation and chromatin relaxation, or chromosome compaction during cell division [7][8][9][10][11][12][13][14]. This suggests, that H3S10ph, rather than directly affecting chromatin structure, acts indirectly, serving as a protein binding platform, or a chromatin signpost, and the effect of H3S10ph on its neighborhood as well as its role, demand context-dependent interpretation. ...
... Role in mitotic chromosome condensation. Inhibition facilitates leukemia cell differentiation [91,92] AURKB Role in mitotic division [142] IKKα NF-κB, inflammation, immune-response [129][130][131]143] JNK Certain oncogenes activation through H3S10ph in their promoters in response to oncogenic stimuli [98,99,110] AKT1 Potential role in gene expression activation in response to oncogenic compounds together with RSK2 [144] MAP3K8 Activation of IEG/oncogene-FOS and cellular transformation in response to DNA damage [145] CDK8 Activation of IEGs/oncogenes [111,112]; downregulation of superenhancer-dependent genes in acute myeloid leukemia [118] MSK1/2 Activation of IEGs/oncogenes FOS and JUN [7,12]; response to carcinogens [16,64,100]; inflammation [134,135] PIM1 Activation of proto-oncogene FOSL1 [63]; oncogenic miR-17-92 [146] cooperation with MYC to induce cellular transformation [62,63]; potential role in mitotic checkpoint regulation [147,148] mitoses [95]. AURKA inhibition-associated genomic instability might be synthetically lethal in cancers with defects in certain DNA repair genes. ...
... Canonical mechanism of inducible H3S10 deposition involves mitogen-activated protein kinase (MAPK) pathway, activated by growth factors, mitogens, serum response, cytokines and cellular stressors. Operating downstream of the ERK1/2 and p38 MAPKs, MSK1 and 2, orthologs of Drosophila Jil-1 kinase, the canonical H3S10 kinase in the fruit fly, induce rapid H3S10 phosphorylation at the promoter and enhancer regions of immediate early genes (IEGs) [7,12]. IEGs are identified by their rapid and transient transcriptional induction, requiring no new protein synthesis. ...
Background
With the discovery that more than half of human cancers harbor mutations in chromatin proteins, deregulation of epigenetic mechanisms has been recognized a hallmark of malignant transformation. Post-translational modifications (PTMs) of histone proteins, as main components of epigenetic regulatory machinery, are also broadly accepted as therapeutic target. Current “epigenetic” therapies target predominantly writers, erasers and readers of histone acetylation and (to a lesser extent) methylation, leaving other types of PTMs largely unexplored. One of them is the phosphorylation of serine 10 on histone H3 (H3S10ph).
Main body
H3S10ph is emerging as an important player in the initiation and propagation of cancer, as it facilitates cellular malignant transformation and participates in fundamental cellular functions. In normal cells this histone mark dictates the hierarchy of additional histone modifications involved in the formation of protein binding scaffolds, transcriptional regulation, blocking repressive epigenetic information and shielding gene regions from heterochromatin spreading. During cell division, this mark is essential for chromosome condensation and segregation. It is also involved in the function of specific DNA–RNA hybrids, called R-loops, which modulate transcription and facilitate chromosomal instability. Increase in H3S10ph is observed in numerous cancer types and its abundance has been associated with inferior prognosis. Many H3S10-kinases, including MSK1/2, PIM1, CDK8 and AURORA kinases, have been long considered targets in cancer therapy. However, since these proteins also participate in other critical processes, including signal transduction, apoptotic signaling, metabolic fitness and transcription, their chromatin functions are often neglected.
Conclusions
H3S10ph and enzymes responsible for deposition of this histone modification are important for chromatin activity and oncogenesis. Epigenetic-drugs targeting this axis of modifications, potentially in combination with conventional or targeted therapy, provide a promising angle in search for knowledge-driven therapeutic strategies in oncology.
... Phosphorylation of histone H3 had gained more attention due to its association with the condensation of chromosomes during mitosis [50][51][52]. Interestingly, H3 phosphorylation was first reported in response to the activation of mitogenic signaling pathways [53]. Phosphorylation of histone H3 on the serine 10 residue is mediated by ribosomal protein S6 kinase 2 (RSK2), which is downstream of several other kinases, including extracellular signal-regulated kinase (ERK), mitogen-and stressactivated protein kinase 1 (MSK1), and the aurora kinase family member increase in ploidy 1 (IPL1) [54][55][56]. ...
... Recent studies also indicate aurora kinases in H3 serine 28 phosphorylation [57]. Histone phosphorylation is a reversible process during which phosphatases remove phosphate groups from the histones [53,58]. Protein phosphatases 1 (PP1) and 2A (PP2A) have been implicated in the regulation of H3 phosphorylation [56,59]. ...
Epigenetic mechanisms, which include DNA methylation, a variety of post-translational modifications of histone proteins (acetylation, phosphorylation, methylation, ubiquitination, sumoylation, serotonylation, dopaminylation), chromatin remodeling enzymes, and long non-coding RNAs, are robust regulators of activity-dependent changes in gene transcription. In the brain, many of these epigenetic modifications have been widely implicated in synaptic plasticity and memory formation. Dysregulation of epigenetic mechanisms has been reported in the aged brain and is associated with or contributes to memory decline across the lifespan. Furthermore, alterations in the epigenome have been reported in neurodegenerative disorders, including Alzheimer’s disease. Here, we review the diverse types of epigenetic modifications and their role in activity- and learning-dependent synaptic plasticity. We then discuss how these mechanisms become dysregulated across the lifespan and contribute to memory loss with age and in Alzheimer’s disease. Collectively, the evidence reviewed here strongly supports a role for diverse epigenetic mechanisms in memory formation, aging, and neurodegeneration in the brain.
... H3 dephosphorylation on serine 10 (H3S10) was shown to be induced in vitro in epithelial cells and to correlate with the repression of inflammatory genes. Although cellular processes, such as entry into mitosis or activation by extracellular signals (EGF, cellular stress, or inflammation) have been shown to be associated with H3S10 phosphorylation, the role of this modification in these processes is not fully understood (Mahadevan et al., 1991;Saccani et al., 2002;Sawicka et al., 2014). Key kinase signaling pathways, including mitogen-activated protein kinase (MAPK) and nuclear factor kB (NF-kB), induce the phosphorylation of histone H3 on residue 10 (H3S10) or 28 (H3S28), which are important for crosstalk with histone H3 acetylation, a canonical mark of transcriptional activation (Drobic et al., 2010;Yamamoto et al., 2003). ...
... This important step allows us to determine that, during infection with S. pneumoniae, PP1-mediated H3S10 dephosphorylation is not necessary for the difference in expression of inflammatory genes between WT and mutant strains. Because H3 phosphorylation is tightly linked to transcriptional regulation (Mahadevan et al., 1991;Saccani et al., 2002;Sawicka et al., 2014), we hypothesize that S. pneumoniae infection may alter the expression of unidentified genes controlled by basal H3S10 phosphorylation in epithelial cells. Further studies would be necessary to identify the genes involved. ...
Pathogenic bacteria can alter host gene expression through post-translational modifications of histones. We show that a natural colonizer, Streptococcus pneumoniae, induces specific histone modifications, including robust dephosphorylation of histone H3 on serine 10 (H3S10), during infection of respiratory epithelial cells. The bacterial pore-forming toxin pneumolysin (PLY), along with the pyruvate oxidase SpxB responsible for H2O2 production, play important roles in the induction of this modification. The combined effects of PLY and H2O2 trigger host signaling that culminates in H3S10 dephosphorylation, which is mediated by the host cell phosphatase PP1. Strikingly, S. pneumoniae infection induces dephosphorylation and subsequent activation of PP1 catalytic activity. Colonization of PP1 catalytically deficient cells results in impaired intracellular S. pneumoniae survival and infection. Interestingly, PP1 activation and H3S10 dephosphorylation are not restricted to S. pneumoniae and appear to be general epigenomic mechanisms favoring intracellular survival of pathogenic bacteria.
... These are further described in subsequent sections and exemplified in their relation to cfos promoter elements in the Figures 3C and 3D. The chemical states of the histone proteins play essential roles in the instigation and termination of transcription (Mahadevan et al., 1991;Tsankova et al., 2004;Tsankova et al., 2007;O'Donnell et al., 2012). ...
Neuroscience began an important new chapter in the 1980s when it was demonstrated that the induction of cFos occurred in response to the stimulation of acetylcholine receptors in neuron like cells. Transcription of cfos commenced within minutes and involved an influx of extracellular Ca2+ through voltage-sensitive calcium channels. Neuronal activity in many neuron types and brain regions led to the induction of many genes on various time scales. The first to be activated were called immediate early genes (IEGs), which include the Fos family cfos, fosB, fra1, fra2, and several isoforms. A short form of fosB called ΔFosB resisted degradation and was thought to play a role in inducing changes in neurons associated with addiction. The protein products of many IEGs act as transcription factors which are important in neurons of the central nervous system for their roles in neuronal plasticity, exemplified by learning and memory, addiction and several neuropsychiatric disorders such as depression. In this article experimental data and the biochemical processes underlying the pathways which lead to such transcription are described as a prelude to modeling.
... These are further described in subsequent sections and exemplified in their relation to cfos promoter elements in the Figures 3C and 3D. The chemical states of the histone proteins play essential roles in the instigation and termination of transcription (Mahadevan et al., 1991;Tsankova et al., 2004;Tsankova et al., 2007;O'Donnell et al., 2012). ...
Neuroscience began an important new chapter in the 1980s when it was demonstrated that the induction of cFos occurred in response to the stimulation of acetylcholine receptors in neuron like cells. Transcription of cfos commenced within minutes and involved an influx of extracellular Ca2+ through voltage-sensitive calcium channels. Neuronal activity in many neuron types and brain regions led to the induction of many genes on various time scales. The first to be activated were called immediate early genes (IEGs), which include the Fos family cfos, fosB, fra1, fra2, and several isoforms. A short form of fosB called ΔFosB resisted degradation and was thought to play a role in inducing changes in neurons associated with addiction. The protein products of many IEGs act as transcription factors which are important in neurons of the central nervous system for their roles in neuronal plasticity, exemplified by learning and memory, addiction and several neuropsychiatric disorders such as depression. In this article experimental data and the biochemical processes underlying the pathways which lead to such transcription are described as a prelude to modeling.
... The cellular response to diverse external stimuli, particularly stress or growth factors, is known to cause a phosphorylation cascade that results in the induction or repression of specific genes. A direct link between the phosphorylation cascade and chromatin modification was demonstrated by Mahadevan et al., 1991 with the observation that both histone H3 (at Serine 10) ...
Hyperacetylation of core histones H3 and H4 has been linked with both the presence of open domains of chromatin and additionally to the promoters/enhancers of actively transcribing genes. In order to elucidate the role(s) of acetylated histones H3 and H4 with respect to these models, their distribution across the embryonic chicken β-globin locus was investigated. Affinity purified polyclonal antibodies responding to the most highly acetylated isoforms of histones H3 and H4 were used in chromatin immunoprecipitation (ChIP) experiments to fractionate mono/dinucleosomes from 15-day chicken embryo erythrocyte nuclei. A map of acetyl-H3 and acetyl-H4 distribution across the β-globin locus was obtained using quantitative PCR analysis of the immunoselected DNA. Widespread, but modulated, acetylation of both H3 and H4 was found across the domain, with peaks at the LCR and promoter of the active βA gene. Acetylation levels across the four globin genes, and particularly their promoters, showed a distribution that closely correlates with their relative rates of transcription. The modulation of H3 acetylation was found to be greater than that of H4, which in contrast, showed a more uniform widespread distribution. This suggests that the roles of acetyl-H3 and acetyl-H4 are separable to a certain extent. The more evident widespread acetylation of H4 may be more closely implicated with the initiation or maintenance an open chromatin domain, whilst the greater modulation of acetyl-H3 may be indicative of a closer association with the process of transcriptional initiation and elongation.
... Due to its link to chromosome condensation during mitosis, histone H3 phosphorylation has attracted considerable attention (Bradbury et al., 1973;Gurley et al., 1974;Gurley et al., 1978). Interestingly, the activation of mitogenic signaling pathways is the origin of H3 phosphorylation (Mahadevan et al., 1991). For example, ribosomal protein S6 kinase 2 (RSK2) is downstream of several other kinases, including extracellular signal-regulated kinase (ERK), aurora kinase family member increase in ploidy 1 (IPL1), and mitogen-and stressactivated protein kinase 1 (MSK1), and RSK2 mediates the phosphorylation of histone H3 on the serine 10 residue (Sassone-Corsi et al., 1999;Thomson et al., 1999;Hsu et al., 2000). ...
With the advancement of technology, increasingly many newborns are receiving general anesthesia at a young age for surgery, other interventions, or clinical assessment. Anesthetics cause neurotoxicity and apoptosis of nerve cells, leading to memory and cognitive impairments. The most frequently used anesthetic in infants is sevoflurane; however, it has the potential to be neurotoxic. A single, short bout of sevoflurane exposure has little impact on cognitive function, but prolonged or recurrent exposure to general anesthetics can impair memory and cognitive function. However, the mechanisms underlying this association remain unknown. Posttranslational modifications (PTMs), which can be described roughly as the regulation of gene expression, protein activity, and protein function, have sparked enormous interest in neuroscience. Posttranslational modifications are a critical mechanism mediating anesthesia-induced long-term modifications in gene transcription and protein functional deficits in memory and cognition in children, according to a growing body of studies in recent years. Based on these recent findings, our paper reviews the effects of sevoflurane on memory loss and cognitive impairment, discusses how posttranslational modifications mechanisms can contribute to sevoflurane-induced neurotoxicity, and provides new insights into the prevention of sevoflurane-induced memory and cognitive impairments.
... Accordingly, flowering-promoting genes, such as AG, AP1, FT, SEP3, and AGL17, were upregulated in both H3.1S28A and H3.3S28A, and the increase was stronger in H3.3S28A than in H3.1S28A. In mammals, H3S28ph is involved in the transcriptional activation of immediate early genes (Mahadevan et al., 1991). Moreover, a genome-wide study in human cells revealed a novel function of H3S28ph in the transcriptional activation of stress-inducible genes (Sawicka et al., 2014). ...
Histone modifications are essential for chromatin activity and play an important role in many biological processes. Trimethylation of histone H3K27 (H3K27me3) is a repressive modification established by Polycomb Repressive Complex 2 (PRC2). Although the presence of the histone H3 serine 28 phosphorylation (H3S28ph) modification at adjacent amino acid residues has both positive and negative effects on Polycomb silencing in mammals, little is known about the effect of H3S28ph on H3K27me3-mediated gene silencing in plants. In this study, we show that mutating H3S28A in Arabidopsis (Arabidopsis thaliana) causes a dominant-negative effect that leads to an early-flowering phenotype by promoting the expression of flowering-promoting genes independently of abnormal cell division. While H3S28ph levels decreased due to the H3S28A mutation, H3K27me3 levels at the same loci did not increase. Moreover, we observed decreased H3K27me3 levels at some known PRC2 target genes in H3.3S28A transgenic lines, rather than the expected enhanced H3K27me3-mediated silencing. In line with the reduced H3K27me3 levels, the expression of the PRC2 catalytic subunits CURLY LEAF (CLF) and SWINGER (SWN) decreased. Taken together, these data demonstrate that H3.3S28 is required for PRC2-dependent H3K27me3-mediated silencing in Arabidopsis, suggesting that H3S28 has a non-canonical function in H3K27me3-mediated gene silencing.
... MSK1/2-mediated phosphorylation of histone H3S10 and H3S28 transduces MAPK activation in response to mitogenic stimulation activation to induction of IEG expression in a wide range of tissues [32,33,55,57]. Until now, a mechanism involving MSKs acting as histone kinases contributing to the induction of IEG and of hypertrophic gene expression via a nucleosomal response has not been demonstrated in CM. ...
Heart failure is a leading cause of death that develops subsequent to deleterious hypertrophic cardiac remodelling. MAPK pathways play a key role in coordinating the induction of gene expression during hypertrophy. Induction of the immediate early gene (IEG) response including activator protein 1 (AP-1) complex factors is a necessary and early event in this process. How MAPK and IEG expression are coupled during cardiac hypertrophy is not resolved. Here, in vitro, in rodent models and in human samples, we demonstrate that MAPK-stimulated IEG induction depends on the mitogen and stress-activated protein kinase (MSK) and its phosphorylation of histone H3 at serine 28 (pH3S28). pH3S28 in IEG promoters in turn recruits Brg1, a BAF60 ATP-dependent chromatin remodelling complex component, initiating gene expression. Without MSK activity and IEG induction, the hypertrophic response is suppressed. These studies provide new mechanistic insights into the role of MAPK pathways in signalling to the epigenome and regulation of gene expression during cardiac hypertrophy.
... Many kinases have been shown to phosphorylate serine and threonine residues of the H3 histone, in particular H3S10 (Sassone-Corsi et al., 1999;Duncan et al., 2006). For a long time phosphorylation of this histone has been known to correlate with actively transcribed genes (Mahadevan et al., 1991), but it also plays a central role in the condensation of chromosomes during mitosis (Wei et al., 1999;Giet & Glover, 2001). However, the precise mechanism of this modification is not known. ...
The nucleosome remodeling and histone deacetylase (NuRD) complex is one of the main epigenetic regulators of the genome. It contributes to the formation and maintenance of the heterochromatin, a tightly packed structure of DNA and proteins that represses transcription. NuRD plays a central role in relevant biological processes such as pluripotency regulation or tumorigenesis. Despite that, its structure and action mechanism remain unknown. This is largely due to the inherent compositional and conformational hetesrogeneity of NuRD. In this PhD work we tried to overcome these challenges using a multidisciplinary approach. We combined affinity purification of endogenous and recombinant NuRD complexes and subunits, cross-linking, electron cryo-microscopy and mass spectrometry. Thanks to that we were able to identify a stable histone deacetylase subcomplex with independent activity (PMMR) and solve its structure at 16.6 Å. We also obtained low resolution structures of several smaller subcomplexes. Studying these subcomplexes allowed us to propose a Holo-NuRD assembly pathway, leading to the publication of a scientific paper.Key words: NuRD, Epigenetic regulation, Chromatin remodeling, Histone deacetylase, Cryo-EM
... The darker staining (condensation) (see black arrow in Figure 2B) indicates an increase in chromosome condensation taking place in mitosis and may point to a higher degree of cell proliferation (Crosio et al., 2003;Goto et al., 1999;Koshland and Strunnikov, 1996). In contrast, the lighter staining may be related to gene expression (transcription) (Labrador and Corces, 2003) at interphase (Chadee et al., 1999;Cheung et al., 2000;Mahadevan et al., 1991) (see white arrow in Figure 2B). No differences in the level of P-H3 related to the ''chromatin condensation type'' (mitosis) were observed. ...
Post-translational epigenetic modifications take place in mouse neurons of the dentate gyrus (DG) with age. Here, we report that age-dependent reduction in H3K9 trimethylation (H3K9me3) is prevented by cyclic induction of the Yamanaka factors used for cell reprogramming. Interestingly, Yamanaka factors elevated the levels of migrating cells containing the neurogenic markers doublecortin and calretinin, and the levels of the NMDA receptor subunit GluN2B. These changes could result in an increase in the survival of newborn DG neurons during their maturation and higher synaptic plasticity in mature neurons. Importantly, these cellular changes were accompanied by an improvement in mouse performance in the object recognition test over long time. We conclude that transient cyclic reprogramming in vivo in the central nervous system could be an effective strategy to ameliorate aging of the central nervous system and neurodegenerative diseases.
... H3S10ph also takes place in interphase, where it has been associated with transcription activation [29][30][31]. It is mediated by several kinases in mammals [32] but mostly by JIL-1 kinase in Drosophila [33][34][35]. ...
Heterochromatin Protein 1 (HP1) is a highly conserved protein that has been used as a classic marker for heterochromatin. HP1 binds to di- and tri-methylated histone H3K9 and regulates heterochromatin formation, functions and structure. Besides the well-established phosphorylation of histone H3 Ser10 that has been shown to modulate HP1 binding to chromatin, several studies have recently highlighted the importance of HP1 post-translational modifications and additional epigenetic features for the modulation of HP1-chromatin binding ability and heterochromatin formation. In this review, we summarize the recent literature of HP1 post-translational modifications that have contributed to understand how heterochromatin is formed, regulated and maintained.
... Histone H3 is a substrate for the MAP Kinase effector RSK-2 , and H3 has also been reported to be a substrate of the kinase MSK . Mahadevan and co-workers have shown stimulation of quiescent mammalian cells with epidermal growth factor (EGF) causes a phoshorylation of histone H3 by Rsk-2 kinase, in addition to the induction of immediate early genes, such as Q-fos (Barratt et al., 1994;Clayton et al., 2000;Mahadevan et al., 1991;. Acétylation and phosphorylation are found in combination at active promoters (Cheung et al., 2000). ...
The Serum Response Element (SRE) is a promoter element important in the transcriptional regulation of many immediate early and muscle specific genes. Serum Response Factor (SRF) and Ternary Complex Factor (TCF) form a complex at the c-fos SRE. TCF is phosphorylated on MAP Kinase activation resulting in activation of transcription. In addition, a TCF independent pathway, dependent on RhoA signalling and depletion of G-actin levels, activates transcription through SRF. The sensitivity of SRF target genes to inhibitors of the actin pathway correlates with the presence of a TCF binding site adjacent to the SRE. c-fos and egr-1 promoters have a TCF binding site adjacent to the SRE and are insensitive to the actin pathway, vinculin and actin promoters do not have a TCF binding site and are sensitive to the actin pathway. In this study vinculin has been used to investigate whether the presence of a TCF binding site is sufficient to render the SRE insensitive to the actin pathway. At the vinculin promoter a TCF site alone is not sufficient to reduce the sensitivity to the actin pathway inhibitor, Latrunculin B. This finding is in contrast to studies using the c-fos promoter, which can be made sensitive to the actin pathway by the removal of the TCF binding site (Murai and Treisman, 2002). The promoter context of the SRE may therefore be important in rendering a promoter insensitive to the actin pathway. However, targeting an altered DNA binding specificity TCF molecule to the SRE can inhibit the actin pathway at the vinculin promoter. In addition, a 1-hybrid screen was conducted to identify factors capable of interacting with SRF, to allow identification of the SRF co-activator of the actin pathway. A clone was identified that is not the co-activator of the actin pathway, but which may have a role in SRF mediated transcriptional activation.
... Chromatin structure is modified as an early response of cells to growth factors, with phosphorylation of histone H3 protein proposed to relax chromatin structure (Mahadevan et al., 1991), and rearrangement of the nucleosomal structure of hormone responsive elements in response to steroid hormone stimulation (reviewed by Beato, 1989). ...
Murine embryonic development relies on the precise temporal and spatial expression of genes, the protein products of which dictate cellular phenotype. Proteins controlling the transcription of genes during early embryogenesis might be expected to be regulated on the differentiation of F9 embryonal carcinoma stem cells. One such regulated activity is DRTF1 (differentiation regulated transcription factor), a transcription factor which can associate with the product of the retinoblastoma tumour suppressor gene (pRb), pi07, and cyclin A. The embryonic expression pattern and biochemical characterisation of DRTF1 are the subject of this study. DRTF1 is a sequence specific DNA binding protein that consists of at least 3 DNA binding activities referred to as la, b, and c. Complexed DRTF1, referred to as DRTF1a, has similar DNA binding specificity and DNA binding polypeptides to DRTF1b/c, which lack pRb and cyclin A. DRTF1b/c are abundant in embryonal carcinoma (EC) and embryonic stem cells (ES), and down regulated on differentiation. In contrast, DRTF1a is not abundant in EC or ES cells, and is not down regulated as EC cells start to differentiate. The binding activity of DRTF1 was investigated during murine embryogenesis by preparing microextracts from tissues and whole embryos at various stages of development. DRTF1b was present in blastocyst stage embryos, and was abundant up to about 14 days of gestation. However, as embryogenesis progressed, the levels of DRTF1 b/c decreased whereas in contrast, the binding activity of DRTF1a increased. DRTF1 b/c were abundant in all tissues examined during early stages of embryogenesis, but became tissue restricted during later stages of development, and were frequently excluded from terminally differentiated tissues, for example liver and brain. In summary, DRTF1 is a group of DNA binding activities which share common DNA binding polypeptides, and which complex with other non-DNA binding polypeptides such as pRb and cyclin A in a developmentally regulated and tissue dependent fashion. The DNA binding polypeptides of DRTF1 were purified by sequence-specific affinity chromatography from F9 EC cells. The purification procedure was modified to incorporate a mutant site affinity column, which improved the purity of DRTF1. A unique 360 nucleotide cDNA was isolated by using degenerate oligonucleotides against peptide sequences derived from one purified polypeptide. The distribution of its mRNA was investigated by Northern blot and RNAse protection assays. These data, combined with a detailed examination of the expression during embryogenesis by in situ hybridisation, indicated that the mRNA expression was developmentally regulated, with high levels of expression at 6.5 to approximately 13 days post coitum, and lower levels of expression at later stages of development. This expression pattern was similar to the quantitative regulation of DRTF1 DNA binding activity, and the cDNA was therefore likely to encode a polypeptide involved in the DRTF1 activity. In conclusion, DRTF1 is a developmentally regulated and tissue-dependent transcription factor, that is subject to different levels of regulation. It is likely that DRTF1 plays an important role during embryonic development.
... This evidence suggested that gene expression or repression can be triggered in early phases of development and be maintained despite cell divisions (Wakimoto 1998). Recent studies in yeast and fruit flies suggest that PEV is achieved with the help of different histone modification mechanisms including methylation and phosphorylation of histones (Mahadevan et al 1991, De Cesare et al 1998, Lee & Karpen 2017, Ahringer & Gasser 2018, O'Kane & Hyland 2019. Hence, the evidences that once helped Waddington to develop his ideas regarding epigenetics are looping back with modern molecular epigenetics (Jamniczky et al 2010, Burggren 2016, Loison 2019. ...
Insects are the largest animal group on Earth both in biomass and diversity. Their outstanding success has inspired genetics and developmental research, allowing the discovery of dynamic process explaining extreme phenotypic plasticity and canalization. Epigenetic molecular mechanisms (EMMs) are vital for several housekeeping functions in multicellular organisms, regulating developmental, ontogenetic trajectories and environmental adaptations. In Insecta, EMMs are involved in the development of extreme phenotypic divergences such as polyphenisms and eusocial castes. Here, we review the history of this research field and how the main EMMs found in insects help to understand their biological processes and diversity. EMMs in insects confer them rapid response capacity allowing insect either to change with plastic divergence or to keep constant when facing different stressors or stimuli. EMMs function both at intra as well as transgenerational scales, playing important roles in insect ecology and evolution. We discuss on how EMMs pervasive influences in Insecta require not only the control of gene expression but also the dynamic interplay of EMMs with further regulatory levels, including genetic, physiological, behavioral, and environmental among others, as was earlier proposed by the Probabilistic Epigenesis model and Developmental System Theory.
... The authors declare no conflict of interest. 3 as FOS and JUN (Cheung et al., 2000, Mahadevan et al., 1991. In this regard, SRF transcriptional activity has 50 been proposed to play an important role in the regulation of muscle mass, as evidenced by loss of 51 overload-induced hypertrophy following inducible targeted deletion of skeletal muscle SRF in mouse 52 (Guerci et al., 2012). ...
Mechanotransduction in skeletal muscle
... The significance of interphase H3S10ph is often underestimated because most H3S10 phosphorylation in asynchronous cell populations stems from mitotic chromatin, where it is deployed by Aurora B kinase 2,3 . Originally, interphase H3S10ph has been associated, in combination with H3K9ac and H3K14ac, with transcriptional activation of immediate early genes upon MAPK activation 4,5 . In Drosophila, interphase H3S10ph is enriched at the body of active genes 6 . ...
In flies, the chromosomal kinase JIL-1 is responsible for most interphase histone H3S10 phosphorylation and has been proposed to protect active chromatin from acquiring heterochromatic marks, such as dimethylated histone H3K9 (H3K9me2) and HP1. Here, we show that JIL-1’s targeting to chromatin depends on a PWWP domain-containing protein JASPer (JIL-1 Anchoring and Stabilizing Protein). JASPer-JIL-1 (JJ)-complex is the major form of kinase in vivo and is targeted to active genes and telomeric transposons via binding of the PWWP domain of JASPer to H3K36me3 nucleosomes, to modulate transcriptional output. JIL-1 and JJ-complex depletion in cycling cells lead to small changes in H3K9me2 distribution at active genes and telomeric transposons. Finally, we identify interactors of the endogenous JJ-complex and propose that JIL-1 not only prevents heterochromatin formation but also coordinates chromatin-based regulation in the transcribed part of the genome. The chromosomal kinase JIL-1 is responsible for interphase histone H3S10 phosphorylation and has been proposed to protect active chromatin from heterochromatinisation. Here, the authors show that JIL-1 is stabilized and anchored to active genes and telomeric transposons by JASPer, which binds to H3K36me3 nucleosomes via its PWWP domain.
... In the context of IEG induction, exemplified by cfos and cjun, these authors, citing Mahadevan et al. (1991), Chadee et al. (1999) and Thomson et al. (1999), pointed out that H3 phosphorylation at Ser10 and acetylation at Lys14 at the promoters of these genes were necessary and sufficient for the nucleosomal response (rapid phosphorylation of H3) and that these modifications occurred on the same histone tail. The phosphorylation of H3 on Ser10 involves the MAPK/ERK signaling pathway which leads to the activation of RSKs and MSKs by means of complicated sequences of phosphorylation. ...
We elaborate upon further details of the mechanisms involved in transcription of Fos family and other immediate early genes in brain. The AP-1 element on the cfos promoter is bound by homodimers of Jun family proteins and heterodimers of Jun family members with Fos family members including ∆FosB. The frequencies of combinations are discussed as well as their activities which may be activating or inhibiting. With acute stimulation by psychostimulants the mRNAs of cfos and fosB are induced within a few minutes to rise to maximum levels in about 30 minutes with a decline to basal levels in a few hours. The many possible mechanisms for the shut down are discussed. Epigenetic modifications are strongly implicated in the instigation and inhibition of transcription, particularly histone modifications which may decompact-ify DNA. Summaries of histone modifications have been related to a histone code. Various general schemes for the steps to transcription have been proposed and three of these are described, followed by more detailed dynamical pictures. Finally, results for transcription measurements in single cells are discussed and some simple mathematical models that have been employed to quantify their stochastic properties are described.
... These are further described in subsequent sections and exemplified in their relation to cfos promoter elements in the Figures 3C and 3D. The chemical states of the histone proteins play essential roles in the instigation and termination of transcription (Mahadevan et al., 1991;Tsankova et al., 2004;Tsankova et al., 2007;O'Donnell et al., 2012). ...
... In spite of that, at the peak of astrocyte proliferation (around 5dpi), the proportion of mitotic (Ki67+/PH3+) cells among all BrdU-labeled cells decrease over the course of aging (Fig. 2E, F), and most of these cells are microglia [16,19,30]. It is thus noteworthy that astrocyte proliferation can be reactivated even in the aged brain parenchyma, as also in old animals some GM astrocytes that were nonproliferative prior to injury start to upregulate expression of the cell cycle regulators [30], like cyclin D1 (CcnD1) (Fig. 2G), as well as the Ki67 protein that is present during all active phases of the cell cycle (G 1 , S, G 2 , and mitosis) [31], and the indicator of mitogenic signaling activation PH3 [32] (Subheadings 3.4 and 3.5). Even though the acquisition of proliferative phenotype in astrocytes at the injury site occurs at a significant lesser extent in old animals, the bias of reactive astrocytes to proliferate at the interface of blood vessels remained unaffected by aging [16], as the majority of astrocytes within the injured GM resume proliferation still at juxtavascular positions ( Fig. 2H). ...
Reactive gliosis is a complicated process involving all types of glial cells and is the therapeutic target of efforts to treat several types of neuropathologies. Parenchymal astrocytes continuously survey their microenvironment to identify even tiny abnormalities in the central nervous system (CNS) homeostasis and react rapidly to brain damage, such as following ischemia, trauma, or neurodegenerative diseases, to prevent propagation of tissue damage. Aging can play causal roles in certain astroglial dysfunctions, however, still little is known to what extent the heterogeneous reaction of astrocytes at the injury site might be impaired over the course of aging. Based on our experience with both in vitro and in vivo experimental paradigms, we describe here in detail the analysis of age-related changes in (1) proliferative response of parenchymal astrocytes within the posttraumatic cerebral cortex grey matter (GM), and (2) repertoire of their cell divisions in adherent cell culture prepared from the injured GM of young and old double transgenic GFAP-mRFP1/(FUCCI)-S/G2/M-mAG-hGeminin mice by single cell time-lapse imaging.
... Phosphorylated histone H3 Ser-10 (p-Histone H3 Ser-10 ) is more precise and contrary to DCX, its expression occurs only in newborn neurons (neuronal progenitor cells, NPCs) [23]. Phosphorylation of N-terminal domain of histone H3 in position Ser-10 and/or Ser-28 destabilizes chromatin, directly preceding replication and transcription [6,7,17]. ...
Introduction:
Adult neurogenesis includes proliferation and differentiation of progenitor cells as well as their migration and maturation. In the adult human brain, two neurogenic regions, the hippocampal dentate gyrus (DG) and the subventricular zone (SVZ) of lateral ventricles, have been identified. In the dentate gyrus, three types of transcriptionally active cells and in the subventricular zone, four types of transcriptionally active cells, including GFAP-positive neural stem cells (NSCs), have been differentiated.
Material and methods:
The aim of the study was to identify and compare density of neurogenic cells between two study groups of patients (7 men, 7 women, mean age 70 ± 6.03) with ischemic stroke and with hemorrhage (6 men, 2 women, mean age 64.75 ± 12.23) and the control group of patients (6 men, 2 women, mean age 64 ± 10.95) free of neuropathologic changes who died suddenly within less than 10 min.
Results:
In both groups, in the hippocampal dentate gyrus and in the subventricular zone of lateral ventricles, the presence of single GFAP-positive neural stem cells and the transcriptionally active cells labelled with phosphorylated histone H3Ser-10 (p-Histone H3Ser-10)/neural progenitor cells (NPCs), was observed. The quantitative analysis of cells with p-Histone H3Ser-10 expression in the hippocampal DG revealed significant differences between the hemorrhage and control groups (p = 0.001, test t). However, in the SVZ, it showed a statistically significant decrease in the density of transcriptionally active cells in the group of patients with ischemic stroke (p = 0.001, test t). A distinct decrease in the density of transcriptionally active cells, proportional to the length of the patients' hospitalization, was observed.
Conclusions:
Hypoxia belongs to pathomechanic factors responsible for ischemic stroke, which can induce neurogenesis. However, hypoxia along with ischemia and other factors implicated in ischemic stroke, such as the patient's age or duration of ischemia can have a decisive influence on the decrease in the density of transcriptionally active cells in this pathologic process.
... The ARKS phosphorylation events feature ejection of repressive factors, recruitment of activating factors (14-3-3, BRD-containing factors), or activation of HATs (GCN5/PCAF) highlighting the general association of histone phosphorylation with active chromatin processes and transcription. Indeed, histone H3 phosphorylation is a common feature of cellular responses to stimulation (Mahadevan et al., 1991). Therefore, kinase pathways can potently and directly regulate chromatin via histone phosphorylation during cellular responses to environmental cues, and this feature likely acts coordinately with the wide array of other kinase-regulated cellular processes, especially activation of DNA-binding transcription factors. ...
... Due to the external factor activity, gene activation of early cellular response occur in parallel with histone H3 phosphorylation, e.g., c-fos genes. The same kinase, mitogen-activated protein kinase (MAPK), is responsible for both processes [7,9,18]. The product of the c-fos gene is a good marker for neuron activation in the mechanism of neuroadaptation [21,25]. ...
In our study expression of phospho-(Ser-10)-histone H3 (pH3S10), a marker for the early stage of neurogenesis, and cellular early response genes were investigated using c-Fos protein as an example of a transcription factor in the neurogenic process in rats. Neurogenesis in the adult brain is regulated by endo- and exogenous factors, which influence the proliferation potential of progenitor cells and accelerate the dendritic development of newborn neurons. D-amphetamine, a psychoactive substance, is one of the exogenous factors able to influence the process of neurogenesis. The rats were injected with D-amphetamine at a dose of 1.5 mg/kg/body weight (b.w.) under one administration scheme. Analysis of the pH3S10 and c-Fos expression levels in the group of D-amphetamine administered rats provided evidence of enhanced expression of these proteins in the regions of neurogenesis occurrence in rats. However, conclusions concerning stimulant effects of amphetamine on neurogenesis should be formulated with great caution, taking into account amphetamine dosage and the administration scheme. It should also be remembered that doses of psychoactive substances used in animal models can be lethal to humans.
... Early work in vitro has shown that the induction of c-Fos and Egr-1 requires the formation of phosphorylation and acetylation marks within histone H3 in their gene promoter regions (Mahadevan et al., 1991;Thomson et al., 1999). This dual histone mark is histone H3 phosphorylated at serine10 (S10) and acetylated at lysine14 (K14), i.e., H3K14ac-S10p. ...
The immediate early genes c-Fos and Egr-1 are rapidly and transiently induced in sparse neurons within the hippocampus after exposure to an acute stressor. The induction of these genes is a critical part of the molecular mechanisms underlying successful behavioural adaptation to stress. Our previous work has shown that transcriptional activation of c-Fos and Egr-1 in the hippocampus requires formation of a dual histone mark within their promoter regions, the phosphorylation of serine 10 and acetylation of lysine 9/14 of histone H3. In the present study, using chromatin immuno-precipitation (ChIP), we found that an increase in the formation of H3K9ac-S10p occurs within the c-Fos and Egr-1 promoters after forced swim stress in vivo and that these histone modifications were located to promotor regions containing cAMP Responsive Elements (CREs), but not in neighbouring regions containing only Serum Responsive Elements (SREs). Surprisingly, however, subsequent ChIP analyses showed no changes in the binding of pCREB or CREB-binding protein (CBP) to the CREs after forced swimming. In fact, pCREB binding to the c-Fos and Egr-1 promoters was already highly enriched under baseline conditions and did not increase further after stress. We suggest that constitutive pCREB binding may keep c-Fos and Egr-1 in a poised state for activation. Possibly, the formation of H3K9ac-S10p in the vicinity of CRE sites may participate in unblocking transcriptional elongation through recruitment of additional epigenetic factors.
... By early metaphase, histone H3 is extensively phosphorylated by Aurora kinase B (AURKB) at S10 and S28 (Wei et al. 1998), a process essential for the initiation of chromosome condensation and transition through mitosis (Van Hooser et al. 1998). Conversely, during interphase, activation of the MAPK pathway by mitogens or stress promotes rapid H3S10 phosphorylation at the promoter and enhancer regions of immediate early genes (Mahadevan et al. 1991;Cheung et al. 2000), stimulating the release of paused RNA POL II and P-TEFb-dependent transcription elongation (Zippo et al. 2009). While several nuclear kinases have been implicated as the downstream effectors of such inducible H3S10 phosphorylation at specific loci, including the JIL-1 homologs RPS6KA5 (also known as MSK1) and RPS6KA4 (also known as MSK2), RPS6KA3 (also known as RSK2), CHUK (also known as IKKα), and PIM1 (Sassone-Corsi et al. 1999; Thomson et al. 1999;Anest et al. 2003;Zippo et al. 2009), a genome-wide view of the dynamics of H3S10ph during the mammalian cell cycle has not been reported. ...
Phosphorylation of histone H3 at serine 10 (H3S10ph) by Aurora kinases plays an important role in mitosis; however, H3S10ph also marks regulatory regions of inducible genes in interphase mammalian cells, implicating mitosis-independent functions. Using the fluorescent ubiquitin-mediated cell cycle indicator (FUCCI), we found that 30% of the genome in interphase mouse embryonic stem cells (ESCs) is marked with H3S10ph. H3S10ph broadly demarcates gene-rich regions in G1 and is positively correlated with domains of early DNA replication timing (RT) but negatively correlated with H3K9me2 and lamin-associated domains (LADs). Consistent with mitosis-independent kinase activity, this pattern was preserved in ESCs treated with Hesperadin, a potent inhibitor of Aurora B/C kinases. Disruption of H3S10ph by expression of nonphosphorylatable H3.3S10A results in ectopic spreading of H3K9me2 into adjacent euchromatic regions, mimicking the phenotype observed in Drosophila JIL-1 kinase mutants. Conversely, interphase H3S10ph domains expand in Ehmt1 (also known as Glp) null ESCs, revealing that H3S10ph deposition is restricted by H3K9me2. Strikingly, spreading of H3S10ph at RT transition regions (TTRs) is accompanied by aberrant transcription initiation of genes co-oriented with the replication fork in Ehmt1-/- and Ehmt2-/- ESCs, indicating that establishment of repressive chromatin on the leading strand following DNA synthesis may depend upon these lysine methyltransferases. H3S10ph is also anti-correlated with H3K9me2 in interphase murine embryonic fibroblasts (MEFs) and is restricted to intragenic regions of actively transcribing genes by EHMT2. Taken together, these observations reveal that H3S10ph may play a general role in restricting the spreading of repressive chromatin in interphase mammalian cells.
... A variety of agents that induce cellular proliferation such as growth factors, for example, stimulate protein kinases that phosphorylate Ser and Tyr residues on many proteins having important biological roles including those in chromatin. 1,2 Post-translational modifications in histones include not only acetylation of Lys residues first reported by Allfrey 3 and phosphorylation of Ser residues but also methylation of Lys and Arg residues 4,5 as well as ubiquitination of Lys residues. These multiple side chain modifications in histones, most of which are on the N-terminal tails, either promote or repress DNA transcription by somehow altering the constraints within the nucleosome structure. ...
The effect of phosphorylation on the basicities of amines in histone H3 peptides and their acetylation kinetics is probed with a mild chemical acetylating agent. Phosphorylation of Ser-10 lowers the rate of chemical acetylation of Lys-9, Lys-14 and Lys-18 by methyl acetyl phosphate in that order consistent with a higher pKa of these Lys residues induced by phosphorylation; basicities increase up to 3 pKa units as a function of distance from Ser-10 phosphate. Enzymic acetylation of Lys residues with high pKa values in nucleosomes is also expected to be enhanced by phosphorylation, consistent with the known mechanism involving binding of protonated amines to N-acetyltransferases; fetal hemoglobin has a related linkage of increased basicity at a specific site, its acetylation, and a resulting decrease in subunit interaction strength. In the absence of a phosphate on Ser-10, the amines of Lys-9, Lys-14 and Lys-18 have lowered pKa values. Chemical acetylation of glycine and glycinamide have analogous kinetic profiles to the histone peptides but the phosphate inductive effect in histone H3 is more potent since the linkage between phosphorylation and acetylation is propagated with a range extending 9-10 amino acids in either direction from the phosphorylation site enhancing protonation of amino groups. We conclude that lysine amine basicities in histone tails are not static but inducible and variable due to a dynamic and immediate interaction between phosphorylation/acetylation that may contribute to inactive heterochromatin by compaction through such Ser phosphate–Lys amine electrostatic interactions and their relaxation by acetylation in euchromatin. This article is protected by copyright. All rights reserved.
... Phosphorylation of histone tails appears to result from signal transduction pathways. For example, phosphorylation of serine 10 on histone 3 (H3S10p) is acquired in genes after response to stimulation, such as growth factors [58], and marks mitotic cells. Acetylation of lysine residues is thought to promote euchromatin formation by reducing the positive charge of lysine side chains, thereby disrupting the electrochemical attraction of the positively charged histones to the negatively charged backbone of the DNA. ...
Epigenetics allows for the inheritance of information in cellular lineages during differentiation, independent of changes to the underlying genetic sequence. This raises the question of whether epigenetic mechanisms also function in post-mitotic neurons. During the long life of the neuron, fluctuations in gene expression allow the cell to pass through stages of differentiation, modulate synaptic activity in response to environmental cues, and fortify the cell through age-related neuroprotective pathways. Emerging evidence suggests that epigenetic mechanisms such as DNA methylation and histone modification permit these dynamic changes in gene expression throughout the life of a neuron. Accordingly, recent studies have revealed the vital importance of epigenetic players in the central nervous system and during neurodegeneration. Here, we provide a review of several of these recent findings, highlighting novel functions for epigenetics in the fields of Rett syndrome, Fragile X syndrome, and Alzheimer’s disease research. Together, these discoveries underscore the vital importance of epigenetics in human neurological disorders.
Extracellular signals are transmitted through kinase cascades to modulate gene expression, but it remains unclear how epigenetic changes regulate this response. Here, we provide evidence that growth factor–stimulated changes in the transcript levels of many responsive genes are accompanied by increases in histone phosphorylation levels, specifically at histone H3 serine-10 when the adjacent lysine-9 is dimethylated (H3K9me2S10). Imaging and proteomic approaches show that epidermal growth factor (EGF) stimulation results in H3K9me2S10 phosphorylation, which occurs in genomic regions enriched for regulatory enhancers of EGF-responsive genes. We also demonstrate that the EGF-induced increase in H3K9me2S10ph is dependent on the nuclear kinase MSK2, and this subset of EGF-induced genes is dependent on MSK2 for transcription. Together, our work indicates that growth factor–induced changes in chromatin state can mediate the activation of downstream genes.
Acute Myeloid Leukemia (AML) is caused by multiple mutations which dysregulate growth and differentiation of myeloid cells. Cells adopt different gene regulatory networks specific to individual mutations, maintaining a rapidly proliferating blast cell population with fatal consequences for the patient if not treated. The most common treatment option is still chemotherapy which targets such cells. However, patients harbour a population of quiescent leukemic stem cells (LSCs) which can emerge from quiescence to trigger relapse after therapy. The processes that allow such cells to re-grow remain unknown. Here, we examine the well characterised t(8;21) AML sub-type as a model to address this question. Using four primary AML samples and a novel t(8;21) patient-derived xenograft model, we show that t(8;21) LSCs aberrantly activate the VEGF and IL-5 signalling pathways. Both pathways operate within a regulatory circuit consisting of the driver oncoprotein RUNX1::ETO and an AP-1/GATA2 axis allowing LSCs to re-enter the cell cycle while preserving self-renewal capacity.
The term epigenetics was coined by Waddington in 1942 and was used to describe the “interactions of genes with their environment that brings the phenotype into being” (Waddington 2012). In general, genetic factors responsible for change in the appearance of an organism or biological functions without changing the actual DNA sequence is referred as epigenetics (Gavery and Roberts 2017). Therefore, only the expression of genes changes but not the genes. At molecular level, epigenetics refers to the addition or deletion of a methyl group to a DNA base, turning the gene on or off, or to packaging of the chromatin structure by silencing or opening regions of the genome by winding or unwinding the DNA around histones. Environmental factors are known to cause the epigenetic changes, and these changes are more common than mutations. It is also known that epigenetic changes caused by environmental exposures can be transmitted down several generations. The widely studied molecular epigenetic mechanisms are as follows:
Epigenetics play important roles during development progress of tumor. The histone modifications are the most important constitutedfield. Recently, accumulating research focused on exploring the roles of those modifications in regulating tumorigenesis. Moreover, the dysregulation of histone modifications is supposed to have vital clinical significance. Numerous histone modifications have the potential to be prognostic biomarkers, monitoring response of therapy, early diagnostic markers. Herein, we review the recent advances of histone modifications involving development of gastric cancer.Gastric cancer (GC) is one of the most frequent tumors in the world. Stomach adenocarcinoma is a heterogeneous tumor, turning the prognosisprediction and patients’ clinical management difficult. Some diagnosis tests for GC are been development using knowledge based in polymorphisms, somatic copy number alteration (SCNA) and aberrant histone methylation. This last event, a posttranslational modification that occurs at the chromatin level, is an important epigenetic alteration seen in several tumors including stomach adenocarcinoma. Histone methyltransferases (HMT) are the proteins responsible for the methylation in specific amino acids residues of histones tails. Here, were presented several HMTs that could be relating to GC process.
Epigenetics play a fundamental role in induced pluripotent stem cell (iPSC) technology due to their effect on iPSC's reprogramming efficiency and their subsequent role in iPSC differentiation toward a specific lineage. Epigenetics can skew the differentiation course of iPSCs toward a specific lineage based on the epigenetic memory of the source cells, or even lead to acquisition of new cell phenotypes, due to its aberrations during reprogramming. This viewpoint discusses key features of the epigenetic process during iPSC reprogramming/differentiation and outlines important epigenetic factors that need to be considered for successful generation and differentiation of iPSCs for downstream applications.
We demonstrate that members of the erk-encoded family of mitogen-activated protein (MAP) kinases (pp44/42mapk/erk) and members of the rsk-encoded protein kinases (RSKs or pp90rsk) are present in the cytoplasm and nucleus of HeLa cells. Addition of growth factors to serum-deprived cells results in increased tyrosine and threonine phosphorylation and in the activation of cytosolic and nuclear MAP kinases. Activated MAP kinases then phosphorylate (serine/threonine) and activate RSKs. Concurrently, a fraction of the activated MAP kinases and RSKs enter the nucleus. In addition, a distinct growth-regulated RSK-kinase activity (an enzyme[s] that phosphorylates recombinant RSK in vitro and that may be another member of the erk-encoded family of MAP kinases) was found associated with a postnuclear membrane fraction. Regulation of nuclear MAP kinase and RSK activities by growth factors and phorbol ester is coordinate with immediate-early gene expression. Indeed, in vitro, MAP kinase and/or RSK phosphorylates histone H3 and the recombinant c-Fos and c-Jun polypeptides, transcription factors phosphorylated in a variety of cells in response to growth stimuli. These in vitro studies raise the possibility that the MAP kinase/RSK signal transduction pathway represents a protein-Tyr/Ser/Thr phosphorylation cascade with the spatial distribution and temporal regulation that can account for the rapid transmission of growth-regulating information from the membrane, through the cytoplasm, and to the nucleus.
To identify trans-acting factors involved in mRNA decay in the yeast Saccharomyces cerevisiae, we have begun to characterize conditional lethal mutants that affect mRNA steady-state levels. A screen of a collection of temperature-sensitive mutants identified ts352, a mutant that accumulated moderately stable and unstable mRNAs after a shift from 23 to 37 degrees C (M. Aebi, G. Kirchner, J.-Y. Chen, U. Vijayraghavan, A. Jacobson, N.C. Martin, and J. Abelson, J. Biol. Chem. 265:16216-16220, 1990). ts352 has a defect in the CCA1 gene, which codes for tRNA nucleotidyltransferase, the enzyme that adds 3' CCA termini to tRNAs (Aebi et al., J. Biol. Chem., 1990). In a shift to the nonpermissive temperature, ts352 (cca1-1) cells rapidly cease protein synthesis, reduce the rates of degradation of the CDC4, TCM1, and PAB1 mRNAs three- to fivefold, and increase the relative number of ribosomes associated with mRNAs and the overall size of polysomes. These results were analogous to those observed for cycloheximide-treated cells and are generally consistent with models that invoke a role for translational elongation in the process of mRNA turnover.
Anisomycin, a translational inhibitor, synergizes with growth factors and phorbol esters to superinduce c-fos and c-jun by a number mechanisms, one of which is its ability to act as a potent signalling agonist, producing strong, prolonged activation of the same nuclear responses as epidermal growth factor or tetradecanoyl phorbol acetate. These responses include the phosphorylation of pp33, which exists in complexed and chromatin-associated forms, and of histone H3 and an HMG-like protein. By peptide mapping and microsequencing, we show here that pp33 is the phosphoprotein S6, present in ribosomes and in preribosomes in the nucleolus. Ablation of epidermal growth factor-, tetradecanoyl phorbol acetate-, or anisomycin-stimulated S6 phosphorylation by using the p70/85S6k inhibitor rapamycin has no effect on histone H3 and HMG-like protein phosphorylation or on the induction and superinduction of c-fos and c-jun. Further, [35S]methionine-labelling and immunoprecipitation studies show that the ablation of S6 phosphorylation has no discernible effect on translation in general or translation of newly induced c-fos transcripts. Finally, we show that anisomycin augments and prolongs S6 phosphorylation not by blocking S6 phosphatases but by sustained activation of p70/85S6k. These results suggest the possible use of anisomycin and rapamycin to define upstream and downstream boundaries of an area of signalling above p70/85S6k which contains a bifurcation that produces histone H3-HMG-like protein phosphorylation and c-fos-c-jun induction in the nucleus.
Complex organisms are able to respond to diverse environmental cues by rapidly inducing specific transcriptional programs comprising a few dozen genes among thousands. The highly complex environment within the nucleus— a crowded milieu containing large genomes tightly condensed with histone proteins in the form of chromatin— makes inducible transcription a challenge for the cell, akin to the proverbial needle in a haystack. The different signaling pathways and transcription factors involved in the transmission of information from the cell surface to the nucleus have been readily explored, but not so much the specific mechanisms employed by the cell to ultimately instruct the chromatin changes necessary for a fast and robust transcription activation. Signaling pathways rely on cascades of protein kinases that, in addition to activating transcription factors can also activate the chromatin template by phosphorylating histone proteins, what we refer to as “signaling- to- chromatin.” These pathways appear to be selectively employed and especially critical for driving inducible transcription in macrophages and likely in diverse other immune cell populations. Here, we discuss signaling- to- chromatin pathways with potential relevance in diverse immune cell populations together with chromatin related mechanisms that help to “solve” the needle in a haystack challenge of robust chromatin activation and inducible transcription.
Years of research has firmly underscored the importance of epigenetic alterations in cervical cancer, which ranks fourth in incidence and mortality among all cancers in the world. The epigenome is characterized by changes to chromatin structure and gene expression brought about by DNA methylation, histone modifications and RNA processing. The chief architects of epigenetic alterations are mediated by opposing classes of enzymes including the DNA methyltransferases and histone modifiers such as the histone deacetylases, histone methyltransferases, histone phosphorylases and histone ubiquitinases. Early changes in the epigenetic landscape following HPV infection has been documented by several studies, alongside the frequent and successive epigenetic alteration leading to cancer progression. Epigenetic changes have been considered as biomarkers and have the potential to be used as diagnostic and prognostic indicators. The reversible nature of epigenetic changes presents an interesting and robust therapeutic target. Several agents have been studied for their potential in modulating the epigenetic machinery and bring about anti-carcinogenic effect. In this review, we comprehensively document the epigenomic alterations in cervical cancer that influence the hallmarks of cancer and the effect of phytochemicals on them. Consolidation of the various in vitro studies highlighting the epigenetic modulatory potential of phytochemicals allow us to understand the progress made thus far and identify areas of further work.
The nucleosome has an unexpectedly complex anatomy. The histone proteins undergo highly selective interactions between themselves and with DNA. Although the basis for this selectivity has not been resolved, several new features have been recently recognized. Most notable is the extended helical structure of the C-terminal domains of the histones. The C-terminal domains of the core histones do not form monomeric globules, but have extensive protein–protein and protein–DNA contacts. The interfaces between histone heterodimers are not extensive, and although specific, offer the possibility of conformational flexibility. Moreover, the stability of the interaction of the histones with DNA depends on the presence of continuous contact with DNA. This may be disrupted by trans-acting factors or RNA polymerase. Conformational changes in the histone octamer may follow from sequestration of linker histones into the nucleosome or from modification or mutation of the tail domains of the core histones. Since these changes are implicated in the regulation of trans-acting factor access to DNA in chromatin, they are likely to be an important area for future investigation.
Aim
Loading‐induced tension development is often assumed to constitute an independent cue to initiate muscle protein synthesis following resistance exercise. However, with traditional physiological models of resistance exercise, changes in loading‐induced tension development also reflect changes in neural activation patterns, and direct evidence for a mechanosensitive mechanism is therefore limited. Here, we sought to examine the importance of excitation and tension development per se on initiation of signaling, gene transcription and protein synthesis in rat skeletal muscle.
Methods
Isolated rat EDL muscles were allocated to the following interventions: (A) Excitation‐induced eccentric contractions (ECC); (B) Passive stretching without excitation (PAS); (C) Excitation with inhibition of contractions (STIM+IMA) and; (D) Excitation in combination with both inhibition of contractions and passive stretching (STIM+IMA+PAS). Assessment of transcriptional and translational signaling, gene transcription and acute muscle protein synthesis was compared in stimulated versus contra‐lateral non‐stimulated control muscle.
Results
Protein synthesis increased solely in muscles subjected to a combination of excitation and tension development (ECC and STIM+IMA+PAS). The same pattern was true for p38MAPK signaling for gene transcription as well as for gene transcription of immediate early genes FOS and JUN. In contrast, mTORC1 signaling for translation initiation increased in all muscles subjected to increased tension development (ECC and STIM+IMA+PAS as well as PAS).
Conclusions
The current study suggest that exercise‐induced increases in protein synthesis as well as transcriptional signaling is dependent on the concomitant effect of excitation and tension development, whereas signaling for translation initiation is only dependent of tension development per se .
The research described in this thesis was focused on the study of signalling pathways that lead to activation of serum response factor (SRF) and its target genes in response to diverse extracellular signals, such as serum, LPA, PDGF or TPA. SRF is able to bind to the serum response element (SRE) which is found in many, but not all immediate early gene promoters. At some promoters, like c-fos and egr-1, in conjunction with SRF, ternary complex factor (TCF) can bind to the SRE. TCF-dependent transcription is regulated by MAP kinases in response to growth factors or stress stimuli, while SRF activity is potentiated by RhoA-dependent signalling pathways in response to serum or LPA. In contrast to transiently transfected templates, an integrated SRF reporter gene is also activated by growth factors or the phorbol ester TPA. Using pharmacological inhibitors it is demonstrated that activation by growth factors and TPA was dependent on PI-3 kinase activity, while activation of SRF by serum and LPA was not. SRF activation by all stimuli was absolutely dependent on RhoA activity and largely independent of MEK activity. Cloning of LIMK, a regulator of the actin treadmilling cycle, as an SRF activator, suggested that actin dynamics are involved in regulating transcriptional activation. Studies with activators and inhibitors of actin polymerisation demonstrated that depletion of the cellular G-actin pool is necessary and sufficient for SRF activation. In contrast, alterations in actin dynamics are neither necessary nor sufficient for activation of TCF. Accordingly, activation of some SRF target genes, such as c-fos and egr-1, was actin and RhoA independent, but MAPK dependent. However, another group of genes, such as srf and vinculin, was actin and RhoA dependent, but largely independent of MEK-ERK signalling. Based on these findings, two classes of SRF target gene can be defined: one regulated by actin dynamics and a second by MEK-ERK signalling. It is proposed that two classes of SRF target gene are regulated in a mutually exclusive manner, and that the presence of TCF may control signalling specificity at SRF target promoters.
Pathogenic bacteria can alter host gene expression through post-translational modifications of histones. We show for the first time that a natural colonizer, Streptococcus pneumoniae, also induces specific histone modifications, including robust dephosphorylation of histone H3 on serine 10, during infection of respiratory epithelial cells. Two bacterial factors are important for the induction of this modification: the bacterial toxin PLY, a pore-forming toxin, and the pyruvate oxidase SpxB, an enzyme responsible for H2O2 production. The combined effects of PLY and H2O2 lead to host signaling which culminates in H3S10 dephosphorylation, mediated by the host cell phosphatase PP1. Strikingly, S. pneumoniae infection induces dephosphorylation and associated activation of PP1 catalytic activity. Colonization of cells, which lacked active PP1, resulted in the impairment of intracellular S. pneumoniae survival. Interestingly, PP1 activation mediating H3S10 dephosphorylation is not restricted to S. pneumoniae and appears to be a general epigenomic mechanism favoring intracellular survival.
Purpose of the Review
The purpose is to provide current knowledge and recent development and understanding of periodontal disease dysbiosis in the perspective of epigenetic changes. Epigenetic changes, where environmental factors modify the gene expression network without changing the DNA sequence, may influence inflammatory diseases such as chronic periodontitis. These chemical modifications of DNA and histone proteins cause epigenetic changes that alter cellular function and host defenses.
Recent Findings
Findings suggest that the methylation of cytosine residues on DNA particularly at CpG Islands is commonly associated with gene silencing, and covalent modifications on histones are associated with chromatin structural integrity and function that play crucial roles in gene expression. In periodontal immune dysbiosis, aberrant DNA methylation and/or histone modifications could potentially play a role in disease state.
Summary
We broadly discuss epigenetic modifications related to immune regulation and comprehensively discuss recent developments in the dynamics of epigenetic changes pertaining to chronic inflammatory periodontal disease.
The mitogen- and stress-activated protein kinases activated by the extracellular-signal-regulated kinase 1/2 and/or stress-activated protein kinase 2/p38 mitogen-activated protein kinase pathways are recruited to the regulatory region of a subset of genes termed immediate-early genes, often leading to their induction. These genes, many of which code for transcription factors, have been directly linked to the phenotypic events in carcinogenesis. In this paper, we focus on the mitogen- and stress-activated protein kinases; their discovery, activation, H3 phosphorylation and recent discoveries in their roles in cancer.
This chapter covers the broad field of transcription and epigenetic regulatory mechanisms to equip the gastrointestinal physiologist with the tools to understand gene expression. This chapter reviews the composition of the nucleic acids and the methods used to study their structure and function. The effect of noncoding RNA species such as microRNA and long noncoding RNAs is discussed. In addition, the impact of histone and DNA modification on gene expression collectively known as epigenetic influences are discussed in the context of how the gut microenvironment.
In eukaryotes, the genome is organized into a complex nucleoprotein structure called chromatin. Despite the simplicity of its monomer, DNA and two copies of four histones, the existence of histone variants opens possibilities of multiple chromatin landscapes and fine-tune regulation of molecular mechanisms for the regulation of gene expression and maintenance of genome stability. However, any defects in these combinations may contribute to disease development and/or progression. Here, I review human histone variants and their chaperones, and discuss how they contribute to pathological conditions.
A rapid and convenient method for peptide mapping of proteins has been developed. The technique, which is especially suitable for analysis of proteins that have been isolated from gels containg sodium dodecyl sulfate, involves partial enzymatic proteolysis in the presence of sodium dodecyl sulfate and analysis of the cleavage products by polyacrylamide gel electrophoresis. The pattern of peptide fragments produced is characteristic of the protein substrate and the proteolytic enzyme and is highly reproducible. Several common proteases have been used including chymotrypsin, Staphylococcus aureus protease, and papain.
The suppression of growth arrest-specific (gas) gene expression by serum appeared to be independent of protein synthesis,
but expression in resting cells was sensitive to 2-aminopurine, an inhibitor of intracellular protein kinases. Although accumulation
of gas gene mRNA was reduced by serum, nuclear transcription of the gas-2, -3, and -5 genes was observed in serum-stimulated
cells, indicating that posttranscriptional events may regulate mRNA levels. Growth induction by serum, on the other hand,
led to suppression of transcription of the gas-1 gene. Cell cycle regulation and the serum response of gas-1 were lost in
ras-transformed cells.
Transcription of the proto-oncogene c-fos is known to be activated by growth factors in serum and subsequently repressed by
the Fos protein. We show that generalized DNase I sensitivity of c-fos chromatin correlates closely with enhancer activity
during induction, repression, and superinduction of the c-fos gene. Within 90 s of serum stimulation, proximal DNA sequences
on both sides of the enhancer exhibit increased DNase I sensitivity. Within 5 min, elevated DNase I sensitivity spreads to
chromatin at the distal 3' end of the c-fos gene. These results suggest that an open state of chromatin is propagated in both
directions from the enhancer. The induced alterations in chromatin structure precede the increased transcriptional activity
of the c-fos gene, suggesting that these changes in chromatin structure potentiate transcription.
In foot-and-mouth disease virus (FMDV)-infected cells, the disappearance of nuclear protein histone H3 and the simultaneous appearance of a new chromatin-associated protein termed Pi can be observed (P. R. Grigera and S. G. Tisminetzky, Virology 136:10-19, 1984). We sequenced the amino terminus of protein Pi and showed that Pi derives from histone H3 by proteolytic cleavage. The 20 N-terminal amino acid residues of histone H3 are specifically cleaved off early during infection. Truncated histone H3 remains chromatin associated. In addition, we showed that the histone H3-Pi transition is catalyzed by the FMDV 3C protease. The only known function of the viral 3C protease was, until now, the processing of the viral polyprotein. The viral 3C protease is the only FMDV protein required to induce the histone H3-Pi transition, as well as being the only viral protein capable of cleaving histone H3. No viral precursor fusion protein is needed for this specific cleavage as was reported for the processing of the poliovirus P1 precursor polyprotein by 3C/D protease. As the deleted part of the histone H3 corresponds to the presumed regulatory domain involved in the regulation of transcriptionally active chromatin in eucaryotes, it seems possible that this specific cleavage of histone H3 is related to the host cell transcription shutoff reported for several picornaviruses.
We have compared transcriptionally competent and repressed genes with respect to their linker histone content and their ability to fold into higher-order structures. Histones were cross-linked covalently to DNA in chicken erythrocyte and oviduct nuclei by UV irradiation, and the DNA that was immunoprecipitated with anti-H1 and (for erythrocytes) anti-H5 antibodies was analysed for particular DNA sequences. None of the sequences investigated was free of H1 (H5). However, in mature erythrocytes the tissue-specific adult beta-globin gene (beta A) appears to be partially depleted of H5, and both the beta-globin gene and the H5 gene (also tissue-specific), as well as the 'housekeeping' beta-actin gene, appear to be partially depleted of H1 relative to inactive genes; in oviduct slight H1-depletion is detected on the ovalbumin gene relative to genes that are inactive in this tissue and the actin gene. Transcriptionally competent erythrocyte chromatin fragments, in contrast to inactive fragments, are unable to self-associate into 'pseudo-higher-order structures'. This is likely to be a consequence of the partial depletion of H5 and/or H1 in active chromatin, resulting in the breakdown of (probably cooperative) interactions between H5 and/or H1 molecules that otherwise mediate the assembly of pseudo-higher-order structures in vitro and a stable 30 nm chromatin filament in vivo.
The inhibitory effect of a marine-sponge toxin, okadaic acid, was examined on type 1, type 2A, type 2B and type 2C protein phosphatases as well as on a polycation-modulated (PCM) phosphatase. Of the protein phosphatases examined, the catalytic subunit of type 2A phosphatase from rabbit skeletal muscle was most potently inhibited. For the phosphorylated myosin light-chain (PMLC) phosphatase activity of the enzyme, the concentration of okadaic acid required to obtain 50% inhibition (ID50) was about 1 nM. The PMLC phosphatase activities of type 1 and PCM phosphatase were also strongly inhibited (ID50 0.1-0.5 microM). The PMCL phosphatase activity of type 2B phosphatase (calcineurin) was inhibited to a lesser extent (ID50 4-5 microM). Similar results were obtained for the phosphorylase a phosphatase activity of type 1 and PCM phosphatases and for the p-nitrophenyl phosphate phosphatase activity of calcineurin. The following phosphatases were not affected by up to 10 microM-okadaic acid: type 2C phosphatase, phosphotyrosyl phosphatase, inositol 1,4,5-trisphosphate phosphatase, acid phosphatases and alkaline phosphatases. Thus okadaic acid had a relatively high specificity for type 2A, type 1 and PCM phosphatases. Kinetic studies showed that okadaic acid acts as a non-competitive or mixed inhibitor on the okadaic acid-sensitive enzymes.
The extensive literature on receptor tyrosine kinases and the intense research effort it currently receives reflects its central role in the regulation of mitosis, aberrations of which result in uncontrolled proliferation of cells and the formation of tumours. This literature is comprehensively summarized and reviewed in several recent articles and is not to be considered here (1 3). This chapter deals with the practical aspects of demonstrating that a particular protein or protein complex is a receptor tyrosine kinase (RTK) which is defined as one in which the binding of a specific ligand results in the direct activation of tyrosine kinase activity intrinsic to the receptor. It is obvious immediately that there are two criteria that must be satisfied. First, the putative receptor must be shown to have the appropriate ligand-binding characteristics. Approaches to demonstrat ing respectable ligand binding (summarized in Section 2.1) are considered in detail in a companion book in this series (25) and are not described here, although a brief outline is given in Appendix 1.
For didactic purposes, the process of protein biosynthesis can be arbitrarily divided into: a) events taking place prior to translation and b) those that take place at the ribosome level.
Contrary to native -containing chromatin where phosphorylation induces local structural changes affecting chromatin condensation, in stripped fibers phosphorylation of the totality of H3 molecules does not affect significantly chromatin conformation and DNA-protein interactions. Modification of H3 causes only a slight increase of flexibility of nucleosomal chains, despite important changes in histone topography revealed by immunochemical reactivity studies.
Histone neighbors in compact and extended chromatin have been investigated by cross-linking histones in nuclei and in nucleohistone extended with 6 M urea, using the bifunctional reversible reagent methyl-4-mercaptobutyrimidate (MMB). Similar histone dimers are found in both conformational states of chromatin. The dimers most frequently found are H2b-H2a, H2b-H3 and H3-H2a; dimers found less frequently are H3-H4, H3-H3 and H2b-H4. More H3-H3 is found in nuclei than in extended chromatin. H1 is found predominantly as poly-H1, although it can be cross-linked to H2b or H3. After reaction with MMB, native compact chromatin is no longer extendable in 6 M urea, which shows that the reagent is capable of linking together histones holding the chromatin in a compact conformation. Thus the histone propinquity in extended chromatin mimics the intimate histone associations in compact chromatin.
Okadaic acid (OA) and 12-O-tetradecanoylphorbol-13-acetate (TPA) are both potent tumor promoters in a mouse skin carcinogenesis experiment. OA was much
more toxic than TPA for murine embryo cell lines such as Swiss 3T3 cells or C3H10T½ cells. TPA is a potent mitogen for 3T3
cells; in contrast OA was unable to stimulate DNA synthesis in these cells. TPA induces a family of primary response genes,
the TPA induced sequence (TIS) genes, in a wide variety of cells. Although OA induced modest levels of TIS mRNA expression,
the time course of the induction of TIS1 and TIS8 mRNA was delayed when compared to induction by TPA or peptide mitogeas such
as fibroblast growth factor (FGF). In addition TPA-mediated down-regulation of protein kinase C attenuated TIS gene induction
by OA, but not by FGF.
A rapid and convenient method for peptide mapping of proteins has been developed. The technique, which is especially suitable for analysis of proteins that have been isolated from gels containg sodium dodecyl sulfate, involves partial enzymatic proteolysis in the presence of sodium dodecyl sulfate and analysis of the cleavage products by polyacrylamide gel electrophoresis. The pattern of peptide fragments produced is characteristic of the protein substrate and the proteolytic enzyme and is highly reproducible. Several common proteases have been used including chymotrypsin, Staphylococcus aureus protease, and papain.
Histone neighbors in compact and extended chromatin have been investigated by cross-linking histones in nuclei and in nucleohistone extended with 6 M urea, using the bifunctional reversible reagent methyl-4-mercaptobutyrimidate (MMB). Similar histone dimers are found in both conformational states of chromatin. The dimers most frequently found are H2b-H2a, H2b-H3 and H3-H2a; dimers found less frequently are H3-H4, H3-H3 and H2b-H4. More H3-H3 is found in nuclei than in extended chromatin. H1 is found predominantly as poly-H1, although it can be cross-linked to H2b or H3. After reaction with MMB, native compact chromatin is no longer extendable in 6 M urea, which shows that the reagent is capable of linking together histones holding the chromatin in a compact conformation. Thus the histone propinquity in extended chromatin mimics and intimate histone associations in compact chromatin.
Epidermal Growth Factor (EGF) and Tetradecanoyl Phorbol Acetate (TPA) initiate signalling cascades in C3H 10T1/2 fibroblasts by primarily activating distinct protein kinases, the EGF receptor tyrosine kinase and protein kinase C respectively; there is no signal crossover at the initiation of signalling. Nevertheless, we show here that both agents rapidly elicit common intracellular responses, including the phosphorylation of complexed and chromatin-associated forms of a 33 kDa phosphoprotein (pp33), that of a 15 kDa chromatin-associated phosphoprotein (pp15), as well as the transcriptional activation of a common subset of genes including the c-fos proto-oncogene. 2-aminopurine specifically abolishes complexed and chromatin-associated pp33 phosphorylation in response to EGF and TPA, as well as the induction of c-fos by both agents. The activation of protein kinase C and the levels of transcription factors that bind to the serum response element (SRE), TPA response element (TRE) or NFkB sites in stimulated cells are relatively unaffected by 2-aminopurine. This, to our knowledge, is the first demonstration that it is possible, by using 2-aminopurine which selectively blocks TPA-stimulated pp33 phosphorylation, to block c-fos induction in TPA-treated cells although protein kinase C remains fully active. Further, we show here that although EGF- and TPA-stimulated induction of c-fos is abolished by 2-aminopurine, the appearance of TRE-binding activity in nuclear extracts of stimulated cells is unaffected, suggesting that EGF- and TPA-stimulated induction of TRE-binding activity utilises existing proteins and is not dependent on fresh c-FOS synthesis. These results imply that 2-aminopurine-sensitive complexed and chromatin-associated pp33 phosphorylation may be crucial to c-fos induction in response to EGF and TPA.
Minichromosomes containing the MMTV hormone responsive element (HRE) exhibit precisely positioned nucleosomes. Chromatin reconstitution of short HRE DNA fragments also results in precise positioning of nucleosomes as revealed by footprinting, which suggests that information for nucleosome phasing is contained within this short sequence. While hormone receptors bind naked DNA and reconstituted nucleosomes with similar affinities (3- to 5-fold difference), NFI, a transcription factor essential for efficient utilization of the MMTV promoter, binds naked DNA very tightly but does not bind the nucleosomally organized promoter. Hormone receptor binding to the MMTV nucleosome does not dissociate the nucleosome but leads to greater accessibility of the promoter-proximal end to exonuclease III. Precise positioning of one nucleosome over the MMTV promoter could repress transcription by preventing NFI binding in the absence of hormone, while still allowing interaction of activated hormone receptor with HRE.
The tumour promoter okadaic acid is a potent and specific inhibitor of protein phosphatases 1 and 2A. Here we review recent studies which demonstrate that this toxin is extremely useful for identifying biological processes that are controlled through the reversible phosphorylation of proteins.
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Studies of phosphatidylinositol signaling pathways are entering a new phase in which molecular genetic techniques are providing powerful tools to dissect the functions of various metabolites and pathways. Studies with phospholipase C are most advanced and clearly indicate that phosphatidylinositol turnover is critical for vision in Drosophila and cell proliferation in various cultured cells. Expression of cDNA constructs and microinjection of PLC or antibodies against it clearly establish a role for PtdIns signaling distinct from its role in calcium mobilization and protein kinase C activation. The importance of inositol cyclic phosphates is also beginning to be realized from the study of cyclic hydrolase using similar techniques. Elucidation of the function of the 3-phosphate inositol phospholipid pathway awaits similar studies. The recent cDNA cloning of inositol monophosphatase (Diehl et al., 1990), Ins(1,4,5)P3 3-kinase (Choi et al., 1990), and inositol polyphosphate 1-phosphatase (York and Majerus, 1991) should provide tools to define further the cell biology of the phosphatidylinositol signaling pathway.
Actively transcribed chromatin is structurally different from bulk inactive chromatin. It has been difficult to define the molecular basis of the difference, however, because purified fractions of active chromatin were not available. We have overcome this problem by releasing oligonucleosomes from the nonmethylated CpG-rich islands (CpG islands) of HeLa cell nuclei using restriction endonucleases. Since CpG islands very often include the promoters and 5' transcribed regions of genes, they represent a model for the "active" chromatin structure. CpG island chromatin differs in three respects from bulk chromatin prepared in the same way: histone H1 is present in very low amounts; histones H3 and H4 are highly acetylated; and nucleosome-free regions are present. Except for the latter regions, the average nucleosomal spacing is similar to that of bulk chromatin.
A new affinity chromatographic procedure for the separation of transcriptionally active nucleosomes has been used to study the changes that take place in chromatin structure along the c-fos and c-myc genes when RNA synthesis is inhibited. Mercury-affinity chromatography separates the sulfhydryl-reactive nucleosomes of transcriptionally active genes from the compactly beaded, non-reactive nucleosomes of transcriptionally inert DNA sequences. The new procedure also discriminates between nucleosomes that have "unfolded" to reveal the previously shielded SH groups of histone H3 and nucleosomes that bind to the mercury column because of their association with thiol-containing non-histone proteins located in the transcription unit. Both classes of Hg-bound nucleosomes contain the c-fos and c-myc sequences, but only when they are being transcribed. We compared the effects of alpha-amanitin and actinomycin D on the transcription of c-fos and c-myc with the effects of each inhibitor on the distribution of the corresponding oncogenic DNA sequences in the chromatographically separated nucleosome fractions. It was found that the inhibition of RNA polymerase II by alpha-amanitin (added at the peaks of c-fos or c-myc expression in serum-stimulated BALB/c 3T3 cells) resulted in a rapid loss of affinity of the oncogene-containing nucleosomes for the mercury column. There was no corresponding effect on the mercury-binding properties of nucleosomes containing 28 S ribosomal gene sequences, which continue to be transcribed by amanitin-resistant RNA polymerase I. Therefore, the binding of the c-fos and c-myc nucleosomes to the mercury column seems to depend upon reversible structural changes associated with their transcription. Surprisingly, there was no corresponding loss of affinity of the c-fos and c-myc nucleosomes for the mercury column when actinomycin D was employed to inhibit RNA synthesis, despite the fact that transcription of both genes had been arrested abruptly. Measurements of [3H]actinomycin D binding show its preferential intercalation into the transcriptionally active nucleosomes. We suggest that the intercalation of actinomycin D into the DNA of active nucleosomes can lock the transcription complex into an "unfolded" but potentially active configuration. This was confirmed by run-off transcription assays showing a restoration of c-fos and c-myc RNA synthesis when actinomycin D was displaced by proflavine.
A mechanism of mitosis-specific enhancement of histone H3 phosphorylation was analyzed in vitro in terms of nucleosome structure. The incorporation of [32P]phosphate into DNA-bound H3 was approximately 5-7 times higher than in DNA-free H3 using the catalytic subunit of cAMP-dependent protein kinase. The two major N-terminal serine sites, including the mitosis-specific site (Ser10) and Ser28, were extensively phosphorylated in the DNA-bound forms. These phosphorylation patterns were identical to those of nucleosomal H3. In contrast, the H3 in DNA-free octamers was very slightly phosphorylated. The major site of H3 phosphorylation in DNA-free H3 was Thr118 in the C-terminus. Results indicate that DNA-binding is essential for the high level of mitosis-specific H3 phosphorylation, and that the nucleosome structure promotes H3 N-terminal phosphorylation in vitro. It also suggests the possibility that H1 prevents H3 phosphorylation during interphase of the cell cycle.
Stimulation of quiescent 3T3 cells with purified growth factors or of the pheochromocytoma cell line PC12 with nerve growth factor results in the rapid transient induction of c-fos, c-myc, and actin gene transcription (M.E. Greenberg and E.B. Ziff, Nature [London] 312:711-716; M.E. Greenberg, L.A. Greene, and E.B. Ziff, J. Biol. Chem. 26:14101-14110). We used protein synthesis inhibitors to investigate whether synthesis of new proteins plays a role in the rapid induction and subsequent repression of the transcription of these genes. Pretreatment of quiescent 3T3 cells with the inhibitor anisomycin before growth factor stimulation caused a superinduction of c-fos and c-myc mRNA levels upon growth factor addition. Nuclear runoff transcription analyses of 3T3 cells indicated that anisomycin potentiated c-fos, c-myc, and also actin expression at the transcriptional level, possibly by inhibiting transcriptional repression. Somewhat different results were obtained when PC12 cells were incubated with either anisomycin or cycloheximide. In PC12 cells protein synthesis inhibitors superinduced nerve growth factor activation of c-fos mRNA production but completely abolished the activation of c-myc. The results suggest that in PC12 cells c-fos transcription is activated by a protein-synthesis-independent mechanism, whereas c-myc stimulation requires new protein synthesis. The difference in the effect of anisomycin on growth factor activation of c-myc expression in 3T3 versus PC12 cells may be due to differential stringency of protein synthesis inhibition in the two cells or could reflect cell type differences in c-myc regulation.
The response of a cell to mitogens and differentiation agents involves the transcriptional induction of several cellular genes. Prominent among these so-called 'immediate early' or 'competence' genes are the nuclear oncogenes fos and myc. Although the precise function of these early response genes in growth control is not understood, it is likely that many of them are involved in the transition from G0 to G1 in the cell cycle. The findings that the products of nuclear proto-oncogenes jun and erbA are transcriptional factors supports the notion of the role of the nuclear oncoproteins in the regulation of gene expression. Recently, it has been reported that the FOS protein is associated in transcriptional complexes with the product of the jun oncogene, the transcription factor AP-1. As the fos gene is induced in response to mitogens during initiation of cell growth, we investigated whether expression of the nuclear transcription factor AP-1 is also inducible. We report that mouse c-jun gene transcription is rapidly induced by serum and phorbol-ester 12-o-tetradecanoyl phorbol 13-acetate (TPA). Furthermore, induction is transient and the mRNA is superinduced by inhibitors of protein synthesis.
When spermatozoa of Arbacia punctulata are labeled with 32P and treated with soluble egg jelly, radiolabel is incorporated into histone H3. The time course of labeling correlates with the period of chromatin decondensation of sperm pronuclei in eggs. Phosphorylation is on serine and may result from increased turnover of phosphate on H3. The macromolecular fraction of egg jelly (and not the peptide fraction) is the inducer of H3 phosphorylation. The reaction is dependent on external Ca2+ and is induced by monensin and A23187. H3 phosphorylation is not induced by the phosphodiesterase inhibitor IBMX and relatively high (250 microM) concentrations of the protein kinase inhibitor H8 are needed to block the reaction, suggesting that it is cAMP independent. A surprising finding is that merely diluting the cells into Na+ free media is the most effective method to induce the radiolabeling of H3. These results are in contrast to findings on the egg jelly induced phosphorylation of histone H1 in S. purpuratus spermatozoa. These species differences must reflect the great evolutionary divergence between these two sea urchin species in the mechanism of regulation of the phosphorylation of nuclear proteins during fertilization.
Immobilon, a membrane of polyvinylidene difluoride to which gel-fractionated proteins can be transferred electrophoretically, was found to be an excellent matrix for the analysis of the phosphoamino acid content of phosphoproteins. Hydrolysis of 32P-labeled proteins bound to Immobilon with 5.7 N HCl resulted in the release of 90% of the 32P in the form of Pi, phosphoamino acids, and phosphopeptides. Two-dimensional electrophoretic analysis of the released phosphoamino acids yielded undistorted patterns. Because direct hydrolysis of proteins transferred to Immobilon eliminated the need for both preparative extraction of proteins from a gel and recovery by precipitation, analysis was rapid and yields of phosphoamino acids were extremely consistent. The yield of phosphoamino acids from proteins bound to Immobilon, unlike that from proteins eluted from gels, was independent of the size of the protein. The detection of 32P-labeled, phosphotyrosine-containing proteins in sodium dodecyl sulfate-polyacrylamide gels has been shown to be substantially improved by incubation of the gel in 1.0 N KOH for 2 h at 55 degrees C. Base hydrolysis of proteins bound to Immobilon proved to be faster and more sensitive than hydrolysis of proteins in gels. Less than 10% of bound protein was lost from Immobilon during the 2-h incubation at 55 degrees C in 1.0 N KOH. The autoradiographic image after alkaline hydrolysis of proteins on Immobilon was sharper than that obtained after hydrolysis of proteins in the gel. In addition, unlike base-treated gels, the dimensions of the Immobilon filter were unaffected by treatment with base.(ABSTRACT TRUNCATED AT 250 WORDS)
Inositol 1,4,5-trisphosphate is a second messenger which regulates intracellular calcium both by mobilizing calcium from internal stores and, perhaps indirectly, by stimulating calcium entry. In these actions it may function with its phosphorylated metabolite, inositol 1,3,4,5-tetrakisphosphate. The subtlety of calcium regulation by inositol phosphates is emphasized by recent studies that have revealed oscillations in calcium concentration which are perhaps part of a frequency-encoded second-messenger system.
Okadaic acid is a polyether derivative of 38-carbon fatty acid, and is implicated as the causative agent of diarrhetic shellfish poisoning. It is a potent tumour promoter that is not an activator of protein kinase C, but is a powerful inhibitor of protein phosphatases-1 and -2A (PP1 and PP2A) in vitro. We report here that okadaic acid rapidly stimulates protein phosphorylation in intact cells, and behaves like a specific protein phosphatase inhibitor in a variety of metabolic processes. Our results indicate that PP1 and PP2A are the dominant protein phosphatases acting on a wide range of phosphoproteins in vivo. We also find that okadaic acid mimics the effect of insulin on glucose transport in adipocytes, which suggests that this process is stimulated by a serine/threonine phosphorylation event.
Rapid, transient induction of the human c-fos proto-oncogene by extracellular signals requires the presence in cis of the serum response element (SRE). Two protein factors that bind to the SRE in vitro are the serum response factor (p67SRF) and polypeptide p62. These polypeptides must interact with one another and the SRE for efficient serum induction of the c-fos gene. Here we use dimethyl sulphate genomic footprinting to establish the in vivo protein contacts on the SRE and flanking sequences. In human A431 cells the patterns of protection and hyper-reactivity that we find are consistent with the presence of p67SRF, p62, and at least one other protein immediately 3' to p67SRF. The protein-DNA contacts we observe within the SRE are present before induction by epidermal growth factor and are unchanged during gene activation and subsequent repression. Our results indicate that a specific DNA-protein architecture may be maintained at the c-fos SRE, regardless of changes in the transcriptional state of the gene. Such established structures could be important generally in rapid transcriptional responses to extracellular signals.
We have recently reported that several purified polypeptide mitogens such as epidermal growth factor, embryonal carcinoma-derived growth factor, basic fibroblast growth factor and Bombesin induce the rapid appearance of a 33 kDa chromatin-associated phosphoprotein in the nuclei of murine fibroblasts. We show here that in both mouse and human cell lines, a second form of the 33 kDa phosphoprotein exists in a detergent-extractable complexed form which may be pelleted by ultracentrifugation. When quiescent [32P]-labelled cells are treated with EGF, both complexed and chromatin-associated forms of the labelled phosphoprotein are detectable within 10 min, the response peaking at about 1 h and being substantially over 3 h after EGF stimulation. By chymotryptic and cyanogen bromide phosphopeptide mapping studies, the two forms of the 33 kDa phosphoprotein are indistinguishable, as are the mouse and human forms of the protein. The protein kinase inhibitor 2-aminopurine, which has recently been shown to block growth factor-stimulated c-fos and c-myc induction, specifically abolishes the mitogen-stimulated appearance of both forms of the 33 kDa phosphoprotein without affecting the phosphorylation of other cellular proteins. The 33 kDa protein has been purified from Hela cells by a combination of sucrose density gradient centrifugation, preparative electrophoresis and reverse-phase HPLC during which the protein is resolved into two closely-eluting peaks which differ markedly in their specific activity. These results are discussed in relation to the possible role of these events in coupling growth factor-receptor interaction at the cell surface to the early responses of transcriptional activation in the nucleus.
Serum and growth factor regulation of c-FOS protooncogene transcription is mediated by the serum response element. A factor, serum response factor, binding to this element has already been identified. We demonstrate that serum response factor is phosphorylated in vivo on serine residues and that phosphatase treatment of this factor in vitro abolishes its DNA-binding activity. These results show phosphorylation of serum response factor to be required for its DNA-binding activity. The importance of serum response factor phosphorylation for the regulation of c-FOS expression is discussed.
Excerpt
Growth factors are implicated in a wide variety of physiological and pathological processes, including embryogenesis, hematopoiesis, wound healing, immune responses, atherosclerosis, and neoplasis (Evered et al. 1985; Sporn and Roberts 1986). An important link between growth factors and their receptors and oncogene products has also been established (Heldin and Westermark 1984; Weinstein 1987). Thus, the elucidation of the mechanism of action of growth factors has emerged as one of the fundamental problems in biology and may prove crucial for understanding the unrestrained proliferation of cancer cells.
Many studis of growth factors have used cultured fibroblasts, such as 3T3 cells, as a model system. These cells cease to proliferate when they deplete the medium of its growth-promoting activity. Such quiescent cells can be stimulated to reinitiate DNA synthesis and cell division either by replenishing the medium with fresh serum or by the addition of growth factors or pharmacological agents in serum-free...
Genes whose expression is growth factor regulated are likely to be important components in the mechanisms controlling cell
proliferation and differentiation. With the aim of identifying some of those genes, a lambda cDNA library was prepared with
poly(A)+ RNA from quiescent NIH 3T3 cells stimulated with serum for 4 h in the presence of cycloheximide. Differential screening
of approximately 200,000 recombinant phage plaques revealed 2,540 clones that cross hybridized preferentially with [32P]cDNA
derived from RNA of stimulated cells rather than with cDNA derived from nonstimulated cells. Cross hybridization of these
clones identified 82 independent sequences, including c-fos and c-myc. Seventy-one clones were further studied. Analysis of
the changes in transcription and mRNA levels after serum stimulation demonstrated that the kinetics and extent of the induction
vary dramatically between the different genes. Cycloheximide in all cases superinduced the mRNA levels by two mechanisms,
inhibiting the shutoff of transcription and prolonging the half-lives of the mRNAs. Our results showed that induction of proliferation
is accompanied by the onset of a complex genetic program.
Lipocortin-like proteins are a family of steroid-induced inhibitors of phospholipase activity with potential anti-inflammatory activity. Related proteins have been detected in a variety of tissues and species. The best characterized form is a protein of relative molecular mass (Mr) approximately 40,000 (40K), which is phosphorylated in vivo by protein tyrosine kinases and by protein serine-threonine kinases. It has been proposed that the phospholipase inhibitory activity of lipocortin can be regulated by its phosphorylation. In the A431 cell line, a protein of approximately 35K is phosphorylated by the protein tyrosine kinase activity of the epidermal growth factor (EGF) receptor. Here we report that human lipocortin is phosphorylated near its amino terminus by the EGF receptor/kinase. By peptide mapping and immunological analyses, we show that lipocortin and the endogenous 35K substrate for the EGF receptor/kinase from A431 cells are the same protein.
The promoter regions of several phorbol diester-(TPA-) inducible genes (collagenase, stromelysin, hMT IIA, and SV40) share a conserved 9 bp motif. Synthetic copies of these closely related sequences conferred TPA inducibility upon heterologous promoters. Footprinting analysis indicated that these TPA-responsive elements (TREs) are recognized by a common cellular protein: the previously described transcription factor AP-1. A point mutation that eliminated the basal and induced activity of the TRE also interfered with its ability to bind AP-1. Treatment of cultured cells with TPA led to a rapid 3- to 4-fold increase in TRE binding activity, by a posttranslational mechanism. These results strongly suggest that AP-1 is at the receiving end of a complex pathway responsible for transmitting the effects of phorbol ester tumor promoters from the plasma membrane to the transcriptional machinery.
Protein kinase C is now known to be a large family of proteins, with multiple subspecies that have subtle individual enzymological characteristics. Some members of the family exhibit distinct patterns of tissue expression and intracellular localization; different kinases probably have distinct functions in the processing and modulation of a variety of physiological and pathological responses to external signals.
We have investigated the covalent modification of the proteins encoded by the murine fos proto-oncogene (c-fos) and that of the corresponding gene product of FBJ murine osteosarcoma virus (v-fos). Both proteins are posttranslationally processed in the cell, resulting in forms with lower electrophoretic mobilities than that of the initial translation product on sodium dodecyl sulfate-polyacrylamide gels. Treatment with alkaline phosphatase indicates that most, if not all, of this electrophoretic shift is due to phosphoesterification of both proteins. These phosphoryl groups stoichiometrically modify the v-fos and c-fos proteins on serine residues and turn over rapidly in vivo in the presence of protein kinase inhibitors (half-life, less than 15 min). Direct quantitative comparison of steady-state labeling studies with L-[35S]methionine and [32P]phosphate reveals that the c-fos protein is four- to fivefold more highly phosphorylated than the v-fos protein is. Comparison of tryptic fragments from [32P]phosphate-labeled proteins indicates that although the two proteins have several tryptic phosphopeptides in common, the c-fos protein contains unique major tryptic phosphopeptides that the v-fos protein lacks. These unique sites of c-fos phosphorylation have been tentatively localized to the carboxy-terminal 20 amino acid residues of the protein. Phosphorylation of the c-fos protein, but not the v-fos protein, can be stimulated at least fivefold in vivo by the addition of either 12-tetradecanoyl-phorbol-13-acetate or serum. This increase in the steady-state degree of phosphorylation of c-fos appears to be independent of protein kinase C since phosphorylation is Ca2+ and diacylglycerol independent. The possible role of phosphorylation of these proteins in cellular transformation is discussed.
Some growth factor-induced genes, such as the c-fos gene, are activated rapidly and transiently without intervening protein synthesis. Others, like the rat transin gene, are activated more slowly but more durably and their activation requires prior protein synthesis. It is tempting to speculate that certain rapidly-activated genes code for transcription factors which interact directly with promoter regions of genes like the transin gene to trigger their expression. Unfortunately, little is known about the majority of primary response genes to support this hypothesis. The proto-oncogene c-jun codes for the transcription factor AP-1 or a closely related protein. We show that epidermal growth factor stimulates transcription of the c-jun gene in fibroblasts as a primary response. This supports the notion that increased expression of genes encoding transcription factors is an important element of the signal transduction mechanism, assuring the long-term transcriptional response of cells to growth factors.
Before quiescent cells can respond to mitogens and progress through the G1 phase of cell growth, new messenger RNA synthesis is required. The G1 phase seems to be a critical point of control in the cell cycle, where normal cells deprived of growth factors halt cycling while transformed cells do not, suggesting that regulatory genes, uncontrolled in the neoplastic phenotype, are expressed during the G0 to G1 transition. Some of these may code for nuclear proteins that participate in the transactivation of genes required for the progression through G1. The observed changes in expression of the proto-oncogenes c-fos and c-myc, following stimulation of fibroblasts with growth factors, support this notion as recent evidence suggests that c-FOS and c-MYC proteins can function as transactivating factors. Moreover, the rapid induction of several genes in fibroblasts coding for putative transacting factors during the G0 to G1 transition has been recently reported. Here we present the nucleotide sequence of a mouse cDNA clone coding for a 334 residue protein which shows 80% similarity with v-JUN and more than 98% similarity with the human c-JUN sequence. We have demonstrated that in quiescent fibroblasts c-jun transcription is rapidly induced during the G0 to G1 transition.
Okadaic acid, a non-TPA-type tumour promoter, induces hyperphosphorylation of a 60-kd protein in primary human fibroblasts. Treatment with TPA-type tumour promoters (e.g. TPA and teleocidin) did not cause this hyperphosphorylation. Phosphorylation of this protein was not seen at times earlier than 90 min after the addition of 75 ng/ml okadaic acid to the proliferating cell cultures. The presence of inhibitors such as actinomycin D and cycloheximide, did not significantly influence the level of hyperphosphorylation induced by okadaic acid treatment. By immunoblotting using an antibody anti-nucleolin, the 60-kd protein was identified as a fragment of nucleolar protein, nucleolin. Similarly, antibodies against the 60-kd protein cross-reacted with nucleolin. Furthermore peptide mapping, using staphylococcal V8 protease, showed that the 60-kd protein phosphorylated by casein kinase II in vitro and the okadaic-acid-induced hyperphosphorylated 60-kd protein exhibited identical phosphopeptide maps, indicating that there is also structural relatedness between N-60 and nucleolin. Hyperphosphorylation of the nucleolin fragment (N-60) was suppressed by anti-tumour promoter retinoic acid.
The c-fos proto-oncogene provides a good system to study the processes underlying messenger RNA degradation. After growth factor stimulation of susceptible cells, the c-fos transcription rate transiently increases from a low basal level by as much as 50-fold, producing a large amount of exceedingly unstable c-fos mRNA that is rapidly degraded. Here, we investigate the c-fos mRNA degradation process, and find that: (1) ongoing translation of the c-fos mRNA itself is required for its degradation; (2) after synthesis, the mRNA poly(A) tail is rapidly removed, in a translation-dependent manner, leading to accumulation of apparently deadenylated RNA; (3) deletion or replacement of an AU-rich sequence at the mRNA 3' end significantly stabilizes the mRNA; (4) deletion of the 3' AU-rich sequences dramatically slows the poly(A) shortening rate. These results suggest that the 3' AU-rich sequences act to destabilize the mRNA by directing rapid removal of the mRNA poly(A) tract.
Regulation of the genes by PDGF has some common features. All are primary response genes, and they can still be expressed in the presence of cycloheximide (Cochran et al. 1983; Kelly et al. 1983; Kruijer et al. 1984; Lau and Nathans 1985). In fact, many of the genes are superinduced when cells are treated with growth factors plus cycloheximide (Greenberg et al. 1986). The genes that have been characterized all contain a sequence motif in their 3'-noncoding sequences that appears to make the message labile (Meijlink et al. 1985; Treisman 1985; Shaw and Kamen 1986). All competence genes so far examined are controlled at least in part at the level of transcription (Cochran et al. 1983; Edwards et al. 1985; Almendral et al. 1988). Differences in regulation of the genes include variations in the time course of induction, ranging from 10 minutes to over 4 hours, and differences in the persistence of the mRNAs after their synthesis (Cochran et al. 1983; Muller et al. 1984; Lau and Nathans 1987). The data presented in this paper strongly suggest that multiple, distinct intracellular signals that lead to the expression of multiple genes are generated when cells are treated with growth factors such as PDGF. The variation in the time course of induction of PDGF-inducible genes suggests several models of signal transduction. Four such models are presented in Figure 5. One possibility (Fig. 5A) is that one signal is generated by the interaction of PDGF with its receptor and that this signal activates the very early genes, such as c-fos.(ABSTRACT TRUNCATED AT 250 WORDS)
The protein kinase inhibitor 2-aminopurine (2AP) blocks the induction of the human beta-interferon gene by virus or poly(I)-poly(C) at the level of transcription. This inhibition is specific, since 2AP does not inhibit induction of either the hsp70 heat-shock gene by high temperature or the metallothionein gene by cadmium or dexamethasone. However, 2AP does block the induction of the c-fos and c-myc proto-oncogenes by serum growth factors or virus, suggesting that a protein kinase may be involved in the regulation of these genes, as well as of the beta-interferon gene. However, different factors must be required for the induction of these three genes, since they are not coordinately regulated by the same inducers in most of the cell lines examined.
To distinguish between mechanisms of eukaryotic transcriptional activation, we tested whether yeast upstream promoter elements can stimulate transcription by a heterologous transcription machinery, bacteriophage T7 RNA polymerase. The gal enhancer-like element recognized by GAL4 protein or the ded1 poly(dA-dT) element was placed upstream of the T7 promoter and his3 structural gene, and T7 RNA polymerase was produced in yeast cells. Under conditions where the gal element would normally be either activating or nonactivating, his3 transcription by T7 RNA polymerase was not stimulated above the level observed in the absence of any upstream element. In contrast, the ded1 poly(dA-dT) element stimulated transcription 7-fold, similar to the enhancement observed on the native ded1 promoter. Activation by the ded1 element thus may involve effects on the chromatin template that facilitate entry of the transcription machinery, whereas activation by the gal element may involve specific contacts between GAL4 and the transcriptional machinery.
The tumor promoter tetradecanoyl phorbol acetate (TPA) is also a potent mitogen for murine 3T3 cells. We have previously described the isolation of variant Swiss 3T3 cell lines unable to proliferative in response to TPA. In this report we sought to identify genes that are stimulated by TPA as 'primary responses', i.e., without intervening protein synthesis. We constructed a cDNA library in lambda gt10, using RNA from quiescent 3T3 cells treated with TPA in the presence of cycloheximide (CHX). Of 50,000 recombinant phages, we identified 50 isolates that demonstrated preferential hybridization to cDNA probes generated from TPA-stimulated cells. One of the clones contains a fragment of the proto-oncogene c-fos. Twenty-nine of the remaining 49 clones fall into six cross-hybridization families. All the characterized clones detected mRNAs that are also inducible by epidermal growth factor, fibroblast growth factor or elevated serum. TPA induction of all the characterized messages is rapid and transient. All these mRNAs are superinduced by a combination of mitogens and CHX. Induction of these messages following TPA addition also occurs in subconfluent 3T3 cultures; expression of these genes is, therefore, not restricted to the G0/G1 transition. Expression of all six clones was also induced by TPA and other mitogens in 3T3-TNR9 cells, a TPA non-proliferative variant.