The Swi/Snf chromatin remodeling complex functions to alter nucleosome positions by either sliding nucleosomes on DNA or the eviction of histones. The presence of histone acetylation and activator-dependent recruitment and retention of Swi/Snf is important for its efficient function. It is not understood, however, why such mechanisms are required to enhance Swi/Snf activity on nucleosomes. Snf2, the catalytic subunit of the Swi/Snf remodeling complex, has been shown to be a target of the Gcn5 acetyltransferase. Our study found that acetylation of Snf2 regulates both recruitment and release of Swi/Snf from stress-responsive genes. Also, the intramolecular interaction of the Snf2 bromodomain with the acetylated lysine residues on Snf2 negatively regulates binding and remodeling of acetylated nucleosomes by Swi/Snf. Interestingly, the presence of transcription activators mitigates the effects of the reduced affinity of acetylated Snf2 for acetylated nucleosomes. Supporting our in vitro results, we found that activator-bound genes regulating metabolic processes showed greater retention of the Swi/Snf complex even when Snf2 was acetylated. Our studies demonstrate that competing effects of (1) Swi/Snf retention by activators or high levels of histone acetylation and (2) Snf2 acetylation-mediated release regulate dynamics of Swi/Snf occupancy at target genes.
Cse4 is the centromeric histone H3 variant in budding yeast. Psh1 is an E3 ubiquitin ligase that controls Cse4 levels through proteolysis. Here we report that Psh1 is phosphorylated by the Cka2 subunit of casein kinase 2 (CK2) to promote its E3 activity for Cse4. Deletion of CKA2 significantly stabilized Cse4. Consistent with phosphorylation promoting the activity of Psh1, Cse4 was stabilized in a Psh1 phosphodepleted mutant strain in which the major phosphorylation sites were changed to alanines. Phosphorylation of Psh1 did not control Psh1-Cse4 or Psh1-Ubc3(E2) interactions. Although Cse4 was highly stabilized in a cka2? strain, mislocalization of Cse4 was mild, suggesting that Cse4 misincorporation was prevented by the intact Psh1-Cse4 association. Supporting this idea, Psh1 was also stabilized in a cka2? strain. Collectively our data suggest that phosphorylation is crucial in Psh1-assisted control of Cse4 levels and that the Psh1-Cse4 association itself functions to prevent Cse4 misincorporation.
Nine genetic diseases arise from expansion of CAG repeats in seemingly unrelated genes. They are referred to as polyglutamine (polyQ) diseases due to the presence of elongated glutamine tracts in the corresponding proteins. The pathologic consequences of polyQ expansion include progressive spinal, cerebellar, and neural degeneration. These pathologies are not identical, however, suggesting that disruption of protein-specific functions is crucial to establish and maintain each disease. A closer examination of protein function reveals that several act as regulators of gene expression. Here we examine the roles these proteins play in regulating gene expression, discuss how polyQ expansion may disrupt these functions to cause disease, and speculate on the neural specificity of perturbing ubiquitous gene regulators.
Histone deacetylases (HDACs) are targets for cancer therapy. Suberoylanilide hydroxamic acid (SAHA) is an HDAC inhibitor approved by the U.S. Food and Drug Administration for the treatment of cutaneous T-cell lymphoma. To obtain a better mechanistic understanding of the Sin3/HDAC complex in cancer, we extended its protein-protein interaction network and identified a mutually exclusive pair within the complex. We then assessed the effects of SAHA on the disruption of the complex network through six homologous baits. SAHA perturbs multiple protein interactions and therefore compromises the composition of large parts of the Sin3/HDAC network. A comparison of the effect of SAHA treatment on gene expression in breast cancer cells to a knockdown of the ING2 subunit indicated that a portion of the anticancer effects of SAHA may be attributed to the disruption of ING2's association with the complex. Our dynamic protein interaction network resource provides novel insights into the molecular mechanism of SAHA action and demonstrates the potential for drugs to rewire networks.
Regulatory information stored in modified histones is functionally translated by effector proteins ('readers'), which identify the histone mark to determine the specificity of the response. A recent study identifying the tumor suppressor protein ZMYND11 as an exclusive reader of methylated histone variant H3.3, throws light on the role of transcription regulation in suppressing tumors.
The Spt-Ada-Gcn5-acetyltransferase (SAGA) chromatin-modifying complex possesses acetyltransferase and deubiquitinase activities. Within this modular complex, Ataxin-7 anchors the deubiquitinase activity to the larger complex. Here we identified and characterized Drosophila Ataxin-7 and found that reduction of Ataxin-7 protein results in loss of components from the SAGA complex. In contrast to yeast, where loss of Ataxin-7 inactivates the deubiquitinase and results in increased H2B ubiquitination, loss of Ataxin-7 results in decreased H2B ubiquitination and H3K9 acetylation without affecting other histone marks. Interestingly, the effect on ubiquitination was conserved in human cells, suggesting a novel mechanism regulating histone deubiquitination in higher organisms. Consistent with this mechanism in vivo, we found that a recombinant deubiquitinase module is active in the absence of Ataxin-7 in vitro. When we examined the consequences of reduced Ataxin-7 in vivo, we found that flies exhibited pronounced neural and retinal degeneration, impaired movement, and early lethality.
The regulation of gene expression at the level of transcription involves the concerted action of several proteins and protein complexes committed to dynamically alter the surrounding chromatin environment of a gene being activated or repressed. ATP-dependent chromatin remodeling complexes are key factors in chromatin remodeling, and the SWI/SNF complex is the founding member. While many studies have linked the action of these complexes to specific transcriptional regulation of a large number of genes and much is known about their catalytic activity, less is known about the nuclear elements that can enhance or modulate their activity. A number of studies have found that certain High Mobility Group (HMG) proteins are able to stimulate ATP-dependent chromatin remodeling activity, but their influence on the different biochemical outcomes of this activity is still unknown. In this work we studied the influence of the yeast Nhp6A, Nhp6B and Hmo1 proteins (HMGB family members) on different biochemical outcomes of yeast SWI/SNF remodeling activity. We found that all these HMG proteins stimulate the sliding activity of ySWI/SNF, while transient exposure of nucleosomal DNA and octamer transfer catalyzed by this complex are only stimulated by Hmo1. Consistently, only Hmo1 stimulates SWI/SNF binding to the nucleosome. Additionally, the sliding activity of another chromatin remodeling complex, ISW1a, is only stimulated by Hmo1. Further analyses show that these differential stimulatory effects of Hmo1 are dependent on the presence of its C-terminal tail, which contains a stretch of acidic and basic residues.
Spinocerebellar ataxia 7 (SCA7) is an incurable disease caused by expansion of CAG trinucleotide sequences within the Ataxin-7 gene. This elongated CAG tract results in an Ataxin-7 protein bearing an expanded polyglutamine (PolyQ) repeat. SCA7 disease is characterized by progressive neural and retinal degeneration leading to ataxia and blindness. Evidence gathered from investigating SCA7 and other PolyQ diseases strongly suggest that misregulation of gene expression contributes to neurodegeneration. In fact, Ataxin-7 is a subunit of the essential Spt-Ada-Gcn5-Acetltransferase (SAGA) chromatin modifying complex that regulates expression of a large number of genes. Here we discuss recent insights into Ataxin-7 function and, considering these findings, propose a model for how polyglutamine expansion of Ataxin-7 may affect Ataxin-7 function to alter chromatin modifications and gene expression.
Histone deacetylases (HDACs) and lysine acetyltransferases (KATs) catalyze dynamic histone acetylation at regulatory and coding regions of transcribed genes. Highly phosphorylated HDAC2 is recruited within corepressor complexes to regulatory regions, while the nonphosphorylated form is associated with the gene body. In this study, we characterized the nonphosphorylated HDAC2 complexes recruited to the transcribed gene body and explored the function of HDAC-complex-mediated dynamic histone acetylation. HDAC1 and 2 were coimmunoprecipitated with several splicing factors, including serine/arginine-rich splicing factor 1 (SRSF1) which has roles in alternative splicing. The co-chromatin immunoprecipitation of HDAC1/2 and SRSF1 to the gene body was RNA-dependent. Inhibition of HDAC activity and knockdown of HDAC1, HDAC2 or SRSF1 showed that these proteins were involved in alternative splicing of MCL1. HDAC1/2 and KAT2B were associated with nascent pre-mRNA in general and with MCL1 pre-mRNA specifically. Inhibition of HDAC activity increased the occupancy of KAT2B and acetylation of H3 and H4 of the H3K4 methylated alternative MCL1 exon 2 nucleosome. Thus, nonphosphorylated HDAC1/2 is recruited to pre-mRNA by splicing factors to act at the RNA level with KAT2B and other KATs to catalyze dynamic histone acetylation of the MCL1 alternative exon and alter the splicing of MCL1 pre-mRNA.
Eukaryotic chromatin is kept flexible and dynamic to respond to environmental, metabolic, and developmental cues through the action of a family of so-called "nucleosome remodeling" ATPases. Consistent with their helicase ancestry, these enzymes experience conformation changes as they bind and hydrolyze ATP. At the same time they interact with DNA and histones, which alters histone-DNA interactions in target nucleosomes. Their action may lead to complete or partial disassembly of nucleosomes, the exchange of histones for variants, the assembly of nucleosomes, or the movement of histone octamers on DNA. "Remodeling" may render DNA sequences accessible to interacting proteins or, conversely, promote packing into tightly folded structures. Remodeling processes participate in every aspect of genome function. Remodeling activities are commonly integrated with other mechanisms such as histone modifications or RNA metabolism to assemble stable, epigenetic states.
How cells ensure that productive transcription from divergent promoters is limited to the downstream protein-coding region is an important question in the transcription field. A recent study in Nature proposed an answer by revealing that the upstream antisense transcripts undergo early termination through the polyadenylation signal-dependent pathway, and the downstream sense transcripts are protected from premature cleavage by U1 small nuclear ribonucleoprotein (snRNP).
C-Jun is a major transcription factor belonging to the activating protein 1 (AP-1) family. Phosphorylation has been shown to be critical for c-Jun activation and stability. Here, we report that Jra, the Drosophila Jun protein, is acetylated in vivo. We demonstrate that the acetylation of Jra leads to its rapid degradation in response to osmotic stress. Intriguingly, we also found that Jra phosphorylation antagonized its acetylation, indicating the opposite roles of acetylation and phosphorylation in Jra degradation process under osmotic stress. Our results provide new insights into how c-Jun proteins are precisely regulated by the interplay of different posttranslational modifications.
The packaging of eukaryotic DNA into nucleosomal arrays permits cells to tightly regulate and fine-tune gene expression. The ordered disassembly and reassembly of these nucleosomes allows RNA polymerase II (RNAPII) conditional access to the underlying DNA sequences. Disruption of nucleosome reassembly following RNAPII passage results in spurious transcription initiation events, leading to the production of non-coding RNA (ncRNA). We review the molecular mechanisms involved in the suppression of these cryptic initiation events and discuss the role played by ncRNAs in regulating gene expression.
Eukaryotic RNA polymerase II (RNAPII) is a 12-subunit enzyme that is responsible for the transcription of messenger RNA. Two of the subunits of RNA polymerase II, Rpb4 and Rpb7, have been shown to dissociate from the enzyme under a number of specific laboratory conditions. However, a biological context for the dissociation of Rpb4 and Rpb7 has not been identified. We have found that Rpb4/7 dissociate from RNAPII upon interaction with specific transcriptional elongation-associated proteins that are recruited to the hyperphosphorylated form of the C-terminal domain. However, the dissociation of Rpb4/7 is likely short lived because a significant level of free Rpb4/7 was not detected by quantitative proteomic analyses. In addition, we have found that RNAPII that is isolated through Rpb7 is depleted in serine 2 C-terminal domain phosphorylation. In contrast to previous reports, these data indicate that Rpb4/7 are dispensable during specific stages of transcriptional elongation in Saccharomyces cerevisiae.
Signaling involves the coordinated action of multiple molecules including stimuli, receptors and enzymes part of which interact with the transcriptional machinery and target chromatin. Signaling systems regulate the cell events responsible for survival, development and homeostasis. Many of the signaling pathways induce target gene activation through interaction with the transcription machinery, including RNA polymerase II, and with histone modifying complexes. These studies are having a broad impact on chromatin biology. Recent studies suggest that chromatin itself receives the signals. Increasing examples are illustrating novel regulatory mechanisms that promote our understanding of development and disease.
Set2 is a RNA polymerase II (RNAPII) associated histone methyltransferase involved in the cotranscriptional methylation of the H3 K36 residue (H3K36me). It is responsible for multiple degrees of methylation (mono-, di-, and trimethylation), each of which has a distinct functional consequence. The extent of methylation and its genomic distribution is determined by different factors that coordinate to achieve a functional outcome. In yeast, the Set2-mediated H3K36me is involved in suppressing histone exchange, preventing hyperacetylation and promoting maintenance of well-spaced chromatin structure over the coding regions. In metazoans, separation of this enzymatic activity affords greater functional diversity extending beyond the control of transcription elongation to developmental gene regulation. This review focuses on the molecular aspects of the Set2 distribution and function, and discusses the role played by H3 K36 methyl mark in organismal development.
A significant challenge in biology is to functionally annotate novel and uncharacterized proteins. Several approaches are available for deducing the function of proteins in silico based upon sequence homology and physical or genetic interaction, yet this approach is limited to proteins with well-characterized domains, paralogs and/or orthologs in other species, as well as on the availability of suitable large-scale data sets. Here, we present a quantitative proteomics approach extending the protein network of core histones H2A, H2B, H3, and H4 in Saccharomyces cerevisiae, among which a novel associated protein, the previously uncharacterized Ydl156w, was identified. In order to predict the role of Ydl156w, we designed and applied integrative bioinformatics, quantitative proteomics and biochemistry approaches aiming to infer its function. Reciprocal analysis of Ydl156w protein interactions demonstrated a strong association with all four histones and also to proteins strongly associated with histones including Rim1, Rfa2 and 3, Yku70, and Yku80. Through a subsequent combination of the focused quantitative proteomics experiments with available large-scale genetic interaction data and Gene Ontology functional associations, we provided sufficient evidence to associate Ydl156w with multiple processes including chromatin remodeling, transcription and DNA repair/replication. To gain deeper insights into the role of Ydl156w in histone biology we investigated the effect of the genetic deletion of ydl156w on H4 associated proteins, which lead to a dramatic decrease in the association of H4 with RNA polymerase III proteins. The implication of a role for Ydl156w in RNA Polymerase III mediated transcription was consequently verified by RNA-Seq experiments. Finally, using these approaches we generated a refined network of Ydl156w-associated proteins.
The Spt-Ada-Gcn5-acetyltransferase (SAGA) transcription coactivator plays multiple roles in regulating transcription because of the presence of functionally independent modules of subunits within the complex. We have recently identified a role for the ubiquitin protease activity of SAGA in regulating tissue-specific gene expression in Drosophila. Here, we discuss the modular nature of SAGA and the different mechanisms through which SAGA is recruited to target promoters. We propose that the genes sensitive to loss of the ubiquitin protease activity of SAGA share functional characteristics that require deubiquitination of monoubiquitinated histone H2B (ubH2B) for full activation. We hypothesize that deubiquitination of ubH2B by SAGA destabilizes promoter nucleosomes, thus enhancing recruitment of RNA polymerase II (Pol II) to weak promoters. In addition, SAGA-mediated deubiquitination of ubH2B may facilitate binding of factors that are important for the transition of paused Pol II into transcription elongation.
The positioning of nucleosomes within the coding regions of eukaryotic genes is aligned with respect to transcriptional start sites. This organization is likely to influence many genetic processes, requiring access to the underlying DNA. Here, we show that the combined action of Isw1 and Chd1 nucleosome-spacing enzymes is required to maintain this organization. In the absence of these enzymes, regular positioning of the majority of nucleosomes is lost. Exceptions include the region upstream of the promoter, the +1 nucleosome, and a subset of locations distributed throughout coding regions where other factors are likely to be involved. These observations indicate that adenosine triphosphate-dependent remodeling enzymes are responsible for directing the positioning of the majority of nucleosomes within the Saccharomyces cerevisiae genome.
Histone H3 lysine 4 trimethylation needed for transcription is mediated by the Set1 methyltransferase and requires prior monoubiquitination of histone H2B. In this issue, Latham et al. (2011) report that dimethylation of the yeast kinetochore protein Dam1 by Set1 similarly requires H2B monoubiquitination. Thus, H2B ubiquitination signals for methylation beyond chromatin.
We have used EM and biochemistry to characterize the structure of NuA4, an essential yeast histone acetyltransferase (HAT) complex conserved throughout eukaryotes, and we have determined the interaction of NuA4 with the nucleosome core particle (NCP). The ATM-related Tra1 subunit, which is shared with the SAGA coactivator complex, forms a large domain joined to a second region that accommodates the catalytic subcomplex Piccolo and other NuA4 subunits. EM analysis of a NuA4-NCP complex shows the NCP bound at the periphery of NuA4. EM characterization of Piccolo and Piccolo-NCP provided further information about subunit organization and confirmed that histone acetylation requires minimal contact with the NCP. A small conserved region at the N terminus of Piccolo subunit enhancer of Polycomb-like 1 (Epl1) is essential for NCP interaction, whereas the subunit yeast homolog of mammalian Ing1 2 (Yng2) apparently positions Piccolo for efficient acetylation of histone H4 or histone H2A tails. Taken together, these results provide an understanding of the NuA4 subunit organization and the NuA4-NCP interactions.
The Spt-Ada-Gcn5-acetyltransferase (SAGA) complex was discovered from Saccharomyces cerevisiae and has been well characterized as an important transcriptional coactivator that interacts both with sequence-specific transcription factors and the TATA-binding protein TBP. SAGA contains a histone acetyltransferase and a ubiquitin protease. In metazoans, SAGA is essential for development, yet little is known about the function of SAGA in differentiating tissue. We analyzed the composition, interacting proteins, and genomic distribution of SAGA in muscle and neuronal tissue of late stage Drosophila melanogaster embryos. The subunit composition of SAGA was the same in each tissue; however, SAGA was associated with considerably more transcription factors in muscle compared with neurons. Consistent with this finding, SAGA was found to occupy more genes specifically in muscle than in neurons. Strikingly, SAGA occupancy was not limited to enhancers and promoters but primarily colocalized with RNA polymerase II within transcribed sequences. SAGA binding peaks at the site of RNA polymerase pausing at the 5 end of transcribed sequences. In addition, many tissue-specific SAGA-bound genes required its ubiquitin protease activity for full expression. These data indicate that in metazoans SAGA plays a prominent post-transcription initiation role in tissue-specific gene expression.
Despite the availability of several large-scale proteomics studies aiming to identify protein interactions on a global scale, little is known about how proteins interact and are organized within macromolecular complexes. Here, we describe a technique that consists of a combination of biochemistry approaches, quantitative proteomics and computational methods using wild-type and deletion strains to investigate the organization of proteins within macromolecular protein complexes. We applied this technique to determine the organization of two well-studied complexes, Spt-Ada-Gcn5 histone acetyltransferase (SAGA) and ADA, for which no comprehensive high-resolution structures exist. This approach revealed that SAGA/ADA is composed of five distinct functional modules, which can persist separately. Furthermore, we identified a novel subunit of the ADA complex, termed Ahc2, and characterized Sgf29 as an ADA family protein present in all Gcn5 histone acetyltransferase complexes. Finally, we propose a model for the architecture of the SAGA and ADA complexes, which predicts novel functional associations within the SAGA complex and provides mechanistic insights into phenotypical observations in SAGA mutants.
Alterations of chromatin structure have been shown to be crucial for response to cell signaling and for programmed gene expression in development. Posttranslational histone modifications influence changes in chromatin structure both directly and by targeting or activating chromatin-remodeling complexes. Histone modifications intersect with cell signaling pathways to control gene expression and can act combinatorially to enforce or reverse epigenetic marks in chromatin. Through their recognition by protein complexes with enzymatic activities cross talk is established between different modifications and with other epigenetic pathways, including noncoding RNAs (ncRNAs) and DNA methylation. Here, we review the functions of histone modifications and their exploitation in the programming of gene expression during several events in development.
Histone modifications not only play important roles in regulating chromatin structure and nuclear processes but also can be passed to daughter cells as epigenetic marks. Accumulating evidence suggests that the key function of histone modifications is to signal for recruitment or activity of downstream effectors. Here, we discuss the latest discovery of histone-modification readers and how the modification language is interpreted.
Heterochromatin protein 1a (HP1a) is well known as a silencing protein and regulates gene expression through its binding to methylated histone H3K9. Despite sharing a nearly identical domain architecture, most eukaryotes have at least three HP1 homologs that have differential localization patterns and functions in heterochromatin and euchromatin. Among the three main HP1 paralogs in Drosophila, HP1c functions the least like the canonical heterochromatic HP1, HP1a. HP1c exclusively localizes to euchromatin. In contrast to a key role of HP1a in heterochromatin formation and gene silencing, recent studies link transcriptional elongation by RNA polymerase II to euchromatic HP1c or HP1?. These findings expand the role for HP1c in targeting euchromatin, suggesting that HP1c acts as a positive regulator in active transcription in euchromatin. Here, we highlight recent data on the specificity and function of HP1c.
Heterochromatin protein 1 (HP1) is a positive regulator of active transcription in euchromatin. HP1 was first identified in Drosophila melanogaster as a major component of heterochromatin. Most eukaryotes have at least three isoforms of HP1, which are conserved in overall structure but localize differentially to heterochromatin and euchromatin. Although initial studies revealed a key role for HP1 in heterochromatin formation and gene silencing, recent progress has shed light on additional roles for HP1 in processes such as euchromatic gene expression. Recent studies have highlighted the importance of HP1-mediated gene regulation in euchromatin. Here, we focus on recent advances in understanding the role of HP1 in active transcription in euchromatin and how modification and localization of HP1 can regulate distinct functions for this protein in different contexts.
The SAGA (Spt-Ada-Gcn5 acetyltransferase) complex is an important chromatin modifying complex that can both acetylate and deubiquitinate histones. Sgf29 is a novel component of the SAGA complex. Here, we report the crystal structures of the tandem Tudor domains of Saccharomyces cerevisiae and human Sgf29 and their complexes with H3K4me2 and H3K4me3 peptides, respectively, and show that Sgf29 selectively binds H3K4me2/3 marks. Our crystal structures reveal that Sgf29 harbours unique tandem Tudor domains in its C-terminus. The tandem Tudor domains in Sgf29 tightly pack against each other face-to-face with each Tudor domain harbouring a negatively charged pocket accommodating the first residue alanine and methylated K4 residue of histone H3, respectively. The H3A1 and K4me3 binding pockets and the limited binding cleft length between these two binding pockets are the structural determinants in conferring the ability of Sgf29 to selectively recognize H3K4me2/3. Our in vitro and in vivo functional assays show that Sgf29 recognizes methylated H3K4 to recruit the SAGA complex to its targets sites and mediates histone H3 acetylation, underscoring the importance of Sgf29 in gene regulation.
The positive link between the SWI/SNF and the Gcn5 histone acetyltransferase in transcriptional activation has been well described. Here we report an inhibitory role for Gcn5 in SWI/SNF targeting. We demonstrate that Gcn5-containing complexes directly acetylate the Snf2 subunit of the SWI/SNF complex in vitro, as well as in vivo. Moreover, the acetylation of Snf2 facilitates the dissociation of the SWI/SNF complex from acetylated histones, and reduces its association with promoters in vivo. These data reveal a novel mechanism by which Gcn5 modulates chromatin structure not only through the acetylation of histones, but also by directly acetylating Snf2.
The use of quantitative proteomics methods to study protein complexes has the potential to provide in-depth information on the abundance of different protein components as well as their modification state in various cellular conditions. To interrogate protein complex quantitation using shotgun proteomic methods, we have focused on the analysis of protein complexes using label-free multidimensional protein identification technology and studied the reproducibility of biological replicates. For these studies, we focused on three highly related and essential multi-protein enzymes, RNA polymerase I, II, and III from Saccharomyces cerevisiae. We found that label-free quantitation using spectral counting is highly reproducible at the protein and peptide level when analyzing RNA polymerase I, II, and III. In addition, we show that peptide sampling does not follow a random sampling model, and we show the need for advanced computational models to predict peptide detection probabilities. In order to address these issues, we used the APEX protocol to model the expected peptide detectability based on whole cell lysate acquired using the same multidimensional protein identification technology analysis used for the protein complexes. Neither method was able to predict the peptide sampling levels that we observed using replicate multidimensional protein identification technology analyses. In addition to the analysis of the RNA polymerase complexes, our analysis provides quantitative information about several RNAP associated proteins including the RNAPII elongation factor complexes DSIF and TFIIF. Our data shows that DSIF and TFIIF are the most highly enriched RNAP accessory factors in Rpb3-TAP purifications and demonstrate our ability to measure low level associated protein abundance across biological replicates. In addition, our quantitative data supports a model in which DSIF and TFIIF interact with RNAPII in a dynamic fashion in agreement with previously published reports.
Heterochromatin protein 1 (HP1) is well known as a silencing protein found at pericentric heterochromatin. Most eukaryotes have at least three isoforms of HP1 that play differential roles in heterochromatin and euchromatin. In addition to its role in heterochromatin, HP1 proteins have been shown to function in transcription elongation. To gain insights into the transcription functions of HP1, we sought to identify novel HP1-interacting proteins. Biochemical and proteomic approaches revealed that HP1 interacts with the histone chaperone complex FACT (facilitates chromatin transcription). HP1c interacts with the SSRP1 (structure-specific recognition protein 1) subunit and the intact FACT complex. Moreover, HP1c guides the recruitment of FACT to active genes and links FACT to active forms of RNA polymerase II. The absence of HP1c partially impairs the recruitment of FACT into heat-shock loci and causes a defect in heat-shock gene expression. Thus, HP1c functions to recruit the FACT complex to RNA polymerase II.
Cumulative evidence indicates that the proteasome, which is mainly known as a protein-degrading machine, is very essential for gene expression. Destructive functions of the proteasome, i.e., ubiquitin-dependent proteolytic activity, are significant for activator localization, activator destruction, co-activator/repressor destruction and PIC disassembly. Non-proteolytic functions of the proteasome are important for recruitment of activators and co-activators to promoters, ubiquitin-dependent histone modification, transcription elongation and possibly maturation of mRNA via the facilitation of mRNA export from the nucleus to the cytoplasm. In this review, we discuss how the proteasome regulates transcription at numerous stages during gene expression. This article is part of a Special Issue entitled The 26S Proteasome: When degradation is just not enough!
The Set2-Rpd3S pathway is important for the control of transcription memory. Mutation of components of this pathway results in cryptic transcription initiation within the coding region of approximately 30% of yeast genes. Specifically, deletion of the Set2 histone methyltransferase or Rco1, a component of the Rpd3S histone deacetylase complex leads to hyperacetylation of certain open reading frames (ORFs). We used this mutant as a system to study the role of histone modifications and co-activator recruitment in preinitiation complex (PIC) formation. Specifically, we looked at the dependence of promoters on the bromodomain-containing RSC complex and the Bdf1 protein. We found that the dependence of cryptic promoters for these proteins varied. Overall, our data indicate that cryptic promoters are independently regulated, and their activation is dependent on factors that govern gene activation at canonical promoters.
Cse4 is a variant of histone H3 that is incorporated into a single nucleosome at each centromere in budding yeast. We have discovered an E3 ubiquitin ligase, called Psh1, which controls the cellular level of Cse4 via ubiquitylation and proteolysis. The activity of Psh1 is dependent on both its RING and zinc finger domains. We demonstrate the specificity of the ubiquitylation activity of Psh1 toward Cse4 in vitro and map the sites of ubiquitylation. Mutation of key lysines prevents ubiquitylation of Cse4 by Psh1 in vitro and stabilizes Cse4 in vivo. While deletion of Psh1 stabilizes Cse4, elimination of the Cse4-specific chaperone Scm3 destabilizes Cse4, and the addition of Scm3 to the Psh1-Cse4 ubiquitylation reaction prevents Cse4 ubiquitylation, together suggesting Scm3 may protect Cse4 from ubiquitylation. Without Psh1, Cse4 overexpression is toxic and Cse4 is found at ectopic locations. Our results suggest Psh1 functions to prevent the mislocalization of Cse4.
In response to extracellular cues, signal transduction activates downstream transcription factors like c-Jun to induce expression of target genes. We demonstrate that the ATAC (Ada two A containing) histone acetyltransferase (HAT) complex serves as a transcriptional cofactor for c-Jun at the Jun N-terminal kinase (JNK) target genes Jra and chickadee. ATAC subunits are required for c-Jun occupancy of these genes and for H4K16 acetylation at the Jra enhancer, promoter, and transcribed sequences. Under conditions of osmotic stress, ATAC colocalizes with c-Jun, recruits the upstream kinases Misshapen, MKK4, and JNK, and suppresses further activation of JNK. Relocalization of these MAPKs and suppression of JNK activation by ATAC are dependent on the CG10238 subunit of ATAC. Thus, ATAC governs the transcriptional response to MAP kinase signaling by serving as both a coactivator of transcription and as a suppressor of upstream signaling.
The rapid activation of gene expression in response to stimuli occurs largely through the regulation of RNA polymerase II-dependent transcription. In this Review, we discuss events that occur during the transcription cycle in eukaryotes that are important for the rapid and specific activation of gene expression in response to external stimuli. In addition to regulated recruitment of the transcription machinery to the promoter, it has now been shown that control steps can include chromatin remodelling and the release of paused polymerase. Recent work suggests that some components of signal transduction cascades also play an integral part in activating transcription at target genes.
Histone acetylation is generally considered a mark involved in activating gene expression by making chromatin structures less compact. In the April 1, 2010, issue of Genes & Development, Xhemalce and Kouzarides (pp. 647-652) demonstrate that the acetylation of histone H3 at Lys 4 (H3K4) plays a role in the formation of repressive heterochromatin in Schizosaccharomyces pombe. H3K4 acetylation mediates a switch of chromodomain proteins associated with methylated H3K9 during heterochromatin assembly.
Methylation of histone H3 by Set1 and Set2 is required for deacetylation of nucleosomes in coding regions by histone deacetylase complexes (HDACs) Set3C and Rpd3C(S), respectively. We report that Set3C and Rpd3C(S) are cotranscriptionally recruited in the absence of Set1 and Set2, but in a manner stimulated by Pol II CTD kinase Cdk7/Kin28. Consistently, Rpd3C(S) and Set3C interact with Ser5-phosphorylated Pol II and histones in extracts, but only the histone interactions require H3 methylation. Moreover, reconstituted Rpd3C(S) binds specifically to Ser5-phosphorylated CTD peptides in vitro. Hence, whereas interaction with methylated H3 residues is required for Rpd3C(S) and Set3C deacetylation activities, their cotranscriptional recruitment is stimulated by the phosphorylated CTD. We further demonstrate that Rpd3, Hos2, and Hda1 have overlapping functions in deacetylating histones and suppressing cotranscriptional histone eviction. A strong correlation between increased acetylation and lower histone occupancy in HDA mutants implies that histone acetylation is important for nucleosome eviction.
One of the most notable features of the vertebrate body plan organization is its bilateral symmetry, evident at the level of vertebrae and skeletal muscles. Here we show that a mutation in Rere (also known as atrophin2) leads to the formation of asymmetrical somites in mouse embryos, similar to embryos deprived of retinoic acid. Furthermore, we also demonstrate that Rere controls retinoic acid signalling, which is required to maintain somite symmetry by interacting with Fgf8 in the left-right signalling pathway. Rere forms a complex with Nr2f2, p300 (also known as Ep300) and a retinoic acid receptor, which is recruited to the retinoic acid regulatory element of retinoic acid targets, such as the Rarb promoter. Furthermore, the knockdown of Nr2f2 and/or Rere decreases retinoic acid signalling, suggesting that this complex is required to promote transcriptional activation of retinoic acid targets. The asymmetrical expression of Nr2f2 in the presomitic mesoderm overlaps with the asymmetry of the retinoic acid signalling response, supporting its implication in the control of somitic symmetry. Misregulation of this mechanism could be involved in symmetry defects of the human spine, such as those observed in patients with scoliosis.
Histone deacetylase (HDAC) inhibitors are in clinical development for several diseases, including cancers and neurodegenerative disorders. HDACs 1 and 2 are among the targets of these inhibitors and are part of multisubunit protein complexes. HDAC inhibitors (HDACis) block the activity of HDACs by chelating a zinc molecule in their catalytic sites. It is not known if the inhibitors have any additional functional effects on the multisubunit HDAC complexes. Here, we find that suberoylanilide hydroxamic acid (SAHA), the first FDA-approved HDACi for cancer, causes the dissociation of the PHD-finger-containing ING2 subunit from the Sin3 deacetylase complex. Loss of ING2 disrupts the in vivo binding of the Sin3 complex to the p21 promoter, an important target gene for cell growth inhibition by SAHA. Our findings reveal a molecular mechanism by which HDAC inhibitors disrupt deacetylase function.
The histone acetyltransferase complex SAGA is well characterized as a coactivator complex in yeast. In this study of Drosophila SAGA (dSAGA), we describe three novel components that include an ortholog of Spt20, a potential ortholog of Sgf73/ATXN7, and a novel histone fold protein, SAF6 (SAGA factor-like TAF6). SAF6, which binds directly to TAF9, functions analogously in dSAGA to TAF6/TAF6L in the yeast and human SAGA complexes, respectively. Moreover, TAF6 in flies is restricted to TFIID. Mutations in saf6 disrupt SAGA-regulated gene expression without disrupting acetylated or ubiquitinated histone levels. Thus, SAF6 is essential for SAGA coactivator function independent of the enzymatic activities of the complex.
Direct binding of WD40 repeat of Embryonic Ectoderm Development (EED) in the Polycomb Repressor Complex 2 (PRC2) to the histone H3 tail regulates the H3K27 methyltrasnferase activity of PRC2. The binding activity is required for the methylation of H3K27 over long distance of a Drosophila chromosome from the UBX (Hox gene) promoter to the PBX upstream enhancer region, implying EED initiates propagation of this repressive mark.
Protein complexes are key molecular machines executing a variety of essential cellular processes. Despite the availability of genome-wide protein-protein interaction studies, determining the connectivity between proteins within a complex remains a major challenge. Here we demonstrate a method that is able to predict the relationship of proteins within a stable protein complex. We employed a combination of computational approaches and a systematic collection of quantitative proteomics data from wild-type and deletion strain purifications to build a quantitative deletion-interaction network map and subsequently convert the resulting data into an interdependency-interaction model of a complex. We applied this approach to a data set generated from components of the Saccharomyces cerevisiae Rpd3 histone deacetylase complexes, which consists of two distinct small and large complexes that are held together by a module consisting of Rpd3, Sin3 and Ume1. The resulting representation reveals new protein-protein interactions and new submodule relationships, providing novel information for mapping the functional organization of a complex.
The reversible acetylation of proteins is mediated by histone acetyltransferases which acetylate proteins and histone deacetylases that remove the acetyl groups. High levels of histone acetylation are correlated with active genes, while hypoacetylation of histones corresponds with gene repression. Importantly, acetylation also occurs on non-histone proteins and this can affect the activity and stability of these proteins. Aberrant epigenetic changes are a common hallmark of tumors and imbalances in the activities of deacetylases have been associated with cancers. Accordingly, inhibitors to the histone deacetylases are in clinical trials for the treatment of several cancer types. These drugs mediate a number of molecular changes and in turn can induce cell cycle arrest, apoptosis or differentiation of cancer cells while displaying limited toxicity in normal cells.
Interaction between acidic activation domains and the activator-binding domains of Swi1 and Snf5 of the yeast SWI/SNF chromatin remodeling complex has previously been characterized in vitro. Although deletion of both activator-binding domains leads to phenotypes that differ from the wild-type, their relative importance for SWI/SNF recruitment to target genes has not been investigated. In the present study, we used chromatin immunoprecipitation assays to investigate the individual and collective importance of the activator-binding domains for SWI/SNF recruitment to genes within the GAL regulon in vivo. We also investigated the consequences of defective SWI/SNF recruitment for target gene activation. We demonstrate that deletion of both activator-binding domains essentially abolishes galactose-induced SWI/SNF recruitment and causes a reduction in transcriptional activation similar in magnitude to that associated with a complete loss of SWI/SNF activity. The activator-binding domains in Swi1 and Snf5 make approximately equal contributions to the recruitment of SWI/SNF to each of the genes studied. The requirement for SWI/SNF recruitment correlates with GAL genes that are highly and rapidly induced by galactose.
Spinocerebellar ataxia (SCA) is a physically devastating, genetically inherited disorder characterized by abnormal brain function that results in the progressive loss of the ability to coordinate movements. There are many types of SCAs as there are various gene mutations that can cause this disease. SCA types 1-3, 6-10, 12, and 17 result from a trinucleotide repeat expansion in the DNA-coding sequence. Intriguingly, recent work has demonstrated that increased trinucleotde expansions in the SCA7 gene result in defect in the function of the SAGA histone acetyltransferase complex. The SCA7 gene encodes a subunit of the SAGA complex. This subunit is conserved in yeast as the SGF73 gene. We demonstrate that Sgf73 is required to recruit the histone deubiquitination module into both SAGA and the related SliK(SALSA) complex, and to maintain levels of histone ubiquitination, which is necessary for regulation of transcription at a number of genes.
The budding yeast CenH3 histone variant Cse4 localizes to centromeric nucleosomes and is required for kinetochore assembly and chromosome segregation. The exact composition of centromeric Cse4-containing nucleosomes is a subject of debate. Using unbiased biochemical, cell-biological, and genetic approaches, we have tested the composition of Cse4-containing nucleosomes. Using micrococcal nuclease-treated chromatin, we find that Cse4 is associated with the histones H2A, H2B, and H4, but not H3 or the nonhistone protein Scm3. Overexpression of Cse4 rescues the lethality of a scm3 deletion, indicating that Scm3 is not essential for the formation of functional centromeric chromatin. We also find that octameric Cse4 nucleosomes can be reconstituted in vitro. Furthermore, Cse4-Cse4 dimerization occurs in vivo at the centromeric nucleosome, and this requires the predicted Cse4-Cse4 dimerization interface. Taken together, our experimental evidence supports the model that the Cse4 nucleosome is an octamer, containing two copies each of Cse4, H2A, H2B, and H4.
Messenger RNA processing is coupled to RNA polymerase II (RNAPII) transcription through coordinated recruitment of accessory proteins to the Rpb1 C-terminal domain (CTD). Dynamic changes in CTD phosphorylation during transcription elongation are responsible for their recruitment, with serine 5 phosphorylation (S5-P) occurring toward the 5 end of genes and serine 2 phosphorylation (S2-P) occurring toward the 3 end. The proteins responsible for regulation of the transition state between S5-P and S2-P CTD remain elusive. We show that a conserved protein of unknown function, Rtr1, localizes within coding regions, with maximum levels of enrichment occurring between the peaks of S5-P and S2-P RNAPII. Upon deletion of Rtr1, the S5-P form of RNAPII accumulates in both whole-cell extracts and throughout coding regions; additionally, RNAPII transcription is decreased, and termination defects are observed. Functional characterization of Rtr1 reveals its role as a CTD phosphatase essential for the S5-to-S2-P transition.
Histone methylation is associated with both transcription activation and repression. However, the functions of different states of methylation remain largely elusive. Here, using methyl-lysine analog technology, we demonstrate that the histone deacetylase complex, Rpd3S, can distinguish the nucleosomes methylated to different extents and that K36me2 is sufficient to target Rpd3S in vitro. Through a genome-wide survey, we identified a few mutants in which the level of K36me3 is significantly reduced, whereas the level of K36me2 is sustained. Transcription analysis and genome-wide histone modification studies on these mutants suggested that K36me2 is sufficient to target Rpd3S in vivo, thereby maintaining a functional Set2-Rpd3S pathway.
Maintenance of ordered chromatin structure over the body of genes is vital for the regulation of transcription. Increased access to the underlying DNA sequence results in the recruitment of RNA polymerase II to inappropriate, promoter-like sites within genes, resulting in unfettered transcription. Two new papers show how the Set2-mediated methylation of histone H3 on Lys36 (H3K36me) maintains chromatin structure by limiting histone dynamics over gene bodies, either by recruiting chromatin remodelers that preserve ordered nucleosomal distribution or by lowering the binding affinity of histone chaperones for histones, preventing their removal.
An abundance of long non-coding RNA (lncRNA) present in most species from yeast to human are involved in transcriptional regulation, dosage compensation and imprinting. This underscores the importance of lncRNA as functional RNA despite the fact that they do not produce proteins. Two recent papers in Cell have demonstrated that transcription of the non-conserved lncRNAs, but not the RNAs themselves, is necessary to introduce co-transcriptional regulatory histone marks to regulate gene expression.
In this issue, Hughes et al. (2012) show that nucleosome positioning is determined not by any single mechanism, but by the coordinated action of multiple factors, including the underlying DNA sequence, species-specific DNA binding proteins, chromatin remodelers, and the transcription machinery.
Here we describe the function of a previously uncharacterized protein, named family with sequence similarity 60 member A (FAM60A) that maps to chromosome 12p11 in humans. We use quantitative proteomics to determine that the main biochemical partners of FAM60A are subunits of the Sin3 deacetylase complex and show that FAM60A resides in active HDAC complexes. In addition, we conduct gene expression pathway analysis and find that FAM60A regulates expression of genes that encode components of the TGF-beta signaling pathway. Moreover, our studies reveal that loss of FAM60A or another component of the Sin3 complex, SDS3, leads to a change in cell morphology and an increase in cell migration. These studies reveal the function of a previously uncharacterized protein and implicate the Sin3 complex in suppressing cell migration.
Eukaryotic genomes are packaged into chromatin, a highly organized structure consisting of DNA and histone proteins. All nuclear processes take place in the context of chromatin. Modifications of either DNA or histone proteins have fundamental effects on chromatin structure and function, and thus influence processes such as transcription, replication or recombination. In this review we highlight histone modifications specifically associated with gene transcription by RNA polymerase II and summarize their genomic distributions. Finally, we discuss how (mis-)regulation of these histone modifications perturbs chromatin organization over coding regions and results in the appearance of aberrant, intragenic transcription. This article is part of a Special Issue entitled: RNA polymerase II Transcript Elongation.
Histone methylation is widely believed to contribute to epigenetic inheritance by persevering through DNA replication and subsequently templating methylation of daughter chromosome regions. However, a report in this issue (Petruk et al.) suggests that chromatin association of the methytransferase complexes themselves persists through replication and re-establishes histone methylation.
Set2-mediated methylation of histone H3 Lys36 (H3K36) is a mark associated with the coding sequences of actively transcribed genes, but it has a negative role during transcription elongation. It prevents trans-histone exchange over coding regions and signals for histone deacetylation in the wake of RNA polymerase II (RNAPII) passage. We have found that in Saccharomyces cerevisiae the Isw1b chromatin-remodeling complex is specifically recruited to open reading frames (ORFs) by H3K36 methylation through the PWWP domain of its Ioc4 subunit in vivo and in vitro. Isw1b acts in conjunction with Chd1 to regulate chromatin structure by preventing trans-histone exchange from taking place over coding regions. In this way, Isw1b and Chd1 are important in maintaining chromatin integrity during transcription elongation by RNAPII.
Set2-mediated methylation of histone H3 at Lys?36 (H3K36me) is a co-transcriptional event that is necessary for the activation of the Rpd3S histone deacetylase complex, thereby maintaining the coding region of genes in a hypoacetylated state. In the absence of Set2, H3K36 or Rpd3S acetylated histones accumulate on open reading frames (ORFs), leading to transcription initiation from cryptic promoters within ORFs. Although the co-transcriptional deacetylation pathway is well characterized, the factors responsible for acetylation are as yet unknown. Here we show that, in yeast, co-transcriptional acetylation is achieved in part by histone exchange over ORFs. In addition to its function of targeting and activating the Rpd3S complex, H3K36 methylation suppresses the interaction of H3 with histone chaperones, histone exchange over coding regions and the incorporation of new acetylated histones. Thus, Set2 functions both to suppress the incorporation of acetylated histones and to signal for the deacetylation of these histones in transcribed genes. By suppressing spurious cryptic transcripts from initiating within ORFs, this pathway is essential to maintain the accuracy of transcription by RNA polymerase?II.
Environments can be ever-changing and stresses are commonplace. In order for organisms to survive, they need to be able to respond to change and adapt to new conditions. Fortunately, many organisms have systems in place that enable dynamic adaptation to immediate stresses and changes within the environment. Much of this cellular response is coordinated by modulating the structure and accessibility of the genome. In eukaryotic cells, the genome is packaged and rolled up by histone proteins to create a series of DNA/histone core structures known as nucleosomes; these are further condensed into chromatin. The degree and nature of the condensation can in turn determine which genes are transcribed. Histones can be modified chemically by a large number of proteins that are thereby responsible for dynamic changes in gene expression. In this Primer we discuss findings from a study published in this issue of PLoS Biology by Weiner et al. that highlight how chromatin structure and chromatin binding proteins alter transcription in response to environmental changes and stresses. Their study reveals the importance of chromatin in mediating the speed and amplitude of stress responses in cells and suggests that chromatin is a critically important component of the cellular response to stress.
Signal transduction pathways alter the gene expression program in response to extracellular or intracellular cues. Mitogen-activated protein kinases (MAPKs) govern numerous cellular processes including cell growth, stress response, apoptosis, and differentiation. In the past decade, MAPKs have been shown to regulate the transcription machinery and associate with chromatin-modifying complexes. Moreover, recent studies demonstrate that several MAPKs bind directly to chromatin at target genes. This review highlights the recent discoveries of MAPK signaling in regard to histone modifications and chromatin regulation. Evidence suggesting that further unknown mechanisms integrate signal transduction with chromatin biology is discussed.
The KDM4 subfamily of JmjC domain-containing demethylases mediates demethylation of histone H3K36me3/me2 and H3K9me3/me2. Several studies have shown that human and yeast KDM4 proteins bind to specific gene promoters and regulate gene expression. However, the genome-wide distribution of KDM4 proteins and the mechanism of genomic-targeting remain elusive. We have previously identified Drosophila KDM4A (dKDM4A) as a histone H3K36me3 demethylase that directly interacts with HP1a. Here, we performed H3K36me3 ChIP-chip analysis in wild type and dkdm4a mutant embryos to identify genes regulated by dKDM4A demethylase activity in vivo. A subset of heterochromatic genes that show increased H3K36me3 levels in dkdm4a mutant embryos overlap with HP1a target genes. More importantly, binding to HP1a is required for dKDM4A-mediated H3K36me3 demethylation at a subset of heterochromatic genes. Collectively, these results show that HP1a functions to target the H3K36 demethylase dKDM4A to heterochromatic genes in Drosophila.
The Saccharomyces cerevisiae SUN-domain protein Mps3 is required for duplication of the yeast centrosome-equivalent organelle, the spindle pole body (SPB), and it is involved in multiple aspects of nuclear organization, including telomere tethering and gene silencing at the nuclear membrane, establishment of sister chromatid cohesion, and repair of certain types of persistent DNA double-stranded breaks. How these diverse SUN protein functions are regulated is unknown. Here we show that the Mps3 N-terminus is a substrate for the acetyltransferase Eco1/Ctf7 in vitro and in vivo and map the sites of acetylation to three lysine residues adjacent to the Mps3 transmembrane domain. Mutation of these residues shows that acetylation is not essential for growth, SPB duplication, or distribution in the nuclear membrane. However, analysis of nonacetylatable mps3 mutants shows that this modification is required for accurate sister chromatid cohesion and for chromosome recruitment to the nuclear membrane. Acetylation of Mps3 by Eco1 is one of the few regulatory mechanisms known to control nuclear organization.
Molybdopterin (MPT) synthase is an essential enzyme involved in the synthesis of the molybdenum cofactor precursor molybdopterin. The molybdenum cofactor biosynthetic pathway is conserved from prokaryotes to Metazoa. CG10238 is the Drosophila homolog of the MoaE protein, a subunit of MPT synthase, and is found in a fusion with the mitogen-activated protein kinase (MAPK)-upstream protein kinase-binding inhibitory protein (MBIP). This fused protein inhibits the activation of c-Jun N-terminal kinase (JNK). dMoaE (CG10238) carries out this function as a subunit of the ATAC histone acetyltransferase complex. In this study, we demonstrate that Drosophila MoaE (CG10238) also interacts with Drosophila MoaD and with itself to form a complex with stoichiometry identical to the MPT synthase holoenzyme in addition to its function in ATAC. We also show that sequence determinants that regulate MAPK signaling are located within the MoaE region of dMoaE (CG10238). Analysis of other metazoan MBIPs reveals that MBIP protein sequences have an N-terminal region that appears to have been derived from the MoaE protein, although it has lost residues responsible for catalytic activity. Thus, intact and modified copies of the MoaE protein may have been conscripted to play a new, noncatalytic role in MAPK signaling in Metazoa as part of the ATAC complex.
ATPases and histone chaperones facilitate RNA polymerase II (pol II) elongation on chromatin. In vivo, the coordinated action of these enzymes is necessary to permit pol II passage through a nucleosome while restoring histone density afterward. We have developed a biochemical system recapitulating this basic process. Transcription through a nucleosome in vitro requires the ATPase remodels structure of chromatin (RSC) and the histone chaperone nucleosome assembly protein 1 (NAP1). In the presence of NAP1, RSC generates a hexasome. Despite the propensity of RSC to evict histones, NAP1 reprograms the reaction such that the hexasome is retained on the template during multiple rounds of transcription. This work has implications toward understanding the mechanism of pol II elongation on chromatin.
Transcription factor C/EBP? is involved in several cellular processes, such as proliferation, differentiation, and energy metabolism. This factor exerts its activity through recruitment of different proteins or protein complexes, including the ATP-dependent chromatin remodeling complex SWI/SNF. The C/EBP? protein is found as three major isoforms, C/EBP?1, -2, and -3. They are generated by translation at alternative AUG initiation codons of a unique mRNA, C/EBP?1 being the full-length isoform. It has been found that C/EBP?1 participates in terminal differentiation processes. Conversely, C/EBP?2 and -3 promote cell proliferation and are involved in malignant progression in a number of tissues. The mechanisms by which C/EBP?2 and -3 promote cell proliferation and tumor progression are not fully understood. In this work, we sought to identify proteins interacting with hC/EBP? using a proteomics approach. We found that all three isoforms interact with hSNF2H and hACF, components of ACF and CHRAC chromatin remodeling complexes, which belong to the imitation switch subfamily. Additional protein-protein interaction studies confirmed this finding and also showed that hC/EBP? directly interacts with hACF1. By overexpressing hC/EBP?, hSNF2H, and hACF1 in HepG2 cells and analyzing variations in expression of cyclin D1 and other C/EBP? target genes, we observed a functional interaction between C/EBP? and SNF2H/ACF1, characterized mainly by suppression of C/EBP? transactivation activity in the presence of SNF2H and ACF1. Consistent with these findings, induction of differentiation of HepG2 cells by 1% DMSO was accompanied by a reduction in the level of cyclin D1 expression and the appearance of hC/EBP?, hSNF2H, and hACF1 on the promoter region of this gene.
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