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Two recent reports by Cramer and Ben-Shem and colleagues present high-resolution structures of the yeast SAGA transcription coactivator complex. These are the first to resolve the stoichiometry and structure of the core. The core contains an octamer-like fold, flexibly links the enzymatic modules, and facilitates TBP loading onto TATA promoters., (Published by Elsevier Ltd.)

Mutations that affect myelodysplasia/myeloid leukemia factor (MLF) proteins are associated with leukemia and several other cancers. However, with no strong homology to other proteins of known function, the role of MLF proteins in the cell has remained elusive. Here, we describe a proteomics approach that identifies MLF as a member of a nuclear chaperone complex containing a DnaJ protein, BCL2-associated anthanogene 2, and Hsc70. This complex associates with chromatin and regulates the expression of target genes. The MLF complex is bound to sites of nucleosome depletion and sites containing active chromatin marks (e.g., H3K4me3 and H3K4me1). Hence, MLF binding is enriched at promoters and enhancers. Additionally, the MLF-chaperone complex functions to regulate transcription factor stability, including the RUNX transcription factor involved in hematopoiesis. Although Hsc70 and other co-chaperones have been shown to play a role in nuclear translocation of a variety of proteins including transcription factors, our findings suggest that MLF and the associated co-chaperones play a direct role in modulating gene transcription., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Chromatin plays a central role in the conversion of energy in cells: alteration of chromatin structure to make DNA accessible consumes energy, and compaction of chromatin preserves energy. Alteration of chromatin structure uses energy sources derived from carbon metabolism such as ATP and acetyl-CoA; conversely, chromatin compaction and epigenetic modification feedback to metabolism and energy homeostasis by controlling gene expression and storing metabolites. Coordination of these dual chromatin events must be flexibly modulated in response to environmental changes such as during development and exposure to stress. Aging also alters chromatin structure and the coordination of metabolism, chromatin dynamics, and other cell processes. Noncoding RNAs and other RNA species that associate directly with chromatin or with chromatin modifiers contribute to spatiotemporal control of transcription and energy conversion. The time required for generating the large amounts of RNAs and chromatin modifiers observed in super-enhancers may be critical for regulation of transcription and may be impacted by aging. Here, taking into account these factors, we review alterations of chromatin that are fundamental to cell responses to metabolic changes due to stress and aging to maintain redox and energy homeostasis. We discuss the relationship between spatiotemporal control of energy and chromatin function, as this emerging concept must be considered to understand how cell homeostasis is maintained. [ABSTRACT FROM AUTHOR]

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.cell.2008.10.036 Byline: Tamaki Suganuma (1), Jerry L. Workman (1) Abstract: Histone modifications play a complex role in the regulation of transcription. Recent studies () reveal that regulation of histone modifications can be functionally linked to reinforce the activation or repression of gene expression. Author Affiliation: (1) Stowers Institute for Medical Research, Kansas City, MO 64110, USA

The mechanisms of epigenetic gene regulation—histone modifications, chromatin remodeling, DNA methylation, and noncoding RNA—use metabolites as enzymatic cofactors and substrates in reactions that allow chromatin formation, nucleotide biogenesis, transcription, RNA processing, and translation. Gene expression responds to demands from cellular processes that use specific metabolites and alters or maintains cellular metabolic status. However, the roles of metabolites—particularly nucleotides—as regulatory molecules in epigenetic regulation and biological processes remain largely unknown. Here we review the crosstalk between gene expression, nucleotide metabolism, and cellular processes, and explore the role of metabolism in epigenetics as a critical regulator of biological events. [ABSTRACT FROM AUTHOR]

Summary The 12-subunit Swi/Snf chromatin remodeling complex is conserved from yeast to humans. It functions to alter nucleosome positions by either sliding nucleosomes on DNA or evicting histones. Interestingly, 20% of all human cancers carry mutations in subunits of the Swi/Snf complex. Many of these mutations cause protein instability and loss, resulting in partial Swi/Snf complexes. Although several studies have shown that histone acetylation and activator-dependent recruitment of Swi/Snf regulate its function, it is less well understood how subunits regulate stability and function of the complex. Using functional proteomic and genomic approaches, we have assembled the network architecture of yeast Swi/Snf. In addition, we find that subunits of the Swi/Snf complex regulate occupancy of the catalytic subunit Snf2, thereby modulating gene transcription. Our findings have direct bearing on how cancer-causing mutations in orthologous subunits of human Swi/Snf may lead to aberrant regulation of gene expression by this complex. [ABSTRACT FROM AUTHOR]

The SAGA complex contains two enzymatic modules, which house histone acetyltransferase (HAT) and deubiquitinase (DUB) activities. USP22 is the catalytic subunit of the DUB module, but two adaptor proteins, ATXN7L3 and ENY2, are necessary for DUB activity toward histone H2Bub1 and other substrates. ATXN7L3B shares 74% identity with the N-terminal region of ATXN7L3, but the functions of ATXN7L3B are not known. Here we report that ATXN7L3B interacts with ENY2 but not other SAGA components. Even though ATXN7L3B localizes in the cytoplasm, ATXN7L3B overexpression increases H2Bub1 levels, while overexpression of ATXN7L3 decreases H2Bub1 levels. In vitro, ATXN7L3B competes with ATXN7L3 to bind ENY2, and in vivo, knockdown of ATXN7L3B leads to concomitant loss of ENY2. Unlike the ATXN7L3 DUB complex, a USP22-ATXN7L3BENY2 complex cannot deubiquitinate H2Bub1 efficiently in vitro. Moreover, ATXN7L3B knockdown inhibits migration of breast cancer cells in vitro and limits expression of ER target genes. Collectively, our studies suggest that ATXN7L3B regulates H2Bub1 levels and SAGA DUB activity through competition for ENY2 binding. [ABSTRACT FROM AUTHOR]

Proteins that respond to DNA damage play critical roles in normal and diseased states in human biology. Studies have suggested that the S. cerevisiae protein CMR1/YDL156w is associated with histones and is possibly associated with DNA repair and replication processes. Through a quantitative proteomic analysis of affinity purifications here we show that the human homologue of this protein, WDR76, shares multiple protein associations with the histones H2A, H2B, and H4. Furthermore, our quantitative proteomic analysis of WDR76 associated proteins demonstrated links to proteins in the DNA damage response like PARP1 and XRCC5 and heterochromatin related proteins like CBX1, CBX3, and CBX5. Co-immunoprecipitation studies validated these interactions. Next, quantitative imaging studies demonstrated that WDR76 was recruited to laser induced DNA damage immediately after induction, and we compared the recruitment of WDR76 to laser induced DNA damage to known DNA damage proteins like PARP1, XRCC5, and RPA1. In addition, WDR76 co-localizes to puncta with the heterochromatin proteins CBX1 and CBX5, which are also recruited to DNA damage but much less intensely than WDR76. This work demonstrates the chromatin and DNA damage protein associations of WDR76 and demonstrates the rapid response of WDR76 to laser induced DNA damage. [ABSTRACT FROM AUTHOR]

The packaging of DNA into strings of nucleosomes is one of the features that allows eukaryotic cells to tightly regulate gene expression. The ordered disassembly of nucleosomes permits RNA polymerase II (Pol II) to access the DNA, whereas nucleosomal reassembly impedes access, thus preventing transcription and mRNA synthesis. Chromatin modifications, chromatin remodellers, histone chaperones and histone variants regulate nucleosomal dynamics during transcription. Disregulation of nucleosome dynamics results in aberrant transcription initiation, producing non-coding RNAs. Ongoing research is elucidating the molecular mechanisms that regulate chromatin structure during transcription by preventing histone exchange, thereby limiting non-coding RNA expression. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 genomictargeting 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. [ABSTRACT FROM AUTHOR]

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. [Copyright &y& Elsevier]

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. [ABSTRACT FROM AUTHOR]

Abstract: 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! [Copyright &y& Elsevier]

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. [ABSTRACT FROM AUTHOR]

The male-specific lethal (MSL) complex upregulates the single male X chromosome to achieve dosage compensation in Drosophila melanogaster. We have proposed that MSL recognition of specific entry sites on the X is followed by local targeting of active genes marked by histone H3 trimethylation (H3K36me3). Here we analyze the role of the MSL3 chromodomain in the second targeting step. Using ChIP-chip analysis, we find that MSL3 chromodomain mutants retain binding to chromatin entry sites but show a clear disruption in the full pattern of MSL targeting in vivo, consistent with a loss of spreading. Furthermore, when compared to wild type, chromodomain mutants lack preferential affinity for nucleosomes containing H3K36me3 in vitro. Our results support a model in which activating complexes, similarly to their silencing counterparts, use the nucleosomal binding specificity of their respective chromodomains to spread from initiation sites to flanking chromatin. [ABSTRACT FROM AUTHOR]

We have programmed human cells to express physiological levels of recombinant RNA polymerase II (RNAPII) subunits carrying tandem affinity purification (TAP) tags. Double-affinity chromatography allowed for the simple and efficient isolation of a complex containing all 12 RNAPII subunits, the general transcription factors TFIIB and TFIIF, the RNAPII phosphatase Fcp1, and a novel 153-kDa polypeptide of unknown function that we named RNApII-associated protein 1 (RPAPI). The TAp-tagged RNAPII complex is functionally active both in vitro and in vivo. A role for RPAP1 in RNAPII transcription was established by shutting off the synthesis of Ydr527wp, a Saccharomyces cerevisiae protein homologous to RPAP1, and demonstrating that changes in global gene expression were similar to those caused by the loss of the yeast RNAPII subunit Rpb11. We also used TAp-tagged Rpb2 with mutations in fork loop 1 and switch 3, two structural elements located strategically within the active center, to start addressing the roles of these elements in the interaction of the enzyme with the template DNA during the transcription reaction. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

KAT6 histone acetyltransferases (HATs) are highly conserved in eukaryotes and have been shown to play important roles in transcriptional regulation. Here, we demonstrate that the Drosophila KAT6 Enok acetylates histone H3 Lys 23 (H3K23) in vitro and in vivo. Mutants lacking functional Enok exhibited defects in the localization of Oskar (Osk) to the posterior end of the oocyte, resulting in loss of germline formation and abdominal segments in the embryo. RNA sequencing (RNA-seq) analysis revealed that spire (spir) and maelstrom (mael), both required for the posterior localization of Osk in the oocyte, were down-regulated in enok mutants. Chromatin immunoprecipitation showed that Enok is localized to and acetylates H3K23 at the spir and mael genes. Furthermore, Gal4-driven expression of spir in the germline can largely rescue the defective Osk localization in enok mutant ovaries. Our results suggest that the Enok-mediated H3K23 acetylation (H3K23Ac) promotes the expression of spir, providing a specific mechanism linking oocyte polarization to histone modification. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Summary: 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. [Copyright &y& Elsevier]

Chromatin regulators play fundamental roles in the regulation of gene expression and chromosome maintenance, but the regions of the genome where most of these regulators function has not been established. We explored the genome-wide occupancy of four different chromatin regulators encoded in Saccharomyces cerevisiae. The results reveal that the histone acetyltransferases Gcn5 and Esa1 are both generally recruited to the promoters of active protein-coding genes. In contrast, the histone deacetylases Hst1 and Rpd3 are recruited to specific sets of genes associated with distinct cellular functions. Our results provide new insights into the association of histone acetyltransferases and histone deacetylases with the yeast genome, and together with previous studies, suggest how these chromatin regulators are recruited to specific regions of the genome. [Copyright &y& Elsevier]

The SWI/SNF complex is required for the expression of many yeast genes. Previous studies have implicated DNA binding transcription activators in targeting SWI/SNF to UASs and promoters. To determine how activators interact with the complex and to examine the importance of these interactions, relative to other potential targeting mechanisms, for SWI/SNF function, we sought to identify and mutate the activator-interaction domains in the complex. Here we show that the N-terminal domain of Snf5 and the second quarter of Swi1 are sites of activation domain contact. Deletion of both of these domains left the SWI/SNF complex intact but impaired its ability to bind activation domains. Importantly, while deletion of either domain alone had minor phenotypic effect, deletion of both resulted in strong SWI/SNF related phenotypes. Thus, two distinct activator-interaction domains play overlapping roles in the targeting activity of SWI/SNF, which is essential for its function in vivo. [Copyright &y& Elsevier]