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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.

Maintenance of a regular chromatin structure over the coding regions of genes occurs co-transcriptionally via the ‘chromatin resetting' pathway. One of the central players in this pathway is the histone methyltransferase Set2. Here we show that the loss of Set2 in yeast, Saccharomyces cerevisiae, results in transcription initiation of antisense RNAs embedded within body of protein-coding genes. These RNAs are distinct from the previously identified non-coding RNAs and cover 11% of the yeast genome. These RNA species have been named Set2-repressed antisense transcripts (SRATs) since the co-transcriptional addition of the H3K36 methyl mark by Set2 over their start sites results in their suppression. Interestingly, loss of chromatin resetting factor Set2 or the subsequent production of SRATs does not affect the abundance of the sense transcripts. This difference in transcriptional outcomes of overlapping transcripts due to a strand-independent addition of H3K36 methylation is a key regulatory feature of interleaved transcriptomes., Maintenance of chromatin structure in coding regions is partially dependent on transcription, with histone methyltransferase Set2 playing a role in this process. Here, the authors provide evidence that Set2 regulates repression of a specific set of antisense RNAs embedded within the coding genes.

Set2 is a RNA Polymerase II (RNAPII) associated histone methyltransferase involved in the co-transcriptional methylation of the H3 K36 residue (H3K36me). It is responsible for multiple degrees of methylation (mono-, di-, tri-), each of which has a distinct functional consequence. The extent of methylation and its genomic distribution is determined by different factors that co-ordinate 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.

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.

Set2-mediated methylation of histone H3 Lys36 (H3K36) is a mark associated with the coding sequences of actively transcribed genes, yet plays 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 and thus maintains chromatin integrity during transcription elongation by RNA polymerase II.

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.

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. Our recent studies show how 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.

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.

Recognition of modified histones by “reader” proteins plays a critical role in the regulation of chromatin1. H3K36 trimethylation (H3K36me3) is deposited onto the nucleosomes in the transcribed regions following RNA polymerase II (Pol II) elongation. In yeast, this mark in turn recruits epigenetic regulators to reset the chromatin to a relatively repressive state thus suppressing cryptic transcription2. However, much less is known about the role of H3K36me3 in transcription regulation in mammals. This is further complicated by the transcription-coupled incorporation of the histone variant H3.3 in gene bodies3. Here we show that the candidate tumor suppressor ZMYND11 specifically recognizes H3K36me3 on H3.3 (H3.3K36me3) and regulates Pol II elongation. Structural studies reveal that in addition to the trimethyl-lysine binding by an aromatic cage within the PWWP domain, the H3.3-dependent recognition is mediated by the encapsulation of the H3.3-specific “Ser31” residue in a composite pocket formed by the tandem bromo-PWWP domains of ZMYND11. ChIP-sequencing analyses reveal a genome-wide colocalization of ZMYND11 with H3K36me3 and H3.3 in gene bodies, and its occupancy requires the pre-deposition of H3.3K36me3. Although ZMYND11 is associated with highly expressed genes, it functions as an unconventional transcription corepressor via modulating Pol II at the elongation stage. ZMYND11 is critical for the repression of a transcriptional program that is essential for tumor cell growth; low expression level of ZMYND11 in breast cancer patients correlates with worse prognosis. Consistently, overexpression of ZMYND11 suppresses cancer cell growth in vitro and tumor formation in mice. Together, this study identifies ZMYND11 as an H3.3-specific reader of H3K36me3 that links the histone variant-mediated transcription elongation control to tumor suppression.

During the process of transcription elongation, the chromatin structure of transcribed sequences can be perturbed, exposing cryptic promoter-like sequences within the body of transcribed genes to function as initiation sites. Re-establishing a stable repressive structure of open reading frames requires histone chaperones, methyltransferases, deacetylases and chromatin remodeling complexes. The Set2/Rpd3S pathway is used by elongating RNA polymerase II to signal for histone deacetylation in its wake. Set2 associates with the elongating form of RNA polymerase II and co-transcriptionally methylates histone H3K36. H3K36 is recognized by the Rpd3S deacetylase complex to deacetylate histones in transcribed sequences. In recent work, we have found that a major source of cotranscriptional histone acetylation is the incorporation of soluble, Rtt109 H3K56-acetylated histones by the Asf1 histone chaperone. Set2 methylation of H3K36 promotes retention of the original histones and suppresses the incorporation of soluble histones by Asf1. By identifying factors that interact with H3K36 methylated nucleosomes, we have found that chromatin remodeling is also required to stabilize the chromatin structures of open reading frames following transcription elongation. Our studies identified the ATP-dependent chromatin remodelers Isw1 and Chd1 as two factors involved in this pathway as deletion of ISW1 and CHD1 enhanced the cryptic transcript phenotype caused by set2. Moreover, loss of ISW1 and CHD1 also enhanced the incorporation of new histones from the soluble pool into chromatin. Thus, retention of original histones, deacetylation of any new ones and their organization by chromatin remodeling are all required to re-establish stable chromatin resistant to cryptic transcription initiation.

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.