Lys Methylation Research Articles

Dynamic Phosphorylation of G9a Regulates its Repressive Activity on Chromatin Accessibility and Mitotic Progression.

Phosphorylation of Ser10 of histone H3 (H3S10p), together with the adjacent methylation of Lys9 (H3K9me), has been proposed to function as a 'phospho-methyl switch' to regulate mitotic chromatin architecture. Despite of immense understanding of the roles of H3S10 phosphorylation, how H3K9me2 are dynamically regulated during mitosis is poorly understood. Here, it is identified that Plk1 kinase phosphorylates the H3K9me1/2 methyltransferase G9a/EHMT2 at Thr1045 (pT1045) during early mitosis, which attenuates its catalytic activity toward H3K9me2. Cells bearing Thr1045 phosphomimic mutant of G9a (T1045E) show decreased H3K9me2 levels, increased chromatin accessibility, and delayed mitotic progression. By contrast, dephosphorylation of pT1045 during late mitosis by the protein phosphatase PPP2CB reactivates G9a activity and upregulates H3K9me2 levels, correlated with decreased levels of H3S10p. Therefore, the results provide a mechanistic explanation of the essential of a 'phospho-methyl switch' and highlight the importance of Plk1 and PPP2CB-mediated dynamic regulation of G9a activity in chromatin organization and mitotic progression.

New insight into the composition of extracellular traps released by macrophages exposed to different types of inducers.

Neutrophil extracellular trap (NET) release plays a key role in many chronic disease settings, including atherosclerosis. They are critical to innate immune defence, but also contribute to disease by promoting thrombosis and inflammation. Macrophages are known to release extracellular traps or "METs", but their composition and role in pathological processes are less well defined. In this study, we examined MET release from human THP-1 macrophages exposed to model inflammatory and pathogenic stimuli, including tumour necrosis factor α (TNFα), hypochlorous acid (HOCl) and nigericin. In each case, there was release of DNA from the macrophages, as visualized by fluorescence microscopy with the cell impermeable DNA binding dye SYTOX green, consistent with MET formation. Proteomic analysis on METs released from macrophages exposed to TNFα and nigericin reveals that they are composed of linker and core histones, together with a range of cytosolic and mitochondrial proteins. These include proteins involved in DNA binding, stress responses, cytoskeletal organisation, metabolism, inflammation, anti-microbial activity, and calcium binding. Quinone oxidoreductase in particular, was highly abundant in all METs but has not been reported previously in NETs. Moreover, there was an absence of proteases in METs in contrast to NETs. Some of the MET histones, contained post-translational modifications, including acetylation and methylation of Lys but not citrullination of Arg. These data provide new insight into the potential implications of MET formation in vivo and their contributions to immune defence and pathology.

WDR5 represents a therapeutically exploitable target for cancer stem cells in glioblastoma.

Glioblastomas (GBMs) are heterogeneous, treatment-resistant tumors driven by populations of cancer stem cells (CSCs). However, few molecular mechanisms critical for CSC population maintenance have been exploited for therapeutic development. We developed a spatially resolved loss-of-function screen in GBM patient-derived organoids to identify essential epigenetic regulators in the SOX2-enriched, therapy-resistant niche and identified WDR5 as indispensable for this population. WDR5 is a component of the WRAD complex, which promotes SET1 family-mediated Lys4 methylation of histone H3 (H3K4me), associated with positive regulation of transcription. In GBM CSCs, WDR5 inhibitors blocked WRAD complex assembly and reduced H3K4 trimethylation and expression of genes involved in CSC-relevant oncogenic pathways. H3K4me3 peaks lost with WDR5 inhibitor treatment occurred disproportionally on POU transcription factor motifs, including the POU5F1(OCT4)::SOX2 motif. Use of a SOX2/OCT4 reporter demonstrated that WDR5 inhibitor treatment diminished cells with high reporter activity. Furthermore, WDR5 inhibitor treatment and WDR5 knockdown altered the stem cell state, disrupting CSC in vitro growth and self-renewal, as well as in vivo tumor growth. These findings highlight the role of WDR5 and the WRAD complex in maintaining the CSC state and provide a rationale for therapeutic development of WDR5 inhibitors for GBM and other advanced cancers.

Histone methyltransferase Dot1L recruits O-GlcNAc transferase to target chromatin sites to regulate histone O-GlcNAcylation

O-GlcNAc transferase (OGT) is the distinctive enzyme responsible for catalyzing O-GlcNAc addition to the serine or threonine residues of thousands of cytoplasmic and nuclear proteins involved in such basic cellular processes as DNA damage repair, RNA splicing, and transcription preinitiation and initiation complex assembly. However, the molecular mechanism by which OGT regulates gene transcription remains elusive. Using proximity labeling-based mass spectrometry, here, we searched for functional partners of OGT and identified interacting protein Dot1L, a conserved and unique histone methyltransferase known to mediate histone H3 Lys79 methylation, which is required for gene transcription, DNA damage repair, cell proliferation, and embryo development. Although this specific interaction with OGT does not regulate the enzymatic activity of Dot1L, we show that it does facilitate OGT-dependent histone O-GlcNAcylation. Moreover, we demonstrate that OGT associates with Dot1L at transcription start sites and that depleting Dot1L decreases OGT associated with chromatin globally. Notably, we also show that downregulation of Dot1L reduces the levels of histone H2B S112 O-GlcNAcylation and histone H2B K120 ubiquitination in vivo, which are associated with gene transcription regulation. Taken together, these results reveal that O-GlcNAcylation of chromatin is dependent on Dot1L.

STEM-14. THE WRAD COMPLEX REPRESENTS A THERAPEUTIC TARGET FOR CANCER STEM CELLS IN GLIOBLASTOMA

Abstract Glioblastoma (GBM) progression and resistance to conventional therapies is driven in part by cells within the tumor with stem cell properties including quiescence, self-renewal and drug efflux potential. It is thought that eliminating these cancer stem cells (CSCs) is a key component to successful clinical management of GBM. However, currently, few known molecular mechanisms driving CSCs can be exploited for therapeutic development. Core transcription factors such as SOX2, OLIG2, OCT4 and NANOG maintain the CSC state in GBM. Our laboratory recently uncovered a self-renewal signaling axis involving RBBP5 that is necessary and sufficient for CSC maintenance through driving expression of these core stem cell maintenance transcription factors. RBBP5 is a component of the WRAD complex, which promotes Lys4 methylation of histone H3 to positively regulate transcription. We hypothesized that targeting RBBP5 could be a means to disrupt epigenetic programs that maintain CSCs in stemness transcriptional states. We found that genetic and pharmacologic inhibition of the WRAD complex reduced CSC growth, self-renewal and tumor initiation potential. WRAD inhibitors partially dissembled the WRAD complex and reduced H3K4 trimethylation both globally and at the promoters of key stem cell maintenance transcription factors. Using a CSC reporter system, we demonstrated that WRAD complex inhibition decreased growth of SOX2/OCT4 expressing CSCs in a concentration-dependent manner as quantified by live imaging. Overall, our studies assess the function of the WRAD complex and the effect of WRAD complex inhibitors in preclinical models and specifically on the stem cell state for the first time in GBM. Studying the functions of the WRAD complex in CSCs may improve understanding of GBM pathogenesis and elucidate how CSCs survive despite aggressive chemotherapy and radiation. Our ongoing studies aim to develop brain penetrant inhibitors targeting the WRAD complex as an anti-CSC strategy that could potentially synergize with standard of care treatments.

Hypoxia-inducible factor-1α cooperates with histone Lys methylation to predict prognosis in esophageal squamous cell carcinoma.

Aim: To investigate hypoxia-inducible factor-1α(HIF-1α) and histone methylation markers as potential indicators of prognosis in esophageal squamous cell carcinoma (ESCC). Patients &methods: The prognostic value of HIF-1α and histone methylation markers levelswas analyzed using Kaplan-Meier survival analysis. Results: HIF-1α protein expression was higher in ESCC tumors than in paracancerous tissues. Histone H3 Lys9 trimethylation (H3K9me3), histone H3 Lys27 trimethylation (H3K27me3), histone H3 Lys4trimethylation (H3K4me3)and histone H4 Lys20 trimethylation (H4K20me3) were significantly upregulated in ESCC tissues. HIF-1α was only positively correlated with H3K9me3 and H3K4me3 expression. Kaplan-Meier analysis indicated that H3K9me3, H3K27me3, H4K20me3 and histone H3 Lys36 trimethylation (H3K36me3) were independent indicators of prognosis for ESCC. Conclusion: This study identified a pattern of epigenetic methylation markers and HIF-1α expression in ESCC, and their combined evaluation might improve survival prediction for ESCC patients.

BRCA1/BARD1 site-specific ubiquitylation of nucleosomal H2A is directed by BARD1.

Mutations in BRCA1/BARD1’s E3 ubiquitin ligase RING domains predispose carriers to breast and ovarian cancers. We present the first structure of the BRCA1/BARD1 RING heterodimer with the E2 enzyme UbcH5c bound to its cellular target, the nucleosome, along with biochemical data that explain how the complex selectively ubiquitylates lysines 125/127/129 in the flexible C-terminal tail of H2A in a fully human system. The structure reveals that a novel BARD1-histone interface couples to a repositioning of UbcH5c compared to the structurally similar PRC1 E3 ligase Ring1b/Bmi1 that ubiquitylates H2A Lys119 in nucleosomes. This interface is sensitive to both H3 Lys79 methylation status and mutations found in cancer patients. Furthermore, NMR reveals an unexpected mode of E3-mediated substrate regulation through modulation of dynamics in the C-terminal tail of H2A. Our findings provide insight into how E3 ligases preferentially target nearby lysine residues in nucleosomes by a steric occlusion and distancing mechanism.

BRAF inhibition in melanoma is associated with the dysregulation of histone methylation and histone methyltransferases

The development of mutant BRAF inhibitors has improved the outcome for melanoma patients with BRAFV600E mutations. Although the initial response to these inhibitors can be dramatic, sometimes resulting in complete tumor regression, the majority of melanomas become resistant. To study resistance to BRAF inhibition, we developed a novel mouse model of melanoma using a tetracycline/doxycycline-regulated system that permits control of mutant BRAF expression. Treatment with doxycycline leads to loss of mutant BRAF expression and tumor regression, but tumors recur after a prolonged period of response to treatment. Vemurafenib, encorafenib and dabrafenib induce cell cycle arrest and apoptosis in BRAF melanoma cell lines; however, a residual population of tumor cells survive. Comparing gene expression in human cell lines and mouse tumors can assist with the identification of novel mechanisms of resistance. Accordingly, we conducted RNA sequencing analysis and immunoblotting on untreated and doxycycline-treated dormant mouse melanomas and human mutant BRAF melanoma cell lines treated with 2 μM vemurafenib for 20 days. We found conserved expression changes in histone methyltransferase genes ASH2, EZH2, PRMT5, SUV39H1, SUV39H2, and SYMD2 in P-ERK low, p-38 high melanoma cells following prolonged BRAF inhibition. Quantitative mass spectrometry, determined a corresponding reduction in histone Lys9 and Lys27 methylation and increase in Lys36 methylation in melanoma cell lines treated with 2 μM vemurafenib for 20 days. Thus, these changes as are part of the initiate response to BRAF inhibition and likely contribute to the survival of melanoma cells.

Proteome-wide Prediction of Lysine Methylation Leads to Identification of H2BK43 Methylation and Outlines the Potential Methyllysine Proteome

Protein Lys methylation plays a critical role in numerous cellular processes, but it is challenging to identify Lys methylation in a systematic manner. Here we present an approach combining in silico prediction with targeted mass spectrometry (MS) to identify Lys methylation (Kme) sites at the proteome level. We develop MethylSight, a program that predicts Kme events solely on the physicochemical properties of residues surrounding the putative methylation sites, which then requires validation by targeted MS. Using this approach, we identify 70 new histone Kme marks with a 90% validation rate. H2BK43me2, which undergoes dynamic changes during stem cell differentiation, is found to be a substrate of KDM5b. Furthermore, MethylSight predicts that Lys methylation is a prevalent post-translational modification in the human proteome. Our work provides a useful resource for guiding systematic exploration of the role of Lys methylation in human health and disease.

The polycomb group protein PCGF6 mediates germline gene silencing by recruiting histone-modifying proteins to target gene promoters

Polycomb group (PcG) proteins are essential for maintenance of lineage fidelity by coordinating developmental gene expression programs. Polycomb group ring finger 6 (PCGF6) has been previously reported to repress expression of lineage-specific genes, especially germ cell-related genes in mouse embryonic stem cells (ESCs) via the noncanonical polycomb repressive complex PRC1.6. However, the molecular mechanism of this repression remains largely unknown. Here, using RNA-Seq, real-time RT-PCR, immunohistochemistry, immunoprecipitation, and ChIP analyses, we demonstrate that PCGF6 plays an essential role in embryonic development, indicated by the partially penetrant embryonic lethality in homozygous PCGF6 (Pcgf6-/-)-deficient mice. We also found that surviving Pcgf6-deficient mice exhibit reduced fertility. Using the Pcgf6-deficient mice, we observed that ablation of Pcgf6 in somatic tissues robustly derepresses germ cell-related genes. We further provide evidence that these genes are direct targets of PCGF6 in ESCs and that endogenous PCGF6 co-localizes with the histone-modifying proteins G9A histone methyltransferase (G9A)/G9a-like protein (GLP) and histone deacetylase 1/2 (HDAC1/2) on the promoters of the germ cell-related genes. Moreover, the binding of these proteins to their target genes correlated with methylation of Lys-9 of histone 3 and with the status of histone acetylation at these genes. Moreover, the recruitment of G9A/GLP and HDAC1/2 to target promoters depended on the binding of PCGF6. Our findings indicate that PCGF6 has a critical role in safeguarding lineage decisions and in preventing aberrant expression of germ cell-related genes.

Macrocyclic Peptides Uncover a Novel Binding Mode for Reversible Inhibitors of LSD1.

Lysine-specific demethylase 1 (LSD1) is an epigenetic enzyme which regulates the methylation of Lys4 of histone 3 (H3) and is overexpressed in certain cancers. We used structures of H3 substrate analogues bound to LSD1 to design macrocyclic peptide inhibitors of LSD1. A linear, Lys4 to Met-substituted, 11-mer (4) was identified as the shortest peptide distinctly interacting with LSD1. It was evolved into macrocycle 31, which was >40 fold more potent (Ki = 2.3 μM) than 4. Linear and macrocyclic peptides exhibited unexpected differences in structure–activity relationships for interactions with LSD1, indicating that they bind LSD1 differently. This was confirmed by the crystal structure of 31 in complex with LSD1-CoREST1, which revealed a novel binding mode at the outer rim of the LSD1 active site and without a direct interaction with FAD. NMR spectroscopy of 31 suggests that macrocyclization restricts its solution ensemble to conformations that include the one in the crystalline complex. Our results provide a solid basis for the design of optimized reversible LSD1 inhibitors.

Structural basis of recognition and destabilization of the histone H2B ubiquitinated nucleosome by the DOT1L histone H3 Lys79 methyltransferase.

DOT1L is a histone H3 Lys79 methyltransferase whose activity is stimulated by histone H2B Lys120 ubiquitination, suggesting cross-talk between histone H3 methylation and H2B ubiquitination. Here, we present cryo-EM structures of DOT1L complexes with unmodified or H2B ubiquitinated nucleosomes, showing that DOT1L recognizes H2B ubiquitin and the H2A/H2B acidic patch through a C-terminal hydrophobic helix and an arginine anchor in DOT1L, respectively. Furthermore, the structures combined with single-molecule FRET experiments show that H2B ubiquitination enhances a noncatalytic function of the DOT1L-destabilizing nucleosome. These results establish the molecular basis of the cross-talk between H2B ubiquitination and H3 Lys79 methylation as well as nucleosome destabilization by DOT1L.

Rpp29 regulates histone H3.3 chromatin assembly through transcriptional mechanisms

The histone H3 variant H3.3 is a highly conserved and dynamic regulator of chromatin organization. Therefore, fully elucidating its nucleosome incorporation mechanisms is essential to understanding its functions in epigenetic inheritance. We previously identified the RNase P protein subunit, Rpp29, as a repressor of H3.3 chromatin assembly. Here, we use a biochemical assay to show that Rpp29 interacts with H3.3 through a sequence element in its own N terminus, and we identify a novel interaction with histone H2B at an adjacent site. The fact that archaeal Rpp29 does not include this N-terminal region suggests that it evolved to regulate eukaryote-specific functions. Oncogenic H3.3 mutations alter the H3.3-Rpp29 interaction, which suggests that they could dysregulate Rpp29 function in chromatin assembly. We also used KNS42 cells, an H3.3(G34V) pediatric high-grade glioma cell line, to show that Rpp29 1) represses H3.3 incorporation into transcriptionally active protein-coding, rRNA, and tRNA genes; 2) represses mRNA, protein expression, and antisense RNA; and 3) represses euchromatic post-translational modifications (PTMs) and promotes heterochromatic PTM deposition (i.e. histone H3 Lys-9 trimethylation (H3K9me3) and H3.1/2/3K27me3). Notably, we also found that K27me2 is increased and K36me1 decreased on H3.3(G34V), which suggests that Gly-34 mutations dysregulate Lys-27 and Lys-36 methylation in cis The fact that Rpp29 represses H3.3 chromatin assembly and sense and antisense RNA and promotes H3K9me3 and H3K27me3 suggests that Rpp29 regulates H3.3-mediated epigenetic mechanisms by processing a transcribed signal that recruits H3.3 to its incorporation sites.

Biochemical and dynamic basis for combinatorial recognition of H3R2K9me2 by dual domains of UHRF1

UHRF1 is a multi-domain protein comprising of a tandem tudor (UHRF1 TTD), a PHD finger, and a SET and RING-associated domain. It is required for the maintenance of CG methylation, heterochromatin formation and DNA repair. Isothermal titration calorimetry binding studies of unmodified and methylated lysine histone peptides establish that the UHRF1 TTD binds dimethylated Lys9 on histone H3 (H3K9me2). Further, MD simulation and binding studies reveal that TTD-PHD of UHRF1 (UHRF1 TTD-PHD) preferentially recognizes dimethyl-lysine status. Importantly, we show that Asp145 in the binding pocket determines the preferential recognition of the dimethyl-ammonium group of H3K9me2. Interestingly, PHD finger of the UHRF1 TTD-PHD has a negligible contribution to the binding affinity for recognition of K9me2 by the UHRF1 TTD. Surprisingly, Lys4 methylation on H3 peptide has an insignificant effect on combinatorial recognition of R2 and K9me2 on H3 by the UHRF1 TTD-PHD. We propose that subtle variations of key residues at the binding pocket determine status specific recognition of histone methyl-lysines by the reader domains.

Widespread transcriptional pausing and elongation control at enhancers.

Regulation by gene-distal enhancers is critical for cell type-specific and condition-specific patterns of gene expression. Thus, to understand the basis of gene activity in a given cell type or tissue, we must identify the precise locations of enhancers and functionally characterize their behaviors. Here, we demonstrate that transcription is a nearly universal feature of enhancers in Drosophila and mammalian cells and that nascent RNA sequencing strategies are optimal for identification of both enhancers and superenhancers. We dissect the mechanisms governing enhancer transcription and discover remarkable similarities to transcription at protein-coding genes. We show that RNA polymerase II (RNAPII) undergoes regulated pausing and release at enhancers. However, as compared with mRNA genes, RNAPII at enhancers is less stable and more prone to early termination. Furthermore, we found that the level of histone H3 Lys4 (H3K4) methylation at enhancers corresponds to transcriptional activity such that highly active enhancers display H3K4 trimethylation rather than the H3K4 monomethylation considered a hallmark of enhancers. Finally, our work provides insights into the unique characteristics of superenhancers, which stimulate high-level gene expression through rapid pause release; interestingly, this property renders associated genes resistant to the loss of factors that stabilize paused RNAPII.

Winged eye Induces Transdetermination of Drosophila Imaginal Disc by Acting in Concert with a Histone Methyltransferase, Su(var)3-9

Drosophila imaginal disc cells exhibit a remarkable ability to convert cell fates in response to various perturbations, a phenomenon called transdetermination (TD). We previously identified winged eye (wge) as a factor that induces eye-to-wing TD upon overexpression in eye imaginal discs, but the molecular mechanisms underlying TD have remained largely unclear. Here, we found that wge induces various histone modifications and enhances the methylation of Lys9 on histone H3 (H3K9), a feature of heterochromatin. A histone methyltransferase, Su(var)3-9, is required for wge-mediated H3K9 methylation and eye-to-wing TD. Su(var)3-9 is also required for classical wound-induced TD but not for normal development, suggesting its involvement in several types of imaginal disc TDs. Transcriptome analysis revealed that wge represses eye identity genes independently of Su(var)3-9 and activates TD-related genes by acting together with Su(var)3-9. These findings provide new insights into diverse types of chromatin regulation at progressive steps of cell-fate conversions.

Functional Redundancy of Variant and Canonical Histone H3 Lysine 9 Modification in Drosophila.

Histone post-translational modifications (PTMs) and differential incorporation of variant and canonical histones into chromatin are central modes of epigenetic regulation. Despite similar protein sequences, histone variants are enriched for different suites of PTMs compared to their canonical counterparts. For example, variant histone H3.3 occurs primarily in transcribed regions and is enriched for "active" histone PTMs like Lys9 acetylation (H3.3K9ac), whereas the canonical histone H3 is enriched for Lys9 methylation (H3K9me), which is found in transcriptionally silent heterochromatin. To determine the functions of K9 modification on variant vs. canonical H3, we compared the phenotypes caused by engineering H3.3K9R and H3K9R mutant genotypes in Drosophila melanogaster Whereas most H3.3K9R , and a small number of H3K9R , mutant animals are capable of completing development and do not have substantially altered protein-coding transcriptomes, all H3.3K9R H3K9R combined mutants die soon after embryogenesis and display decreased expression of genes enriched for K9ac. These data suggest that the role of K9ac in gene activation during development can be provided by either H3 or H3.3. Conversely, we found that H3.3K9 is methylated at telomeric transposons and that this mark contributes to repressive chromatin architecture, supporting a role for H3.3 in heterochromatin that is distinct from that of H3. Thus, our genetic and molecular analyses demonstrate that K9 modification of variant and canonical H3 have overlapping roles in development and transcriptional regulation, though to differing extents in euchromatin and heterochromatin.

A cryptic Tudor domain links BRWD2/PHIP to COMPASS-mediated histone H3K4 methylation.

Histone H3 Lys4 (H3K4) methylation is a chromatin feature enriched at gene cis-regulatory sequences such as promoters and enhancers. Here we identify an evolutionarily conserved factor, BRWD2/PHIP, which colocalizes with histone H3K4 methylation genome-wide in human cells, mouse embryonic stem cells, and Drosophila Biochemical analysis of BRWD2 demonstrated an association with the Cullin-4-RING ubiquitin E3 ligase-4 (CRL4) complex, nucleosomes, and chromatin remodelers. BRWD2/PHIP binds directly to H3K4 methylation through a previously unidentified chromatin-binding module related to Royal Family Tudor domains, which we named the CryptoTudor domain. Using CRISPR-Cas9 genetic knockouts, we demonstrate that COMPASS H3K4 methyltransferase family members differentially regulate BRWD2/PHIP chromatin occupancy. Finally, we demonstrate that depletion of the single Drosophila homolog dBRWD3 results in altered gene expression and aberrant patterns of histone H3 Lys27 acetylation at enhancers and promoters, suggesting a cross-talk between these chromatin modifications and transcription through the BRWD protein family.

Post-translational Modifications in Regulation of Chloroplast Function: Recent Advances.

Post-translational modifications (PTMs) of proteins enable fast modulation of protein function in response to metabolic and environmental changes. Phosphorylation is known to play a major role in regulating distribution of light energy between the Photosystems (PS) I and II (state transitions) and in PSII repair cycle. In addition, thioredoxin-mediated redox regulation of Calvin cycle enzymes has been shown to determine the efficiency of carbon assimilation. Besides these well characterized modifications, recent methodological progress has enabled identification of numerous other types of PTMs in various plant compartments, including chloroplasts. To date, at least N-terminal and Lys acetylation, Lys methylation, Tyr nitration and S-nitrosylation, glutathionylation, sumoylation and glycosylation of chloroplast proteins have been described. These modifications impact DNA replication, control transcriptional efficiency, regulate translational machinery and affect metabolic activities within the chloroplast. Moreover, light reactions of photosynthesis as well as carbon assimilation are regulated at multiple levels by a number of PTMs. It is likely that future studies will reveal new metabolic pathways to be regulated by PTMs as well as detailed molecular mechanisms of PTM-mediated regulation.

The Eaf3/5/7 Subcomplex Stimulates NuA4 Interaction with Methylated Histone H3 Lys-36 and RNA Polymerase II

NuA4 is the only essential lysine acetyltransferase complex in Saccharomyces cerevisiae, where it has been shown to stimulate transcription initiation and elongation. Interaction with nucleosomes is stimulated by histone H3 Lys-4 and Lys-36 methylation, but the mechanism of this interaction is unknown. Eaf3, Eaf5, and Eaf7 form a subcomplex within NuA4 that may also function independently of the lysine acetyltransferase complex. The Eaf3/5/7 complex and the Rpd3C(S) histone deacetylase complex have both been shown to bind di- and trimethylated histone H3 Lys-36 stimulated by Eaf3. We investigated the role of the Eaf3/5/7 subcomplex in NuA4 binding to nucleosomes. Different phenotypes of eaf3/5/7Δ mutants support functions for the complex as both part of and independent of NuA4. Further evidence for Eaf3/5/7 within NuA4 came from mutations in the subcomplex leading to ∼40% reductions in H4 acetylation in bulk histones, probably caused by binding defects to both nucleosomes and RNA polymerase II. In vitro binding assays showed that Eaf3/5/7 specifically stimulates NuA4 binding to di- and trimethylated histone H3 Lys-36 and that this binding is important for NuA4 occupancy in transcribed ORFs. Consistent with the role of NuA4 in stimulating transcription elongation, loss of EAF5 or EAF7 resulted in a processivity defect. Overall, these results reveal the function of Eaf3/5/7 within NuA4 to be important for both NuA4 and RNA polymerase II binding.