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Loss of the short arm of chromosome 3 (3p) occurs early in >95% of clear cell renal cell carcinoma (ccRCC). Nearly ubiquitous 3p loss in ccRCC suggests haploinsufficiency for 3p tumor suppressors as early drivers of tumorigenesis. We previously reported methyltransferase SETD2, which trimethylates H3 histones on lysine 36 (H3K36me3) and is located in the 3p deletion, to also trimethylate microtubules on lysine 40 (αTubK40me3) during mitosis, with αTubK40me3 required for genomic stability. We now show that monoallelic, Setd2-deficient cells retaining H3K36me3, but not αTubK40me3, exhibit a dramatic increase in mitotic defects and micronuclei count, with increased viability compared with biallelic loss. In SETD2-inactivated human kidney cells, rescue with a pathogenic SETD2 mutant deficient for microtubule (αTubK40me3), but not histone (H3K36me3) methylation, replicated this phenotype. Genomic instability (micronuclei) was also a hallmark of patient-derived cells from ccRCC. These data show that the SETD2 tumor suppressor displays a haploinsufficiency phenotype disproportionately impacting microtubule methylation and serves as an early driver of genomic instability.Significance: Loss of a single allele of a chromatin modifier plays a role in promoting oncogenesis, underscoring the growing relevance of tumor suppressor haploinsufficiency in tumorigenesis. Cancer Res; 78(12); 3135–46. ©2018 AACR.

SETD2 loss promotes chromosomal abnormalities.

fig legends for sup figures

Histone post-translational modifications (PTMs) are important forregulating various DNA-templated processes. Here, we report theexistence of a histone PTM in mammalian cells, namely histone H3 withhydroxylation of proline at residue 16 (H3P16oh), which is catalyzed by theproline hydroxylase EGLN2. We show that H3P16oh enhances direct bindingof KDM5A to its substrate, histone H3 with trimethylation at the fourthlysine residue (H3K4me3), resulting in enhanced chromatin recruitmentof KDM5A and a corresponding decrease of H3K4me3 at target genes.Genome- and transcriptome-wide analyses show that the EGLN2–KDM5Aaxis regulates target gene expression in mammalian cells. Specifically, ourdata demonstrate repression of the WNT pathway negative regulator DKK1through the EGLN2-H3P16oh-KDM5A pathway to promote WNT/β-cateninsignaling in triple-negative breast cancer (TNBC). This study characterizesa regulatory mark in the histone code and reveals a role for H3P16oh inregulating mammalian gene expression

The Chromosome Passenger Complex (CPC) generates chromosome autonomous signals that regulate mitotic events critical for genome stability. Tip60 is a lysine acetyltransferase that is a tumor suppressor and is targeted for proteasomal degradation by oncogenic papilloma viruses. Mitotic regulation requires the localization of the CPC to inner centromeres, which is driven by the Haspin kinase phosphorylating histone H3 on threonine 3 (H3T3ph). Here we describe how Tip60 acetylates histone H3 at lysine 4 (H3K4ac) to block both the H3T3ph writer and the reader to ensure that this mitotic signaling cannot begin before prophase. Specifically, H3K4ac inhibits Haspin phosphorylation of H3T3 and prevents binding of the Survivin subunit to H3T3ph. Tip60 acetylates H3K4 during S/G2 at centromeres. Inhibition of Tip60 allows the CPC to bind centromeres in G2 cells, and targeting of Tip60 to centromeres prevents CPC localization in mitosis. The H3K4ac mark is removed in prophase by HDAC3 to initiate the CPC localization cascade. Together, our results suggest that Tip60 and HDAC3 temporally control H3K4 acetylation to precisely time the targeting of the CPC to inner centromeres.

Mono-methylation of histone H4 lysine 20 (H4K20me1) is catalyzed by Set8/KMT5A and regulates numerous aspects of genome organization and function. Loss-of-function mutations in Drosophila melanogaster Set8 or mammalian KMT5A prevent H4K20me1 and disrupt development. Set8/KMT5A also has non-histone substrates, making it difficult to determine which developmental functions of Set8/KMT5A are attributable to H4K20me1 and which to other substrates or to non-catalytic roles. Here, we show that human KMT5A can functionally substitute for Set8 during Drosophila development and that the catalytic SET domains of the two enzymes are fully interchangeable. We also uncovered a role in eye development for the N-terminal domain of Set8 that cannot be complemented by human KMT5A. Whereas Set8null mutants are inviable, we found that an R634G mutation in the SET domain predicted to ablate catalytic activity resulted in viable adults, suggesting important non-catalytic functions of Set8. Similarly, flies that were engineered to express only unmodifiable H4 histones (H4K20A) can also complete development, but they are phenotypically distinct from H4K20R, Set8null, and Set8R634G animals. Taken together, our results demonstrate functional conservation of KMT5A and Set8 enzymes, as well as distinct roles for Set8 and H4K20me1 in Drosophila development.

Trimethylated histone H3 lysine 27 (H3K27me3) regulates gene repression, cell-fate determination and differentiation. We report that a conserved bromo-adjacent homology (BAH) module of BAHCC1 (BAHCC1BAH) 'recognizes' H3K27me3 specifically and enforces silencing of H3K27me3-demarcated genes in mammalian cells. Biochemical, structural and integrated chromatin immunoprecipitation-sequencing-based analyses demonstrate that direct readout of H3K27me3 by BAHCC1 is achieved through a hydrophobic trimethyl-L-lysine-binding 'cage' formed by BAHCC1BAH, mediating colocalization of BAHCC1 and H3K27me3-marked genes. BAHCC1 is highly expressed in human acute leukemia and interacts with transcriptional corepressors. In leukemia, depletion of BAHCC1, or disruption of the BAHCC1BAH-H3K27me3 interaction, causes derepression of H3K27me3-targeted genes that are involved in tumor suppression and cell differentiation, leading to suppression of oncogenesis. In mice, introduction of a germline mutation at Bahcc1 to disrupt its H3K27me3 engagement causes partial postnatal lethality, supporting a role in development. This study identifies an H3K27me3-directed transduction pathway in mammals that relies on a conserved BAH 'reader'.

Human Microrchidia 4 (MORC4) is associated with acute and chronic pancreatitis, inflammatory disorders and cancer but it remains largely uncharacterized. Here, we describe the structure–function relationship of MORC4 and define the molecular mechanism for MORC4 activation. Enzymatic and binding assays reveal that MORC4 has ATPase activity, which is dependent on DNA-binding functions of both the ATPase domain and CW domain of MORC4. The crystal structure of the ATPaseCW cassette of MORC4 and mutagenesis studies show that the DNA-binding site and the histone/ATPase binding site of CW are located on the opposite sides of the domain. The ATPase and CW domains cooperate in binding of MORC4 to the nucleosome core particle (NCP), enhancing the DNA wrapping around the histone core and impeding binding of DNA-associated proteins, such as transcription factors, to the NCP. In cells, MORC4 mediates formation of nuclear bodies in the nucleus and has a role in the progression of S-phase of the cell cycle, and both these functions require CW and catalytic activity of MORC4. Our findings highlight the mechanism for MORC4 activation, which is distinctly different from the mechanisms of action observed in other MORC family members., Human Microrchidia 4 (MORC4) ATPase has been implicated in acute and chronic pancreatitis, inflammatory disorders and cancer. Here the authors describe the structure–function relationship of MORC4 and define the molecular mechanism for MORC4 activation.

In nucleosomes, histone N-terminal tails exist in dynamic equilibrium between free/accessible and collapsed/DNA-bound states. The latter state is expected to impact histone N-termini availability to the epigenetic machinery. Notably, H3 tail acetylation (e.g., K9ac, K14ac, K18ac) is linked to increased H3K4me3 engagement by the BPTF PHD finger, but it is unknown if this mechanism has broader extension. Here we show that H3 tail acetylation promotes nucleosomal accessibility to other H3K4 methyl readers, and importantly, extends to H3K4 writers, notably methyltransferase MLL1. This regulation is not observed on peptide substrates yet occurs on thecisH3 tail, as determined with fully-defined heterotypic nucleosomes.In vivo, H3 tail acetylation is directly and dynamically coupled withcisH3K4 methylation levels. Together, these observations reveal an acetylation ‘chromatin switch’ on the H3 tail that modulates read-write accessibility in nucleosomes and resolve the long-standing question of why H3K4me3 levels are coupled with H3 acetylation.

The assembly and function of the yeast general transcription factor TFIID complex requires specific contacts between its Taf14 and Taf2 subunits, however, the mechanism underlying these contacts remains unclear. Here, we determined the molecular and structural basis by which the YEATS and ET domains of Taf14 bind to the C-terminal tail of Taf2 and identified a unique DNA-binding activity of the linker region connecting the two domains. We show that in the absence of ligands the linker region of Taf14 is occluded by the surrounding domains, and therefore the DNA binding function of Taf14 is autoinhibited. Binding of Taf2 promotes a conformational rearrangement in Taf14, resulting in a release of the linker for the engagement with DNA and the nucleosome. Genetic in vivo data indicate that the association of Taf14 with both Taf2 and DNA is essential for transcriptional regulation. Our findings provide a basis for deciphering the role of individual TFIID subunits in mediating gene transcription.

How transcription programs rapidly adjust to changing metabolic and cellular cues remains poorly defined. Here, we reveal a function for the Yaf9 component of the SWR1-C and NuA4 chromatin regulatory complexes in maintaining timely transcription of metabolic genes across the yeast metabolic cycle (YMC). By reading histone acetylation during the oxidative and respiratory phase of the YMC, Yaf9 recruits SWR1-C and NuA4 complexes to deposit H2A.Z and acetylate H4, respectively. Increased H2A.Z and H4 acetylation during the oxidative phase promotes transcriptional initiation and chromatin machinery occupancy and is associated with reduced RNA polymerase II levels at genes—a pattern reversed during transition from oxidative to reductive metabolism. Prevention of Yaf9-H3 acetyl reading disrupted this pattern of transcriptional and chromatin regulator recruitment and impaired the timely transcription of metabolic genes. Together, these findings reveal that Yaf9 contributes to a dynamic chromatin and transcription initiation factor signature that is necessary for the proper regulation of metabolic gene transcription during the YMC. They also suggest that unique regulatory mechanisms of transcription exist at distinct metabolic states.

Histone post-translational modifications (PTMs) are crucial for many cellular processes including mitosis, transcription, and DNA repair. The cellular readout of histone PTMs is dependent on both the chemical modification and histone site, and the array of histone PTMs on chromatin is dynamic throughout the eukaryotic life cycle. Accordingly, methods that report on the presence of PTMs are essential tools for resolving open questions about epigenetic processes and for developing therapeutic diagnostics. Reader domains that recognize histone PTMs have shown potential as advantageous substitutes for anti-PTM antibodies, and engineering efforts aimed at enhancing reader domain affinities would advance their efficacy as antibody alternatives. Here we describe engineered chromodomains from Drosophila melanogaster and humans that bind more tightly to H3K9 methylation (H3K9me) marks and result in the tightest reported reader-H3K9me interaction to date. Point mutations near the binding interface of the HP1 chromodomain were screened in a combinatorial fashion, and a triple mutant was found that binds 20-fold tighter than the native scaffold without any loss in PTM-site selectivity. The beneficial mutations were then translated to a human homologue, CBX1, resulting in an even tighter interaction with H3K9me3. Furthermore, we show that these engineered readers (eReaders) increase detection of H3K9me marks in several biochemical assays and outperform a commercial anti-H3K9me antibody in detecting H3K9me-containing nucleosomes in vitro, demonstrating the utility of eReaders to complement antibodies in epigenetics research.

Acetylation of histone H3K23 has emerged as an essential posttranslational modification associated with cancer and learning and memory impairment, yet our understanding of this epigenetic mark remains insufficient. Here, we identify the native MORF complex as a histone H3K23-specific acetyltransferase and elucidate its mechanism of action. The acetyltransferase function of the catalytic MORF subunit is positively regulated by the DPF domain of MORF (MORFDPF). The crystal structure of MORFDPF in complex with crotonylated H3K14 peptide provides mechanistic insight into selectivity of this epigenetic reader and its ability to recognize both histone and DNA. ChIP data reveal the role of MORFDPF in MORF-dependent H3K23 acetylation of target genes. Mass spectrometry, biochemical and genomic analyses show co-existence of the H3K23ac and H3K14ac modifications in vitro and co-occupancy of the MORF complex, H3K23ac, and H3K14ac at specific loci in vivo. Our findings suggest a model in which interaction of MORFDPF with acylated H3K14 promotes acetylation of H3K23 by the native MORF complex to activate transcription., Acetylation of histone H3K23 has emerged as an essential posttranslational modification, yet this epigenetic mark remains poorly understood. Here, the authors identify the native MORF complex as a histone H3K23-specific acetyltransferase and show that interaction of the MORF subunit with acylated H3K14 promotes acetylation of H3K23 by this complex to activate transcription.

Histone post-translational modifications contribute to chromatin function largely through the recruitment of effector proteins that contain specialized “reader” domains. While a significant number of reader domains have been characterized for their histone binding specificities, many of these domains remain poorly characterized. Peptide microarrays have been widely employed for the characterization of histone readers, as well as modifying enzymes and histone antibodies. While powerful, this platform has limitations in terms of its sensitivity and they frequently miss low affinity reader domain interactions. Here, we provide several technical changes that improve reader domain detection of low-affinity interactions. We show that 1% non-fat milk in 1X PBST as the blocking reagent during incubation improved reader-domain interaction results. Further, coupling this with post-binding high-salt washes and a brief, low-percentage formaldehyde cross-linking step prior to the high-salt washes provided the optimal balance between resolving specific low-affinity interactions and minimizing background or spurious signals. We expect this improved methodology will lead to the elucidation of previously unreported reader-histone interactions that will be important for chromatin function.

DNA methylation and trimethylated histone H4 Lysine 20 (H4K20me3) constitute two important heterochromatin-enriched marks that frequently cooperate in silencing repetitive elements of the mammalian genome. However, it remains elusive how these two chromatin modifications crosstalk. Here, we report that DNA methyltransferase 1 (DNMT1) specifically ‘recognizes’ H4K20me3 via its first bromo-adjacent-homology domain (DNMT1BAH1). Engagement of DNMT1BAH1-H4K20me3 ensures heterochromatin targeting of DNMT1 and DNA methylation at LINE-1 retrotransposons, and cooperates with the previously reported readout of histone H3 tail modifications (i.e., H3K9me3 and H3 ubiquitylation) by the RFTS domain to allosterically regulate DNMT1’s activity. Interplay between RFTS and BAH1 domains of DNMT1 profoundly impacts DNA methylation at both global and focal levels and genomic resistance to radiation-induced damage. Together, our study establishes a direct link between H4K20me3 and DNA methylation, providing a mechanism in which multivalent recognition of repressive histone modifications by DNMT1 ensures appropriate DNA methylation patterning and genomic stability., How histone modifications crosstalk with DNA methylation to regulate epigenomic patterning and genome stability in mammals remains elusive. Here, the authors show that DNA methyltransferase DNMT1 is a reader for histone H4K20 trimethylation via its BAH1 domain, which leads to optimal maintenance of DNA methylation at repetitive LINE-1 elements.

DNA double-strand breaks (DSBs) are among the deleterious lesions that are both endogenous and exogenous in origin and are repaired by nonhomologous end joining or homologous recombination. However, the molecular mechanisms responsible for maintaining genome stability remain incompletely understood. Here, we investigate the role of two E3 ligases, Dma1 and Dma2 (homologs of human RNF8), in the maintenance of genome stability in budding yeast. Using yeast spotting assays, chromatin immunoprecipitation and plasmid and chromosomal repair assays, we establish that Dma1 and Dma2 act in a redundant and a catalysis-dependent manner in the maintenance of genome stability, as well as localize to transcribed regions of the genome and increase in abundance upon phleomycin treatment. In addition, Dma1 and Dma2 are required for the normal kinetics of histone H4 acetylation under DNA damage conditions, genetically interact with RAD9 and SAE2, and are in a complex with Rad53 and histones. Taken together, our results demonstrate the requirement of Dma1 and Dma2 in regulating DNA repair pathway choice, preferentially affecting homologous recombination over nonhomologous end joining, and open up the possibility of using these candidates in manipulating the repair pathways toward precision genome editing.

Measuring protein-protein interaction (PPI) affinities is fundamental to biochemistry. Yet, conventional methods rely upon the law of mass action and cannot measure many PPIs due to a scarcity of reagents and limitations in the measurable affinity ranges. Here we present a novel technique that leverages the fundamental concept of friction to produce a mechanical signal that indicates binding. The mechanically transduced immunosorbent (METRIS) assay utilizes rolling magnetic probes to measure PPI interaction affinities. METRIS measures the translational displacement of protein-coated particles on a protein-functionalized substrate. The translational displacement scales with the effective friction induced by a PPI, thus producing a mechanical signal when a binding event occurs. The METRIS assay uses as little as 20 pmols of reagents to measure a wide range of affinities while exhibiting a high resolution and sensitivity. Here we use METRIS to measure several PPIs that were previously inaccessible using traditional methods, providing new insights into epigenetic recognition.

From phenotype to structure Much insight has come from structures of macromolecular complexes determined by methods such as crystallography or cryo–electron microscopy. However, looking at transient complexes remains challenging, as does determining structures in the context of the cellular environment. Braberg et al. used an integrative approach in which they mapped the phenotypic profiles of a comprehensive set of mutants in a protein complex in the context of gene deletions or environmental perturbations (see the Perspective by Wang). By associating the similarity between phenotypic profiles with the distance between residues, they determined structures for the yeast histone H3-H4 complex, subunits Rpb1-Rpb2 of yeast RNA polymerase II, and subunits RpoB-RpoC of bacterial RNA polymerase. Comparison with known structures shows that the accuracy is comparable to structures determined based on chemical cross-links. Science , this issue p. eaaz4910 ; see also p. 1269

The chromatin-binding E3 ubiquitin ligase ubiquitin-like with PHD and RING finger domains 1 (UHRF1) contributes to the maintenance of aberrant DNA methylation patterning in cancer cells through multivalent histone and DNA recognition. The tandem Tudor domain (TTD) of UHRF1 is well-characterized as a reader of lysine 9 di- and tri-methylation on histone H3 (H3K9me2/me3) and, more recently, lysine 126 di- and tri-methylation on DNA ligase 1 (LIG1K126me2/me3). However, the functional significance and selectivity of these interactions remain unclear. In this study, we used protein domain microarrays to search for additional readers of LIG1K126me2, the preferred methyl state bound by the UHRF1 TTD. We show that the UHRF1 TTD binds LIG1K126me2 with high affinity and selectivity compared to other known methyllysine readers. Notably, and unlike H3K9me2/me3, the UHRF1 plant homeodomain (PHD) and its N-terminal linker (L2) do not contribute to multivalent LIG1K126me2 recognition along with the TTD. To test the functional significance of this interaction, we designed a LIG1K126me2 cell-penetrating peptide (CPP). Consistent with LIG1 knockdown, uptake of the CPP had no significant effect on the propagation of DNA methylation patterning across the genomes of bulk populations from high-resolution analysis of several cancer cell lines. Further, we did not detect significant changes in DNA methylation patterning from bulk cell populations after chemical or genetic disruption of lysine methyltransferase activity associated with LIG1K126me2 and H3K9me2. Collectively, these studies identify UHRF1 as a selective reader of LIG1K126me2 in vitro and further implicate the histone and non-histone methyllysine reader activity of the UHRF1 TTD as a dispensable domain function for cancer cell DNA methylation maintenance.

In mammals, repressive histone modifications such as trimethylation of histone H3 Lys9 (H3K9me3), frequently coexist with DNA methylation, producing a more stable and silenced chromatin state. However, it remains elusive how these epigenetic modifications crosstalk. Here, through structural and biochemical characterizations, we identified the replication foci targeting sequence (RFTS) domain of maintenance DNA methyltransferase DNMT1, a module known to bind the ubiquitylated H3 (H3Ub), as a specific reader for H3K9me3/H3Ub, with the recognition mode distinct from the typical trimethyl-lysine reader. Disruption of the interaction between RFTS and the H3K9me3Ub affects the localization of DNMT1 in stem cells and profoundly impairs the global DNA methylation and genomic stability. Together, this study reveals a previously unappreciated pathway through which H3K9me3 directly reinforces DNMT1-mediated maintenance DNA methylation.

[Image: see text] A hallmark of human immunodeficiency type-1 (HIV) infection is the integration of the viral genome into host chromatin, resulting in a latent reservoir that persists despite antiviral therapy or immune response. Thus, key priorities toward eradication of HIV infection are to understand the mechanisms that allow HIV latency and to develop latency reversal agents (LRAs) that can facilitate the clearance of latently infected cells. The repressive H3K27me3 histone mark, catalyzed by the PRC2 complex, plays a pivotal role in transcriptional repression at the viral promoter in both cell line and primary CD4+ T cell models of latency. EZH2 inhibitors which block H3K27 methylation have been shown to act as LRAs, suggesting other PRC2 components could also be potential targets for latency reversal. EED, a core component of PRC2, ensures the propagation of H3K27me3 by allosterically activating EZH2 methyltransferase activity. Therefore, we sought to investigate if inhibition of EED would also reverse latency. Inhibitors of EED, EED226 and A-395, demonstrated latency reversal activity as single agents, and this activity was further enhanced when used in combination with other known LRAs. Loss of H3K27me3 following EED inhibition significantly increased the levels of H3K27 acetylation globally and at the HIV LTR. These results further confirm that PRC2 mediated repression plays a significant role in the maintenance of HIV latency and suggest that EED may serve as a promising new target for LRA development.