H3K4 Methyltransferase Research Articles

Histone methyltransferase SUV39H1 regulates the Golgi complex via the nuclear envelope-spanning LINC complex.

Cell motility is related to the higher-order structure of chromatin. Stimuli that induce cell migration change chromatin organization; such stimuli include elevated histone H3 lysine 9 trimethylation (H3K9me3). We previously showed that depletion of histone H3 lysine 9 methyltransferase, SUV39H1, suppresses directional cell migration. However, the molecular mechanism underlying this association between chromatin and cell migration remains elusive. The Golgi apparatus is a cell organelle essential for cell motility. In this study, we show that loss of H3K9 methyltransferase SUV39H1 but not SETDB1 or SETDB2 causes dispersion of the Golgi apparatus throughout the cytoplasm. The Golgi dispersion triggered by SUV39H1 depletion is independent of transcription, centrosomes, and microtubule organization, but is suppressed by depletion of any of the following three proteins: LINC complex components SUN2, nesprin-2, or microtubule plus-end-directed kinesin-like protein KIF20A. In addition, SUN2 is closely localized to H3K9me3, and SUV39H1 affects the mobility of SUN2 in the nuclear envelope. Further, inhibition of cell motility caused by SUV39H1 depletion is restored by suppression of SUN2, nesprin-2, or KIF20A. In summary, these results show the functional association between chromatin organization and cell motility via the Golgi organization regulated by the LINC complex.

Cxxc finger protein 1 maintains homeostasis and function of intestinal group 3 innate lymphoid cells with aging.

Aging is accompanied by homeostatic and functional dysregulation of multiple immune cell subsets. Group 3 innate lymphoid cells (ILC3s) constitute a heterogeneous cell population that plays pivotal roles in intestinal immunity. In this study, we found that ILC3s in aged mice exhibited dysregulated homeostasis and function, leading to bacterial and fungal infection susceptibility. Moreover, our data revealed that the enrichment of the H3K4me3 modification in effector genes of aged gut CCR6+ ILC3s was specifically decreased compared to young mice counterparts. Disruption of Cxxc finger protein 1 (Cxxc1) activity, a key subunit of H3K4 methyltransferase, in ILC3s led to similar aging-related phenotypes. An integrated analysis revealed Kruppel-like factor 4 (Klf4) as a potential Cxxc1 target. Klf4 overexpression partially restored the differentiation and functional defects seen in both aged and Cxxc1-deficient intestinal CCR6+ ILC3s. Therefore, these data suggest that targeting intestinal ILC3s may provide strategies to protect against age-related infections.

HMGB1 neuroimmune signaling and REST-G9a gene repression contribute to ethanol-induced reversible suppression of the cholinergic neuron phenotype.

Adolescent binge drinking increases Toll-like receptor 4 (TLR4), receptor for advanced glycation end products (RAGE), the endogenous TLR4/RAGE agonist high-mobility group box 1 (HMGB1), and proinflammatory neuroimmune signaling in the adult basal forebrain in association with persistent reductions of basal forebrain cholinergic neurons (BFCNs). In vivo preclinical adolescent intermittent ethanol (AIE) studies find anti-inflammatory interventions post-AIE reverse HMGB1-TLR4/RAGE neuroimmune signaling and loss of BFCNs in adulthood, suggesting proinflammatory signaling causes epigenetic repression of the cholinergic neuron phenotype. Reversible loss of BFCN phenotype in vivo is linked to increased repressive histone 3 lysine 9 dimethylation (H3K9me2) occupancy at cholinergic gene promoters, and HMGB1-TLR4/RAGE proinflammatory signaling is linked to epigenetic repression of the cholinergic phenotype. Using an ex vivo basal forebrain slice culture (FSC) model, we report EtOH recapitulates the in vivo AIE-induced loss of ChAT+IR BFCNs, somal shrinkage of the remaining ChAT+ neurons, and reduction of BFCN phenotype genes. Targeted inhibition of EtOH-induced proinflammatory HMGB1 blocked ChAT+IR loss while disulfide HMBG1-TLR4 and fully reduced HMGB1-RAGE signaling decreased ChAT+IR BFCNs. EtOH increased expression of the transcriptional repressor RE1-silencing transcription factor (REST) and the H3K9 methyltransferase G9a that was accompanied by increased repressive H3K9me2 and REST occupancy at promoter regions of the BFCN phenotype genes Chat and Trka as well as the lineage transcription factor Lhx8. REST expression was similarly increased in the post-mortem human basal forebrain of individuals with alcohol use disorder, which is negatively correlated with ChAT expression. Administration of REST siRNA and the G9a inhibitor UNC0642 blocked and reversed the EtOH-induced loss of ChAT+IR BFCNs, directly linking REST-G9a transcriptional repression to suppression of the cholinergic neuron phenotype. These data suggest that EtOH induces a novel neuroplastic process involving neuroimmune signaling and transcriptional epigenetic gene repression resulting in the reversible suppression of the cholinergic neuron phenotype.

TONSOKU is required for the maintenance of repressive chromatin modifications in Arabidopsis

The stability of eukaryotic genomes relies on the faithful transmission of DNA sequences and the maintenance of chromatin states through DNA replication. Plant TONSOKU (TSK) and its animal ortholog TONSOKU-like (TONSL) act as readers for newly synthesized histones and preserve DNA integrity via facilitating DNA repair at post-replicative chromatin. However, whether TSK/TONSL regulate the maintenance of chromatin states remains elusive. Here, we show that TSK is dispensable for global histone and nucleosome accumulation but necessary for maintaining repressive chromatin modifications, including H3K9me2, H2A.W, H3K27me3, and DNA methylation. TSK physically interacts with H3K9 methyltransferases and Polycomb proteins. Moreover, TSK mutation strongly enhances defects in Polycomb pathway mutants. TSK is intended to only associate with nascent chromatin until it starts to mature. We propose that TSK ensures the preservation of chromatin states by supporting the recruitment of chromatin modifiers to post-replicative chromatin in a critical short window of time following DNA replication.

Histone bivalency regulates the timing of cerebellar granule cell development.

Developing neurons undergo a progression of morphological and gene expression changes as they transition from neuronal progenitors to mature neurons. Here we used RNA-seq and H3K4me3 and H3K27me3 ChIP-seq to analyze how chromatin modifications control gene expression in a specific type of CNS neuron: the mouse cerebellar granule cell (GC). We found that in proliferating GC progenitors (GCPs), H3K4me3/H3K27me3 bivalency is common at neuronal genes and undergoes dynamic changes that correlate with gene expression during migration and circuit formation. Expressing a fluorescent sensor for bivalent domains revealed subnuclear bivalent foci in proliferating GCPs. Inhibiting H3K27 methyltransferases EZH1 and EZH2 in vitro and in organotypic cerebellar slices dramatically altered the expression of bivalent genes, induced the down-regulation of migration-related genes and up-regulation of synaptic genes, inhibited glial-guided migration, and accelerated terminal differentiation. Thus, histone bivalency is required to regulate the timing of the progression from progenitor cells to mature neurons.

Overcoming the H4K20me3 epigenetic barrier improves somatic cell nuclear transfer reprogramming efficiency in mice.

Epigenetic reprogramming during fertilization and somatic cell nuclear transfer (NT) is required for cell plasticity and competent development. Here, we characterize the epigenetic modification pattern of H4K20me3, a repressive histone signature in heterochromatin, during fertilization and NT reprogramming. Importantly, the dynamic H4K20me3 signature identified during preimplantation development in fertilized embryos differed from NT and parthenogenetic activation (PA) embryos. In fertilized embryos, only maternal pronuclei carried the canonical H4K20me3 peripheral nucleolar ring-like signature. H4K20me3 disappeared at the 2-cell stage and reappeared in fertilized embryos at the 8-cell stage and in NT and PA embryos at the 4-cell stage. H4K20me3 intensity in 4-cell, 8-cell, and morula stages of fertilized embryos was significantly lower than in NT and PA embryos, suggesting aberrant regulation of H4K20me3 in PA and NT embryos. Indeed, RNA expression of the H4K20 methyltransferase Suv4-20h2 in 4-cell fertilized embryos was significantly lower than NT embryos. Knockdown of Suv4-20h2 in NT embryos rescued the H4K20me3 pattern similar to fertilized embryos. Compared to control NT embryos, knockdown of Suv4-20h2 in NT embryos improved blastocyst development ratios (11.1% vs. 30.5%) and full-term cloning efficiencies (0.8% vs. 5.9%). Upregulation of reprogramming factors, including Kdm4b, Kdm4d, Kdm6a, and Kdm6b, as well as ZGA-related factors, including Dux, Zscan4, and Hmgpi, was observed with Suv4-20h2 knockdown in NT embryos. Collectively, these are the first findings to demonstrate that H4K20me3 is an epigenetic barrier of NT reprogramming and begin to unravel the epigenetic mechanisms of H4K20 trimethylation in cell plasticity during natural reproduction and NT reprogramming in mice.

HGG-14. RAS PATHWAY MEDIATED REGULATION OF HISTONE METHYLTRANSFERASE AND TUMOR SUPPRESSOR SETD2 IN PEDIATRIC HIGH GRADE GLIOMA

Abstract BACKGROUND Pediatric High-Grade Gliomas (pHGG) are a significant cause of cancer-associated deaths in children. Hemispheric pHGG have frequent loss of the NF1 tumor suppressor (15%) causing aberrant RAS pathway activation. Intriguingly, loss of SETD2, a H3K36 methyltransferase, significantly co-occurs with loss of NF1. We found that nearly 80% SETD2-mutant pHGG have inactive NF1 (Pedcbio, 18/23 cases, 78%; odds-ratio 25; FDR adjusted P-value <0.001), suggesting a functional link between SETD2 and RAS signaling. NF1 is a GTPase-activating protein that negatively regulates the RAS oncogenic pathway. SETD2 is an epigenetic regulator and tumor suppressor that tri-methylates H3K36 on active gene bodies during transcription. We found that RAS pathway activation regulates SETD2 function. METHODS First, we used mass spectrometry to identify phosphorylation sites on SETD2 and generated phospho-mimetics/mutants to determine if these sites alter H3K36me3 levels. Second, we assessed if modulating downstream RAS effector pathways with MEK and PI3K inhibitors altered the phosphorylation of identified sites using the PhosTag gel electrophoresis assay. RESULTS We found that T2441 phosphorylation was upregulated upon MEK inhibition and that expressing T2441D phospho-mimetic and T2441A phospho-mutants had a differential effect on global H3K36me3 levels, implicating T2441 as an important regulatory node. Additionally, we identified that Cyclin Dependent Kinases are likely responsible for T2441 phosphorylation and are now investigating how RAS regulates these kinases. CONCLUSION To our knowledge it is the first time a functional connection between RAS pathway activation and SETD2 function has been found. It remains to be seen how of if RAS pathway activation alters the tumor suppressor function of SETD2 in pHGG.

Discovery of SDS-347 as a specific peptide competitive inhibitor of G9a with promising anti-cancer potential

BackgroundG9a is a histone H3K9 methyltransferase enzyme found highly upregulated in many cancers. H3 binds to the rigid I-SET domain and the cofactor, S-adenosyl methionine, binds to the flexible post-SET domain of G9a. Inhibition of G9a is known to inhibit the growth of cancer cell-lines. MethodsRecombinant G9a and H3 were used to develop radioisotope-based inhibitor screening assay. The identified inhibitor was evaluated for isoform selectivity. The mode of enzymatic inhibition was studied by enzymatic assays and bioinformatics. Anti-proliferative activity of the inhibitor was studied in cancer cell lines by utilizing MTT assay. The mechanism of cell death was studied by western blotting and microscopy. ResultsWe developed a robust G9a inhibitor screening assay that led to the discovery of SDS-347 as a potent G9a inhibitor with IC50 of 3.06 μM. It was shown to reduce the levels of H3K9me2 in cell-based assay. The inhibitor was found to be peptide competitive and highly specific as it did not show any significant inhibition of other histone methyltransferases and DNA methyltransferase. Docking studies showed that SDS-347 could form direct bonding interaction with Asp1088 of the peptide-binding site. SDS-347 showed anti-proliferative effect against various cancer cell lines especially the K562 cells. Our data suggested that SDS-347 mediated antiproliferative action via ROS generation, induction of autophagy and apoptosis. ConclusionOverall, the findings of the current study include development of a new G9a inhibitor screening assay and identification of SDS-347, as a novel, peptide competitive and highly specific G9a inhibitor with promising anticancer potential.

METTL14 regulates chromatin bivalent domains in mouse embryonic stem cells

METTL14 (methyltransferase-like 14) is an RNA-binding protein that partners with METTL3 to mediate N6-methyladenosine (m6A) methylation. Recent studies identified a function for METTL3 in heterochromatin in mouse embryonic stem cells (mESCs), but the molecular function of METTL14 on chromatin in mESCs remains unclear. Here, we show that METTL14 specifically binds and regulates bivalent domains, which are marked by trimethylation of histone H3 lysine 27 (H3K27me3) and lysine 4 (H3K4me3). Knockout of Mettl14 results in decreased H3K27me3 but increased H3K4me3 levels, leading to increased transcription. We find that bivalent domain regulation by METTL14 is independent of METTL3 or m6A modification. METTL14 enhances H3K27me3 and reduces H3K4me3 by interacting with and probably recruiting the H3K27 methyltransferase polycomb repressive complex 2 (PRC2) and H3K4 demethylase KDM5B to chromatin. Our findings identify an METTL3-independent role of METTL14 in maintaining the integrity of bivalent domains in mESCs, thus indicating a mechanism of bivalent domain regulation in mammals.

Role of histone H3K4 methyltransferase in regulating Monascus pigments production by red light-coupled magnetic field.

Light, magnetic field, and methylation affected the growth and secondary metabolism of fungi. The regulation effect of the three factors on the growth and Monascus pigments (MPs) synthesis of Monascus purpureus was investigated in this study. 5-azacytidine (5-AzaC), DNA methylation inhibitor, was used to treat M. purpureus (wild-type, WT). Twenty micromolar 5-AzaC significantly promoted the growth, development, and MPs yield. Moreover, 250 lux red light and red light coupled magnetic field (RLCMF) significantly promoted the biomass. For WT, red light, and RLCMF significantly promoted MPs yield. But compared with red light treatment, only 0.2 mT RLCMF promoted the alcohol-soluble MPs yield. For histone H3K4 methyltransferase complex subunit Ash2 gene knockout strain (ΔAsh2), only 0.2 mT RLCMF significantly promoted water-soluble MPs yield. Yet red light, 1.0 and 0.2 mT RLCMF significantly promoted alcohol-soluble MPs yield. This indicated that methylation affected the MPs biosynthesis. Red light and weaker MF had a synergistic effect on the growth and MPs synthesis of ΔAsh2. This result was further confirmed by the expression of related genes. Therefore, histone H3K4 methyltransferase was involved in the regulation of the growth, development, and MPs synthesis of M. purpureus by the RLCMF.

Nematode-induced trap formation regulated by the histone H3K4 methyltransferase AoSET1 in the nematode-trapping fungus Arthrobotrys oligospora.

The methylation of lysine 4 of histone H3 (H3K4), catalyzed by the histone methyltransferase KMT2/SET1, has been functionally identified in many pathogenic fungi but remains unexplored in nematode-trapping fungi (NTFs). Here, we report a regulatory mechanism of an H3K4-specific SET1 orthologue, AoSET1, in the typical nematode-trapping fungus Arthrobotrys oligospora. When the fungus is induced by the nematode, the expression of AoSET1 is up-regulated. Disruption of AoSet1 led to the abolishment of H3K4me. Consequently, the yield of traps and conidia of ΔAoSet1 was significantly lower than that of the WT strain, and the growth rate and pathogenicity were also compromised. Moreover, H3K4 trimethylation was enriched mainly in the promoter of two bZip transcription factor genes (AobZip129 and AobZip350) and ultimately up-regulated the expression level of these two transcription factor genes. In the ΔAoSet1 and AoH3K4A strains, the H3K4me modification level was significantly decreased at the promoter of transcription factor genes AobZip129 and AobZip350. These results suggest that AoSET1-mediated H3KEme serves as an epigenetic marker of the promoter region of the targeted transcription factor genes. Furthermore, we found that AobZip129 negatively regulates the formation of adhesive networks and the pathogenicity of downstream AoPABP1 and AoCPR1. Our findings confirm that the epigenetic regulatory mechanism plays a pivotal role in regulating trap formation and pathogenesis in NTFs, and provide novel insights into the mechanisms of interaction between NTFs and nematodes.

Epigenetic plasticity safeguards heterochromatin configuration in mammals.

Heterochromatin is a key architectural feature of eukaryotic chromosomes critical for cell type-specific gene expression and genome stability. In the mammalian nucleus, heterochromatin segregates from transcriptionally active genomic regions and exists in large, condensed, and inactive nuclear compartments. However, the mechanisms underlying the spatial organization of heterochromatin need to be better understood. Histone H3 lysine 9 trimethylation (H3K9me3) and lysine 27 trimethylation (H3K27me3) are two major epigenetic modifications that enrich constitutive and facultative heterochromatin, respectively. Mammals have at least five H3K9 methyltransferases (SUV39H1, SUV39H2, SETDB1, G9a and GLP) and two H3K27 methyltransferases (EZH1 and EZH2). In this study, we addressed the role of H3K9 and H3K27 methylation in heterochromatin organization using a combination of mutant cells for five H3K9 methyltransferases and an EZH1/2 dual inhibitor, DS3201. We showed that H3K27me3, which is normally segregated from H3K9me3, was redistributed to regions targeted by H3K9me3 after the loss of H3K9 methylation and that the loss of both H3K9 and H3K27 methylation resulted in impaired condensation and spatial organization of heterochromatin. Our data demonstrate that the H3K27me3 pathway safeguards heterochromatin organization after the loss of H3K9 methylation in mammalian cells.

Hi-TrAC detects active sub-TADs and reveals internal organizations of super-enhancers.

The spatial folding of eukaryotic genome plays a key role in genome function. We report here that our recently developed method, Hi-TrAC, which specializes in detecting chromatin loops among accessible genomic regions, can detect active sub-TADs with a median size of 100kb, most of which harbor one or two cell specifically expressed genes and regulatory elements such as super-enhancers organized into nested interaction domains. These active sub-TADs are characterized by highly enriched histone mark H3K4me1 and chromatin-binding proteins, including Cohesin complex. Deletion of selected sub-TAD boundaries have different impacts, such as decreased chromatin interaction and gene expression within the sub-TADs or compromised insulation between the sub-TADs, depending on the specific chromatin environment. We show that knocking down core subunit of the Cohesin complex using shRNAs in human cells or decreasing the H3K4me1 modification by deleting the H3K4 methyltransferase Mll4 gene in mouse Th17 cells disrupted the sub-TADs structure. Our data also suggest that super-enhancers exist as an equilibrium globule structure, while inaccessible chromatin regions exist as a fractal globule structure. In summary, Hi-TrAC serves as a highly sensitive and inexpensive approach to study dynamic changes of active sub-TADs, providing more explicit insights into delicate genome structures and functions.

The histone methyltransferase Whsc1 regulates TGFβ-driven macrophage to myofibroblast transition

Abstract In normal wound healing, macrophages regulate early and late inflammation, allowing for resolution of tissue injury and subsequent healing. During normal tissue repair, macrophages undergo transition to myofibroblasts, which promote wound healing. While this critical transition is impaired in diabetic wounds, the underlying mechanisms responsible are unknown. Our group and others have identified that epigenetic regulation of macrophages controls their phenotype in wounds. Since TGFβ drives macrophage to myofibroblast transition, we performed an epigenetic superarray to identify key chromatin modifying enzymes in macrophages (CD11b+CD3− CD19-NK1.1-Ly6G-) from normal diet (ND) and diabetic-induced obese (DIO) murine wounds treated ex vivo with TGFβ. The histone methyltransferase Whsc1, which mediates di- and tri-methylation at H3K36, was significantly increased in DIO macrophages with TGFβ treatment compared to ND macrophages (p<0.05). siRNA knockdown of Whsc1 in BMDMs decreased TGFβ-dependent expression of fibrotic (Acta2, Col1a1, Col3a1) and inflammatory genes (IL-1b, IL-6, and IL-12) (p<0.05). In ChIP experiments, Whsc1 was enriched at Col1a1 and Col3a1 promoters in ND but not DIO BMDMs, and TGFβ inhibited Whsc1 binding as well as H3K36me2 and H3K36me3 at fibrotic gene promoters. Additionally, H3K36me2 and H3K36me3 were decreased in Whsc1-knockdown BMDMs at Col1a1 and Col3a1, validating Whsc1 as an H3K36 methyltransferase that regulates myofibroblast gene expression. These results suggest that TGFβ regulates macrophage to myofibroblast conversion via Whsc1, which we identify as a critical epigenetic switch in tissue repair and potentially an important therapeutic target in diabetic wounds. F32 DK131799-01, 2022 American College of Surgeons Resident Research Scholarship, 2022 VESS/Medtronic Resident Research Award

The DOT1L-MLLT10 complex regulates male fertility and promotes histone removal during spermiogenesis.

Histone modifications regulate chromatin remodeling and gene expression in development and diseases. DOT1L, the sole histone H3K79 methyltransferase, is essential for embryonic development. Here, we report that DOT1L regulates male fertility in mouse. DOT1L associates with MLLT10 in testis. DOT1L and MLLT10 localize to the sex chromatin in meiotic and post-meiotic germ cells in an inter-dependent manner. Loss of either DOT1L or MLLT10 leads to reduced testis weight, decreased sperm count and male subfertility. H3K79me2 is abundant in elongating spermatids, which undergo the dramatic histone-to-protamine transition. Both DOT1L and MLLT10 are essential for H3K79me2 modification in germ cells. Strikingly, histones are substantially retained in epididymal sperm from either DOT1L- or MLLT10-deficient mice. These results demonstrate that H3K79 methylation promotes histone replacement during spermiogenesis.

Caf1 regulates the histone methyltransferase activity of Ash1 by sensing unmodified histone H3

Histone modifications are one of the many key mechanisms that regulate gene expression. Ash1 is a histone H3K36 methyltransferase and is involved in gene activation. Ash1 forms a large complex with Mrg15 and Caf1/p55/Nurf55/RbAp48 (AMC complex). The Ash1 subunit alone exhibits very low activity due to the autoinhibition, and the binding of Mrg15 releases the autoinhibition. Caf1 is a scaffolding protein commonly found in several chromatin modifying complexes and has two histone binding pockets: one for H3 and the other for H4. Caf1 has the ability to sense unmodified histone H3K4 residues using the H3 binding pocket. However, the role of Caf1 in the AMC complex has not been investigated. Here, we dissected the interaction among the AMC complex subunits, revealing that Caf1 uses the histone H4 binding pocket to interact with Ash1 near the histone binding module cluster. Furthermore, we showed that H3K4 methylation inhibits AMC HMTase activity via Caf1 sensing unmodified histone H3K4 to regulate the activity in an internucleosomal manner, suggesting that crosstalk between H3K4 and H3K36 methylation. Our work revealed a delicate mechanism by which the AMC histone H3K36 methyltransferase complex is regulated.

The histone H4K20 methyltransferase SUV4-20H1/KMT5B is required for multiciliated cell differentiation in Xenopus.

H4 lysine 20 dimethylation (H4K20me2) is the most abundant histone modification in vertebrate chromatin. It arises from sequential methylation of unmodified histone H4 proteins by the mono-methylating enzyme PR-SET7/KMT5A, followed by conversion to the dimethylated state by SUV4-20H (KMT5B/C) enzymes. We have blocked the deposition of this mark by depleting Xenopus embryos of SUV4-20H1/H2 methyltransferases. In the larval epidermis, this results in a severe loss of cilia in multiciliated cells (MCC), a key component of mucociliary epithelia. MCC precursor cells are correctly specified, amplify centrioles, but ultimately fail in ciliogenesis because of the perturbation of cytoplasmic processes. Genome-wide transcriptome profiling reveals that SUV4-20H1/H2-depleted ectodermal explants preferentially down-regulate the expression of several hundred ciliogenic genes. Further analysis demonstrated that knockdown of SUV4-20H1 alone is sufficient to generate the MCC phenotype and that its catalytic activity is needed for axoneme formation. Overexpression of the H4K20me1-specific histone demethylase PHF8/KDM7B also rescues the ciliogenic defect in a significant manner. Taken together, this indicates that the conversion of H4K20me1 to H4K20me2 by SUV4-20H1 is critical for the formation of cilia tufts.

NSD1 deposits histone H3 lysine 36 dimethylation to pattern non-CG DNA methylation in neurons

During postnatal development, the DNA methyltransferase DNMT3A deposits high levels of non-CG cytosine methylation in neurons. This methylation is critical for transcriptional regulation, and loss of this mark is implicated in DNMT3A-associated neurodevelopmental disorders (NDDs). Here, we show in mice that genome topology and gene expression converge to shape histone H3 lysine 36 dimethylation (H3K36me2) profiles, which in turn recruit DNMT3A and pattern neuronal non-CG methylation. We show that NSD1, an H3K36 methyltransferase mutated in NDD, is required for the patterning of megabase-scale H3K36me2 and non-CG methylation in neurons. We find that brain-specific deletion of NSD1 causes altered DNA methylation that overlaps with DNMT3A disorder models to drive convergent dysregulation of key neuronal genes that may underlie shared phenotypes in NSD1- and DNMT3A-associated NDDs. Our findings indicate that H3K36me2 deposited by NSD1 is important for neuronal non-CG DNA methylation and suggest that the H3K36me2-DNMT3A-non-CG-methylation pathway is likely disrupted in NSD1-associated NDDs.

Bidirectional regulation of postmitotic H3K27me3 distributions underlie cerebellar granule neuron maturation dynamics.

The functional maturation of neurons is a prolonged process that extends past the mitotic exit and is mediated by the chromatin-dependent orchestration of gene transcription programs. We find that expression of this maturation gene program in mouse cerebellar granule neurons (CGNs) requires dynamic changes in the genomic distribution of histone H3 lysine 27 trimethylation (H3K27me3), demonstrating a function for this chromatin modification beyond its role in cell fate specification. The developmental loss of H3K27me3 at promoters of genes activated as CGNs mature is facilitated by the lysine demethylase and ASD-risk gene, Kdm6b. Interestingly, inhibition of the H3K27 methyltransferase EZH2 in newborn CGNs not only blocks the repression of progenitor genes but also impairs the induction of mature CGN genes, showing the importance of bidirectional H3K27me3 regulation across the genome. These data demonstrate that H3K27me3 turnover in developing postmitotic neurons regulates the temporal coordination of gene expression programs that underlie functional neuronal maturation.

Abstract 3935: CRISPR/Cas9 screens to identify proliferation and resistance mechanisms in uveal melanoma

Abstract Purpose: Uveal (ocular) melanoma (UM) is a highly aggressive and frequently fatal cancer of the eye. While only 2-4% of patients have metastases at diagnosis, up to 50% of develop metastatic disease, overwhelmingly to the liver. Almost all UM harbor an initiating mutation in the Gαq signaling pathway, most commonly in GNAQ. Most UM also contain secondary mutations that drive tumor progression, the most common being BAP1 loss. Targeted therapies such as MEK inhibitors have been utilized in UM, but tumors rapidly become refractory. The aims of this study were to identify novel mechanisms specifically activated in UM cells that drive proliferation and escape from MEK inhibitor therapy. Procedures: We performed CRISPR-Cas9 mediated gene disruption screening of UM cell lines 92.1, Mel202 and Mel270 using the Brunello sgRNA library targeting the whole genome. Cells were transduced at 0.3 MOI and 500X coverage then split into two groups to compare treatment with the MEK inhibitor (MEKi) trametinib (6nM) vs. untreated control cells. Cell pellets were collected at day 2 (baseline) and after every 3 cell divisions for 21 cell divisions (treated and control). In addition, we generated 92.1 and Mel270 cell lines stably expressing Cas9 and transduced cells at 1000x coverage with a sgRNA library targeting either 192 epigenetic regulators or 482 kinases. At least two replicates for each screen were performed. sgRNA representation between samples was compared using MAGeCK. Genes specifically essential in UM cell lines were distinguished from commonly essential genes using the Broad institute dependency map (DepMap). These hits were validated by individual sgRNAs and pharmacological inhibition. Results: Whole genome screens revealed a set of 84 genetic dependencies unique for UM cells that were required for proliferation. Among these genes were those required for the lipoic acid biosynthesis pathway: MCAT, MECR, LIPT2, LIAS as well as genes encoding the MITF-SOX10-TFAP2 transcription factors, the H3K9 methyltransferase SETDB1, and members of the MLL3/4 complex. Screening of UM cell lines with a focused guide RNA library targeting epigenetic regulators revealed that BRD2, TRIM28, KMT2D, SETD1B and HDAC1 were specifically required for UM survival. Screens with a sgRNA library targeting the kinome revealed CDK2, CDK4 and TRRAP were required for UM survival. After 1 week of treatment with MEKi trametinib, we identified 112 genes whose disruption increased the anti-proliferative effects of trametinib in Mel202 and Mel270 cells and 10 of these genes were essential only in UM. These genes included metabolic enzymes critical for fatty and lipoic acid synthesis such as MECR, MCAT and TAZ. By contrast disruption of TP53, NF1, or PTEN led to MEKi resistance. Conclusions: These results identify novel mechanisms of proliferation and therapy resistance specifically required in GNAQ mutant UM cells that may represent new therapeutic targets. Citation Format: Richard L. Bennett, Darby Monagle, Katelyn R. Raburn Smith, Amin Sobh, Keiran S. Smalley, J William Harbour, Jonathan D. Licht. CRISPR/Cas9 screens to identify proliferation and resistance mechanisms in uveal melanoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3935.