Family Of Histone Methyltransferases Research Articles

NSD3 in Cancer: Unraveling Methyltransferase-Dependent and Isoform-Specific Functions.

NSD3 (nuclear receptor-binding SET domain protein 3) is a member of the NSD histone methyltransferase family of proteins. In recent years, it has been identified as a potential oncogene in certain types of cancer. The NSD3 gene encodes three isoforms, the long version (NSD3L), a short version (NSD3S) and the WHISTLE isoforms. Importantly, the NSD3S isoform corresponds to the N-terminal region of the full-length protein, lacking the methyltransferase domain. The chromosomal location of NSD3 is frequently amplified across cancer types, such as breast, lung, and colon, among others. Recently, this amplification has been correlated to a chromothripsis event, that could explain the different NSD3 alterations found in cancer. The fusion proteins containing NSD3 have also been reported in leukemia (NSD3-NUP98), and in NUT (nuclear protein of the testis) midline carcinoma (NSD3-NUT). Its role as an oncogene has been described by modulating different cancer pathways through its methyltransferase activity, or the short isoform of the protein, through protein interactions. Specifically, in this review we will focus on the functions that have been characterized as methyltransferase dependent, and those that have been correlated with the expression of the NSD3S isoform. There is evidence that both the NSD3L and NSD3S isoforms are relevant for cancer progression, establishing NSD3 as a therapeutic target. However, further functional studies are needed to differentiate NSD3 oncogenic activity as dependent or independent of the catalytic domain of the protein, as well as the contribution of each isoform and its clinical significance in cancer progression.

361MO Characterizing the role of NSD family of histone methyltransferases in the head and neck squamous cell carcinoma tumor immune microenvironment

361MO Characterizing the role of NSD family of histone methyltransferases in the head and neck squamous cell carcinoma tumor immune microenvironment

Regulation of the SUV39H Family Methyltransferases: Insights from Fission Yeast.

Histones, which make up nucleosomes, undergo various post-translational modifications, such as acetylation, methylation, phosphorylation, and ubiquitylation. In particular, histone methylation serves different cellular functions depending on the location of the amino acid residue undergoing modification, and is tightly regulated by the antagonistic action of histone methyltransferases and demethylases. The SUV39H family of histone methyltransferases (HMTases) are evolutionarily conserved from fission yeast to humans and play an important role in the formation of higher-order chromatin structures called heterochromatin. The SUV39H family HMTases catalyzes the methylation of histone H3 lysine 9 (H3K9), and this modification serves as a binding site for heterochromatin protein 1 (HP1) to form a higher-order chromatin structure. While the regulatory mechanism of this family of enzymes has been extensively studied in various model organisms, Clr4, a fission yeast homologue, has made an important contribution. In this review, we focus on the regulatory mechanisms of the SUV39H family of proteins, in particular, the molecular mechanisms revealed by the studies of the fission yeast Clr4, and discuss their generality in comparison to other HMTases.

Parallel functional annotation of cancer-associated missense mutations in histone methyltransferases

Using exome sequencing for biomarker discovery and precision medicine requires connecting nucleotide-level variation with functional changes in encoded proteins. However, for functionally annotating the thousands of cancer-associated missense mutations, or variants of uncertain significance (VUS), purifying variant proteins for biochemical and functional analysis is cost-prohibitive and inefficient. We describe parallel functional annotation (PFA) of large numbers of VUS using small cultures and crude extracts in 96-well plates. Using members of a histone methyltransferase family, we demonstrate high-throughput structural and functional annotation of cancer-associated mutations. By combining functional annotation of paralogs, we discovered two phylogenetic and clustering parameters that improve the accuracy of sequence-based functional predictions to over 90%. Our results demonstrate the value of PFA for defining oncogenic/tumor suppressor functions of histone methyltransferases as well as enhancing the accuracy of sequence-based algorithms in predicting the effects of cancer-associated mutations.

Set2 family regulates mycotoxin metabolism and virulence via H3K36 methylation in pathogenic fungus Aspergillus flavus

ABSTRACT Aspergillus flavus infects various crops with aflatoxins, and leads to aspergillosis opportunistically. Though H3K36 methylation plays an important role in fungal toxin metabolism and virulence, no data about the biological function of H3K36 methylation in A. flavus virulence has been reported. Our study showed that the Set2 histone methyltransferase family, AshA and SetB, involves in morphogenesis and mycotoxin anabolism by regulating related transcriptional factors, and they are important for fungal virulence to crops and animals. Western-blotting and double deletion analysis revealed that AshA mainly regulates H3K36me2, whereas SetB is mainly responsible for H3K36me3 in the nucleus. By construction of domain deletion A. flavus strain and point mutation strains by homologous recombination, the study revealed that SET domain is indispensable in mycotoxin anabolism and virulence of A. flavus, and N455 and V457 in it are the key amino acid residues. ChIP analysis inferred that the methyltransferase family controls fungal reproduction and regulates the production of AFB1 by directly regulating the production of the transcriptional factor genes, including wetA, steA, aflR and amylase, through H3K36 trimethylation in their chromatin fragments, based on which this study proposed that, by H3K36 trimethylation, this methyltransferase family controls AFB1 anabolism through transcriptional level and substrate utilization level. This study illuminates the epigenetic mechanism of the Set2 family in regulating fungal virulence and mycotoxin production, and provides new targets for controlling the virulence of the fungus A. flavus. AUTHOR SUMMARY The methylation of H3K36 plays an important role in the fungal secondary metabolism and virulence, but no data about the regulatory mechanism of H3K36 methylation in the virulence of A. flavus have been reported. Our study revealed that, in the histone methyltransferase Set2 family, AshA mainly catalyzes H3K36me2, and involves in the methylation of H3K36me1, and SetB mainly catalyzes H3K36me3 and H3K36me1. Through domain deletion and point mutation analysis, this study also revealed that the SET domain was critical for the normal biological function of the Set2 family and that N455 and V457 in the domain were critical for AshA. By ChIP-seq and ChIP-qPCR analysis, H3K36 was found to be trimethylation modified in the promotors and ORF positions of wetA, steA, aflR and the amylase gene (AFLA_084340), and further qRT-PCR results showed that these methylation modifications regulate the expression levels of these genes. According to the results of ChIP-seq analysis, we proposed that, by H3K36 trimethylation, this methyltransferase family controls the metabolism of mycotoxin through transcriptional level and substrate utilization level. All the results from this study showed that Set2 family is essential for fungal secondary metabolism and virulence, which lays a theoretical groundwork in the early prevention and treatment of A. flavus pollution, and also provides an effective strategy to fight against other pathogenic fungi.

Abstract 3274: Targeting NSD family histone methyltransferase activity with irreversible inhibitors

Abstract Nuclear SET domain containing (NSD) family member proteins are histone methyltransferases that mono- and di-methylate histone 3 at lysine 36 (H3K36) to alter gene expression in a context-dependent manner. NSD1 and NSD3 are two members of this family which share greater than 80 percent sequence similarity in the catalytic SET domain. NSD1 is found as a fusion with NUP98 in aggressive cases of pediatric leukemia, and the lysine methyltransferase (KMTase) activity of NUP98-NSD1 drives bone marrow progenitor proliferation. Similarly, high levels of enzymatically active NSD3 mRNA are correlated with reduced disease-free survival in breast cancer and catalytic NSD3 expression is sufficient to rescue tumor growth in a xenograft model of triple negative breast cancer. Selective and potent inhibitors of NSD1 and NSD3 KMTase activity are needed to further investigate the therapeutic potential of targeting this function in leukemia and/or solid cancers. Here, we report the development of irreversible small molecule inhibitors of the NSD1 and NSD3 SET domains. We utilized a NMR-based fragment screen of the NSD1 SET domain to identify the hit BT1, which binds reversibly to the SET domain of NSD1. BT1 was extensively optimized into a series of potent irreversible ligands. Mass spectrometry and structural studies revealed that these compounds react with a cysteine near the autoinhibitory loop, providing a rational for the strong inhibition of KMTase activity we observe in biochemical assays. These inhibitors also bind to the NSD1 and NSD3 SET domains in cells and exhibit potent anti-proliferative effects against leukemia cells containing NUP98-NSD1 and breast cancer cells with high levels of NSD3 expression. In summary, we present novel irreversible inhibitors of NSD1 and NSD3 KMTase activity which represent important chemical probes that can be used to investigate the therapeutic potential of targeting NSD family member catalytic activity in cancers. Citation Format: Christina Howard, Sergei Zari, Shuangjiang Li, Huang Huang, EunGi Kim, Se Ra Park, Trupta Purohit, Hongzhi Miao, Xiaotian Zhang, Caroline Nikolaidis, Hao Li, Juliano Ndoj, HyoJe Cho, Jolanta Grembecka, Tomasz Cierpicki. Targeting NSD family histone methyltransferase activity with irreversible inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3274.

Abstract B002: Modeling evolutionary therapy for GI malignancies

Abstract Despite development of personalized treatments GI cancer like pancreatic ductal adenocarcinoma (PDAC) and colorectal cancer (CRC) remain dismal outcomes. While some patients are potentially curable with surgery and chemotherapy, most unresectable cases are invariably lethal. To advance patient care, we rely on clinical trials to test new therapeutic approaches. Here, we assume that the highest dose of a drug that does not cause severe toxicities is optimal for cancer treatment. However, this treatment strategy, in many cases, may be more harmful for the patient. Adopting principles from the field of evolutionary biology and pest management could offer more effective strategies that can be used for patients with metastatic PDAC and CRC. Resistance mechanisms are assumed to come with an energy cost, making the organism/cell less fit than others in the population. When high doses of treatment are applied, it results in a significant selection gradient by eliminating sensitive populations, freeing resources and facilitating resistant population growth. Such evolutionary dynamics and interactions within the tumor microenvironment can be modeled and exploited, in the form of adaptive strategies, for treatment of CRC and PDAC. It is our central hypothesis that integration of evolutionary dynamics and into PDAC and CRC treatment will delay emergence of resistance, enhance quality of life, and improve clinical outcomes. Our preliminary observations in co-cultured drug sensitive and resistant cell containing spheroids show that a higher proportion of drug sensitive cells inhibits the proliferation of drug resistant cells over time, in the presence of chemotherapeutic agents 5-FU and Oxaliplatin. We also demonstrate a role for the MLL family of histone methyltransferases in the epigenetic regulation of genes involved in chemoresistance. RNA-seq data has shown differential gene expression in metabolic genes regulated by histone methyltransferase KMT2A, KM2TB and KMT2D knockout (KO) cells including Gstt1, CYP26B1 and PYGL. In addition to this, KMT2A, KM2TB and KMT2D KOs show differential sensitivity to chemotherapeutic agents Gemcitabine, SN-38, 5-FU and Oxaliplatin. Further, analysis of KMT2A, KM2TB and KMT2D KO cells will provide mechanistic insight of the epigenetic regulation of metabolic patterns in drug resistant cells. This strategy would provide an approach for reducing exposure to toxic drugs, improving patient quality of life and renewing hope for prolonged control of metastatic PDAC and CRC. This proposal represents a bedside-to-bench-to-bedside project; identifying a shortcoming in how we currently treat PDAC and CRC patients, developing solutions in the lab and ultimately translating such strategies to benefit patients. Citation Format: Glancis Luzeena Raja Arul, Ezequiel Tolosa, Ryan M. Carr, Martin E. Fernandez-Zapico. Modeling evolutionary therapy for GI malignancies [abstract]. In: Proceedings of the AACR Special Conference on the Evolutionary Dynamics in Carcinogenesis and Response to Therapy; 2022 Mar 14-17. Philadelphia (PA): AACR; Cancer Res 2022;82(10 Suppl):Abstract nr B002.

Rotational dynamics of the MLL complex on nucleosome and its implication in heterogeneity of the epigenetic landscape

The MLL family histone methyltransferases deposit mono‐, di‐, tri‐methylation of histone H3 lysine 4 (H3K4me). Epigenomic studies highlight the discrete distribution of H3K4me3 and H3K4me1 at gene promoters and distal enhancers, respectively. However, how this is achieved remains unclear. We have performed single particle cryo‐EM studies for the MLL1 core complex on the nucleosome core particles (NCP). We revealed a surprisingly dynamic nature of the MLL1 complex on the NCP. We show that DPY30 and the intrinsically disordered regions (IDRs) of ASH2L work together to restrict the rotational dynamics of the MLL1 complex, which is necessary for dramatic increase of processivity and activity of the MLL1 complex. The DPY30 and ASH2L‐IDR dependent regulation applies to all members of the MLL/SET1 family enzymes. We further show that DPY30 is causal for de novo establishment of H3K4me3 in cells and its preferred localization at gene promoters may be the primary reason for selective enrichment of H3K4me3. Our study provides a new paradigm of how discrete H3K4me state is regulated on chromatin.

NSD2 activates the E2F transcription factor 1/Y-box binding protein 2 axis to promote the malignant development of oral squamous cell carcinoma

ObjectiveNuclear receptor-binding SET domain protein 2 (NSD2) belongs to the SET histone methyltransferase family with potential oncogenic roles. This study aimed to probe the roles of NSD2 and its relevant molecules in oral squamous cell carcinoma (OSCC). MethodsAn OSCC-related GSE138206 dataset was analyzed to identify potential key genes implicated in OSCC development. NSD2 expression in OSCC tissues and cells was examined. NSD2 silencing was administrated in OSCC cells to examine its function in cell growth in vitro and in vivo. Downstream molecules mediated by NSD2 were predicted using bioinformatic tools. Rescue experiments of E2F transcription factor 1 (E2F1) and Y-box binding protein 2 (YBX2) were performed to validate their participation in NSD2-regulated events. ResultsNSD2 was highly expressed in OSCC tissues and cells, along with elevated expression of H3K36me2, a major target of NSD2-mediated methylation. NSD2 silencing significantly reduced proliferation, invasion and epithelial to mesenchymal transition of OSCC cells but induced cell apoptosis, and it reduced growth of xenograft tumors in nude mice. Downregulation of NSD2 led to transcriptional suppression of E2F1 by inhibiting H3K36me2 modification at the E2F1 promoter, while E2F1 transcriptionally activated YBX2. Either upregulation of E2F1 or YBX2 restored the malignant behaviors of OSCC cells suppressed by NSD2 silencing. ConclusionThis work demonstrates that NSD2 plays an oncogenic role in OSCC by activating E2F1 and YBX2. Silencing of NSD2, E2F1 or YBX2 may help suppress the progression of OSCC.

Essential Functions of MLL1 and MLL2 in Retinal Development and Cone Cell Maintenance.

MLL1 (KMT2A) and MLL2 (KMT2B) are homologous members of the mixed-lineage leukemia (MLL) family of histone methyltransferases involved in epigenomic transcriptional regulation. Their sequence variants have been associated with neurological and psychological disorders, but little is known about their roles and mechanism of action in CNS development. Using mouse retina as a model, we previously reported MLL1’s role in retinal neurogenesis and horizontal cell maintenance. Here we determine roles of MLL2 and MLL1/MLL2 together in retinal development using conditional knockout (CKO) mice. Deleting Mll2 from Chx10+ retinal progenitors resulted in a similar phenotype as Mll1 CKO, but removal of both alleles produced much more severe deficits than each single CKO: 1-month double CKO mutants displayed null light responses in electroretinogram; thin retinal layers, including shorter photoreceptor outer segments with impaired phototransduction gene expression; and reduced numbers of M-cones, horizontal and amacrine neurons, followed by fast retinal degeneration. Despite moderately reduced progenitor cell proliferation at P0, the neurogenic capacity was largely maintained in double CKO mutants. However, upregulated apoptosis and reactive gliosis were detected during postnatal retinal development. Finally, the removal of both MLLs in fated rods produced a normal phenotype, but the CKO in M-cones impaired M-cone function and survival, indicating both cell non-autonomous and autonomous mechanisms. Altogether, our results suggest that MLL1/MLL2 play redundant roles in maintaining specific retinal neurons after cell fate specification and are essential for establishing functional neural networks.

Structure, Activity and Function of the NSD3 Protein Lysine Methyltransferase.

NSD3 is one of six H3K36-specific lysine methyltransferases in metazoans, and the methylation of H3K36 is associated with active transcription. NSD3 is a member of the nuclear receptor-binding SET domain (NSD) family of histone methyltransferases together with NSD1 and NSD2, which generate mono- and dimethylated lysine on histone H3. NSD3 is mutated and hyperactive in some human cancers, but the biochemical mechanisms underlying such dysregulation are barely understood. In this review, the current knowledge of NSD3 is systematically reviewed. Finally, the molecular and functional characteristics of NSD3 in different tumor types according to the current research are summarized.

Polycomb repressive complex 2 mutations predict survival benefit in advanced cancer patients treated with immune checkpoint inhibitors.

BackgroundNumerous biomarkers are being tested to enhance the ability of clinicians to predict responses and prognosis after treatment with immune checkpoint inhibitors (ICIs). Polycomb repressive complex 2 (PRC2) is a histone methyltransferase family that plays a major role in chromatin silencing. Preclinical evidence implicates PRC2 components such as enhancer of zeste homolog 2 (EZH2) in immune resistance. This study aimed to assess the clinical relevance of PRC2 mutations in the clinical outcome of ICI-treated patients.Materials and methodsNext-generation sequencing (NGS) data from tumor samples of patients treated with ICIs (anti-PD-1/PD-L1, anti-CTLA-4 or both) were interrogated for alterations in PRC2-related genes. The Kaplan–Meier method was used to assess the association between altered and unaltered PRC2-related genes with overall survival.ResultsSomatic NGS data from 1662 advanced-stage, ICI-treated patients with various primaries (lung, melanoma, bladder, kidney, head neck, esophagogastric, glioma, colorectal, breast, unknown primary) were examined. Seventy patients (4%) harbored truncating or missense mutations or fusions in EZH2 (2.4%), EZH1 (1.2%), SUZ12 (0.9%) or EED (0.7%) genes. Patients carrying alterations in PRC2 genes had significantly longer median overall survival (44 months) compared with those with unaltered tumors (18 months, log-rank P=0.0174). These findings were validated in two additional cohorts of patients (n=313) with various primaries (melanoma, lung, bladder, head neck, anal, sarcoma) who were treated with ICIs.ConclusionsInactivating mutations in the PRC2 chromatin silencing machinery, although rare, may predict favorable outcomes in ICI-treated patients with metastatic cancers. This warrants prospective confirmation, and suggests that epigenetic regulators could serve as surrogate markers to guide ICI treatment decisions.

Molecular mechanics and dynamic simulations of well-known Kabuki syndrome-associated KDM6A variants reveal putative mechanisms of dysfunction

BackgroundKabuki syndrome is a genetic disorder that affects several body systems and presents with variations in symptoms and severity. The syndrome is named for a common phenotype of faces resembling stage makeup used in a Japanese traditional theatrical art named kabuki. The most frequent cause of this syndrome is mutations in the H3K4 family of histone methyltransferases while a smaller percentage results from genetic alterations affecting the histone demethylase, KDM6A. Because of the rare presentation of the latter form of the disease, little is known about how missense changes in the KDM6A protein sequence impact protein function.ResultsIn this study, we use molecular mechanic and molecular dynamic simulations to enhance the annotation and mechanistic interpretation of the potential impact of eleven KDM6A missense variants found in Kabuki syndrome patients. These variants (N910S, D980V, S1025G, C1153R, C1153Y, P1195L, L1200F, Q1212R, Q1248R, R1255W, and R1351Q) are predicted to be pathogenic, likely pathogenic or of uncertain significance by sequence-based analysis. Here, we demonstrate, for the first time, that although Kabuki syndrome missense variants are found outside the functionally critical regions, they could affect overall function by significantly disrupting global and local conformation (C1153R, C1153Y, P1195L, L1200F, Q1212R, Q1248R, R1255W and R1351Q), chemical environment (C1153R, C1153Y, P1195L, L1200F, Q1212R, Q1248R, R1255W and R1351Q), and/or molecular dynamics of the catalytic domain (all variants). In addition, our approaches predict that many mutations, in particular C1153R, could allosterically disrupt the key enzymatic interactions of KDM6A.ConclusionsOur study demonstrates that the KDM6A Kabuki syndrome variants may impair histone demethylase function through various mechanisms that include altered protein integrity, local environment, molecular interactions and protein dynamics. Molecular dynamics simulations of the wild type and the variants are critical to gain a better understanding of molecular dysfunction. This type of comprehensive structure- and MD-based analyses should help develop improved impact scoring systems to interpret the damaging effects of variants in this protein and other related proteins as well as provide detailed mechanistic insight that is not currently predictable from sequence alone.

Molecular basis of nucleosomal H3K36 methylation by NSD methyltransferases.

The NSD family histone methyltransferases, including NSD1, NSD2 and NSD3, play crucial roles in chromatin regulation and are implicated in oncogenesis1,2. NSD enzymes exhibit an auto-inhibitory state that is relieved by nucleosome engagement, allowing for H3K36 di-methylation catalysis3–7. However, the molecular basis underlying this mechanism is largely unknown. Here, we have solved the cryo-EM structures of NSD2 and NSD3 bound to mononucleosomes at atomic resolution. We find that NSD2/3 mononucleosome engagement causes DNA near the linker region to unwrap, which facilitates insertion of their catalytic core in-between the histone octamer and the unwrapped segment of DNA. A network of DNA- and histone-specific contacts between the nucleosome and NSD2/3 precisely define the enzymes’ position on the nucleosome, explaining the methylation specificity for H3K36. Further, NSD-nucleosome intermolecular contacts are altered by several recurrent cancer-associated NSD2/3 mutations. NSDs harboring these mutations are catalytically hyperactive in vitro and in cells, and their ectopic expression promotes cancer cell proliferation and xenograft tumor growth. Together, our research provides molecular insights into the nucleosome-based recognition and modification mechanisms of NSD2 and NSD3, which should uncover strategies for therapeutic targeting of the NSD family of proteins.

Identifying novel B-cell targets for chronic inflammatory autoimmune disease by screening of chemical probes in a patient-derived cell assay

B-cell secretion of autoantibodies drives autoimmune diseases, including systemic lupus erythematosus and idiopathic inflammatory myositis. Few therapies are presently available for treatment of these patients, often resulting in unsatisfactory effects and helping only some of the patients. We developed a screening assay for evaluation of novel targets suspending B-cell maturation into antibody secreting cells, which could contribute to future drug development. The assay was employed for testing 43 high quality chemical probes and compounds inhibiting under-explored protein targets, using primary cells from patients with autoimmune disease. Probes inhibiting bromodomain family proteins and histone methyl transferases demonstrated abrogation of B-cell functions to a degree comparable to a positive control, the JAK inhibitor tofacitinib. Inhibition of each target rendered a specific functional cell and potential disease modifying effect, indicating specific epigenetic protein targets as potential new intervention points for future drug discovery and development efforts.

Covalent inhibition of NSD1 histone methyltransferase.

The NSD family of histone methyltransferases is associated with various malignancies, including aggressive acute leukemia with NUP98-NSD1 translocation. While NSD proteins represent attractive drug targets, their catalytic SET domains exist in autoinhibited conformation, presenting significant challenges for inhibitor development. Here, we employed a fragment-based screening strategy followed by chemical optimization, which resulted in development of the first-in-class irreversible small molecule inhibitors of the NSD1 SET domain. The crystal structure of NSD1 in complex with covalently bound ligand reveals conformational change in the autoinhibitory loop of the SET domain and formation of a channel-like pocket suitable for targeting with small molecules. Our covalent lead, compound BT5, demonstrates on-target activity in NUP98-NSD1 leukemia cells, including inhibition of H3K36 dimethylation and downregulation of target genes, and impairs colony formation in NUP98-NSD1 patient sample. This study will facilitate development of the next generation of potent and selective inhibitors of the NSD histone methyltransferases.

H3K4 Methylation Status and Lysine Specific Methyltransferase KMT2C Expression Correlate with Prognosis in Lung Adenocarcinoma.

Genetic events cannot account for the complexity of human carcinogenesis alone. Mutations of epigenetic regulators and aberrations of their expression patterns have been detected in various human malignancies. Methylation of histone H3 at lysine 4 (H3K4me), is an evolutionarily conserved histone modification that marks regions of active transcription and regulates cell growth, migration, and invasion. The MLL/KMT2 family of histone methyltransferases specifically methylate histone H3 at lysine 4. The aim of this study was to explore the role of KMT2C/MLL3 as well as key histone modification activating markers, such as H3K4me2 and H3K4me3 in a cohort of surgically resected human lung adenocarcinomas in an effort to reveal possible biomarkers for lung adenocarcinoma diagnosis and prognosis and potential therapeutic targets. The immunohistochemical expression of KMT2C/MLL3, H3K4me2 and H3K4me3 was analyzed in formalin fixed paraffin embedded tissue from 96 patients with lung adenocarcinoma. Results were associated with clinicopathologic parameters and patient's prognosis. Nuclear expression of KMT2C/MLL3 in epithelial cells was independently associated with shorter overall survival. Cytoplasmic H3K4me2 expression was associated withT stage and nuclear H3K4me2 expression was associated with female gender and patients' prognosis. The latter association persisted after multivariate analysis. No association was found between H3K4me3 expression and clinicopathological data or disease outcome in our cohort of patients. These results suggest that the pattern of histone modifications and KMT2C/MLL3 expression can be used as an independent prognostic factor in lung adenocarcinoma, revealing that chromatin remodeling is criticallyinvolved in cancer progression.

Emerging mechanistic insights into mTOR‐PASK signaling pathway that connects nutrient signaling to chromatin network in stem cells

Stem cell fate in the tissue niche is intimately connected with intracellular metabolic state and the extra‐cellular hormonal stimulations. We have shown that a sensory kinase, PAS domain Kinase (PASK) phosphorylates Wdr5, a member of COMPASS family of histone methyltransferases, to activate the stem cell differentiation program in multiple differentiation paradigms, in vivo and in vitro (eLife, 2016). PASK is expressed highly and exclusively in stem cells, yet the differentiation signaling cues are required to activate PASK and its downstream functions, suggesting PASK remains inaccessible to bind Wdr5 in proliferating stem cells. Here, we show that the mechanistic Target of Rapamycin (mTOR) phosphorylates PASK to promote Wdr5 recruitment and myogenesis in response to nutrient and hormonal signaling. How mTOR phosphorylation stimulates PASK‐Wdr5 interactions remains unknown but is a key mechanistic question that could help understand how nutrient and metabolic signaling can acutely control stem cell differentiation on demand. By using multi‐disciplinary approaches, we show that the PAS domain of PASK inhibits catalytic activity of the kinase domain. Interestingly, mTOR stimulated phosphorylation induces a conformational change resulting in the increased Wdr5 binding, and catalytic activity of PASK. Thus, our data show how information pertaining to nutrient availability is communicated to epigenetic complexes via sequential activation of the mTORC1‐PASK‐Wdr5 pathway.Support or Funding InformationNIH R01: Epigenetic Control of muscle stem cell function by PASK‐Wd5 signaling pathway.1R01AR073906‐01A1

The conserved and divergent roles of Prdm3 and Prdm16 in zebrafish and mouse craniofacial development

The formation of the craniofacial skeleton is a highly dynamic process that requires proper orchestration of various cellular processes in cranial neural crest cell (cNCC) development, including cell migration, proliferation, differentiation, polarity and cell death. Alterations that occur during cNCC development result in congenital birth defects and craniofacial abnormalities such as cleft lip with or without cleft palate. While the gene regulatory networks facilitating neural crest development have been extensively studied, the epigenetic mechanisms by which these pathways are activated or repressed in a temporal and spatially regulated manner remain largely unknown. Chromatin modifiers can precisely modify gene expression through a variety of mechanisms including histone modifications such as methylation. Here, we investigated the role of two members of the PRDM (Positive regulatory domain) histone methyltransferase family, Prdm3 and Prdm16 in craniofacial development using genetic models in zebrafish and mice. Loss of prdm3 or prdm16 in zebrafish causes craniofacial defects including hypoplasia of the craniofacial cartilage elements, undefined posterior ceratobranchials, and decreased mineralization of the parasphenoid. In mice, while conditional loss of Prdm3 in the early embryo proper causes mid-gestation lethality, loss of Prdm16 caused craniofacial defects including anterior mandibular hypoplasia, clefting in the secondary palate and severe middle ear defects. In zebrafish, prdm3 and prdm16 compensate for each other as well as a third Prdm family member, prdm1a. Combinatorial loss of prdm1a, prdm3, and prdm16 alleles results in severe hypoplasia of the anterior cartilage elements, abnormal formation of the jaw joint, complete loss of the posterior ceratobranchials, and clefting of the ethmoid plate. We further determined that loss of prdm3 and prdm16 reduces methylation of histone 3 lysine 9 (repression) and histone 3 lysine 4 (activation) in zebrafish. In mice, loss of Prdm16 significantly decreased histone 3 lysine 9 methylation in the palatal shelves but surprisingly did not change histone 3 lysine 4 methylation. Taken together, Prdm3 and Prdm16 play an important role in craniofacial development by maintaining temporal and spatial regulation of gene regulatory networks necessary for proper cNCC development and these functions are both conserved and divergent across vertebrates.

Cryo-EM structure of the human MLL1 core complex bound to the nucleosome

Mixed lineage leukemia (MLL) family histone methyltransferases are enzymes that deposit histone H3 Lys4 (K4) mono-/di-/tri-methylation and regulate gene expression in mammals. Despite extensive structural and biochemical studies, the molecular mechanisms whereby the MLL complexes recognize histone H3K4 within nucleosome core particles (NCPs) remain unclear. Here we report the single-particle cryo-electron microscopy (cryo-EM) structure of the NCP-bound human MLL1 core complex. We show that the MLL1 core complex anchors to the NCP via the conserved RbBP5 and ASH2L, which interact extensively with nucleosomal DNA and the surface close to the N-terminal tail of histone H4. Concurrent interactions of RbBP5 and ASH2L with the NCP uniquely align the catalytic MLL1SET domain at the nucleosome dyad, thereby facilitating symmetrical access to both H3K4 substrates within the NCP. Our study sheds light on how the MLL1 complex engages chromatin and how chromatin binding promotes MLL1 tri-methylation activity.