Histone methyltransferase MLL4 protects against pressure overload-induced heart failure via a THBS4-mediated protection in ER stress.

Pressure overload-induced pathological cardiac hypertrophy eventually leads to heart failure (HF). Unfortunately, lack of effective targeted therapies for HF remains a challenge in clinical management. Mixed-lineage leukemia 4 (MLL4) is a member of the SET family of histone methyltransferase enzymes, which possesses histone H3 lysine 4 (H3K4)-specific methyltransferase activity. However, whether and how MLL4 regulates cardiac function is not reported in adult HF. Here we report that MLL4 is required for endoplasmic reticulum (ER) stress homeostasis of cardiomyocytes and protective against pressure overload-induced cardiac hypertrophy and HF. We observed that MLL4 is increased in the heart tissue of HF mouse model and HF patients. The cardiomyocyte-specific deletion of Mll4 (Mll4-cKO) in mice leads to aggravated ER stress and cardiac dysfunction following pressure overloading. MLL4 knockdown neonatal rat cardiomyocytes (NRCMs) also display accelerated decompensated ER stress and hypertrophy induced by phenylephrine (PE). The combined analysis of Cleavage Under Targets and Tagmentation sequencing (CUT&Tag-seq) and RNA sequencing (RNA-seq) data reveals that, silencing of Mll4 alters the chromatin landscape for H3K4me1 modification and gene expression patterns in NRCMs. Interestingly, the deficiency of MLL4 results in a marked reduction of H3K4me1 and H3K27ac occupations on Thrombospondin-4 (Thbs4) gene loci, as well as Thbs4 gene expression. Mechanistically, MLL4 acts as a transcriptional activator of Thbs4 through mono-methylation of H3K4 and further regulates THBS4-dependent ER stress response, ultimately plays a role in HF. Our study indicates that pharmacologically targeting MLL4 and ER stress might be a valid therapeutic approach to protect against cardiac hypertrophy and HF.

PI 3-Kinase and the Histone Methyl-Transferase KMT2D Collaborate to Induce Arp2/3-Dependent Migration of Mammary Epithelial Cells.

Breast cancer develops upon sequential acquisition of driver mutations in mammary epithelial cells; however, how these mutations collaborate to transform normal cells remains unclear in most cases. We aimed to reconstitute this process in a particular case. To this end, we combined the activated form of the PI 3-kinase harboring the H1047R mutation with the inactivation of the histone lysine methyl-transferase KMT2D in the non-tumorigenic human mammary epithelial cell line MCF10A. We found that PI 3-kinase activation promoted cell-cycle progression, especially when growth signals were limiting, as well as cell migration, both in a collective monolayer and as single cells. Furthermore, we showed that KMT2D inactivation had relatively little influence on these processes, except for single-cell migration, which KMT2D inactivation promoted in synergy with PI 3-kinase activation. The combination of these two genetic alterations induced expression of the ARPC5L gene that encodes a subunit of the Arp2/3 complex. ARPC5L depletion fully abolished the enhanced migration persistence exhibited by double-mutant cells. Our reconstitution approach in MCF10A has thus revealed both the cell function and the single-cell migration, and the underlying Arp2/3-dependent mechanism, which are synergistically regulated when KMT2D inactivation is combined with the activation of the PI 3-kinase.

Regulation of T helper cell differentiation by the interplay between histone modification and chromatin interaction.

The development and function of the immune system are controlled by temporospatial gene expression programs, which are regulated by cis-regulatory elements, chromatin structure, and trans-acting factors. In this study, we cataloged the dynamic histone modifications and chromatin interactions at regulatory regions during T helper (Th) cell differentiation. Our data revealed that the H3K4me1 landscape established by MLL4 in naive CD4+ T cells is critical for restructuring the regulatory interaction network and orchestrating gene expression during the early phase of Th differentiation. GATA3 plays a crucial role in further configuring H3K4me1 modification and the chromatin interaction network during Th2 differentiation. Furthermore, we demonstrated that HSS3-anchored chromatin loops function to restrict the activity of the Th2 locus control region (LCR), thus coordinating the expression of Th2 cytokines. Our results provide insights into the mechanisms of how the interplay between histone modifications, chromatin looping, and trans-acting factors contributes to the differentiation of Th cells.

MLL4 binds TET3.

Human mixed lineage leukemia 4 (MLL4), also known as KMT2D, regulates cell type specific transcriptional programs through enhancer activation. Along with the catalytic methyltransferase domain, MLL4 contains seven less characterized plant homeodomain (PHD) fingers. Here, we report that the sixth PHD finger of MLL4 (MLL4PHD6) binds to the hydrophobic motif of ten-eleven translocation 3 (TET3), a dioxygenase that converts methylated cytosine into oxidized derivatives. The solution NMR structure of the TET3-MLL4PHD6 complex and binding assays show that, like histone H4 tail, TET3 occupies the hydrophobic site of MLL4PHD6, and that this interaction is conserved in the seventh PHD finger of homologous MLL3 (MLL3PHD7). Analysis of genomic localization of endogenous MLL4 and ectopically expressed TET3 in mouse embryonic stem cells reveals a high degree overlap on active enhancers and suggests a potential functional relationship of MLL4 and TET3.

Histone H4K16ac Binding Function of the Triple PHD Finger Cassette of MLL4.

The human methyltransferase MLL4 plays a critical role in embryogenesis and development, and aberrant activity of MLL4 is linked to neurodegenerative and developmental disorders and cancer. MLL4 contains the catalytic SET domain that catalyzes mono methylation of lysine 4 of histone H3 (H3K4me1) and seven plant homeodomain (PHD) fingers, six of which have not been structurally and functionally characterized. Here, we demonstrate that the triple PHD finger cassette of MLL4, harboring its fourth, fifth and sixth PHD fingers (MLL4PHD456) forms an integrated module, maintains the binding selectivity of the PHD6 finger toward acetylated lysine 16 of histone H4 (H4K16ac), and is capable of binding to DNA. Our findings highlight functional correlation between H4K16ac and H3K4me1, two major histone modifications that are recognized and written, respectively, by MLL4.

LncRNA SEMA3B-AS1 suppresses the tumor-initiating characteristics of triple negative breast cancer via engaging in MLL4-mediated H3K4 trimethylation.

Long noncoding RNAs (lncRNAs) are crucial regulators of tumor-initiating cells (TICs) and hold particular importance in triple negative breast cancer (TNBC). Yet, the precise mechanisms by which TIC-associated lncRNAs influence TNBC remain unclear. Our research utilized The Cancer Genome Atlas Breast Cancer (BC) data set to identify prognostic lncRNAs. We then conducted extensive assays to explore their impact on the tumor-initiating phenotype of TNBC cells and the underlying mechanisms. Notably, we found that low expression of lncRNA SEMA3B-AS1 correlated with unfavorable survival in BC patients. SEMA3B-AS1 was also downregulated in TNBC and linked to advanced tumor stage. Functional experiments confirmed its role as a TIC-suppressing lncRNA, curtailing mammosphere formation, ALDH + TIC cell proportion, and impairing clonogenicity, migration, and invasion. Mechanistic insights unveiled SEMA3B-AS1's nuclear localization and interaction with MLL4 (mixed-lineage leukemia 4), triggering H3K4 methylation-associated transcript activation and thus elevating the expression of SEMA3B, a recognized tumor suppressor gene. Our findings emphasize SEMA3B-AS1's significance as a TNBC-suppressing lncRNA that modulates TIC behavior. This study advances our comprehension of lncRNA's role in TNBC progression, advocating for their potential as therapeutic targets in this aggressive BC subtype.

KMT2D preferentially binds mRNAs of the genes it regulates, suggesting a role in RNA processing.

Histone lysine methyltransferases (HKMTs) perform vital roles in cellular life by controlling gene expression programs through the posttranslational modification of histone tails. Since many of them are intimately involved in the development of different diseases, including several cancers, understanding the molecular mechanisms that control their target recognition and activity is vital for the treatment and prevention of such conditions. RNA binding has been shown to be an important regulatory factor in the function of several HKMTs, such as the yeast Set1 and the human Ezh2. Moreover, many HKMTs are capable of RNA binding in the absence of a canonical RNA binding domain. Here, we explored the RNA binding capacity of KMT2D, one of the major H3K4 monomethyl transferases in enhancers, using RNA immunoprecipitation followed by sequencing. We identified a broad range of coding and non-coding RNAs associated with KMT2D and confirmed their binding through RNA immunoprecipitation and quantitative PCR. We also showed that a separated RNA binding region within KMT2D is capable of binding a similar RNA pool, but differences in the binding specificity indicate the existence of other regulatory elements in the sequence of KMT2D. Analysis of the bound mRNAs revealed that KMT2D preferentially binds co-transcriptionally to the mRNAs of the genes under its control, while also interacting with super enhancer- and splicing-related non-coding RNAs. These observations, together with the nuclear colocalization of KMT2D with differentially phosphorylated forms of RNA Polymerase II suggest a so far unexplored role of KMT2D in the RNA processing of the nascent transcripts.

SETting up the genome: KMT2D and KDM6A genomic function in the Kabuki syndrome craniofacial developmental disorder.

BACKGROUND: Kabuki syndrome is a congenital developmental disorder that is characterized by distinctive facial gestalt and skeletal abnormalities. Although rare, the disorder shares clinical features with several related craniofacial syndromes that manifest from mutations in chromatin-modifying enzymes. Collectively, these clinical studies underscore the crucial, concerted functions of chromatin factors in shaping developmental genome structure and driving cellular transcriptional states. Kabuki syndrome predominantly results from mutations in KMT2D, a histone H3 lysine 4 methylase, or KDM6A, a histone H3 lysine 27 demethylase. AIMS: In this review, we summarize the research efforts to model Kabuki syndrome in vivo to understand the cellular and molecular mechanisms that lead to the craniofacial and skeletal pathogenesis that defines the disorder. DISCUSSION: As several studies have indicated the importance of KMT2D and KDM6A function through catalytic-independent mechanisms, we highlight noncanonical roles for these enzymes as recruitment centers for alternative chromatin and transcriptional machinery.

May the force be with you: Nuclear condensates function beyond transcription control: Potential nongenetic functions of nuclear condensates in physiological and pathological conditions.

Over the past decade, research has revealed biomolecular condensates' relevance in diverse cellular functions. Through a phase separation process, they concentrate macromolecules in subcompartments shaping the cellular organization and physiology. In the nucleus, biomolecular condensates assemble relevant biomolecules that orchestrate gene expression. We here hypothesize that chromatin condensates can also modulate the nongenetic functions of the genome, including the nuclear mechanical properties. The importance of chromatin condensates is supported by the genetic evidence indicating that mutations in their members are causative of a group of rare Mendelian diseases named chromatinopathies (CPs). Despite a broad spectrum of clinical features and the perturbations of the epigenetic machinery characterizing the CPs, recent findings highlighted negligible changes in gene expression. These data argue in favor of possible noncanonical functions of chromatin condensates in regulating the genome's spatial organization and, consequently, the nuclear mechanics. In this review, we discuss how condensates may impact nuclear mechanical properties, thus affecting the cellular response to mechanical cues and, eventually, cell fate and identity. Chromatin condensates organize macromolecules in the nucleus orchestrating the transcription regulation and mutations in their members are responsible for rare diseases named chromatinopathies. We argue that chromatin condensates, in concert with the nuclear lamina, may also govern the nuclear mechanical properties affecting the cellular response to external cues.

Loss of MLL3/4 decouples enhancer H3K4 monomethylation, H3K27 acetylation, and gene activation during embryonic stem cell differentiation.

BACKGROUND: Enhancers are essential in defining cell fates through the control of cell-type-specific gene expression. Enhancer activation is a multi-step process involving chromatin remodelers and histone modifiers including the monomethylation of H3K4 (H3K4me1) by MLL3 (KMT2C) and MLL4 (KMT2D). MLL3/4 are thought to be critical for enhancer activation and cognate gene expression including through the recruitment of acetyltransferases for H3K27. RESULTS: Here we test this model by evaluating the impact of MLL3/4 loss on chromatin and transcription during early differentiation of mouse embryonic stem cells. We find that MLL3/4 activity is required at most if not all sites that gain or lose H3K4me1 but is largely dispensable at sites that remain stably methylated during this transition. This requirement extends to H3K27 acetylation (H3K27ac) at most transitional sites. However, many sites gain H3K27ac independent of MLL3/4 or H3K4me1 including enhancers regulating key factors in early differentiation. Furthermore, despite the failure to gain active histone marks at thousands of enhancers, transcriptional activation of nearby genes is largely unaffected, thus uncoupling the regulation of these chromatin events from transcriptional changes during this transition. These data challenge current models of enhancer activation and imply distinct mechanisms between stable and dynamically changing enhancers. CONCLUSIONS: Collectively, our study highlights gaps in knowledge about the steps and epistatic relationships of enzymes necessary for enhancer activation and cognate gene transcription.

Cross-Talk Between Histone Methyltransferases and Demethylases Regulate REST Transcription During Neurogenesis.

The RE1 Silencing Transcription Factor (REST) is a major regulator of neurogenesis and brain development. Medulloblastoma (MB) is a pediatric brain cancer characterized by a blockade of neuronal specification. REST gene expression is aberrantly elevated in a subset of MBs that are driven by constitutive activation of sonic hedgehog (SHH) signaling in cerebellar granular progenitor cells (CGNPs), the cells of origin of this subgroup of tumors. To understand its transcriptional deregulation in MBs, we first studied control of Rest gene expression during neuronal differentiation of normal mouse CGNPs. Higher Rest expression was observed in proliferating CGNPs compared to differentiating neurons. Interestingly, two Rest isoforms were expressed in CGNPs, of which only one showed a significant reduction in expression during neurogenesis. In proliferating CGNPs, higher MLL4 and KDM7A activities opposed by the repressive polycomb repressive complex 2 (PRC2) and the G9A/G9A-like protein (GLP) complex function allowed Rest homeostasis. During differentiation, reduction in MLL4 enrichment on chromatin, in conjunction with an increase in PRC2/G9A/GLP/KDM7A activities promoted a decline in Rest expression. These findings suggest a lineage-context specific paradoxical role for KDM7A in the regulation of Rest expression in CGNPs. In human SHH-MBs (SHH-α and SHH-ß) where elevated REST gene expression is associated with poor prognosis, up- or downregulation of KDM7A caused a significant worsening in patient survival. Our studies are the first to implicate KDM7A in REST regulation and in MB biology.

A New Pathologic KMT2B Variant Associated with Childhood Onset Dystonia Presenting as Variable Phenotypes among Family Members.

Background: KMT2B-related dystonia is a primarily childhood-onset movement disorder characterized by progressive dystonia, spasticity, and developmental delay. A minority of individuals possess an inherited KMT2B variant. Case Report: As a child, the proband experienced mild developmental delay and laryngeal dystonia which progressed to generalized dystonia. Patellar hyperreflexia, postural tremor, and everted gait were documented. Whole exome sequencing identified a heterozygous pathogenic KMT2B variant in the proband, proband's sister, and proband's mother who had milder presentations. Discussion: This novel KMT2B variant reflects intrafamilial variable expressivity in KMT2B-related dystonia. Further identification of variants will allow for better appreciation of the phenotypic spectrum.

Cancer-epigenetic function of the histone methyltransferase KMT2D and therapeutic opportunities for the treatment of KMT2D-deficient tumors.

Epigenetic mechanisms are central to understanding the molecular basis underlying tumorigenesis. Aberrations in epigenetic modifiers alter epigenomic landscapes and play a critical role in tumorigenesis. Notably, the histone lysine methyltransferase KMT2D (a COMPASS/ Set1 family member; also known as MLL4, ALR, and MLL2) is among the most frequently mutated genes in many different types of cancer. Recent studies have demonstrated how KMT2D loss induces abnormal epigenomic reprograming and rewires molecular pathways during tumorigenesis. These findings also have clinical and therapeutic implications for cancer treatment. In this review, we summarize recent advances in understanding the role of KMT2D in regulating tumorigenesis and discuss therapeutic opportunities for the treatment of KMT2D-deficient tumors.

RORα autoregulates its transcription via MLL4-associated enhancer remodeling in the liver.

AIMS: In hepatocytes, the retinoic acid receptor-related orphan receptor α (RORα) regulates the transcription of diverse genes encoding lipogenic enzymes, antioxidant enzymes, and mitochondrial factors via the regulation of the transcriptional activity of their promoters. The coordination of the expression of RORα by driving its transcription would provide better aspects for managing liver homeostasis. MAIN METHODS: The transcriptional expression of RORα was measured after treatment of RORα agonists on primary hepatocytes and liver. The histone status of Rora gene bodies was examined by analyzing ChIP-seq database. To elucidate molecular mechanism for RORα autoregulation, broad ChIP assays for promoters and enhancers with histone and RORα antibodies were performed. KEY FINDINGS: We report that natural and synthetic RORα agonists, cholesterol sulfate and JC1-40, respectively, increased the transcriptional expression of RORα in primary hepatocytes. An analysis of histone status around the Rora gene body identified promoter and enhancer regions of RORα. We found that RORα indirectly increased histone acetylation of H3K9 at the promoter region and directly enhanced histone monomethylation of H3K4 by binding to enhancer regions. Interestingly, disturbance of mixed-lineage leukemia 4 (MLL4), a histone methyltransferase for enhancers, abolished the JC1-40-induced activation of RORα via a decrease in H3K4me1. Finally, we observed that the MLL4-mediated autoregulation of RORα also occurred in human liver cancer cell lines. SIGNIFICANCE: The ability of RORα to modulate its own transcription is crucial for liver homeostasis, and ligand-dependent autoregulation could amplify the therapeutic effects of RORα in fatty liver diseases.

The KMT2D Kabuki syndrome histone methylase controls neural crest cell differentiation and facial morphology.

Kabuki syndrome (KS) is a congenital craniofacial disorder resulting from mutations in the KMT2D histone methylase (KS1) or the UTX histone demethylase (KS2). With small cohorts of KS2 patients, it is not clear whether differences exist in clinical manifestations relative to KS1. We mutated KMT2D in neural crest cells (NCCs) to study cellular and molecular functions in craniofacial development with respect to UTX. Similar to UTX, KMT2D NCC knockout mice demonstrate hypoplasia with reductions in frontonasal bone lengths. We have traced the onset of KMT2D and UTX mutant NCC frontal dysfunction to a stage of altered osteochondral progenitor differentiation. KMT2D NCC loss-of-function does exhibit unique phenotypes distinct from UTX mutation, including fully penetrant cleft palate, mandible hypoplasia and deficits in cranial base ossification. KMT2D mutant NCCs lead to defective secondary palatal shelf elevation with reduced expression of extracellular matrix components. KMT2D mutant chondrocytes in the cranial base fail to properly differentiate, leading to defective endochondral ossification. We conclude that KMT2D is required for appropriate cranial NCC differentiation and KMT2D-specific phenotypes may underlie differences between Kabuki syndrome subtypes.

Disordered Regions of Mixed Lineage Leukemia 4 (MLL4) Protein Are Capable of RNA Binding.

Long non-coding RNAs (lncRNAs) are emerging as important regulators of cellular processes and are extensively involved in the development of different cancers; including leukemias. As one of the accepted methods of lncRNA function is affecting chromatin structure; lncRNA binding has been shown for different chromatin modifiers. Histone lysine methyltransferases (HKMTs) are also subject of lncRNA regulation as demonstrated for example in the case of Polycomb Repressive Complex 2 (PRC2). Mixed Lineage Leukemia (MLL) proteins that catalyze the methylation of H3K4 have been implicated in several different cancers; yet many details of their regulation and targeting remain elusive. In this work we explored the RNA binding capability of two; so far uncharacterized regions of MLL4; with the aim of shedding light to the existence of possible regulatory lncRNA interactions of the protein. We demonstrated that both regions; one that contains a predicted RNA binding sequence and one that does not; are capable of binding to different RNA constructs in vitro. To our knowledge, these findings are the first to indicate that an MLL protein itself is capable of lncRNA binding.

MLL4 Is Required to Maintain Broad H3K4me3 Peaks and Super-Enhancers at Tumor Suppressor Genes.

Super-enhancers are large clusters of enhancers that activate gene expression. Broad trimethyl histone H3 lysine 4 (H3K4me3) often defines active tumor suppressor genes. However, how these epigenomic signatures are regulated for tumor suppression is little understood. Here we show that brain-specific knockout of the H3K4 methyltransferase MLL4 (a COMPASS-like enzyme, also known as KMT2D) in mice spontaneously induces medulloblastoma. Mll4 loss upregulates oncogenic Ras and Notch pathways while downregulating neuronal gene expression programs. MLL4 enhances DNMT3A-catalyzed DNA methylation and SIRT1/BCL6-mediated H4K16 deacetylation, which antagonize expression of Ras activators and Notch pathway components, respectively. Notably, Mll4 loss downregulates tumor suppressor genes (e.g., Dnmt3a and Bcl6) by diminishing broad H3K4me3 and super-enhancers and also causes widespread impairment of these epigenomic signatures during medulloblastoma genesis. These findings suggest an anti-tumor role for super-enhancers and provide a unique tumor-suppressive mechanism in which MLL4 is necessary to maintain broad H3K4me3 and super-enhancers at tumor suppressor genes.

Histone H3 lysine 4 methyltransferase KMT2D.

Histone-lysine N-methyltransferase 2D (KMT2D), also known as MLL4 and MLL2 in humans and Mll4 in mice, belongs to a family of mammalian histone H3 lysine 4 (H3K4) methyltransferases. It is a large protein over 5500 amino acids in size and is partially functionally redundant with KMT2C. KMT2D is widely expressed in adult tissues and is essential for early embryonic development. The C-terminal SET domain is responsible for its H3K4 methyltransferase activity and is necessary for maintaining KMT2D protein stability in cells. KMT2D associates with WRAD (WDR5, RbBP5, ASH2L, and DPY30), NCOA6, PTIP, PA1, and H3K27 demethylase UTX in one protein complex. It acts as a scaffold protein within the complex and is responsible for maintaining the stability of UTX. KMT2D is a major mammalian H3K4 mono-methyltransferase and co-localizes with lineage determining transcription factors on transcriptional enhancers. It is required for the binding of histone H3K27 acetyltransferases CBP and p300 on enhancers, enhancer activation and cell-type specific gene expression during differentiation. KMT2D plays critical roles in regulating development, differentiation, metabolism, and tumor suppression. It is frequently mutated in developmental diseases, such as Kabuki syndrome and congenital heart disease, and various forms of cancer. Further understanding of the mechanism through which KMT2D regulates gene expression will reveal why KMT2D mutations are so harmful and may help generate novel therapeutic approaches.

A UTX-MLL4-p300 Transcriptional Regulatory Network Coordinately Shapes Active Enhancer Landscapes for Eliciting Transcription.

Enhancer activation is a critical step for gene activation. Here we report an epigenetic crosstalk at enhancers between the UTX (H3K27 demethylase)-MLL4 (H3K4 methyltransferase) complex and the histone acetyltransferase p300. We demonstrate that UTX, in a demethylase activity-independent manner, facilitates conversion of inactive enhancers in embryonic stem cells to an active (H3K4me1+/H3K27ac+) state by recruiting and coupling the enzymatic functions of MLL4 and p300. Loss of UTX leads to attenuated enhancer activity, characterized by reduced levels of H3K4me1 and H3K27ac as well as impaired transcription. The UTX-MLL4 complex enhances p300-dependent H3K27 acetylation through UTX-dependent stimulation of p300 recruitment, while MLL4-mediated H3K4 monomethylation, reciprocally, requires p300 function. Importantly, MLL4-generated H3K4me1 further enhances p300-dependent transcription. This work reveals a previously unrecognized cooperativity among enhancer-associated chromatin modulators, including a unique function for UTX, in establishing an "active enhancer landscape" and defines a detailed mechanism for the joint deposition of H3K4me1 and H3K27ac.