H3K9 Dimethylation Research Articles

PHD-BAH Domain in ASH1L Could Recognize H3K4 Methylation and Regulate the Malignant Behavior of Cholangiocarcinoma.

Histone methyltransferase absent, small, or homeotic discs1-like (ASH1L) is composed of su(var)3-9, enhancer of zeste, trithorax (SET) domain, pleckstrin homology domain (PHD) domain, middle (MID) domain, and bromo adjacent homology (BAH) domain. The SET domain of ASH1L is known to mediate mediate H3K36 dimethylation (H3K36me2) modification. However, the specific functions of the PHD-BAH domain remain largely unexplored. This study aimed to explore the biological function of the PHD-BAH domain in ASH1L. We employed a range of techniques, including a prokaryotic fusion protein expression purification system, pull-down assay, Isothermal Titration Calorimetry (ITC), polymerase chain reaction (PCR), and sitedirected mutagenesis, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR-Cas9) gene editing, cell culture experiment, western blot, cell proliferation assay, and cell apoptosis test. The PHD-BAH domain in ASH1L preferentially binds to the H3K4me2 peptide over H3K4 monomethylation (H3K4me1) and H3K4 trimethylation (H3K4me3) peptide. Notably, the W2603A mutation within the PHD-BAH domain could disrupt the interaction with H3K4me2 in vitro. Compared with wild-type Cholangiocarcinoma (CHOL) cells, deletion of the PHD-BAH domain in ASH1L led to increased CHOL cell apoptosis and reduced cell proliferation (P < 0.001). Additionally, the W2603A mutation affected the regulation of the proteasome 20S subunit beta (PSMB) family gene set. W2603A mutation was crucial for the interaction between the PHD-BAH domain and the H3K4me2 peptide. ASH1L regulated CHOL cell survival and proliferation through its PHD-BAH domain by modulating the expression of the PSMB family gene set.

Decreased histone H3K9 dimethylation in synergy with DNA demethylation of Spi-1 binding site contributes to ADAMTS-5 expression in articular cartilage of osteoarthritis mice.

Osteoarthritis (OA) is defined by articular cartilage degeneration, synovial membrane inflammation, and abnormal bone remodeling. Recent study has discovered that OA development is linked to an aberrant epigenetic modification of OA-related genes. Our previous research showed that DNA demethylation in ADAMTS-5 promoter region had a substantial impact on ADAMTS-5 expression in the mouse OA model. This process facilitated the binding of Spi-1 to ADAMTS-5 promoter. While alterations in histone methylation have been documented during embryonic development and cancer development, there is a paucity of data on the change in OA pathogenesis. Even no data have been reported on the role of histone modifications in ADAMTS-5 activation in OA. Following our previous study on the role of DNA methylation, we aimed to examine the contribution of histone H3K9 dimethylation in ADAMTS-5 activation in OA. Additionally, we aimed to elucidate the molecular mechanisms underlying the cooperative interaction between DNA methylation and histone H3K9 dimethylation. The potential for anti-OA intervention therapy which is based on modulating histone H3K9 dimethylation is also explored. We demonstrated that a reduction in histone H3K9 dimethylation, along with DNA demethylation of the Spi-1 binding site, had a role in ADAMTS-5 activation in the articular cartilage of OA mice. Significantly, the conditional deletion of histone demethylase to be identified as lysine-specific demethylase 1 (LSD1) in articular cartilage could alleviate the degenerative features of OA mice. Our study demonstrates the direct impact of histone H3K9 dimethylation on gene expression, which in turn contributes to OA development. This research enhances our understanding of the underlying causes of OA.

GGNBP2 regulates histone ubiquitination and methylation in spermatogenesis

ABSTRACT Gametogenetin binding protein 2 (GGNBP2) was indispensable in normal spermatids for transformation into mature spermatozoa in mice, and when Gametogenetin binding protein 2 is bound to BRCC36 and RAD51, the complex participates in repairing DNA double-strand breaks (DSB) during the meiotic progression of spermatocytes. Ggnbp2 knockout resulted in the up-regulation of H2AK119ubi and down-regulation of H2BK120ubi in GC-2 cells (mouse spermatogonia-derived cell line) and postnatal day 18 testis lysate. Our results also demonstrated that Gametogenetin binding protein 2 inducedASXL1 to activate the deubiquitinating enzyme BAP1 in deubiquitinating H2A, while Gametogenetin binding protein 2 knockout disrupted the interaction between ASXL1 and BAP1, resulting in BAP1 localization change. Furthermore, the Gametogenetin binding protein 2 deletion reduced H2B ubiquitination by affecting E2 enzymes and E3 ligase binding. Gametogenetin binding protein 2 regulated H2A and H2B ubiquitination levels and controlled H3K27 and H3K79 methylation by PRC2 subunits and histone H3K79 methyltransferase. Altogether, our results suggest that Ggnbp2 knockout increased DNA damage response by promoting H2A ubiquitination and H3K27trimethylation (H3K27me3) and reduced nucleosome stability by decreasing H2B ubiquitination and H3K79 dimethylation (H3K79me2), revealing new mechanisms of epigenetic phenomenon during spermatogenesis. Gametogenetin binding protein 2 seems critical in regulating histone modification and chromatin structure in spermatogenesis.

Distinct roles of COMPASS subunits to Drosophila heart development.

The multiprotein complexes known as the complex of proteins associated with Set1 (COMPASS) play a crucial role in the methylation of histone 3 lysine 4 (H3K4). In Drosophila, the COMPASS series complexes comprise core subunits Set1, Trx, and Trr, which share several common subunits such as ash2, Dpy30-L1, Rbbp5, and wds, alongside their unique subunits: Wdr82 for Set1/COMPASS, Mnn1 for Trx/COMPASS-like, and Ptip for Trr/COMPASS-like. Our research has shown that flies deficient in any of these common or unique subunits exhibited high lethality at eclosion (the emergence of adult flies from their pupal cases) and significantly shortened lifespans of the few adults that do emerge. Silencing these common or unique subunits led to severe heart morphological and functional defects. Moreover, specifically silencing the unique subunits of the COMPASS series complexes, Wdr82, Mnn1, and Ptip, in the heart results in decreased levels of H3K4 monomethylation and dimethylation, consistent with effects observed from silencing the core subunits Set1, Trx, and Trr. These findings underscore the critical roles of each subunit of the COMPASS series complexes in regulating histone methylation during heart development and provide valuable insights into their potential involvement in congenital heart diseases, thereby informing ongoing research in heart disease.

Unravelling the role of Set2 protein domains in H3K36 methylation in Saccharomyces cerevisiae

Histone methylation plays a crucial role in gene expression and chromatin structure regulation. In Saccharomyces cerevisiae, the Set2 protein is responsible for the methylation of histone H3 at lysine 36 (H3K36), which is associated with transcriptional regulation, RNA processing, and DNA repair. This study investigates the specific functions of individual domains within the yeast Set2 protein by utilizing PCR-based domain deletions and subsequent western blot analysis to assess their impact on H3K36 methylation status. The results demonstrate that the SET domain alone is sufficient for H3K36 dimethylation, while optimal trimethylation necessitates the presence of additional domains, including the central autoinhibitory domain. Furthermore, the SRI domain is found to be essential for both di- and trimethylation when considering the full-length Set2 protein. These findings underscore the critical role of Set2 domains in modulating Set2 enzymatic activity.

Abstract 2140: The Tgfb-Whsc1 Axis Drives Macrophage To Myofibroblast Transition During Wound Repair

During the proliferative phase of tissue repair macrophages can transition to myofibroblasts, which promote wound contraction and closure. The mechanisms that control this transition remain unknown and are not well studied. Here, using a combination of murine transgenic models of disease, loss of function approaches, pharmacologic inhibition in vivo, and single cell RNA sequencing (scRNA-seq) in human wounds, we identified that a histone methyltransferase, Whsc1, regulates the fibrotic gene program in macrophages during wound repair. Specifically, we identified that Whsc1 was upregulated in wound macrophages (CD3 - CD19 - NK1.1 - Ly6G - CD11b + ) in response to TGFβ. Genetic loss of TGFβ-mediated signaling as well as siRNA knockdown of Whsc1 in bone marrow derived macrophages (BMDMs) decreased TGFβ-dependent expression of Acta2, Col1a1, Col3a1 (p&lt;0.05). Further, TGFβ stimulation resulted in increased Whsc1 and H3K36 dimethylation at fibrotic gene promoters in wound macrophages (p&lt;0.05). Importantly, local pharmacologic inhibition of Whsc1 in vivo significantly impaired wound healing, suggesting that Whsc1 is critical for normal wound repair. We next examined the role of Whsc1 in pathologic healing conditions (i.e. diabetes) where the macrophage-myofibroblast transition is impaired. Whsc1 expression was decreased in human macrophages analyzed by scRNA-seq of diabetic wounds and in murine wound macrophages from a diabetic model (DIO) (p&lt;0.05). Interestingly, in DIO BMDMs, Whsc1 did not traffic to fibrotic promoters after TGFβ treatment, but rather remained at NFkB-regulated inflammatory gene promoters ( Il1b, Il6, Tnf ). On further examination of this, using co-immunoprecipitation and proximity ligation assay in BMDMs, we demonstrated increased physical interaction between RelA and Whsc1 in diabetic macrophages. Knockdown of RelA in DIO macrophages increased Whsc1 enrichment at fibrotic promoters, suggesting RelA sequestered Whsc1 at inflammatory gene promoters (p&lt;0.05). The identification of TGFβ-Whsc1 as a driver of macrophage-myofibroblast transition in normal and pathological tissues allows for development of novel therapies to improve tissue repair, and targeting RelA in macrophages may improve diabetic wound healing.

The emerging role of DOT1L in cell proliferation and differentiation: Friend or foe.

Cell proliferation and differentiation are the basic physiological activities of cells. Mistakes in these processes may affect cell survival, or cause cell cycle dysregulation, such as tumorigenesis, birth defects and degenerative diseases. In recent years, it has been found that histone methyltransferase DOT1L is the only H3 lysine 79 methyltransferase, which plays an important role in the process of cell fate determination through monomethylation, dimethylation and trimethylation of H3K79. DOT1L has a pro-proliferative effect in leukemia cells; however, loss of heart-specific DOT1L leads to increased proliferation of cardiac tissue. Additionally, DOT1L has carcinogenic or tumor suppressive effects in different neoplasms. At present, some DOT1L inhibitors for the treatment of MLL-driven leukemia have achieved promising results in clinical trials, but completely blocking DOT1L will also bring some side effects. Thus, this uncertainty suggests that DOT1L has a unique function in cell physiology. In this review, we summarize the primary findings of DOT1L in regulating cell proliferation and differentiation. Correlations between DOT1L and cell fate specification might suggest DOT1L as a therapeutic target for diseases.

H3K4 methylation regulates development, DNA repair, and virulence in Mucorales

Mucorales are basal fungi that opportunistically cause a potentially fatal infection known as mucormycosis (black fungus disease), which poses a significant threat to human health due to its high mortality rate and its recent association with SARS-CoV-2 infections. On the other hand, histone methylation is a regulatory mechanism with pleiotropic effects, including the virulence of several pathogenic fungi. However, the role of epigenetic changes at the histone level never has been studied in Mucorales. Here, we dissected the functional role of Set1, a histone methyltransferase that catalyzes the methylation of H3K4, which is associated with the activation of gene transcription and virulence. A comparative analysis of the Mucor lusitanicus genome (previously known as Mucor circinelloides f. lusitanicus) identified only one homolog of Set1 from Candida albicans and Saccharomyces cerevisiae that contains the typical SET domain. Knockout strains in the gene set1 lacked H3K4 monomethylation, dimethylation, and trimethylation enzymatic activities. These strains also showed a significant reduction in vegetative growth and sporulation. Additionally, set1 null strains were more sensitive to SDS, EMS, and UV light, indicating severe impairment in the repair process of the cell wall and DNA lesions and a correlation between Set1 and these processes. During pathogen-host interactions, strains lacking the set1 gene exhibited shortened polar growth within the phagosome and attenuated virulence both in vitro and in vivo. Our findings suggest that the histone methyltransferase Set1 coordinates several cell processes related to the pathogenesis of M. lusitanicus and may be an important target for future therapeutic strategies against mucormycosis.

Inhibition of EHMT1/2 rescues synaptic damage and motor impairment in a PD mouse model

Epigenetic dysregulation that leads to alterations in gene expression and is suggested to be one of the key pathophysiological factors of Parkinson’s disease (PD). Here, we found that α-synuclein preformed fibrils (PFFs) induced histone H3 dimethylation at lysine 9 (H3K9me2) and increased the euchromatic histone methyltransferases EHMT1 and EHMT2, which were accompanied by neuronal synaptic damage, including loss of synapses and diminished expression levels of synaptic-related proteins. Furthermore, the levels of H3K9me2 at promoters in genes that encode the synaptic-related proteins SNAP25, PSD95, Synapsin 1 and vGLUT1 were increased in primary neurons after PFF treatment, which suggests a linkage between H3K9 dimethylation and synaptic dysfunction. Inhibition of EHMT1/2 with the specific inhibitor A-366 or shRNA suppressed histone methylation and alleviated synaptic damage in primary neurons that were treated with PFFs. In addition, the synaptic damage and motor impairment in mice that were injected with PFFs were repressed by treatment with the EHMT1/2 inhibitor A-366. Thus, our findings reveal the role of histone H3 modification by EHMT1/2 in synaptic damage and motor impairment in a PFF animal model, suggesting the involvement of epigenetic dysregulation in PD pathogenesis.

Chronic alcohol-induced long-lasting working memory deficits are associated with altered histone H3K9 dimethylation in the prefrontal cortex.

Epigenetic modifications have emerged as key contributors to the enduring behavioral, molecular and epigenetic neuroadaptations during withdrawal from chronic alcohol exposure. The present study investigated the long-term consequences of chronic alcohol exposure on spatial working memory (WM) and associated changes of transcriptionally repressive histone H3 lysine 9 dimethylation (H3K9me2) in the prefrontal cortex (PFC). Male C57BL/6 mice were allowed free access to either 12% (v/v) ethanol for 5 months followed by a 3-week abstinence period or water. Spatial WM was assessed through the spontaneous alternation T-maze test. Alcoholic and water mice received daily injections of GABAB agonist baclofen or saline during alcohol fading and early withdrawal. Global levels of histone modifications were determined by immunohistochemistry. Withdrawal mice displayed WM impairments along with reduced prefrontal H3K9me2 levels, compared to water-drinking mice. The withdrawal-induced decrease of H3K9me2 occurred concomitantly with increased level of permissive H3K9 acetylation (H3K9ac) in the PFC. Baclofen treatment rescued withdrawal-related WM deficits and fully restored prefrontal H3K9me2 and H3K9ac. Alcohol withdrawal induced brain region-specific changes of H3K9me2 and H3K9ac after testing, with significant decreases of both histone marks in the dorsal hippocampus and no changes in the amygdala and dorsal striatum. Furthermore, the magnitude of H3K9me2 in the PFC, but not the hippocampus, significantly and positively correlated with individual WM performances. No correlation was observed between H3K9ac and behavioral performance. Results also indicate that pre-testing intraperitoneal injection of UNC0642, a selective inhibitor of histone methyltransferase G9a responsible for H3K9me2, led to WM impairments in water-drinking and withdrawal-baclofen mice. Collectively, our results demonstrate that alcohol withdrawal induced brain-region specific alterations of H3K9me2 and H3K9ac, an effect that persisted for at least three weeks after cessation of chronic alcohol intake. The findings suggest a role for long-lasting decreased H3K9me2 specifically in the PFC in the persistent WM impairments related to alcohol withdrawal.

Di- and tri-methylation of histone H3K36 play distinct roles in DNA double-strand break repair.

Histone H3 Lys36 (H3K36) methylation and its associated modifiers are crucial for DNA double-strand break (DSB) repair, but the mechanism governing whether and how different H3K36 methylation forms impact repair pathways is unclear. Here, we unveil the distinct roles of H3K36 dimethylation (H3K36me2) and H3K36 trimethylation (H3K36me3) in DSB repair via non-homologous end joining (NHEJ) or homologous recombination (HR). Yeast cells lacking H3K36me2 or H3K36me3 exhibit reduced NHEJ or HR efficiency. yKu70 and Rfa1 bind H3K36me2- or H3K36me3-modified peptides and chromatin, respectively. Disrupting these interactions impairs yKu70 and Rfa1 recruitment to damaged H3K36me2- or H3K36me3-rich loci, increasing DNA damage sensitivity and decreasing repair efficiency. Conversely, H3K36me2-enriched intergenic regions and H3K36me3-enriched gene bodies independently recruit yKu70 or Rfa1 under DSB stress. Importantly, human KU70 and RPA1, the homologs of yKu70 and Rfa1, exclusively associate with H3K36me2 and H3K36me3 in a conserved manner. These findings provide valuable insights into how H3K36me2 and H3K36me3 regulate distinct DSB repair pathways, highlighting H3K36 methylation as a critical element in the choice of DSB repair pathway.

Loss of NSD2 causes dysregulation of synaptic genes and altered H3K36 dimethylation in mice.

Background: Epigenetic disruptions have been implicated in neurodevelopmental disorders. NSD2 is associated with developmental delay/intellectual disability; however, its role in brain development and function remains unclear. Methods: We performed transcriptomic and epigenetic analyses using Nsd2 knockout mice to better understand the role of NSD2 in the brain. Results and discussion: Transcriptomic analysis revealed that the loss of NSD2 caused dysregulation of genes related to synaptic transmission and formation. By analyzing changes in H3 lysine 36 dimethylation (H3K36me2), NSD2-mediated H3K36me2 mainly marked quiescent state regions and the redistribution of H3K36me2 occurred at transcribed genes and enhancers. By integrating transcriptomic and epigenetic data, we observed that H3K36me2 changes in a subset of dysregulated genes related to synaptic transmission and formation. These results suggest that NSD2 is involved in the regulation of genes important for neural function through H3K36me2. Our findings provide insights into the role of NSD2 and improve our understanding of epigenetic regulation in the brain.

Distinct Roles for COMPASS Core Subunits Set1, Trx, and Trr in the Epigenetic Regulation of Drosophila Heart Development.

Highly evolutionarily conserved multiprotein complexes termed Complex of Proteins Associated with Set1 (COMPASS) are required for histone 3 lysine 4 (H3K4) methylation. Drosophila Set1, Trx, and Trr form the core subunits of these complexes. We show that flies deficient in any of these three subunits demonstrated high lethality at eclosion (emergence of adult flies from their pupal cases) and significantly shortened lifespans for the adults that did emerge. Silencing Set1, trx, or trr in the heart led to a reduction in H3K4 monomethylation (H3K4me1) and dimethylation (H3K4me2), reflecting their distinct roles in H3K4 methylation. Furthermore, we studied the gene expression patterns regulated by Set1, Trx, and Trr. Each of the COMPASS core subunits controls the methylation of different sets of genes, with many metabolic pathways active early in development and throughout, while muscle and heart differentiation processes were methylated during later stages of development. Taken together, our findings demonstrate the roles of COMPASS series complex core subunits Set1, Trx, and Trr in regulating histone methylation during heart development and, given their implication in congenital heart diseases, inform research on heart disease.

2-Hydroxyglutarate modulates histone methylation at specific loci and alters gene expression via Rph1 inhibition.

2-Hydroxyglutarate (2-HG) is an oncometabolite that accumulates in certain cancers. Gain-of-function mutations in isocitrate dehydrogenase lead to 2-HG accumulation at the expense of alpha-ketoglutarate. Elevated 2-HG levels inhibit histone and DNA demethylases, causing chromatin structure and gene regulation changes with tumorigenic consequences. We investigated the effects of elevated 2-HG levels in Saccharomyces cerevisiae, a yeast devoid of DNA methylation and heterochromatin-associated histone methylation. Our results demonstrate genetic background-dependent gene expression changes and altered H3K4 and H3K36 methylation at specific loci. Analysis of histone demethylase deletion strains indicated that 2-HG inhibits Rph1 sufficiently to induce extensive gene expression changes. Rph1 is the yeast homolog of human KDM4 demethylases and, among the yeast histone demethylases, was the most sensitive to the inhibitory effect of 2-HG in vitro. Interestingly, Rph1 deficiency favors gene repression and leads to further down-regulation of already silenced genes marked by low H3K4 and H3K36 trimethylation, but abundant in H3K36 dimethylation. Our results provide novel insights into the genome-wide effects of 2-HG and highlight Rph1 as its preferential demethylase target.

DOT1L is a barrier to histone acetylation during reprogramming to pluripotency.

Embryonic stem cells (ESCs) have transcriptionally permissive chromatin enriched for gene activation-associated histone modifications. A striking exception is DOT1L-mediated H3K79 dimethylation (H3K79me2) that is considered a positive regulator of transcription. We find that ESCs are depleted for H3K79me2 at shared locations of enrichment with somatic cells, which are highly and ubiquitously expressed housekeeping genes, and have lower RNA polymerase II (RNAPII) at the transcription start site (TSS) despite greater nascent transcription. Inhibiting DOT1L increases the efficiency of reprogramming of somatic to induced pluripotent stem cells, enables an ESC-like RNAPII pattern at the TSS, and functionally compensates for enforced RNAPII pausing. DOT1L inhibition increases H3K27 methylation and RNAPII elongation-enhancing histone acetylation without changing the expression of the causal histone-modifying enzymes. Only the maintenance of elevated histone acetylation is essential for enhanced reprogramming and occurs at loci that are depleted for H3K79me2. Thus, DOT1L inhibition promotes the hyperacetylation and hypertranscription pluripotent properties.

The proteasome component PSMD14 drives myelomagenesis through a histone deubiquitinase activity

While 19S proteasome regulatory particle (RP) inhibition is a promising new avenue for treating bortezomib-resistant myeloma, the anti-tumor impact of inhibiting 19S RP component PSMD14 could not be explained by a selective inhibition of proteasomal activity. Here, we report that PSMD14 interacts with NSD2 on chromatin, independent of 19S RP. Functionally, PSMD14 acts as a histone H2AK119 deubiquitinase, facilitating NSD2-directed H3K36 dimethylation. Integrative genomic and epigenomic analyses revealed the functional coordination of PSMD14 and NSD2 in transcriptional activation of target genes (e.g., RELA) linked to myelomagenesis. Reciprocally, RELA transactivates PSMD14, forming a PSMD14/NSD2-RELA positive feedback loop. Remarkably, PSMD14 inhibitors enhance bortezomib sensitivity and fosters anti-myeloma synergy. PSMD14 expression is elevated in myeloma and inversely correlated with overall survival. Our study uncovers an unappreciated function of PSMD14 as an epigenetic regulator and a myeloma driver, supporting the pursuit of PSMD14 as a therapeutic target to overcome the treatment limitation of myeloma.

The Role of Acetylation and Methylation of Rat Hippocampal Histone H3 in the Mechanism of Aluminum-Induced Neurotoxicity.

Aluminum is a known neurotoxin and a major environmental contributor to neurodegenerative diseases such as Alzheimer's disease (AD). We uesd a subchronic aluminum chloride exposure model in offspring rats by continuously treating them with AlCl3 solution from the date of birth until day 90 in this research. Then evaluated the neurobehavioral changes in rats, observed the ultrastructural changes of hippocampal synapses and neurons, and examined the level of hippocampal acetylated histone H3 (H3ac), the activity and protein expression of hippocampal HAT1 and G9a, and the protein expression level of H3K9 dimethylation (H3K9me2). The findings demonstrated that aluminum-treated offspring rats had impaired learning and memory abilities as well as ultrastructural alterations in hippocampal synapses and neurons. The level of histone H3ac was decreased along with decreased protein expression and activity of HAT1, while level of H3K9me2 was increased along with increased protein expression and activity of G9a.

H3K36 Di-Methylation Marks, Mediated by Ash1 in Complex with Caf1-55 and MRG15, Are Required during Drosophila Heart Development.

Methyltransferases regulate transcriptome dynamics during development and aging, as well as in disease. Various methyltransferases have been linked to heart disease, through disrupted expression and activity, and genetic variants associated with congenital heart disease. However, in vivo functional data for many of the methyltransferases in the context of the heart are limited. Here, we used the Drosophila model system to investigate different histone 3 lysine 36 (H3K36) methyltransferases for their role in heart development. The data show that Drosophila Ash1 is the functional homolog of human ASH1L in the heart. Both Ash1 and Set2 H3K36 methyltransferases are required for heart structure and function during development. Furthermore, Ash1-mediated H3K36 methylation (H3K36me2) is essential for healthy heart function, which depends on both Ash1-complex components, Caf1-55 and MRG15, together. These findings provide in vivo functional data for Ash1 and its complex, and Set2, in the context of H3K36 methylation in the heart, and support a role for their mammalian homologs, ASH1L with RBBP4 and MORF4L1, and SETD2, during heart development and disease.

LSD1 knockdown confers protection against osteoclast formation by reducing histone 3 lysine 9 monomethylation and dimethylation in ITGB3 promoter

ITGB3, an osteoclast marker, is involved in osteoclast formation. Nevertheless, its related mechanism remains poorly characterized. Herein, this study examines the mechanisms affecting osteoclast formation with the involvement of ITGB3. Osteoclast formation was induced with macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor-kappa B ligand (RANKL), followed by measurement of the mRNA and protein expression of ITGB3 and LSD1. After gain- and loss-of-function assays, cell viability and the expression of osteoclast marker genes (NFATc1, ACP5, and CTSK) were assessed, and osteoclast formation was evaluated with TRAP staining. ChIP assays were used to examine histone 3 lysine 9 (H3K9) monomethylation (H3K9me1) and H3K9 dimethylation (H3K9me2) modifications and LSD1 protein enrichment in the ITGB3 promoter. During osteoclast formation, ITGB3 and LSD1 were gradually augmented. Knockdown of LSD1 or ITGB3 curbed cell viability, the expression of osteoclast marker genes, and osteoclast formation. Moreover, overexpression of ITGB3 nullified the suppressive impact of LSD1 knockdown on osteoclast formation. Mechanistically, LSD1 promoted ITGB3 expression by reducing H3K9 levels in the ITGB3 promoter. LSD1 enhanced ITGB3 expression by decreasing H3K9me1 and H3K9me2 levels in ITGB3 promoter to boost osteoclast formation.

MLL methyltransferases regulate H3K4 methylation to ensure CENP-A assembly at human centromeres.

The active state of centromeres is epigenetically defined by the presence of CENP-A interspersed with histone H3 nucleosomes. While the importance of dimethylation of H3K4 for centromeric transcription has been highlighted in various studies, the identity of the enzyme(s) depositing these marks on the centromere is still unknown. The MLL (KMT2) family plays a crucial role in RNA polymerase II (Pol II)-mediated gene regulation by methylating H3K4. Here, we report that MLL methyltransferases regulate transcription of human centromeres. CRISPR-mediated down-regulation of MLL causes loss of H3K4me2, resulting in an altered epigenetic chromatin state of the centromeres. Intriguingly, our results reveal that loss of MLL, but not SETD1A, increases co-transcriptional R-loop formation, and Pol II accumulation at the centromeres. Finally, we report that the presence of MLL and SETD1A is crucial for kinetochore maintenance. Altogether, our data reveal a novel molecular framework where both the H3K4 methylation mark and the methyltransferases regulate stability and identity of the centromere.