Histone Demethylase KDM7A Contributes to the Development of Hepatic Steatosis by Targeting Diacylglycerol Acyltransferase 2

Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease. While the development of NAFLD is correlated with aberrant histone methylation, modifiers of histone methylation involved in this event remain poorly understood. Here, we studied the functional role of the histone demethylase KDM7A in the development of hepatic steatosis. KDM7A overexpression in AML12 cells upregulated diacylglycerol acyltransferase 2 (DGAT2) expression and resulted in increased intracellular triglyceride (TG) accumulation. Conversely, KDM7A knockdown reduced DGAT2 expression and TG accumulation, and significantly reversed free fatty acids-induced TG accumulation. Additionally, adenovirus-mediated overexpression of KDM7A in mice resulted in hepatic steatosis, which was accompanied by increased expression of hepatic DGAT2. Furthermore, KDM7A overexpression decreased the enrichment of di-methylation of histone H3 lysine 9 (H3K9me2) and H3 lysine 27 (H3K27me2) on the promoter of DGAT2. Taken together, these results indicate that KDM7A overexpression induces hepatic steatosis through upregulation of DGAT2 by erasing H3K9me2 and H3K27me2 on the promoter.     

Traceless Semisynthesis of a Set of Histone 3 Species Bearing Specific Lysine Methylation Marks

Considerable mechanistic insight into the function of histone post‐translational modifications and the enzymes that install and remove them derives from in vitro experiments with modified histones, often embedded in nucleosomes. We report the first semisyntheses of native‐like histone 3 (H3) bearing tri‐ and dimethyllysines at position 79 and trimethyllysine at position 36, as well as more facile and traceless semisyntheses of K9 and K27 trimethylated species. These semisyntheses are practical on a multi‐milligram scale and can also generate H3 with combinations of marks. Each of these modifications has distinct functional consequences, although the pathways by which H3K36me3 and H3K79me2/3 act have not been entirely mapped. To this end, we demonstrated that our semisynthetic histones, when reconstituted into nucleosomes, are valuable affinity reagents for unbiased binding partner discovery and compare them to their methyllysine analogue (MLA) counterparts at the nucleosome level.     

The Functions of the Demethylase JMJD3 in Cancer

Cancer is a major cause of death worldwide. Epigenetic changes in response to external (diet, sports activities, etc.) and internal events are increasingly implicated in tumor initiation and progression. In this review, we focused on post-translational changes in histones and, more particularly, the tri methylation of lysine from histone 3 (H3K27me3) mark, a repressive epigenetic mark often under- or overexpressed in a wide range of cancers. Two actors regulate H3K27 methylation: Jumonji Domain-Containing Protein 3 demethylase (JMJD3) and Enhancer of zeste homolog 2 (EZH2) methyltransferase. A number of studies have highlighted the deregulation of these actors, which is why this scientific review will focus on the role of JMJD3 and, consequently, H3K27me3 in cancer development. Data on JMJD3’s involvement in cancer are classified by cancer type: nervous system, prostate, blood, colorectal, breast, lung, liver, ovarian, and gastric cancers.     

Kynurenic Acid and Its Analog SZR104 Exhibit Strong Antiinflammatory Effects and Alter the Intracellular Distribution and Methylation Patterns of H3 Histones in Immunochallenged Microglia-Enriched Cultures of Newborn Rat Brains

Kynurenic acid (KYNA) is implicated in antiinflammatory processes in the brain through several cellular and molecular targets, among which microglia-related mechanisms are of paramount importance. In this study, we describe the effects of KYNA and one of its analogs, the brain-penetrable SZR104 (N-(2-(dimethylamino)ethyl)-3-(morpholinomethyl)-4-hydroxyquinoline-2-carboxamide), on the intracellular distribution and methylation patterns of histone H3 in immunochallenged microglia cultures. Microglia-enriched secondary cultures made from newborn rat forebrains were immunochallenged with lipopolysaccharide (LPS). The protein levels of selected inflammatory markers C–X–C motif chemokine ligand 10 (CXCL10) and C–C motif chemokine receptor 1 (CCR1), histone H3, and posttranslational modifications of histone H3 lys methylation sites (H3K9me3 and H3K36me2, marks typically associated with opposite effects on gene expression) were analyzed using quantitative fluorescent immunocytochemistry and western blots in control or LPS-treated cultures with or without KYNA or SZR104. KYNA and SZR104 reduced levels of the inflammatory marker proteins CXCL10 and CCR1 after LPS-treatment. Moreover, KYNA and SZR104 favorably affected histone methylation patterns as H3K9me3 and H3K36me2 immunoreactivities, and histone H3 protein levels returned toward control values after LPS treatment. The cytoplasmic translocation of H3K9me3 from the nucleus indicated inflammatory distress, a process that could be inhibited by KYNA and SZR104. Thus, KYNA signaling and metabolism, and especially brain-penetrable KYNA analogs such as SZR104, could be key targets in the pathway that connects chromatin structure and epigenetic mechanisms with functional consequences that affect neuroinflammation and perhaps neurodegeneration.     

SDG102, a H3K36-Methyltransferase-Encoding Gene,Plays Pleiotropic Roles in Growth and Development of Maize (Zea mays L.)

Although histone lysine methylation has been studied in thale cress (Arabidopsis thaliana (L.) Heynh.) and rice (Oryza sativa L.) in recent years, its function in maize (Zea mays L.) remains poorly characterized. To better understand the function of histone lysine methylation in maize, SDG102, a H3 lysine 36 (H3K36) methylase, was chosen for functional characterization using overexpressed and knockout transgenic plants. SDG102-deficiency in maize caused multiple phenotypes including yellow leaves in seedlings, late-flowering, and increased adult plant height, while the overexpression of SDG102 led to reduced adult plant height. The key flowering genes, ZCN8/ZCN7 and MADS4/MADA67, were downregulated in SDG102-deficient plants. Chromatin immunoprecipitation (ChIP) experiments showed that H3 lysine 36 trimethylation (H3K36me3) levels were reduced at these loci. Perturbation of SDG102 expression caused the misexpression of multiple genes. Interestingly, the overexpression or knockout of SDG102 also led to genome-wide decreases and increases in the H3K36me3 levels, respectively. Together, our results suggest that SDG102 is a methyltransferase that catalyzes the trimethylation of H3K36 of many genes across the maize genome, which are involved in multiple biological processes including those controlling flowering time.     

Comparison of Histone H3K4me3 between IVF and ICSI Technologies and between Boy and Girl Offspring

Epigenetics play a vital role in early embryo development. Offspring conceived via assisted reproductive technologies (ARTs) have a three times higher risk of epigenetic diseases than naturally conceived children. However, investigations into ART-associated placental histone modifications or sex-stratified analyses of ART-associated histone modifications remain limited. In the current study, we carried out immunohistochemistry, chip-sequence analysis, and a series of in vitro experiments. Our results demonstrated that placentas from intra-cytoplasmic sperm injection (ICSI), but not in vitro fertilization (IVF), showed global tri-methylated-histone-H3-lysine-4 (H3K4me3) alteration compared to those from natural conception. However, for acetylated-histone-H3-lysine-9 (H3K9ac) and acetylated-histone-H3-lysine-27 (H3K27ac), no significant differences between groups could be found. Further, sex -stratified analysis found that, compared with the same-gender newborn cord blood mononuclear cell (CBMC) from natural conceptions, CBMC from ICSI-boys presented more genes with differentially enriched H3K4me3 (n = 198) than those from ICSI-girls (n = 79), IVF-girls (n = 5), and IVF-boys (n = 2). We also found that varying oxygen conditions, RNA polymerase II subunit A (Polr2A), and lysine demethylase 5A (KDM5A) regulated H3K4me3. These findings revealed a difference between IVF and ICSI and a difference between boys and girls in H3K4me3 modification, providing greater insight into ART-associated epigenetic alteration.     

Dual Inhibition of H3K9me2 and H3K27me3 Promotes Tumor Cell Senescence without Triggering the Secretion of SASP

Chemotherapy remains the most common cancer treatment. Although chemotherapeutic drugs induce tumor cell senescence, they are often associated with post-therapy tumor recurrence by inducing the senescence-associated secretory phenotype (SASP). Therefore, it is important to identify effective strategies to induce tumor cell senescence without triggering SASP. In this study, we used the small molecule inhibitors, UNC0642 (G9a inhibitor) and UNC1999 (EZH2 inhibitor) alone or in combination, to inhibit H3K9 and H3K27 methylation in different cancer cells. Dual inhibition of H3K9me2 and H3K27me3 in highly metastatic tumor cells had a stronger pro-senescence effect than either inhibitor alone and did not trigger SASP in tumor cells. Dual inhibition of H3K9me2 and H3K27me3 suppressed the formation of cytosolic chromatin fragments, which inhibited the cGAS-STING-SASP pathway. Collectively, these data suggested that dual inhibition of H3K9 and H3K27 methylation induced senescence of highly metastatic tumor cells without triggering SASP by inhibiting the cGAS-STING-SASP pathway, providing a new mechanism for the epigenetics-based therapy targeting H3K9 and H3K27 methylation.     

Mechanistic Insights into the Allosteric Regulation of the Clr4 Protein Lysine Methyltransferase by Autoinhibition and Automethylation

Clr4 is a histone H3 lysine 9 methyltransferase in Schizosaccharomyces pombe that is essential for heterochromatin formation. Previous biochemical and structural studies have shown that Clr4 is in an autoinhibited state in which an autoregulatory loop (ARL) blocks the active site. Automethylation of lysine residues in the ARL relieves autoinhibition. To investigate the mechanism of Clr4 regulation by autoinhibition and automethylation, we exchanged residues in the ARL by site-directed mutagenesis leading to stimulation or inhibition of automethylation and corresponding changes in Clr4 catalytic activity. Furthermore, we demonstrate that Clr4 prefers monomethylated (H3K9me1) over unmodified (H3K9me0) histone peptide substrates, similar to related human enzymes and, accordingly, H3K9me1 is more efficient in overcoming autoinhibition. Due to enzyme activation by automethylation, we observed a sigmoidal dependence of Clr4 activity on the AdoMet concentration, with stimulation at high AdoMet levels. In contrast, an automethylation-deficient mutant showed a hyperbolic Michaelis–Menten type relationship. These data suggest that automethylation of the ARL could act as a sensor for AdoMet levels in cells and regulate the generation and maintenance of heterochromatin accordingly. This process could connect epigenome modifications with the metabolic state of cells. As other human protein lysine methyltransferases (for example, PRC2) also use automethylation/autoinhibition mechanisms, our results may provide a model to describe their regulation as well.     

Regulation of Epigenetic Modifications in the Placenta during Preeclampsia: PPARγ Influences H3K4me3 and H3K9ac in Extravillous Trophoblast Cells

The aim of this study was to analyze the expression of peroxisome proliferator-activated receptor γ (PPARγ) and retinoid X receptor α (RxRα), a binding heterodimer playing a pivotal role in the successful trophoblast invasion, in the placental tissue of preeclamptic patients. Furthermore, we aimed to characterize a possible interaction between PPARγ and H3K4me3 (trimethylated lysine 4 of the histone H3), respectively H3K9ac (acetylated lysine 9 of the histone H3), to illuminate the role of histone modifications in a defective trophoblast invasion in preeclampsia (PE). Therefore, the expression of PPARγ and RxRα was analyzed in 26 PE and 25 control placentas by immunohistochemical peroxidase staining, as well as the co-expression with H3K4me3 and H3K9ac by double immunofluorescence staining. Further, the effect of a specific PPARγ-agonist (Ciglitazone) and PPARγ-antagonist (T0070907) on the histone modifications H3K9ac and H3K4me3 was analyzed in vitro. In PE placentas, we found a reduced expression of PPARγ and RxRα and a reduced co-expression with H3K4me3 and H3K9ac in the extravillous trophoblast (EVT). Furthermore, with the PPARγ-antagonist treated human villous trophoblast (HVT) cells and primary isolated EVT cells showed higher levels of the histone modification proteins whereas treatment with the PPARγ-agonist reduced respective histone modifications. Our results show that the stimulation of PPARγ-activity leads to a reduction of H3K4me3 and H3K9ac in trophoblast cells, but paradoxically decreases the nuclear PPARγ expression. As the importance of PPARγ, being involved in a successful trophoblast invasion has already been investigated, our results reveal a pathophysiologic connection between PPARγ and the epigenetic modulation via H3K4me3 and H3K9ac in PE.     

Targeted Induction of Endogenous VDUP1 by Small Activating RNA Inhibits the Growth of Lung Cancer Cells

Recent studies have reported that small double-strand RNAs (dsRNAs) can activate endogenous genes via an RNA-based promoter targeting mechanism termed RNA activation (RNAa). In the present study, we showed that dsVDUP1-834, a novel small activating RNA (saRNA) targeting promoter of vitamin D₃ up-regulated protein 1 (VDUP1) gene, up-regulated expression of VDUP1 at both mRNA and protein levels in A549 lung cancer cells. We also demonstrated that dsVDUP1-834 inhibited cell proliferation in A549 lung cancer cells. Further studies showed that dsVDUP1-834 induced cell-cycle arrest by increasing p27 and p53 and decreasing cyclin A and cyclin B1. In addition, knockdown of VDUP1 abrogated dsVDUP1-834-induced up-regulation of VDUP1 gene expression and related effects. The activation of VDUP1 by dsVDUP1-834 was accompanied by an increase in dimethylation of histone 3 at lysine 4 (H3K4me2) and acetylation of histone 3 (H3ac) and a decrease in dimethylation of histone 3 at lysine 9 (H3K9me2) at the target site of VDUP1 promoter. Moreover, the enrichment of Ago2 was detected at the dsVDUP1-834 target site, and Ago2 knockdown significantly suppressed dsVDUP1-834-mediated inhibition of cell proliferation and modulation of cell-cycle regulators. Taken together, the results presented in this report demonstrate that dsVDUP1-834 induces VDUP1 gene expression by epigenetic changes, resulting in cell growth inhibition and cell-cycle arrest. Our results suggest that targeted induction of VDUP1 by dsVDUP1-834 might be a promising therapeutic strategy for the treatment of lung cancer.     

The SUV4-20H Histone Methyltransferases in Health and Disease

The post-translational modification of histone tails is a dynamic process that provides chromatin with high plasticity. Histone modifications occur through the recruitment of nonhistone proteins to chromatin and have the potential to influence fundamental biological processes. Many recent studies have been directed at understanding the role of methylated lysine 20 of histone H4 (H4K20) in physiological and pathological processes. In this review, we will focus on the function and regulation of the histone methyltransferases SUV4-20H1 and SUV4-20H2, which catalyze the di- and tri-methylation of H4K20 at H4K20me2 and H4K20me3, respectively. We will highlight recent studies that have elucidated the functions of these enzymes in various biological processes, including DNA repair, cell cycle regulation, and DNA replication. We will also provide an overview of the pathological conditions associated with H4K20me2/3 misregulation as a result of mutations or the aberrant expression of SUV4-20H1 or SUV4-20H2. Finally, we will critically analyze the data supporting these functions and outline questions for future research.