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.     

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.     

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.     

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.     

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.     

Synthesis and Biological Evaluation of Tripartin,a Putative KDM4 Natural Product Inhibitor,and 1‐Dichloromethylinden‐1‐ol Analogues

The natural product tripartin has been reported to inhibit the N‐methyl‐lysine histone demethylase KDM4A. A synthesis of tripartin starting from 3,5‐dimethoxyphenylacrylic acid was developed, and the enantiomers were separated by chiral HPLC. We observed that both tripartin enantiomers manifested an apparent increase in H3K9me3 levels when dosed in cells, as measured by western blot analysis. Thus, there is no enantiomeric discrimination toward this natural product in terms of its effects on cellular histone methylation status. Interestingly, tripartin did not inhibit isolated KDM4A–E under our assay conditions (IC₅₀>100 μm ). Tripartin analogues with a dichloromethylcarbinol group derived from the indanone scaffold were synthesized and found to be inactive against isolated recombinant KDM4 enzymes and in cell‐based assays. Although the precise cellular mode of action of tripartin is unclear, our evidence suggests that it may affect histone methylation status via a mechanism other than direct inhibition of the KDM4 histone demethylases.     

Photoaffinity Labeling-Based Chemoproteomic Strategy Reveals RBBP4 as a Cellular Target of Protopanaxadiol against Colorectal Cancer Cells

Protopanaxadiol (PPD), a main ginseng metabolite, exerts powerful anticancer effects against multiple types of cancer; however, its cellular targets remain elusive. Here, we synthesized a cell-permeable PPD probe via introducing a bifunctional alkyne-containing diazirine photo-crosslinker and performed a photoaffinity labeling-based chemoproteomic study. We identified retinoblastoma binding protein 4 (RBBP4), a chromatin remodeling factor, as an essential cellular target of PPD in HCT116 colorectal cancer cells. PPD significantly decreased RBBP4-dependent trimethylation at lysine 27 of histone H3 (H3K27me3), a crucial epigenetic marker that correlates with histologic signs of colorectal cancer aggressiveness, and PPD inhibition of proliferation and migration of HCT116 cells was antagonized by RBBP4 RNA silencing. Collectively, our study highlights a previously undisclosed anti-colorectal cancer cellular target of the ginseng metabolite and advances the fundamental understanding of RBBP4 functions via a chemical biology strategy.     

Lysine Ethylation by Histone Lysine Methyltransferases

Biomedicinally important histone lysine methyltransferases (KMTs) catalyze the transfer of a methyl group from S-adenosylmethionine (AdoMet) cosubstrate to lysine residues in histones and other proteins. Herein, experimental and computational investigations on human KMT-catalyzed ethylation of histone peptides by using S-adenosylethionine (AdoEth) and Se-adenosylselenoethionine (AdoSeEth) cosubstrates are reported. MALDI-TOF MS experiments reveal that, unlike monomethyltransferases SETD7 and SETD8, methyltransferases G9a and G9a-like protein (GLP) do have the capacity to ethylate lysine residues in histone peptides, and that cosubstrates follow the efficiency trend AdoMet>AdoSeEth>AdoEth. G9a and GLP can also catalyze AdoSeEth-mediated ethylation of ornithine and produce histone peptides bearing lysine residues with different alkyl groups, such as H3K9meet and H3K9me2et. Molecular dynamics and free energy simulations based on quantum mechanics/molecular mechanics potential supported the experimental findings by providing an insight into the geometry and energetics of the enzymatic methyl/ethyl transfer process.     

Tuning HP1α chromodomain selectivity for di- and trimethyllysine

Histone lysine methylation is a critical marker for controlling gene expression. The position and extent of methylation (mono-, di-, or tri-) controls the binding of effector proteins that determine whether the associated DNA is expressed or not. Dysregulation of histone protein methylation has been associated with a number of types of cancer, and development of inhibitors for the effector proteins is becoming an active area of research. For this reason, understanding the mechanism by which effector proteins obtain selectivity for the different methylation states of lysine is of great interest. To this end, we have performed mutation studies on the Drosophila HP1α chromodomain, which binds H3K9Me(2) and H3K9Me(3) with approximately equal affinities. The selectivity of HP1α chromodomain for H3K9Me(3) over H3K9Me(2) was investigated by mutating E52 to remove or weaken the hydrogen bond to K9Me(2) while maintaining affinity for K9Me(3,) including E52F, E52I, E52V, E52D, an E52Q. The E52Q mutant exhibited the greatest degree of selectivity for KMe3, with 3.5-fold weaker binding to the dimethylated peptide (K(D) =52 μM) compared to the trimethylated peptide (K(D) =15 μM). These studies provide insight into the role of electrostatic interactions and hydrogen bonding in the differentiation of methylation states and have implications regarding the evolutionary pressure for selectivity in this protein-protein interaction. Moreover, the information from this study may help guide inhibitor development for this class of proteins.