Epigenetic balance ensures mechanistic control of MLL amplification and rearrangement.

MLL/KMT2A amplifications and translocations are prevalent in infant, adult, and therapy-induced leukemia. However, the molecular contributor(s) to these alterations are unclear. Here, we demonstrate that histone H3 lysine 9 mono- and di-methylation (H3K9me1/2) balance at the MLL/KMT2A locus regulates these amplifications and rearrangements. This balance is controlled by the crosstalk between lysine demethylase KDM3B and methyltransferase G9a/EHMT2. KDM3B depletion increases H3K9me1/2 levels and reduces CTCF occupancy at the MLL/KMT2A locus, in turn promoting amplification and rearrangements. Depleting CTCF is also sufficient to generate these focal alterations. Furthermore, the chemotherapy doxorubicin (Dox), which associates with therapy-induced leukemia and promotes MLL/KMT2A amplifications and rearrangements, suppresses KDM3B and CTCF protein levels. KDM3B and CTCF overexpression rescues Dox-induced MLL/KMT2A alterations. G9a inhibition in human cells or mice also suppresses MLL/KMT2A events accompanying Dox treatment. Therefore, MLL/KMT2A amplifications and rearrangements are controlled by epigenetic regulators that are tractable drug targets, which has clinical implications.

Cross-talk between Lysine-Modifying Enzymes Controls Site-Specific DNA Amplifications.

Acquired chromosomal DNA amplifications are features of many tumors. Although overexpression and stabilization of the histone H3 lysine 9/36 (H3K9/36) tri-demethylase KDM4A generates transient site-specific copy number gains (TSSGs), additional mechanisms directly controlling site-specific DNA copy gains are not well defined. In this study, we uncover a collection of H3K4-modifying chromatin regulators that function with H3K9 and H3K36 regulators to orchestrate TSSGs. Specifically, the H3K4 tri-demethylase KDM5A and specific COMPASS/KMT2 H3K4 methyltransferases modulate different TSSG loci through H3K4 methylation states and KDM4A recruitment. Furthermore, a distinct chromatin modifier network, MLL1-KDM4B-KDM5B, controls copy number regulation at a specific genomic locus in a KDM4A-independent manner. These pathways comprise an epigenetic addressing system for defining site-specific DNA rereplication and amplifications.

Epigenetic modulators provide a path to understanding disease and therapeutic opportunity.

The discovery of epigenetic modulators (writers, erasers, readers, and remodelers) has shed light on previously underappreciated biological mechanisms that promote diseases. With these insights, novel biomarkers and innovative combination therapies can be used to address challenging and difficult to treat disease states. This review highlights key mechanisms that epigenetic writers, erasers, readers, and remodelers control, as well as their connection with disease states and recent advances in associated epigenetic therapies.

KDM4A lysine demethylase induces site-specific copy gain and rereplication of regions amplified in tumors.

Acquired chromosomal instability and copy number alterations are hallmarks of cancer. Enzymes capable of promoting site-specific copy number changes have yet to be identified. Here, we demonstrate that H3K9/36me3 lysine demethylase KDM4A/JMJD2A overexpression leads to localized copy gain of 1q12, 1q21, and Xq13.1 without global chromosome instability. KDM4A-amplified tumors have increased copy gains for these same regions. 1q12h copy gain occurs within a single cell cycle, requires S phase, and is not stable but is regenerated each cell division. Sites with increased copy number are rereplicated and have increased KDM4A, MCM, and DNA polymerase occupancy. Suv39h1/KMT1A or HP1γ overexpression suppresses the copy gain, whereas H3K9/K36 methylation interference promotes gain. Our results demonstrate that overexpression of a chromatin modifier results in site-specific copy gains. This begins to establish how copy number changes could originate during tumorigenesis and demonstrates that transient overexpression of specific chromatin modulators could promote these events.

The Histone Deacetylase SIRT6 Restrains Transcription Elongation via Promoter-Proximal Pausing.

Transcriptional regulation in eukaryotes occurs at promoter-proximal regions wherein transcriptionally engaged RNA polymerase II (Pol II) pauses before proceeding toward productive elongation. The role of chromatin in pausing remains poorly understood. Here, we demonstrate that the histone deacetylase SIRT6 binds to Pol II and prevents the release of the negative elongation factor (NELF), thus stabilizing Pol II promoter-proximal pausing. Genetic depletion of SIRT6 or its chromatin deficiency upon glucose deprivation causes intragenic enrichment of acetylated histone H3 at lysines 9 (H3K9ac) and 56 (H3K56ac), activation of cyclin-dependent kinase 9 (CDK9)-that phosphorylates NELF and the carboxyl terminal domain of Pol II-and enrichment of the positive transcription elongation factors MYC, BRD4, PAF1, and the super elongation factors AFF4 and ELL2. These events lead to increased expression of genes involved in metabolism, protein synthesis, and embryonic development. Our results identified SIRT6 as a Pol II promoter-proximal pausing-dedicated histone deacetylase.

TET1 and TDG Suppress Inflammatory Response in Intestinal Tumorigenesis: Implications for Colorectal Tumors With the CpG Island Methylator Phenotype.

BACKGROUND & AIMS: Aberrant DNA methylation is frequent in colorectal cancer (CRC), but underlying mechanisms and pathologic consequences are poorly understood. METHODS: We disrupted active DNA demethylation genes Tet1 and/or Tdg from ApcMin mice and characterized the methylome and transcriptome of colonic adenomas. Data were compared to human colonic adenocarcinomas (COAD) in The Cancer Genome Atlas. RESULTS: There were increased numbers of small intestinal adenomas in ApcMin mice expressing the TdgN151A allele, whereas Tet1-deficient and Tet1/TdgN151A-double heterozygous ApcMin colonic adenomas were larger with features of erosion and invasion. We detected reduction in global DNA hypomethylation in colonic adenomas from Tet1- and Tdg-mutant ApcMin mice and hypermethylation of CpG islands in Tet1-mutant ApcMin adenomas. Up-regulation of inflammatory, immune, and interferon response genes was present in Tet1- and Tdg-mutant colonic adenomas compared to control ApcMin adenomas. This up-regulation was also seen in murine colonic organoids and human CRC lines infected with lentiviruses expressing TET1 or TDG short hairpin RNA. A 127-gene inflammatory signature separated colonic adenocarcinomas into 4 groups, closely aligned with their microsatellite or chromosomal instability and characterized by different levels of DNA methylation and DNMT1 expression that anticorrelated with TET1 expression. Tumors with the CpG island methylator phenotype (CIMP) had concerted high DNMT1/low TET1 expression. TET1 or TDG knockdown in CRC lines enhanced killing by natural killer cells. CONCLUSIONS: Our findings reveal a novel epigenetic regulation, linked to the type of genomic instability, by which TET1/TDG-mediated DNA demethylation decreases methylation levels and inflammatory/interferon/immune responses. CIMP in CRC is triggered by an imbalance of methylating activities over demethylating activities. These mice represent a model of CIMP CRC.

Hypoxia drives transient site-specific copy gain and drug-resistant gene expression.

Copy number heterogeneity is a prominent feature within tumors. The molecular basis for this heterogeneity remains poorly characterized. Here, we demonstrate that hypoxia induces transient site-specific copy gains (TSSGs) in primary, nontransformed, and transformed human cells. Hypoxia-driven copy gains are not dependent on HIF1α or HIF2α; however, they are dependent on the KDM4A histone demethylase and are blocked by inhibition of KDM4A with a small molecule or the natural metabolite succinate. Furthermore, this response is conserved at a syntenic region in zebrafish cells. Regions with site-specific copy gain are also enriched for amplifications in hypoxic primary tumors. These tumors exhibited amplification and overexpression of the drug resistance gene CKS1B, which we recapitulated in hypoxic breast cancer cells. Our results demonstrate that hypoxia provides a biological stimulus to create transient site-specific copy alterations that could result in heterogeneity within tumors and cell populations. These findings have major implications in our understanding of copy number heterogeneity and the emergence of drug resistance genes in cancer.

LOX out, histones: a new enzyme is nipping at your tails.

In the current issue of Molecular Cell, Herranz et al. (2012) demonstrate that LOXL2 deaminates trimethylated histone 3 lysine 4 (H3K4me3), which uncovers a new chromatin modification and a new enzymatic mechanism with the potential to regulate additional lysine residues.

Histone lysine methylation dynamics: establishment, regulation, and biological impact.

Histone lysine methylation has emerged as a critical player in the regulation of gene expression, cell cycle, genome stability, and nuclear architecture. Over the past decade, a tremendous amount of progress has led to the characterization of methyl modifications and the lysine methyltransferases (KMTs) and lysine demethylases (KDMs) that regulate them. Here, we review the discovery and characterization of the KMTs and KDMs and the methyl modifications they regulate. We discuss the localization of the KMTs and KDMs as well as the distribution of lysine methylation throughout the genome. We highlight how these data have shaped our view of lysine methylation as a key determinant of complex chromatin states. Finally, we discuss the regulation of KMTs and KDMs by proteasomal degradation, posttranscriptional mechanisms, and metabolic status. We propose key questions for the field and highlight areas that we predict will yield exciting discoveries in the years to come.

Conserved antagonism between JMJD2A/KDM4A and HP1γ during cell cycle progression.

The KDM4/JMJD2 family of histone demethylases is amplified in human cancers. However, little is known about their physiologic or tumorigenic roles. We have identified a conserved and unappreciated role for the JMJD2A/KDM4A H3K9/36 tridemethylase in cell cycle progression. We demonstrate that JMJD2A protein levels are regulated in a cell cycle-dependent manner and that JMJD2A overexpression increased chromatin accessibility, S phase progression, and altered replication timing of specific genomic loci. These phenotypes depended on JMJD2A enzymatic activity. Strikingly, depletion of the only C. elegans homolog, JMJD-2, slowed DNA replication and increased ATR/p53-dependent apoptosis. Importantly, overexpression of HP1γ antagonized JMJD2A-dependent progression through S phase, and depletion of HPL-2 rescued the DNA replication-related phenotypes in jmjd-2(-/-) animals. Our findings describe a highly conserved model whereby JMJD2A regulates DNA replication by antagonizing HP1γ and controlling chromatin accessibility.

Conserved role of SIRT1 orthologs in fasting-dependent inhibition of the lipid/cholesterol regulator SREBP.

The sterol regulatory element-binding protein (SREBP) transcription factor family is a critical regulator of lipid and sterol homeostasis in eukaryotes. In mammals, SREBPs are highly active in the fed state to promote the expression of lipogenic and cholesterogenic genes and facilitate fat storage. During fasting, SREBP-dependent lipid/cholesterol synthesis is rapidly diminished in the mouse liver; however, the mechanism has remained incompletely understood. Moreover, the evolutionary conservation of fasting regulation of SREBP-dependent programs of gene expression and control of lipid homeostasis has been unclear. We demonstrate here a conserved role for orthologs of the NAD(+)-dependent deacetylase SIRT1 in metazoans in down-regulation of SREBP orthologs during fasting, resulting in inhibition of lipid synthesis and fat storage. Our data reveal that SIRT1 can directly deacetylate SREBP, and modulation of SIRT1 activity results in changes in SREBP ubiquitination, protein stability, and target gene expression. In addition, chemical activators of SIRT1 inhibit SREBP target gene expression in vitro and in vivo, correlating with decreased hepatic lipid and cholesterol levels and attenuated liver steatosis in diet-induced and genetically obese mice. We conclude that SIRT1 orthologs play a critical role in controlling SREBP-dependent gene regulation governing lipid/cholesterol homeostasis in metazoans in response to fasting cues. These findings may have important biomedical implications for the treatment of metabolic disorders associated with aberrant lipid/cholesterol homeostasis, including metabolic syndrome and atherosclerosis.

Regulation of Transient Site-specific Copy Gain by MicroRNA.

Intra-tumor copy number heterogeneity is commonly observed in cancer; however, the molecular mechanisms that contribute to heterogeneity remain poorly understood. Up-regulation of the histone demethylase KDM4A promotes transient site-specific copy gain (TSSG) in cells; therefore, uncovering how KDM4A levels are controlled is important for understanding the regulation of copy number heterogeneity. Here, we demonstrate that KDM4A is regulated by hsa-mir-23a-3p, hsa-mir-23b-3p, and hsa-mir-137. Altering expression of these microRNAs (miRNAs) regulates KDM4A-dependent TSSG. miRNA inhibition promoted copy gains and increased expression of the drug-resistant oncogene CKS1B, which was further substantiated in primary breast tumors. Consistent with increased CKS1B expression, miRNA inhibition reduced breast cancer cell sensitivity to cisplatin. Our data identify these miRNAs as regulators of TSSG and copy gains of a drug resistance gene.

Examining the impact of gene variants on histone lysine methylation.

In recent years, there has been a boom in the amount of genome-wide sequencing data that has uncovered important and unappreciated links between certain genes, families of genes and enzymatic processes and diseases such as cancer. Such studies have highlighted the impact that chromatin modifying enzymes could have in cancer and other genetic diseases. In this review, we summarize characterized mutations and single nucleotide polymorphisms (SNPs) in histone lysine methyltransferases (KMTs), histone lysine demethylases (KDMs) and histones. We primarily focus on variants with strong disease correlations and discuss how they could impact histone lysine methylation dynamics and gene regulation.

Tipping the lysine methylation balance in disease.

Genomic instability is a major contributing factor to the development and onset of diseases such as cancer. Emerging evidence has demonstrated that maintaining the proper balance of histone lysine methylation is critical to preserve genomic integrity. Genome-wide association studies, gene sequencing, and genome-wide mapping approaches have helped identify mutations, copy number changes, and aberrant gene regulation of lysine methyltransferases (KMTs) and demethylases (KDMs) associated with cancer and cognitive disorders. Structural analysis of KMTs and KDMs has demonstrated the drugability of these enzymes and has led to the discovery of small molecule inhibitors. Use of these inhibitors has allowed better understanding of the biochemical properties of KMTs and KDMs and demonstrated potential for therapeutic use. This review will highlight the methyl modifications, KMTs and KDMs associated with cancer and neurological disorders and how KMT and KDM and the potential for treatment of these conditions with small molecule inhibitors.

A histone H3 lysine 27 demethylase regulates animal posterior development.

The recent discovery of a large number of histone demethylases suggests a central role for these enzymes in regulating histone methylation dynamics. Histone H3K27 trimethylation (H3K27me3) has been linked to polycomb-group-protein-mediated suppression of Hox genes and animal body patterning, X-chromosome inactivation and possibly maintenance of embryonic stem cell (ESC) identity. An imbalance of H3K27 methylation owing to overexpression of the methylase EZH2 has been implicated in metastatic prostate and aggressive breast cancers. Here we show that the JmjC-domain-containing related proteins UTX and JMJD3 catalyse demethylation of H3K27me3/2. UTX is enriched around the transcription start sites of many HOX genes in primary human fibroblasts, in which HOX genes are differentially expressed, but is selectively excluded from the HOX loci in ESCs, in which HOX genes are largely silent. Consistently, RNA interference inhibition of UTX led to increased H3K27me3 levels at some HOX gene promoters. Importantly, morpholino oligonucleotide inhibition of a zebrafish UTX homologue resulted in mis-regulation of hox genes and a striking posterior developmental defect, which was partially rescued by wild-type, but not by catalytically inactive, human UTX. Taken together, these findings identify a small family of H3K27 demethylases with important, evolutionarily conserved roles in H3K27 methylation regulation and in animal anterior-posterior development.

The SKP1-Cul1-F-box and leucine-rich repeat protein 4 (SCF-FbxL4) ubiquitin ligase regulates lysine demethylase 4A (KDM4A)/Jumonji domain-containing 2A (JMJD2A) protein.

Chromatin-modifying enzymes play a fundamental role in regulating chromatin structure so that DNA replication is spatially and temporally coordinated. For example, the lysine demethylase 4A/Jumonji domain-containing 2A (KDM4A/JMJD2A) is tightly regulated during the cell cycle. Overexpression of JMJD2A leads to altered replication timing and faster S phase progression. In this study, we demonstrate that degradation of JMJD2A is regulated by the proteasome. JMJD2A turnover is coordinated through the SKP1-Cul1-F-box ubiquitin ligase complex that contains cullin 1 and the F-box and leucine-rich repeat protein 4 (FbxL4). This complex interacted with JMJD2A. Ubiquitin overexpression restored turnover and blocked the JMJD2A-dependent faster S phase progression in a cullin 1-dependent manner. Furthermore, increased ubiquitin levels decreased JMJD2A occupancy and BrdU incorporation at target sites. This study highlights a finely tuned mechanism for regulating histone demethylase levels and emphasizes the need to tightly regulate chromatin modifiers so that the cell cycle occurs properly.

The conserved NAD(H)-dependent corepressor CTBP-1 regulates Caenorhabditis elegans life span.

CtBP (C-terminal binding protein) is an evolutionarily conserved NAD(H)-dependent transcriptional corepressor, whose activity has been shown to be regulated by the NAD/NADH ratio. Although recent studies have provided significant new insights into mechanisms by which CtBP regulates transcription, the biological function of CtBP remains incompletely understood. Here, we report that genetic inactivation of the Caenorhabditis elegans homolog, ctbp-1, results in life span extension, which is suppressed by reintroduction of the ctbp-1 genomic DNA encoding wild-type but not NAD(H)-binding defective CTBP-1 protein. We show that CTBP-1 possibly modulates aging through the insulin/IGF-1 signaling pathway, dependent on the forkhead transcription factor DAF-16, but independent of the NAD-dependent histone deacetylase SIR-2.1. Genome-wide microarray analysis identifies >200 potential CTBP-1 target genes. Importantly, RNAi inhibition of a putative triacylglycerol lipase gene lips-7(C09E8.2) but not another lipase suppresses the life span extension phenotype. Consistently, metabolic analysis shows that the triacylglycerol level is reduced in the ctbp-1 deletion mutant, which is restored to the wild-type level by RNAi inhibition of lips-7. Taken together, our data suggest that CTBP-1 controls life span probably through the regulation of lipid metabolism.

Protocol to isolate cells in four stages of S phase for high-resolution replication-timing sequencing.

Traditional replication timing (RT) experiments divide S phase into two phases: early and late. However, there is an increasing awareness that variation in RT can occur during the course of S phase and impact our understanding of RT patterns and regulation. Here, we describe a RT protocol in RPE-1 cells for collecting four phases within S and the library preparation that takes advantage of a commercial kit for methyl-DNA. This step allows BrdU-labeled DNA sequencing and assessment of RT genome wide. For complete details on the use and execution of this protocol, please refer to Van Rechem et al. (2021).

A cell-sorting-based protocol for cell cycle small-scale ChIP sequencing.

Classic approaches to characterizing cell cycle leverage chemicals or altered nucleotide pools, which could impact chromatin states at specific phases of the cell cycle. Such approaches could induce metabolic alterations and/or DNA damage, which could reshape protein recruitment and histone modifications. In this protocol, we describe ways to fix and sort cells across the cell cycle based on their DNA content. We further detail immunoprecipitation and library preparation, allowing analysis of the epigenome by chromatin immunoprecipitation sequencing (ChIP-seq) for small numbers of cells. For complete details on the use and execution of this protocol, please refer to Van Rechem et al. (2021).