Chromatin Dysfunction Research Articles

Oncohistones: Hijacking the histone code.

Chromatin dysfunction has been implicated in a growing number of cancers especially in children and young adults. In addition to chromatin modifying and remodeling enzymes, mutations in histone genes are linked to human cancers. Since the first reports of hotspot missense mutations affecting key residues at histone H3 tail, studies have revealed how these so-called "oncohistones" dominantly (H3K27M and H3K36M) or locally (H3.3G34R/W) inhibit corresponding histone methyltransferases and misregulate epigenome and transcriptome to promote tumorigenesis. More recently, widespread mutations in all four core histones are identified in diverse cancer types. Furthermore, an "oncohistone-like" protein EZHIP has been implicated in driving childhood ependymomas through a mechanism highly reminiscent of H3K27M mutation. We will review recent progresses on understanding the biochemical, molecular and biological mechanisms underlying the canonical and novel histone mutations. Importantly, these mechanistic insights have identified therapeutic opportunities for oncohistone-driven tumors.

Short H2A histone variants are expressed in cancer

Short H2A (sH2A) histone variants are primarily expressed in the testes of placental mammals. Their incorporation into chromatin is associated with nucleosome destabilization and modulation of alternate splicing. Here, we show that sH2As innately possess features similar to recurrent oncohistone mutations associated with nucleosome instability. Through analyses of existing cancer genomics datasets, we find aberrant sH2A upregulation in a broad array of cancers, which manifest splicing patterns consistent with global nucleosome destabilization. We posit that short H2As are a class of “ready-made” oncohistones, whose inappropriate expression contributes to chromatin dysfunction in cancer.

Hyperinsulinemia promotes aberrant histone acetylation in triple-negative breast cancer

BackgroundHyperinsulinemia, the presence of excess insulin relative to glucose in the blood, is considered to be a poor prognostic indicator for patients with triple-negative breast cancer (TNBC). mTOR, a downstream effector of insulin, enhances mitochondrial biogenesis and activity, thereby increasing acetyl-CoA precursors. Increased acetyl-CoA can, in turn, be utilized by nuclear acetyltransferases for histone acetylation, a critical feature of genome regulation. While signaling pathways downstream of insulin have been established for sometime, the effect of insulin on chromatin remains unclear. We hypothesized that hyperinsulinemia-induced metabolic changes lead to genome-wide changes in histone acetylation in TNBC.ResultsMDA-MB-231 cells were xenografted into hyperinsulinemic and wild-type mice. Tumors in the hyperinsulinemic mice displayed elevated levels of histone acetylation compared to tumors in normal insulin conditions. We show that insulin treatment in vitro leads to global increase in chromatin-associated histone acetylation, in particular at H3K9, through the PI3K/AKT/mTOR pathway. Genome-wide analyses revealed that most promoter regions have an increase in histone acetylation upon insulin treatment. In addition, insulin induces higher levels of reactive oxygen species and DNA damage foci in cells.ConclusionsThese results demonstrate the impact of hyperinsulinemia on altered gene regulation through chromatin and the importance of targeting hyperinsulinemia-induced processes that lead to chromatin dysfunction in TNBC.

Hyperinsulinemia‐Induced Changes In Chromatin Acetylation In Triple Negative Breast Cancer

Obesity‐associated hyperinsulinemia or insulin‐resistance is considered as a poor prognostic factor for triple negative breast cancer (TNBC), a particularly aggressive breast cancer subtype, constituting about 15–20% of all breast cancers. As a growth factor, insulin can activate the PI3K/AKT/mTOR pathway that is associated with the increased proliferation rate of TNBC cells. While this association has been recognized for some time, the mechanistic role of insulin‐resistance in promoting TNBC remains unclear. mTOR signaling enhances mitochondrial biogenesis and activity thereby potentially increasing acetyl‐CoA precursors; pyruvate and citrate. Acetyl‐CoA, in turn, can be utilized by nuclear acetyltransferases for histone acetylation. We hypothesize that hyperinsulinemia‐induced metabolic changes lead to altered chromatin structure in TNBC through genome‐wide changes in histone acetylation. To test this hypothesis, we used quantitative ChIP‐seq (ChIP‐Rx), RNA‐seq, western blot and immunofluorescence analyses on MDA‐MB‐231 (TNBC) cells treated with insulin. Insulin treatment leads to global increase in chromatin‐associated histone acetylation levels, particularly at H3K9. Using inhibitors targeting kinases in the PI3K/AKT/mTOR signaling pathway, we demonstrate its role in insulin‐induced histone hyperacetylation. ChIP‐Rx confirmed the global increase in histone acetylation at gene promoters that could potentially alter chromatin accessibility and/or gene expression. Comparison of ChIP‐seq and RNA‐seq data revealed that insulin upregulated genes accumulate higher levels of H3K9ac as compared to downregulated genes after insulin treatment. In addition, insulin induces higher levels of reactive oxygen species and DNA damage foci in cells. Moreover, peripheral blood mononuclear cells from insulin‐resistant women show higher levels of H3K9ac and DNA damage marker gamma‐H2AX relative to those from insulin‐sensitive women. These results demonstrate the significance of targeting hyperinsulinemia‐induced processes that lead to chromatin dysfunction through mitochondria in TNBC.Support or Funding InformationThis work was supported by the NCI (R01 CA220693) and a City of Hope CCSG pilot award.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Chromatin's rising tide

At the SciBX Summit on Innovation in Drug Discovery, participants laid out a road map for how to capitalize on the full range of possible chromatin targets in and beyond oncology. They determined that industry and academia must develop tools that can flesh out how and when chromatin dysfunction drives disease.

Aberrant Histone Methylation in Myeloma: What Are the Rules?

Abstract SCI-5Aberrant regulation of histone methylation is a recurrent theme in multiple myeloma, lymphoma, and other B-cell malignancies. MMSET Multiple myeloma SET domain (MMSET) is a histone methyltransferase (HMT) overexpressed as a result of the translocation t(4;14) and is present in about 15 percent of multiple myeloma patients. MMSET is a nuclear protein with multiple domains critical for gene regulation, including the SET domain, which encodes histone methyltransferase activity, and protein and DNA interaction domains, including PHD and PWWP domains. Overexpression of MMSET induces a global increase in H3K36 methylation with concomitant loss of global H3K27 methylation. Kinetic studies using isotopic labeling and mass spectroscopy demonstrate that this change in methylation is due to both an increase in the rate of methylation of H3K36 and an increase in the demethylation of H3K27. These changes cause physical loosening of the chromatin structure, demonstrated by an increase in micrococcal nuclease accessibility, changes in DNA damage response, and aberrant gene expression. The HMT activity of MMSET is essential for growth stimulation by MMSET, as shown by the fact that reexpression of MMSET in a t(4;14) myeloma cell line, in which the rearranged MMSET allele was disrupted by homologous recombination (KMS11-TKO), rescued growth only when the HMT activity of the protein was intact. The complete H3K36/H3K27 switch mediated by MMSET requires all PHD finger domains of the protein, the second PWWP domain, and the functional SET domain. For example, a single point mutation in one PHD domain abrogated chromatin binding, histone methylation, and growth stimulation by the protein. Furthermore, deletion of the PHD domain 4 was able to increase H3K36 methylation but unable to reduce H3K37 methylation, leading to only partial growth stimulation. Despite the global change in histone methylation in response to MMSET, microarray and RNA-Seq analysis showed that only ∼1000 genes are appreciably changed in response to MMSET. The basis of the specificity of differential gene expression is under investigation. For example, many genes activated by MMSET display a peak of H3K27me3 near the transcription start site in MMSET-low cells, which is absent in MMSET-overexpressing cells, displaced by a broad pattern of H3K36me2 modification. We also found a subset of genes repressed in response to MMSET overexpression. While H3K27 methylation is decreased on a genome-wide basis in MMSET-overexpressing cells, H3K27me3 levels at repressed genes were increased in association with increased occupancy by EZH2. These regions did not show an increase in H3K36 methylation and are enriched with GC-rich elements, representing putative polycomb complex recruitment sites. We hypothesize that the global increase in H3K36me2 and drop of H3K27me3 levels on many genes leads to the displacement of the PRC2 complex from lower-affinity sites to such higher-affinity loci. These modes of action likely considerably diverge from the normal role of MMSET and EZH2 in gene regulation. Similarly, EZH2 point mutations in lymphoma lead to global chromatin dysfunction and aberrant regulation of specific sets of genes, only some of which represent previously identified EZH2 targets. Collectively, oncogenic lesions in histone-modifying enzymes in myeloma and other lymphoid neoplasms need to be understood on their own terms, as the lessons learned from the normal function of these enzymes may not predict their activity in malignancy. Disclosures:Licht:Epizyme, Inc: Research Funding.

Comparative study of antioxidant status in women of different age groups with type-2 diabetes

The purpose of this study was to evaluate the metabolic processes in women of different age groups with type-2 diabetes. It was found that hyperglycemia in aging women is characterized by the development of pronounced oxidative stress. The depletion of reduced glutathione pool is associated with a high risk of genotoxicity, since it correlates with the activation of mitochondrial and chromatin dysfunction, as well as with DNA fragmentation. In addition, polyunsaturated fatty acid hydroperoxide formation leads to necrosis and apoptosis. It can be assumed that type-2 diabetes mellitus triggers the process of apoptosis, which leads to the activation of aging programs and an increase in the mortality of patients. It is obvious that the change in the concentration of thiol antioxidants, as well as the change in concentration of molecular products of lipid peroxidation can a criterion for evaluating aging and the efficiency of the treatment of patients.

ATRX Partners with Cohesin and MeCP2 and Contributes to Developmental Silencing of Imprinted Genes in the Brain

Human developmental disorders caused by chromatin dysfunction often display overlapping clinical manifestations, such as cognitive deficits, but the underlying molecular links are poorly defined. Here, we show that ATRX, MeCP2, and cohesin, chromatin regulators implicated in ATR-X, RTT, and CdLS syndromes, respectively, interact in the brain and colocalize at the H19 imprinting control region (ICR) with preferential binding on the maternal allele. Importantly, we show that ATRX loss of function alters enrichment of cohesin, CTCF, and histone modifications at the H19 ICR, without affecting DNA methylation on the paternal allele. ATRX also affects cohesin, CTCF, and MeCP2 occupancy within the Gtl2/Dlk1 imprinted domain. Finally, we show that loss of ATRX interferes with the postnatal silencing of the maternal H19 gene along with a larger network of imprinted genes. We propose that ATRX, cohesin, and MeCP2 cooperate to silence a subset of imprinted genes in the postnatal mouse brain.

Oxidative stress and DNA fragmentation in hepatic injury of the bile duct-ligated rats: modulatory effect of ursodeoxycholic acid

Event Abstract Back to Event Oxidative stress and DNA fragmentation in hepatic injury of the bile duct-ligated rats: modulatory effect of ursodeoxycholic acid Maja Jovic1, Marko Jovic1* and Dusan Sokolovic1 1 University of Nis, Institute of Biochemistry, Serbia Introduction. Accumulation of toxic hydrophobic bile acids (BA) during cholestasis can activate Fas death receptors directly and induce oxidative damage that causes mitochondrial dysfunction and hepatocyte apoptosis. Reactive oxygen species (ROS) induce chromatin dysfunction such as single- and double-strand DNA fragmentation. The major beneficial effects of treatment with ursodeoxycholic acid (UDCA) are protection against cytotoxicity of BA, stimulation of hepatobiliary secretion, antioxidative activity and blocking the apoptotic signal (inhibition of mitochondrial cytochrome C releasing). Aim of the present study was to evaluate the effect of UDCA on oxidative stress and DNA fragmentation in liver injury of bile duct-ligated (BDL) rats. Material and Methods. Three groups of Wistar rats were investigated: I-sham-operated, II-BDL and III-BDL+UDCA (30mg/kgBW,i.g.). The animals were killed after 7 days. Results: Lipid peroxidation (MDA) level and oxidative modification of proteins (carbonyl group content) significantly increased in BDL group (4.75±0.59 vs. sham-operated 2.84±0.31 nmol/mg prot, p<0.001 and 48.21±6.37 vs. sham-operated 39.63±5.69µmol/g prot,p<0.05). Activity of catalase, the free radicals scavenging enzymes was decreased in liver of BDL rats (5.8±2.11 vs. sham-operated 8.4±2.05 U/mg prot,p<0.001). Xanthine oxidase (XO), major enzyme of ROS production was increased in cholestatic group (2.25±0.26 vs. sham-operated 1.48±0.46 U/g prot;p<0.001). The apoptotic effect in cholestasis is probably triggered by the increase activation of alkaline-DNase I (caspase 3-activated) and acid-DNase II (3.19±0.61 vs. sham-operated 1.06±0.19 U/g prot. and 2.93±0.91 vs. sham-operated 1.53±0.24 U/g prot,p<0.001). UDCA was found to be effective in liver of BDL rats:(1) decreased MDA levels (3.26±0.67,p<0.01),(2) no significantly decreased carbonyl group (45.62±6.69), (3) increase activity of catalase (7.94±1.34, p<0.001), (4) reduces activity of XO (1.77±0.68,p<0.05). (5) effect on terminal apoptotic reaction, because of the decrease in DNase I and DNase II activity (2.37±0.34 and 1.92±0.42,p<0.05), compared with cholestatic liver. Conclusion. These results support a novel effect for UDCA in the control of endonuclease activity as a final down-stream mediator of apoptosis and therefore in the DNA stability, beside established role in reducing oxidative damage and maintained antioxidant defenses in cholestatic liver. Keywords: Cholestasis, Oxidative Stress, Ursodeoxycholic Acid, DNA, Rats Conference: 8th Southeast European Congress on Xenobiotic Metabolism and Toxicity - XEMET 2010, Thessaloniki, Greece, 1 Oct - 5 Oct, 2010. Presentation Type: Poster Topic: Xenobiotic toxicity Citation: Jovic M, Jovic M and Sokolovic D (2010). Oxidative stress and DNA fragmentation in hepatic injury of the bile duct-ligated rats: modulatory effect of ursodeoxycholic acid. Front. Pharmacol. Conference Abstract: 8th Southeast European Congress on Xenobiotic Metabolism and Toxicity - XEMET 2010. doi: 10.3389/conf.fphar.2010.60.00133 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 28 Oct 2010; Published Online: 04 Nov 2010. * Correspondence: Dr. Marko Jovic, University of Nis, Institute of Biochemistry, Nis, Serbia, markojovic10@yahoo.com Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Maja Jovic Marko Jovic Dusan Sokolovic Google Maja Jovic Marko Jovic Dusan Sokolovic Google Scholar Maja Jovic Marko Jovic Dusan Sokolovic PubMed Maja Jovic Marko Jovic Dusan Sokolovic Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Glutathione depletion‐induced chromosomal DNA fragmentation associated with apoptosis and necrosis

Chromosomal DNA and mitochondrial dysfunctions play a role on mammalian cell death induced by oxidative stress. The major biochemical dysfunction of chromosome is the presence of an ordered cleavage of the DNA backborn, which is separated and visualized as an electrophoretic pattern of fragments. Oxidative stress provides chromatin dysfunction such as single strand and double strand DNA fragmentation leading to cell death. More than 1 Mb of giant DNA, 200-800 kb or 50-300 kb high molecular weight (HMW) DNA and internucleosomal DNA fragments are produced during apoptosis or necrosis induced by oxidative stress such as glutathione (GSH) depletion in several types of mammalian cells. Reactive oxygen species (ROS)-mediated DNA fragmentation is enhanced by polyunsaturated fatty acids including arachidonic acid or their hydroperoxides, leading to necrosis. Mitochondrial dysfunction on decrease of trans membrane potential, accumulation of ROS, membrane permeability transition and release of apoptotic factors during apoptosis or necrosis has been implicated. This review refers to the correlation of chromosomal DNA fragmentation and apoptosis or necrosis induced by GSH depletion, and the possible mechanisms of oxidative stress-induced cell death.

Chromosomal DNA fragmentation in apoptosis and necrosis induced by oxidative stress

Chromosomal DNA dysfunction plays a role in mammalian cell death. Oxidative stress producing reactive oxygen species (ROS) induces chromatin dysfunction such as single- and double-strand DNA fragmentation leading to cell death through apoptosis or necrosis. More than 1 Mbp giant DNA, 200–800 or 50–300 kbp high molecular weight (HMW) DNA and internucleosomal DNA fragments are produced by oxidative stress and by some agents producing ROS during apoptosis or necrosis in several types of mammalian cells. Some nucleases involved in the chromosomal DNA fragmentation in apoptosis or necrosis are classified. ROS-mediated DNA fragmentation is caused and enhanced by polyunsaturated fatty acids (PUFAs) or their hydroperoxides through lipid peroxidation. A reduction of intracellular GSH levels induced by the inhibition of cystein transport or GSH biosynthesis leads to cell death through over production and accumulation of ROS in some types of mammalian cells. The ROS accumulation system has been used as a model of oxidative stress to discuss whether ROS-mediated DNA fragmentation associated with cell death is based on apoptosis or necrosis.