Polycomb Complex Research Articles

Expression of ANRIL-Polycomb Complexes-CDKN2A/B/ARF Genes in Breast Tumors: Identification of a Two-Gene (EZH2/CBX7) Signature with Independent Prognostic Value.

ANRIL, a long noncoding RNA (lncRNA), has recently been reported to have a direct role in recruiting polycomb repressive complexes PRC2 and PRC1 to regulate the expression of the p15/CDKN2B-p16/CDKN2A-p14/ARF gene cluster. Expression analysis of ANRIL, EZH2, SUZ12, EED, JARID2, CBX7, BMI1, p16, p15, and p14/ARF genes was evaluated in a large cohort of invasive breast carcinomas (IBC, n = 456) by qRT-PCR and immunohistochemistry (IHC) was performed on CBX7, EZH2, p14, p15, p16, H3K27me3, and H3K27ac. We observed significant overexpression in IBCs of ANRIL (19.7%) and EZH2 (77.0%) and an underexpression of CBX7 (39.7%). Correlations were identified between these genes, their expression patterns, and several classical clinical and pathologic parameters, molecular subtypes, and patient outcomes, as well as with proliferation, epithelial-mesenchymal transition, and breast cancer stem cell markers. Multivariate analysis revealed that combined EZH2/CBX7 status is an independent prognostic factor (P = 0.001). In addition, several miRNAs negatively associated with CBX7 underexpression and EZH2 overexpression. These data demonstrate a complex pattern of interactions between lncRNA ANRIL, several miRNAs, PRC2/PRC1 subunits, and p15/CDKN2B-p16/CDKN2A-p14/ARF locus and suggest that their expression should be considered together to evaluate antitumoral drugs, in particular the BET bromodomain inhibitors. This study suggests that the global pattern of expression rather than expression of individual family members should be taken into account when defining functionality of repressive Polycomb complexes and therapeutic targeting potential. Mol Cancer Res; 14(7); 623-33. ©2016 AACR.

Highlights of This Issue

Increasingly, long non-coding RNA (lncRNA) functions are being attributed to many critical biological processes in cancer. This study demonstrates a complex pattern of interactions between lncRNA ANRIL (Antisense Non-coding RNA in the INK4 Locus),several miRs, PRC2/PRC1 subunits and p15/CDKN2Bp16/CDKN2A-p14/ARF locus in a large cohort of invasive breast carcinomas. Significant overexpression of ANRIL and EZH2 and under-expression of CBX7 was observed. Correlations were identified between these genes, their expression patterns and clinicopathological parameters, molecular subtypes and patient outcomes. Multivariate analysis revealed that combined EZH2/CBX7 status is an independent prognostic factor. These data suggest that global expression patterns should be accounted for when defining functionality of repressive Polycomb complexes and therapeutic targeting potential.In this issue, new evidence suggests a mechanism for cancer's resistance to TRAIL-based therapies. For years, the thought has been death receptor activation using a synthetic mimic of TRAIL would selectively kill cancer. However, like most targeted agents used to fight pancreatic cancer, TRAIL-targeted therapies performed poorly in trials. Here, HuR (Hu antigen R) was demonstrated to be a key regulator of TRAIL resistance, which becomes especially active when pancreatic cancer cells are treated with TRAIL. Treated cells activate HuR, which reduces the expression of a death receptor available for TRAIL-induced apoptosis. Small molecule or siRNA inhibition of HuR, sensitized pancreatic cancer cells to TRAIL-drug combinations as compared to TRAIL alone. Upon further validation, this approach could help resurrect a disappointing anti-pancreatic cancer agent.McAdam and colleagues highlight the importance of damage to the DNA repair proteome by demonstrating that DNA repair is impaired in oxidatively stressed cultured human cells. In particular, they show that oxidative stress caused by UVA radiation or by interventions that deplete cellular antioxidants damages nucleotide excision repair (NER) proteins and inhibits the removal of the sunlight-induced DNA lesions that are implicated in skin cancer development. Because UVA comprises around 95% of incident ultraviolet radiation, oxidative inactivation of NER has implications for skin carcinogenesis. DNA repair also attenuates the effectiveness of anticancer drugs and its inhibition by protein oxidation may increase therapeutic efficacy.This novel study introduces for the first time the embryological WNT5a/ROR2 signaling pathway in the pathogenesis of Barrett's-associated esophageal adenocarcinoma. By analyzing human esophageal specimens and esophageal cells, Lyros and colleagues present evidence that early loss of WNT5a expression along with upregulation of ROR2-receptor contribute to tumor growth of esophageal adenocarcinoma.These findings highlight the significance of the WNT5a/ROR2 signaling pathway in the development and malignant transformation of Barrett'esophagus into esophageal adenocarcinoma and suggest a number of novel therapeutic opportunities at all stages of the metaplasia-dysplasia-adenocarcinoma sequence.

Polycomb complex PRC1 as gatekeeper of intestinal stem cell identity

Intestinal stem cells (ISCs) are adult multipotent cells essential for the maintenance of intestinal epithelial homeostasis. Wnt signaling activity ensures that the pool of ISCs at the basis of the intestinal crypts is preserved. Dysregulation of the Wnt pathway is often observed in cancer and supports malignant progression. Chiacchiera and colleagues recently demonstrated the implication of the polycomb complex PRC1 in the regulation of the Wnt pathway in adult ISCs. The authors show that PRC1 maintains intestinal homeostasis by repressing the expression of ZICs, a family of transcription factors inactivating the β-catenin/TCF complex. Importantly, interfering with PRC1 activity completely inhibits the formation of Wnt-dependent tumors. These findings reveal a new layer of epigenetic regulation of the Wnt pathway and open novel opportunities for cancer stem cell targeted therapy.

The dynamic interactome and genomic targets of Polycomb complexes during stem-cell differentiation.

While the core subunits of Polycomb group (PcG) complexes are well characterized, little is known about the dynamics of these protein complexes during cellular differentiation. We used quantitative interaction proteomics and genome-wide profiling to study PcG proteins in mouse embryonic stem cells (ESCs) and neural progenitor cells (NPCs). We found the stoichiometry and genome-wide binding of PRC1 and PRC2 to be highly dynamic during neural differentiation. Intriguingly, we observed a downregulation and loss of PRC2 from H3K27me3-marked chromatin during differentiation, whereas PRC1 was retained at these sites. Additionally, we found PRC1 at enhancer and promoter regions independent of PRC2 binding and H3K27me3. Finally, overexpression of NPC-specific PRC1 interactors in ESCs led to increased Ring1b binding to and decreased expression of NPC-enriched Ring1b target genes. In summary, our integrative analyses have uncovered dynamic PcG subcomplexes and widespread co-localization with active chromatin marks during differentiation.

A balancing act in modifying chromatin

Epigenetics Chromatin modifiers add chemical groups to histones, the proteins that package DNA. These modifications are central to cellular development, and mutations in their molecular machinery are linked to a variety of human diseases. Piunti and Shilatifard review the balance between the prototypic chromatin modifiers Polycomb and COMPASS complexes and their role in gene regulation and normal development. Although originally identified as indispensible regulators of fruit fly development, related roles have been identified in other organisms. Furthermore, mutations in human homologs have been implicated in various cancers. As such, these complexes may serve as effective targets for epigenetic therapies. Science , this issue p. [10.1126/science.aad9780][1] [1]: /lookup/doi/10.1126/science.aad9780

RBFox2 Binds Nascent RNA to Globally Regulate Polycomb Complex 2 Targeting in Mammalian Genomes

Increasing evidence suggests that diverse RNA binding proteins (RBPs) interact with regulatory RNAs toregulate transcription. RBFox2 is a well-characterized pre-mRNA splicing regulator, but we now encounter an unexpected paradigm where depletion of this RBP induces widespread increase in nascent RNA production in diverse cell types. Chromatin immunoprecipitation sequencing (ChIP-seq) reveals extensive interaction of RBFox2 with chromatin in a nascent RNA-dependent manner. Bayesian network analysis connects RBFox2 to Polycomb complex 2 (PRC2) and H3K27me3, and biochemical experiments demonstrate the ability of RBFox2 to directly interact with PRC2. Strikingly, RBFox2 inactivation eradicates PRC2 targeting on the majority of bivalent gene promoters and leads to transcriptional de-repression. Together, these findings uncover a mechanism underlying the enigmatic association of PRC2 with numerous active genes, highlight the importance of gene body sequences to gauge transcriptional output, and suggest nascent RNAs as critical signals for transcriptional feedback control to maintain homeostatic gene expression in mammalian genomes.

Drosophila O-GlcNAcase Deletion Globally Perturbs Chromatin O-GlcNAcylation

Gene expression during Drosophila development is subject to regulation by the Polycomb (Pc), Trithorax (Trx), and Compass chromatin modifier complexes. O-GlcNAc transferase (OGT/SXC) is essential for Pc repression suggesting that the O-GlcNAcylation of proteins plays a key role in regulating development. OGT transfers O-GlcNAc onto serine and threonine residues in intrinsically disordered domains of key transcriptional regulators; O-GlcNAcase (OGA) removes the modification. To pinpoint genomic regions that are regulated by O-GlcNAc levels, we performed ChIP-chip and microarray analysis after OGT or OGA RNAi knockdown in S2 cells. After OGA RNAi, we observed a genome-wide increase in the intensity of most O-GlcNAc-occupied regions including genes linked to cell cycle, ubiquitin, and steroid response. In contrast, O-GlcNAc levels were strikingly insensitive to OGA RNAi at sites of polycomb repression such as the Hox and NK homeobox gene clusters. Microarray analysis suggested that altered O-GlcNAc cycling perturbed the expression of genes associated with morphogenesis and cell cycle regulation. We then produced a viable null allele of oga (oga(del.1)) in Drosophila allowing visualization of altered O-GlcNAc cycling on polytene chromosomes. We found that trithorax (TRX), absent small or homeotic discs 1 (ASH1), and Compass member SET1 histone methyltransferases were O-GlcNAc-modified in oga(del.1) mutants. The oga(del.1) mutants displayed altered expression of a distinct set of cell cycle-related genes. Our results show that the loss of OGA in Drosophila globally impacts the epigenetic machinery allowing O-GlcNAc accumulation on RNA polymerase II and numerous chromatin factors including TRX, ASH1, and SET1.

S6K1ing to ResTOR Adipogenesis with Polycomb

Signal-directed chromatin recruitment of mammalian Polycomb complexes is a fundamental component of epigenetic regulation. In this issue, Yi etal. (2016) reveal how mTORC1 activation deploys the ribosomal serine/threonine kinase S6K1 and Polycomb proteins at genomic regulatory regions to repress expression of anti-adipogenic developmental regulators.

Targeting EZH2-mediated methylation of H3K27 inhibits proliferation and migration of Synovial Sarcoma in vitro.

Synovial sarcoma is an aggressive soft tissue sarcoma genetically defined by the fusion oncogene SS18-SSX. It is hypothesized that either SS18-SSX disrupts SWI/SNF complex inhibition of the polycomb complex 2 (PRC2) methyltransferase Enhancer of Zeste Homologue 2 (EZH2), or that SS18-SSX is able to directly recruit PRC2 to aberrantly silence target genes. This is of potential therapeutic value as several EZH2 small molecule inhibitors are entering early phase clinical trials. In this study, we first confirmed EZH2 expression in the 76% of human synovial sarcoma samples. We subsequently investigated EZH2 as a therapeutic target in synovial sarcoma in vitro. Knockdown of EZH2 by shRNA or siRNA resulted in inhibition of cell growth and migration across a series of synovial sarcoma cell lines. The EZH2 selective small-molecule inhibitor EPZ005687 similarly suppressed cell proliferation and migration. These data support the hypothesis that targeting EZH2 may be a promising therapeutic strategy in the treatment of synovial sarcoma; clinical trials are initiating enrollment currently.

124 The Polycomb Complex PRC2 Regulates the Intestinal Stem Cell Niche and Maturation of the Enterocytes

124 The Polycomb Complex PRC2 Regulates the Intestinal Stem Cell Niche and Maturation of the Enterocytes

MtVRN2 is a Polycomb VRN2-like gene which represses the transition to flowering in the model legume Medicago truncatula.

Optimising the timing of flowering contributes to successful sexual reproduction and yield in agricultural plants. FLOWERING LOCUS T (FT) genes, first identified in Arabidopsis thaliana (Arabidopsis), promote flowering universally, but the upstream flowering regulatory pathways can differ markedly among plants. Flowering in the model legume, Medicago truncatula (Medicago) is accelerated by winter cold (vernalisation) followed by long day (LD) photoperiods leading to elevated expression of the floral activator, FT-like gene FTa1. However, Medicago, like some other plants, lacks the activator CONSTANS (CO) and the repressor FLOWERING LOCUS C (FLC) genes which directly regulate FT and are key to LD and vernalisation responses in Arabidopsis. Conversely, Medicago has a VERNALISATION2-LIKE VEFS-box gene (MtVRN2). In Arabidopsis AtVRN2 is a key member of a Polycomb complex involved in stable repression of Arabidopsis FLC after vernalisation. VRN2-like genes have been identified in other eudicot plants, but their function has never been reported. We show that Mtvrn2 mutants bypass the need for vernalisation for early flowering in LD conditions in Medicago. Investigation of the underlying mechanism by transcriptome analysis reveals that Mtvrn2 mutants precociously express FTa1 and other suites of genes including floral homeotic genes. Double-mutant analysis indicates that early flowering is dependent on functional FTa1. The broad significance of our study is that we have demonstrated a function for a VRN2-like VEFS gene beyond the Brassicaceae. In particular, MtVRN2 represses the transition to flowering in Medicago by regulating the onset of expression of the potent floral activator, FTa1.

Anti‐proliferative Effect of Curcumin is Mediated by Down‐regulation of Polycomb Complex Protein, BMI1

BMI1 encodes a 37‐kDa protein that is a key regulatory component of PRC1 (polycomb regulatory complex‐1). BMI1 functions as an oncogene, induces cell growth and promotes tumor growth in in vitro and in vivo animal models. BMI1 is currently recognized as a gene of interest in terms of pharmaceutical drug development. Curcumin, a major phytochemical in turmeric, inhibits the proliferation of many types of cancer cells, both in vitro and in vivo. Previously, we observed that DLD‐1 colorectal cancer cells had a suppressed BMI1 expression following curcumin treatment. In addition, there was an inhibition of cell proliferation. This statistically significant finding indicates that curcumin potentially inhibits DLD‐1 cell proliferation by targeting BMI1. Curcumin has potential to be used in colorectal cancer cell therapy, however, it has been reported to have poor bioavailability. The purpose of this study is to determine whether or not modified curcumin compounds (dimethoxycurcumin, bisdemethoxycurcumin) and curcumin metabolite (tetrahydrocurcumin), with a higher bioavailability, have the same mechanism of action as curcumin in DLD‐1 colorectal cancer cells. DLD1 cells were treated with curcumin, dimethoxycurcumin, bisdemethoxycurcumin, and tetrahydrocurcumin. After 48 hours, BMI1 gene expression and protein levels will be measured. Preliminary results suggested that dimethoxycurcumin reduced BMI1 protein expression more than curcumin.Support or Funding InformationTAA and KS are supported by the DeNardo Education and Research Foundation.

Neuronal activity controls Bdnf expression via Polycomb de-repression and CREB/CBP/JMJD3 activation in mature neurons.

It has been recently described that in embryonic stem cells, the expression of some important developmentally regulated genes is repressed, but poised for fast activation under the appropriate stimuli. In this work we show that Bdnf promoters are repressed by Polycomb Complex 2 in mature hippocampal neurons, and basal expression is guaranteed by the coexistence with activating histone marks. Neuronal stimulation triggered by N-methyl-D-aspartate application induces the transcription of these promoters by H3K27Me3 demethylation and H3K27Me3 phosphorylation at Serine 28 leading to displacement of EZH2, the catalytic subunit of Polycomb Repressor Complex 2. Our data show that the fast transient expression of Bdnf promoters II and VI after neuronal stimulation is dependent on acetylation of histone H3K27 by CREB-p/CBP. Thus, regulatory mechanisms established during development seem to remain after differentiation controlling genes induced by different stimuli, as would be the case of early memory genes in mature neurons.

Abstract A39: Epigenetic upregulation of lncRNA HOTAIR expression in three-dimensional culture of triple-negative breast cancer cells

Abstract lncRNA HOX transcript antisense RNA (HOTAIR) represses gene expression via recruitment of the chromatin modifying complex polycomb repressive complex 2 (PRC2) and its overexpression mediates invasion and metastasis in breast cancer. In an apparent paradox we observed silencing of HOTAIR expression in MDA-MB231 cells, an invasive and metastatic triple-negative breast cancer cell line in two dimensional monolayer culture (2-D). This observation prompted us to question limitations of 2-D culture as a model system to investigate the role of HOTAIR in breast cancer. Thus we examined HOTAIR expression in reconstituted basement membrane matrix Matrigel-based three dimensional organotypic culture (rBM 3-D) because MDA-MB231 cells undergo stellate morphogenesis, a phenomenon that is characteristic of invasive cancer cells. In MDA-MB231 cells HOTAIR expression in rBM 3-D culture and the grafted tumors exhibited ~130-fold and ~60-fold over than that in 2-D culture, respectively. Induction of HOTAIR in rBM 3-D culture involved transcription from an uncharacterized promoter located in the 1st intron of the HOXC11 gene. However HOXC11 did not exhibit difference in its expression between 2-D and rBM 3-D cultures. Induction of HOTAIR in rBM 3-D culture was congruent to a wide spread increase in expression of the protein coding HOX genes. Moreover stellate morphogenesis was abrogated by the HOTAIR-specific siRNA and replaced by mass morphogenesis that is characteristic of non-invasive cells. To validate induction of HOTAIR as a response to extracellular matrix (ECM), we knocked down a major ECM receptor integrin α2 using lentiviral vectors expressing integrin α2-specific shRNA. As expected knockdown of integrin α2 resulted in a substantial decrease of HOTAIR expression. Because the HOX genes are tightly regulated by CpG methylation, we compared CpG methylation of the promoters of HOTAIR and HOXA9 in 2-D and rBM 3-D cultures. Indeed the promoters of HOTAIR and HOXA9 exhibited lower CpG methylation in rBM 3-D culture than that in 2-D culture. Moreover 5-Azacytidinewe up-regulated the expression of HOTAIR and HOXA9 in 2-D culture of MDA-mb231 cells. Moreover induction of HOTAIR was associated with increased binding of BRD4 to the HOTAIR promoter region located in the HOXC11 1st intron. The BRD4-specific siRNA substantially reduced the expression of HOTAIR in rBM 3-D culture. Taken together our findings suggest that ECM up-regulate the expression of HOTAIR via reduced CpG methylation and increased BRD4 binding in a human triple-negative breast cancer cell line. Note: This abstract was not presented at the conference. Citation Format: Miao Li, Xi Li, Bin Shan. Epigenetic upregulation of lncRNA HOTAIR expression in three-dimensional culture of triple-negative breast cancer cells. [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer: Mechanisms to Medicines ; 2015 Dec 4-7; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2016;76(6 Suppl):Abstract nr A39.

Targeting of BMI-1 with PTC-209 shows potent anti-myeloma activity and impairs the tumour microenvironment.

BackgroundThe polycomb complex protein BMI-1 (BMI-1) is a putative oncogene reported to be overexpressed in multiple myeloma (MM). Silencing of BMI-1 was shown to impair the growth and survival of MM cells. However, therapeutic agents specifically targeting BMI-1 were not available so far. Here, we investigated PTC-209, a novel small molecule inhibitor of BMI-1, for its activity in MM.MethodsBMI-1 expression was analysed in human MM cell lines and primary MM cells by using publically available gene expression profiling (GEP) data. The anti-MM activity of PTC-209 was investigated by viability testing, cell cycle analysis, annexin V and 7-AAD staining, quantification of cleaved poly(ADP-ribose) polymerase (PARP), JC-1 as well as colony formation assays. Deregulation of central myeloma growth and survival genes was studied by quantitative PCR and flow cytometry, respectively. In addition, the impact of PTC-209 on in vitro osteoclast, osteoblast and tube formation was analysed.ResultsWe confirmed overexpression of BMI-1 in MM patients by using publically available GEP datasets. Of note, BMI-1 expression was further increased at relapse which translated into significantly shorter overall survival in relapsed/refractory patients treated with bortezomib or dexamethasone.Treatment with PTC-209 significantly decreased viable cell numbers in human MM cell lines, induced a G1 cell cycle arrest, promoted apoptosis and demonstrated synergistic activity with pomalidomide and carfilzomib. The anti-MM activity of PTC-209 was accompanied by a significant decrease of cyclin D1 (CCND1) and v-myc avian myelocytomatosis viral oncogene homolog (MYC) expression as well as upregulation of cyclin-dependent kinase inhibitor 1A (CDKN1A) and cyclin-dependent kinase inhibitor 1B (CDKN1B). We also observed upregulation of NOXA (up to 3.6 ± 1.2-fold induction, P = 0.009) and subsequent downregulation of myeloid cell leukemia 1 (MCL-1) protein levels, which likely mediates the apoptotic effects of PTC-209. Importantly, the anti-MM activity was upheld in the presence of stromal support or myeloma growth factors insulin-like growth factor 1 (IGF-1) and interleukin 6 (IL-6).In the MM microenvironment, PTC-209 impaired tube formation, impaired osteoclast development and decreased osteoblast formation in a dose-dependent manner (P < 0.01 at 1 μM, respectively). The latter might be attributed to an induction of DKK1 and was reversed by concurrent anti-DKK1 antibody treatment.ConclusionsWe confirmed overexpression of BMI-1 in MM highlighting its role as an attractive drug target and reveal therapeutic targeting of BMI-1 by PTC-209 as a promising novel therapeutic intervention for MM.Electronic supplementary materialThe online version of this article (doi:10.1186/s13045-016-0247-4) contains supplementary material, which is available to authorized users.

Bmi1 Is a Key Epigenetic Barrier to Direct Cardiac Reprogramming

Direct reprogramming of induced cardiomyocytes (iCMs) suffers from low efficiency and requires extensive epigenetic repatterning, although the underlying mechanisms are largely unknown. To address these issues, we screened for epigenetic regulators of iCM reprogramming and found that reducing levels of the polycomb complex gene Bmi1 significantly enhanced induction of beating iCMs from neonatal and adult mouse fibroblasts. The inhibitory role of Bmi1 in iCM reprogramming is mediated through direct interactions with regulatory regions of cardiogenic genes, rather than regulation of cell proliferation. Reduced Bmi1 expression corresponded with increased levels of the active histone mark H3K4me3 and reduced levels of repressive H2AK119ub at cardiogenic loci, and de-repression of cardiogenic gene expression during iCM conversion. Furthermore, Bmi1 deletion could substitute for Gata4 during iCM reprogramming. Thus, Bmi1 acts as a critical epigenetic barrier to iCM production. Bypassing this barrier simplifies iCM generation and increases yield, potentially streamlining iCM production for therapeutic purposes.

Role of Polycomb RYBP in Maintaining the B-1-to-B-2 B-Cell Lineage Switch in Adult Hematopoiesis.

Polycomb chromatin modifiers regulate hematopoietic pluripotent stem and progenitor cell self-renewal and expansion. Polycomb complex redundancy and biochemical heterogeneity complicate the unraveling of the functional contributions of distinct components. We have studied the hematopoietic activity of RYBP, a direct interactor and proposed modulator of RING1A/RING1B-dependent histone H2A monoubiquitylation (H2AUb). Using a mouse model to conditionally inactivate Rybp in adult hematopoiesis, we have found that RYBP deletion results in a reversion of B-1-to-B-2 B-cell progenitor ratios, i.e., of the innate (predominantly fetal) to acquired (mostly adult) immunity precursors. Increased numbers of B-1 progenitors correlated with a loss of pre-proB cells, the B-2 progenitors. RYBP-deficient stem and progenitor cell populations (LKS) and isolated common lymphoid progenitors (CLP) gave rise to increased numbers of B-1 progenitors in vitro. Rybp inactivation, however, did not result in changes of global H2AUb and did not interact genetically with Ring1A or Ring1B deletions. These results show that a sustained regulation of the B-1-to-B-2 switch is needed throughout adult life and that RYBP plays an important role in keeping B-2 dominance, most likely independently of its Polycomb affiliation.

Linking Cancer Metabolism to DNA Repair and Accelerated Senescence.

Conventional wisdom ascribes metabolic reprogramming in cancer to meeting increased demands for intermediates to support rapid proliferation. Prior models have proposed benefits toward cell survival, immortality, and stress resistance, although the recent discovery of oncometabolites has shifted attention to chromatin targets affecting gene expression. To explore further effects of cancer metabolism and epigenetic deregulation, DNA repair kinetics were examined in cells treated with metabolic intermediates, oncometabolites, and/or metabolic inhibitors by tracking resolution of double-strand breaks (DSB) in irradiated MCF7 breast cancer cells. Disrupting cancer metabolism revealed roles for both glycolysis and glutaminolysis in promoting DSB repair and preventing accelerated senescence after irradiation. Targeting pathways common to glycolysis and glutaminolysis uncovered opposing effects of the hexosamine biosynthetic pathway (HBP) and tricarboxylic acid (TCA) cycle. Treating cells with the HBP metabolite N-acetylglucosamine (GlcNAc) or augmenting protein O-GlcNAcylation with small molecules or RNAi targeting O-GlcNAcase each enhanced DSB repair, while targeting O-GlcNAc transferase reversed GlcNAc's effects. Opposing the HBP, TCA metabolites including α-ketoglutarate blocked DSB resolution. Strikingly, DNA repair could be restored by the oncometabolite 2-hydroxyglutarate (2-HG). Targeting downstream effectors of histone methylation and demethylation implicated the PRC1/2 polycomb complexes as the ultimate targets for metabolic regulation, reflecting known roles for Polycomb group proteins in nonhomologous end-joining DSB repair. Our findings that epigenetic effects of cancer metabolic reprogramming may promote DNA repair provide a molecular mechanism by which deregulation of metabolism may not only support cell growth but also maintain cell immortality, drive therapeutic resistance, and promote genomic instability. By defining a pathway from deregulated metabolism to enhanced DNA damage response in cancer, these data provide a rationale for targeting downstream epigenetic effects of metabolic reprogramming to block cancer cell immortality and overcome resistance to genotoxic stress.

Painting a Clearer Picture of Chromatin

Elucidating chromatin's 3D shape is critical to understanding its function, but the fine structure of chromatin domains remains poorly resolved. In a recent report in Nature, Boettiger etal. (2016) visualize chromatin in super-resolution, gaining unprecedented insight into chromatin architecture.

Epigenomic Co-localization and Co-evolution Reveal a Key Role for 5hmC as a Communication Hub in the Chromatin Network of ESCs.

Epigenetic communication through histone and cytosine modifications is essential for gene regulation and cell identity. Here, we propose a framework that is based on a chromatin communication model to get insight on the function of epigenetic modifications in ESCs. The epigenetic communication network was inferred from genome-wide location data plus extensive manual annotation. Notably, we found that 5-hydroxymethylcytosine (5hmC) is the most-influential hub of this network, connecting DNA demethylation to nucleosome remodeling complexes and to key transcription factors of pluripotency. Moreover, an evolutionary analysis revealed a central role of 5hmC in the co-evolution of chromatin-related proteins. Further analysis of regions where 5hmC co-localizes with specific interactors shows that each interaction points to chromatin remodeling, stemness, differentiation, or metabolism. Our results highlight the importance of cytosine modifications in the epigenetic communication of ESCs.