H3K9me3 In Cells Research Articles

Whole-genome doubling drives oncogenic loss of chromatin segregation

Whole-genome doubling (WGD) is a recurrent event in human cancers and it promotes chromosomal instability and acquisition of aneuploidies1–8. However, the three-dimensional organization of chromatin in WGD cells and its contribution to oncogenic phenotypes are currently unknown. Here we show that in p53-deficient cells, WGD induces loss of chromatin segregation (LCS). This event is characterized by reduced segregation between short and long chromosomes, A and B subcompartments and adjacent chromatin domains. LCS is driven by the downregulation of CTCF and H3K9me3 in cells that bypassed activation of the tetraploid checkpoint. Longitudinal analyses revealed that LCS primes genomic regions for subcompartment repositioning in WGD cells. This results in chromatin and epigenetic changes associated with oncogene activation in tumours ensuing from WGD cells. Notably, subcompartment repositioning events were largely independent of chromosomal alterations, which indicates that these were complementary mechanisms contributing to tumour development and progression. Overall, LCS initiates chromatin conformation changes that ultimately result in oncogenic epigenetic and transcriptional modifications, which suggests that chromatin evolution is a hallmark of WGD-driven cancer.

RNA-dependent chromatin localization of KDM4D lysine demethylase promotes H3K9me3 demethylation.

The JmjC-containing lysine demethylase, KDM4D, demethylates di-and tri-methylation of histone H3 on lysine 9 (H3K9me3). How KDM4D is recruited to chromatin and recognizes its histone substrates remains unknown. Here, we show that KDM4D binds RNA independently of its demethylase activity. We mapped two non-canonical RNA binding domains: the first is within the N-terminal spanning amino acids 115 to 236, and the second is within the C-terminal spanning amino acids 348 to 523 of KDM4D. We also demonstrate that RNA interactions with KDM4D N-terminal region are critical for its association with chromatin and subsequently for demethylating H3K9me3 in cells. This study implicates, for the first time, RNA molecules in regulating the levels of H3K9 methylation by affecting KDM4D association with chromatin.

Abstract 123: The breast cancer metastasis suppressor RRP1B modulates metastasis through regulation of histone methylation

Abstract Ribosomal RNA Processing 1 Homolog B (Rrp1b) is a germline breast cancer metastasis suppressor that regulates transcription to induce a highly prognostic gene expression signature. Although RRP1B interacts with chromatin- and mRNA splicing-associated factors, the mechanisms by which RRP1B modulates metastasis-associated gene expression are unclear. In this study, we examined the chromatin-binding properties of RRP1B to determine the mechanism by which it regulates metastasis-associated gene transcription. Genome-wide chromatin binding sites for endogenous RRP1B were defined using chromatin immunoprecipitation-sequencing (ChIP-seq) in Hela and MDA-MB-231 cells. ChIP-seq analysis of these samples identified 689 RRP1B binding regions in Hela and 339 binding regions in MDA-MB-231 cells. Among these, 136 regions were common to both cell lines. The expression of 40 genes located at or near these binding regions was examined by qPCR in MDA-MB-231 cells stably over-expressing RRP1B. These experiments demonstrated that RRP1B binding is primarily associated with transcriptional repression. Co-occupancy of RRP1B with the heterochromatin-associated proteins TRIM28 and HP1α at these loci were observed through ChIP-reChIP assays. Interaction between RRP1B and TRIM28 and HP1α was confirmed through co-immunoprecipitation. TRIM28 and HP1α have been shown to induce trimethylation of histone H3 at the lysine-9 position (H3K9me3) via the histone methyltransferase, SETDB1. Increases in H3K9me3 are typically associated with heterochromatinization and transcriptional repression. Given that RRP1B interacts with both TRIM28 and HP1α, and binds at common chromatin regions with these factors, we hypothesized that RRP1B is also implicated in increasing H3K9me3 levels at these loci. To examine the effect of RRP1B on H3K9me3, ChIP assays were performed in MDA-MB-231 cells stably over-expressing RRP1B and control cells with an antibody against H3K9me3. ChIP-qPCR demonstrated that chromatin regions that displayed co-occupancy of RRP1B with TRIM28 and HP1α have increased levels of H3K9me3 compared to that of the control. Additionally, ChIP-seq for H3K9me3 in cells stably over-expressing RRP1B demonstrated that RRP1B causes a global increase in H3K9me3 levels compared to control. These data collectively demonstrate that RRP1B regulates gene expression at the transcriptional level through an association with the transcriptional repressors TRIM28 and HP1α. Furthermore, the prominent mechanism that the metastasis suppressor RRP1B uses to regulate transcription is through epigenetic modifications, such as histone methylation. Citation Format: Minnkyong Lee, Amy M. Dworkin, Jens Lichtenberg, Shashank J. Patel, Derek Gildea, David M. Bodine, Nigel PS Crawford. The breast cancer metastasis suppressor RRP1B modulates metastasis through regulation of histone methylation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 123. doi:10.1158/1538-7445.AM2014-123

Abstract A56: H3K9me3-based ChIP-seq analysis unveils global reprogramming in Cyclin D1-driven cellular senescence

Abstract Senescence is a permanent G1 cell-cycle arrest that can be triggered by acute stresses such as activated oncogenes and chemotherapeutic agents, and serves as a barrier to cancer development. Senescent cells exhibit distinct chromatin features compared with proliferating cells, known as senescence-associated heterochromatin foci (SAHF). These foci are enriched with histone H3 trimethylated at lysine 9 (H3K9me3), and are thought to impose a repressive state on regional genes. We performed genome-wide identification of elements associated with H3K9me3 by ChIP-seq technology, using an in vivo model of senescent pineal cells driven by Cyclin D1 oncogenic signaling, and compared them to non-senescent (Cyclin D1 expressing but p53-null) counterparts. We identified a set of genes whose H3K9me3 enrichment level was significantly changed (>2-fold). We also analyzed gene expression profiles on Affymetrix transcriptome chips. We found that about 50% of genes associated with H3K9me3 exhibited significant reduction in expression, while other genes were either upregulated or showed no change in expression. Gene ontology analysis clustered the genes into several functional groups, including cell-cycle control, transcription regulation, protein metabolism, cellular differentiation, and cellular adhesion. Notably, the gene ontology clustering showed that genes associated with H3K9me3 in senescent cells clustered into distinctly different functional programs than those associated with H3K9me3 in cells that had bypassed senescence. Our data indicate that chromatin changes in cellular senescence are informative regarding a distinct functional phenotype of the senescent condition. The mechanisms by which such changes contribute to tumor suppression are under active investigation. Citation Format: Mohammad Harajly, Noel Ghanem, Raya Saab. H3K9me3-based ChIP-seq analysis unveils global reprogramming in Cyclin D1-driven cellular senescence. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Jun 19-22, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2013;73(13 Suppl):Abstract nr A56.