Breast cancer cell lines treated with EZH2 inhibitor compounds
ABSTRACT: Loss of H3K27me3 repressive chromatin histone marks, maintained by the histone methyltransferase (HKMT) EZH2, may lead to reversal of epigenetic silencing in tumor cells and have therapeutic potential. Using a cell-based assay, we have identified three compounds from a HKMT inhibitor chemical library which re-express H3K27me3 mediated, silenced genes. Chromatin immunoprecipitation verified a decrease in silencing marks (H3K27me3, H3K9me3) and importantly an increase in active marks (H3K4me2/3, H3K27ac) at the promoter of re-expressed genes. Compound treated breast tumor cells induced enrichment for genome-wide changes in expression of known target genes for EZH2 and induced cell growth inhibition: with most sensitive breast tumor cell lines having low EZH2 protein expression, while a normal epithelial breast line was least sensitive. Agilent SurePrint G3 Human 8x60k two-colour microarrays were used to profile gene expression changes induced by treatment with drug compounds in MDA MB-231 cells, both at 24h and 48h. 4 replicates were used for each drug, time combination. A separate untreated control sample was used for comparison with each replicate.
Project description:Binding of Polycomb repressive complex 2 (PRC2) and chromatin composition of the inactive X (Xi) before, during and after X chromosome inactivation reveal that spreading is driven by a combination of Xi-specific strong and moderate Ezh2 sites. Sequence context of these sites shows a moderate enrichment of SINEs and simple repeats. The general pattern of Ezh2 and H3K27me3 distribution over the chromosome reflect a graded concentration originating from strong Ezh2 sites, around which moderate sites are clustered, suggesting a hierarchy of Ezh2 sites govern spreading. ChIP-seq of Ezh2 and H3K27me3 as well as the three active marks H3K4me3, H3K36me3 and RNA-POLII-serine5 phosphorylation (RNA-polII-S5P) in female cell lines: undifferentiated embryonic stem (ES) cells (d0), differentiating ES cells (d7) and a transformed embryonic fibroblast cell line (MEF).
Project description:Overexpression of EZH2 in estrogen receptor negative (ER-) breast cancer promotes metastasis. EZH2 has been mainly studied as the catalytic component of the Polycomb Repressive Complex 2 (PRC2) that mediates gene repression by trimethylating histone H3 at lysine 27 (H3K27me3). However, how EZH2 drives metastasis despite the low H3K27me3 levels observed in ER- breast cancer is unknown. We have shown that in human invasive carcinomas and distant metastases, cytoplasmic EZH2 phosphorylated at T367 is significantly associated with ER- disease and low H3K27me3 levels. Here, we explore the interactome of EZH2 and of a phosphodeficient mutant EZH2_T367A. We identified novel interactors of EZH2, and identified interactions that are dependent on the phosphorylation and cellular localization of EZH2 that may play a role in EZH2 dependent metastatic progression.
Project description:Mammary development is characterized by the proliferation and progressive differentiation of alveolar epithelium during pregnancy, culminating in lactation. These processes are largely controlled by hormones through transcription factors. We now explore the contributions of histone methyltransferases, which establish H3K27me3 marks, in the temporally-regulated differentiation of mammary epithelium. Loss of EZH2, but not EZH1, resulted in precocious mammary differentiation, which was facilitated by STAT5 binding to specific target genes and their activation. Mammary stem cells were not compromised in the absence of EZH2. Genome-wide H3K27me3 patterns remained intact in the absence of EZH2. Mammary-specific loci were devoid of H3K27me3 marks in mammary progenitor and mature cells, suggesting no regulatory role for this repressive mark. Lastly, the combined absence of EZH1 and EZH2 inhibited the formation of alveoli. Taken together, EZH2 controls temporally-restricted differentiation of mammary epithelium through H3K27me3-independent mechanisms. mRNA-seq and ChIP-seq in MMTV-Cre (Control), E1-/- (E1KO), E1+/-;E2f/f;control (E1+/-E2KO) and Ezh2f/f;control (E2KO) mammary gland tissues or MECs (purified mammary epithelial cells). H3K27me3 and STAT5 ChIP-seqs in mammary tissues at p13; H3K4me3 ChIP-seq in MECs (mammary epithelial cells) at p13; RNA-seqs at mature virgin (with/without prolactin injection), p13 and p18 mammary tissues.
Project description:The EZH2 histone methyltransferase is highly expressed in germinal center (GC) B-cells and targeted by somatic mutations in B-cell lymphomas. Here we find that EZH2 deletion or pharmacologic inhibition suppresses GC formation and functions in mice. EZH2 represses proliferation checkpoint genes and helps establish bivalent chromatin domains at key regulatory loci to transiently suppress GC B-cell differentiation. Somatic mutations reinforce these physiological effects through enhanced silencing of EZH2 targets in B-cells, and in human B-cell lymphomas. Conditional expression of mutant EZH2 in mice induces GC hyperplasia and accelerated lymphomagenesis in cooperation with BCL2. GCB-type DLBCLs are mostly addicted to EZH2, regardless of mutation status, but not the more differentiated ABC-type DLBCLs, thus clarifying the therapeutic scope of EZH2 targeting. RNA sequencing and H3K27me3 ChIP sequencing of human DLBCL cell lines and murine BCL1 cell line. RNA sequencing, H3K27me3 and H3K4me3 ChIP sequencing of B cells from de-identified human tonsills.
Project description:Fibromyalgia (FM) is a syndrome characterized by widespread muscular pain, fatigue and functional symptoms, which is known to be difficult to diagnose as the various symptoms overlap with many other conditions. Currently, there are no biomarkers for FM, and the diagnosis is made subjectively by the clinicians. We have performed shotgun proteomics on cerebrospinal fluid (CSF) from FM patients and non-pain controls to find potential biomarker candidates for this syndrome.
Project description:Congenital heart disease is among the most frequent major birth defects. Epigenetic marks are crucial for organogenesis, but their role in heart development is poorly understood. Polycomb Repressive Complex 2 (PRC2) trimethylates histone H3 at lysine 27, establishing H3K27me3 repressive epigenetic marks that promote tissue-specific differentiation by silencing ectopic gene programs. We studied the function of the catalytic subunit of PRC2, EZH2, in murine heart development. Early EZH2 inactivation by Nkx2-5Cre caused lethal congenital heart malformations, but slightly later EZH2 inactivation by cTNT-Cre did not. To study how the cardiomyocytes gene expression program is properly established in the early heart development, we combined the technologies of RNA sequencing and chromatin immunoprecipitation sequencing to identify the functional target genes directly repressed by EZH2. Intriguingly, these were enriched for transcriptional regulators of non-cardiac expression programs, such as transcription factors that regulate neuronal (Pax6) and cardiac progenitor genes (Isl1 and Six1). EZH2 was also required to maintain spatiotemporal regulation of cardiac gene expression, as Hcn4, Mlc2a, and Bmp10 were inappropriately upregulated in ventricular RNA. Furthermore, EZH2 was required for normal cardiomyocyte proliferation, establishing H3K27me3 epigenetic marks at cell cycle inhibitors Ink4a/b and repressing their expression. Our study reveals a previously undescribed role of EZH2 in regulating heart formation and shows that perturbation of the epigenetic landscape early cardiogenesis has sustained disruptive effects at later developmental stages. 8 E12.5 heart apex were used for RNA preparation each group.
Project description:Gene expression profiling has uncovered the transcription factor Sox4 with up-regulated activity during TGFβ-induced EMT in normal and cancerous breast epithelial cells. Sox4 is indispensable for EMT and cell survival in vitro and for primary tumor growth and metastasis in vivo. Among several EMT-relevant genes, Sox4 directly regulates the expression of Ezh2, encoding the Polycomb group histone methyltransferase that trimethylates histone 3 lysine 27 (H3K27me3) for gene repression. Ablation of Ezh2 expression prevents EMT, while forced expression of Ezh2 restores EMT in Sox4-deficient cells. Ezh2-mediated H3K27me3 marks associate with key EMT genes, representing an epigenetic EMT signature that predicts patient survival. Our results identify Sox4 as a master regulator of EMT by governing the expression of the epigenetic modifier Ezh2. Our Dataset comprises of 12 ChIP-seq samples using chromatin from NMuMG cells which was immunoprecipitated using H3K27me3-specific antibody during TGFβ-induced EMT (2ng/ml) at 6 different stages (day 0, 1, 4, 7, 10, 20).
Project description:Wild-type mouse embryonic stem cells are compared with mutants for components of PRC2 including Ezh2-/-, Eed-/-, and Jarid2-/- cells. Chromatin modifications, Gene expression, Pol-II, small-RNA sequencing, and DNA methylation are compared for both cell types. To study genomic and epigentic control of PRC2 in mESCs, we designed gene expression analysis (RNA-Seq and small RNA-Seq), combining with ChIP-Seq analysis of several factors and histone marks from wild-type and distinct PRC2 mutants including wild-type, Ezh2-/-, Eed-/-, and Jarid2-/-.
Project description:Oltipraz is an activator of Nrf2 but is also an activator of other pathways including those mediated by constitutive activated receptor (CAR). To identify genes regulated by oltipraz that were Nrf2-dependent, we compared gene expression after exposure in wild-type and Nrf2-null mice. Wild-type or Nrf2-null mice were treated each day for 4 days with 75 mg/kg/day/day oltipraz in corn oil or corn oil alone. There were 4 biological replicates used for each of the 4 genotype-treatment groups. Gene expression in the livers of the mice was evaluated using Affymetrix mouse exon arrays (MoEx-1_0-st-v1).
Project description:The present study revealed dynamic reposition of localizations of silencing histone marks including H3K27me3 in hepatocyte differentiation, of which target genes reflect the cell identities in connection to the cellular functions. Our results shed light on the process of hepatocyte terminal differentiation regulated by epigenetic mechanisms. Comparison of gene expression in E13.5 liver cells from WT and Ezh2 KO mice.