Project description:Epigenomic profiling by ChIP-seq is a prevailing methodology used to investigate chromatin-based regulation in biological systems, such as human disease, yet the lack of an empirical methodology to normalize amongst experiments has limited the usefulness of this technique. Here we describe a “spike-in” normalization method that allows the quantitative comparison of histone modification status across cell populations using defined quantities of a reference epigenome. We demonstrate the utility of this method in measuring epigenomic changes following chemical perturbations and show how control normalization of ChIP-seq experiments enables discovery of disease-relevant changes in histone modification occupancy. ChIP-Seq of histone modifications H3K79me2 and H3K4me3 in human samples treated with EPZ-5676 with/without reference epigenome spike-in.
Project description:Estrogen Receptor alpha (ERα), a nuclear receptor with transcriptional activity, is a master regulator of estrogen signaling, widely known as therapeutic target in hormone-responsive breast cancer (BC). Moreover, ERα is highly expressed in approximately 80% of High Grade Serous Ovarian Cancer (HGSOC), the most common epithelial ovarian carcinoma. Despite some promising clinical trials evaluating endocrine therapy in this type of tumor, the role of ERα is still unknown. Epigenetic changes, such as DNA methylation, are emerging as key contributing factors to carcinogenesis. Disruptor of telomeric silencing-1-like (DOT1L), the only known histone methyl transferase capable to produce H3K79 mono, di and tri-methylation, modulates ERα actions in hormone-responsive BC. Considering this evidence, ERα-DOT1L association was confirmed in ERα-positive OC cells, PEO1 and PEO4, by Co-IP. DOT1L pharmacological inhibition by EPZ004777 (EPZ) revealed the involvement of this epigenetic enzyme in cell proliferation, cell cycle progression and apoptosis. Transcriptome profiling after ICI (a Selective Estrogen Receptor Degrader) and EPZ treatment, in both cell lines, has underlined a deep impact of both compounds on ERα-modulated genes, including the down-regulation of ERα itself. On the other hand, functional analysis showed that commonly affected transcripts are involved in different cellular processes, such as cancer cell survival, chemoresistance and cell cycle progression. Moreover, ChIP-qPCR performed on ERα promoter highlighted ERα and DOT1L co-localization, both in PEO1 and in PEO4 cells, which was reduced after EPZ treatment, suggesting a role of this complex on receptor transcriptional activity. In addition, drug combination studies performed with EPZ and ICI showed an additive effect in cell growth inhibition. Taken together, these results suggest DOT1L as a potential therapeutic target in the treatment of OC.
Project description:Estrogen Receptor αlpha (ERα) is the master regulator of estrogen signaling in hormone-responsive breast cancer (BC), however epigenetic mechanisms, including DNA methylation, are emerging as key processes for regulation of critical cell functions including tumorigenesis. We have recently reported the epigenetic writer DOT1L (DOT1 Like Histone Lysine Methyltransferase) to associated to ERα, part of chromatin bound multiprotein complex and that the pharmacological inhibition of this enzyme reduces the transcription rate of several genes involved in ERα-mediated signaling leading to inhibition of BC cell proliferation. Here, we investigated the functional impact of DOT1L inhibition on methylome changes in BC and its possible contribution to deregulation of transcriptional pathways associated to the progression of this disease.
Project description:The impact of drugs inhibiting DNA methylation (5-aza-2'-deoxycytodine, DAC) and EZH2 (EPZ-6438) on H3K27me3 coverage was analyzed in two neuroblastoma cell lines. Parallel analyses investigated associated changes in RNA expression and DNA methylation. The neuroblastoma cell lines Be(2)-C and IMR5-75 were treated with a combination of DAC and EPZ-6438. Controls were treated with solvent (DMSO). H3K27me3 ChIP seq was done to investigate treatment-related changes of this mark. In addition, H3K4me3 and H3K27ac ChIP seq was done in DMSO treated samples to identify putative regulatory regions.
Project description:Estrogen Receptor subtypes (ERα and ERβ) are transcription factors sharing similar structure, however, they often perform opposite roles in breast cancer’s cell proliferation and tumor progression. Besides the well-characterized genomic actions of ERs upon ligand binding, rapid non-genomic cytoplasmic changes together with the recently discovered ligand-free action of ERs are emerging as key regulators of tumorigenesis. The identification of cytoplasmic interaction partners of unliganded ERα and ERβ may help characterize the molecular basis of the extra-nuclear mechanism of action of these receptors, revealing novel mechanisms to explain their role in breast cancer response or resistance to endocrine therapy. To this aim, in this study, cytoplasmic extracts from stably expressing TAP-ERα and -ERβ MCF-7 cell clones were subjected to interaction proteomics in the absence of estrogen stimulation, leading to the identification of 84 and 142 proteins associated with unliganded ERα and ERβ, respectively. Functional analyses of ER subtype-specific interactomes revealed significant differences in the molecular pathways associated to each receptor in the cytoplasm. This work reports the first identification of the unliganded ERα and ERβ cytoplasmic interactomes in breast cancer cells, providing novel experimental evidence on the non-genomic effects of ERs in the absence of hormonal stimulus.
Project description:H3K79me2 ChIP-seq in mouse proximal intestinal Lgr5(hi) stem cells and villus cells Examination of H3K79me2 modifications between Lgr5(hi) stem cells and differentiated villus cells
Project description:Gene expression array of human breast cancer cells MCF7 with DMSO-EPZ- EPZ+E2 and shDOT1L+E2 and ZR-75-1 after treatment with DMSO and EPZ