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Title: Differential Global Effects of Selective Estrogen Receptor Modulators on Estrogen Receptor Binding and Transcriptional Regulation
Authors: LEE YEW KOK
Keywords: Estrogen Receptor, SERMs, Breast Cancer, Estrogen, Array
Issue Date: 10-Jul-2009
Citation: LEE YEW KOK (2009-07-10). Differential Global Effects of Selective Estrogen Receptor Modulators on Estrogen Receptor Binding and Transcriptional Regulation. ScholarBank@NUS Repository.
Abstract: Selective estrogen receptor modulators (SERMs) are used clinically to treat breast cancer as they inhibit estrogen both in promoting cell proliferation and expressing ER-mediated gene expression. SERMs are compound that block the effect of estrogen on estrogen receptor (ER). However, the complexity of estrogen receptor biology hinders an effective drug design. Our lab is interested in examining the global ER binding sites and the corresponding gene expression profiles upon treatment of ER by different SERMs. Chromatin Immunoprecipitation assay (ChIP) was performed on MCF-7 breast tumor cells in the presence or absence of E2/SERMs or a combination of E2+SERMs and immunoprecipitated with ERa antibodies. Global studies investigating genome-wide binding sites through customized tiling array containing more than 40,000 mapped and putative ER binding sites from Nimblegen were initiated. Genome-wide binding sites profiles with the customised array were obtained for different drug treatments (E2 and SERMs), different antibodies (ERa, H3K4Me1, FOXA1 and GATA3), different experiments (ChIP and Formaldehyde-Assisted Isolation of Regulatory Elements (FAIRE)) and different cell lines (MCF-7 and Ishikawa cell lines). Variable Factor Linear Model (VFLM) was developed and implemented, which detected 6482 ER binding sites for ChIP-chip experiment immunoprecipitated with ERa antibody in E2 treatment. We also obtained genome-wide gene expression profiles with the Affymetrix array (HG-U133 Plus) for different drugs treatments (E2, T, R and I) at different time points (0, 3, 6, 9, 12, 24 and 48 hours). For Affymetrix experiments, the Pooled Variance Meta-analysis methods was used, followed by applying a Data-driven Smoothness Enhanced Variance Ratio Test (dSEVRAT) method for assessing the smoothness of the expression of gene across time point. We selected regulated genes based on 3 criterias: P-value=0.05, smoothness score=200 and fold change=1.5. Nimblegen high-density arrays with 2.1 millons probes have been designed, tiling the entire chromosome 21 & 22 arrays with additional selected regions throughout human genome. Lastly, emerging evidences show that regulatory genetic variations have an influence on gene regulation like changing binding site recognition. For the functional studies, we have concentrated on Single Nucleotide Polymorphisms (SNPs) present within transcriptional binding sites and its biological functions. Since breast cancer cell lines for different binding sites polymorphism are not readily available, the polymorphism studies were performed in lymphoblastoid cell lines. ChIP analysis was performed on 8 different cell lines with different genotypes to assess the binding affinity. Furthermore, the binding characteristics associated with homogygous genotype were also carried out in an allele-specific Taqman assay. Interestingly, the SNPs have different regulation of the target gene PRKAG2 through expression studies and AMPK proteins through western blots. These studies above discovered and confirmed a functional SNP within binding site that exhibits an allele-specific transcription factor binding. Together, the information from binding sites, gene expression profiles upon drug treatments, nucleosome profiles and the information from studying regulatory genetic variations will help to decipher the mechanism of Estrogen Receptor gene regulation over time and over several pharmacologic interventions: E2 and SERMS.
Appears in Collections:Ph.D Theses (Open)

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