Progesterone receptor ChIP-seq within the mouse mammary gland
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ABSTRACT: Progesterone (P) acting through its cognate nuclear receptors (PRs) plays an essential role in driving pregnancy-associated branching morphogenesis of the mammary gland. However, the fundamental mechanisms, including global cistromic and acute genomic transcriptional responses that are required to elicit active branching morphogenesis in response to P, have not been elucidated. We used chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) to identify P-regulated genes that directly recruit PRs in the mouse mammary gland after acute P treatment. Two replicate PR ChIP samples and two replicate input DNA control samples from mouse mammary glands after mice are treated subcutaneously with 17?-Estradiol for 24 hours and then 17?-Estradiol plus Progesterone for 6 hours.
Project description:Progesterone (P) acting through its cognate nuclear receptors (PRs) plays an essential role in driving pregnancy-associated branching morphogenesis of the mammary gland. However, the fundamental mechanisms, including global cistromic and acute genomic transcriptional responses that are required to elicit active branching morphogenesis in response to P, have not been elucidated. We used microarray analysis to identify global gene expression signatures that are acutely regulated by PRs in the mouse mammary gland after acute P treatment. Mammary gland gene expression data from 10-week-old ovariectomized wildtype and progesterone receptor null mice treated subcutaneously with 17β-Estradiol for 24 hours and then 17β-Estradiol plus Progesterone for 8 or 24 hours. Three replicate pools were tested with three mice per pool.
Project description:Progesterone (P) acting through its cognate nuclear receptors (PRs) plays an essential role in driving pregnancy-associated branching morphogenesis of the mammary gland. However, the fundamental mechanisms, including global cistromic and acute genomic transcriptional responses that are required to elicit active branching morphogenesis in response to P, have not been elucidated. We used chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) to identify P-regulated genes that directly recruit PRs in the mouse mammary gland after acute P treatment.
Project description:Progesterone (P) acting through its cognate nuclear receptors (PRs) plays an essential role in driving pregnancy-associated branching morphogenesis of the mammary gland. However, the fundamental mechanisms, including global cistromic and acute genomic transcriptional responses that are required to elicit active branching morphogenesis in response to P, have not been elucidated. We used microarray analysis to identify global gene expression signatures that are acutely regulated by PRs in the mouse mammary gland after acute P treatment.
Project description:P190B RhoGAP is required for mammary gland development, and its overexpression disrupts mammary gland branching morphogenesis. To better understand the mechanisms by which p190B regulates mammary gland development we performed gene expression microarray analysis on mammary epithelial cells isolated from p190B overexpressing transgenic mice compared to control mice. The mice used in this study were previously developed to inducibly overexpress p190B selectively in the mammary gland (Vargo-Gogola 2006). FVB/N mice carrying the MMTV-rtTA and TetO-p190B-IRES-luciferase transgenes or the MMTV-rtTA transgene only (Gunther EJ, FASEB) were fed doxycycline (Dox) chow (2 g/kg) for 7 d prior to removal of mammary glands and isolation of primary mammary epithelial cells at 6 weeks of age.
Project description:Both ovarian and pituitary hormones are required for the pubertal development of the mouse mammary gland. Estradiol directs ductal elongation and branching within the adipose stroma of the adolescent mouse mammary gland, while progesterone leads to tertiary branching and alveolar development. The purpose of this investigation was to identify the estrogen-responsive genes that are associated with estrogen-stimulated ductal elongation and branching in the mouse mammary gland in the absence of other ovarian hormones. We also wanted to determine if estrogen-responsive gene regulation at early stages of ductal elongation (ie. when ductal growth was minimal) was similar to those regulated after significant ductal elongation had occurred. To identify estrogen-regulated genes, ovariectomized prepubertal mice were exposed to 17beta-estradiol for four weeks, and mammary gland global gene expression analyzed by microarray analysis at various points during this time course. We determined that while many genes are regulated in all weeks of treatment, there remained a subset of genes that was uniquely regulated at each time-point. This observation was reflected in the biological functions of these genes; some categories were represented in all weeks of treatment while others were specific to only certain time-points. We have also identified estradiol-responsive genes in the mouse mammary gland that co-express with Estrogen Receptor alpha in human breast cancer, which may represent novel effectors of estrogen action and/or biomarkers for the progression of estrogen-dependent cancers and other estrogen-driven diseases.
Project description:Branching morphogenesis of the mammary gland is driven by the highly motile terminal end bud (TEB) throughout pubertal development. The stem cell enriched, proliferative TEB branches as it invades the mammary fat pad to create a complex network of ducts. The gene expression programs specific to the TEB and the differentiated duct are poorly understood. We conducted a time course analysis of gene expression in the TEB and duct throughout branching morphogenesis. Additionally, we determined the gene regulatory networks coordinated by the Co-factor of LIM domains (CLIM/LDB) transcriptional regulators and determined an essential function for CLIMs in branching morphogenesis by maintaining basal mammary epithelial stem cells and promoting cell proliferation. We used laser capture microdissection to isolate TEB and duct cells throughout branching morphogenesis. We then profiled gene expression in these cells to determine gene regulatory networks involved in branching morphogenesis, and specifically those regulated by CLIM transcriptional regulators.
Project description:Branching morphogenesis of the mammary gland is driven by the highly motile terminal end bud (TEB) throughout pubertal development. The stem cell enriched, proliferative TEB branches as it invades the mammary fat pad to create a complex network of ducts. The gene expression programs specific to the TEB and the differentiated duct are poorly understood. We conducted a time course analysis of gene expression in the TEB and duct throughout branching morphogenesis. Additionally, we determined the gene regulatory networks coordinated by the Co-factor of LIM domains (CLIM/LDB) transcriptional regulators and determined an essential function for CLIMs in branching morphogenesis by maintaining basal mammary epithelial stem cells and promoting cell proliferation. We used laser capture microdissection to isolate TEB and duct cells throughout branching morphogenesis. We then profiled gene expression in these cells to determine gene regulatory networks involved in branching morphogenesis, and specifically those regulated by CLIM transcriptional regulators. Mouse mammary glands from 4, 6, 8, and 10 week old mice (early puberty through early adulthood) were used for laser capture microdissection of TEB and duct cells from WT and K14-DN-Clim transgenic mice. RNA was isolated (Qiagen) and hybridized to Affymetrix MouseGene 1.0 ST arrays. In addition, basal (CD29HiCD24+Lin-) and Luminal (CD29LoCD24+Lin-) cells were sorted and RNA collected for hybridization to Affymetrix MouseGene 1.0ST arrays.
Project description:Transcriptional profiling of different mouse mammary cellular compartments (basal, luminal and stromal) under define hormone treatments: estrogen, progesterone, estrogen plus progesterone and the vehicle control. Goal was to determine the effect of ovarian hormones on mammary cellular compartment gene expression. Four-condition experiment within each cellular compartment. vehicle vs. estrogen, progesterone and estrogen plus progesterone. Biological replicates: 3 vehicle control, 4 estrogen treatment, 3 progesterone treatment, 4 estrogen plus progesterone treatment in each epithelial compartment (luminal, basal). 3 vehicle control, 3 estrogen, 3 progesterone, 3 estrogen plus progesterone in the stromal compartment.
Project description:We are using the ACI rat model of 17beta-estradiol induced mammary cancer to define the mechanisms through which estrogens contribute to breast cancer development; identify and functionally characterize the genetic variants that determine susceptibility; and define the hormone-gene-environment interactions that influence development of mammary cancer in this physiologically relevant rat model. Female ACI rats are uniquely susceptible to development of mammary cancer when treated continuously with physiologic levels of 17beta-estradiol. Induction of mammary cancer in female ACI rats occurs through a mechanism that is largely dependent upon estrogen receptor-alpha. Interval mapping analyses of progeny generated in intercrosses between susceptible ACI rats and resistant Brown Norway (BN) rats revealed seven quantitative trait loci (QTL), designated Emca3 (Estrogen-induced mammary cancer) through Emca9, each of which harbors one or more genetic determinants of mammary cancer susceptibility. Genes that reside within Emca8 on RNO5 and were differentially expressed between 17beta-estradiol treated ACI and ACI.BN-Emca8 congenic rats were identified as Emca8 candidates. Two groups of 17beta-estradiol treated female rats were compared. Five ACI and five BN.ACI-Emca8 rats were treated with 17beta-estradiol for 12 weeks. Total RNA was isolated from the mammary glands of these animals, labeled, and hybridized to Affymetrix Rat Genome 230 2.0 Arrays (Affymetrix Inc.). Significantly differentially expressed genes were found between these groups.
Project description:In this project we examined in-vitro effect of female sex hormones, estradiol and progesterone at average physiological concentration level on Chlamydia trachomatis gene expression level. Regulation of chlamydial gene expression by the female sex hormones oestradiol and progesterone was examined. A total of 16 chlamydial arrays were analysed with the 4 culture conditions (no hormone, E, P, E+P) x four replicates. Bacterial samples were grown in non-hormone treated culture were used as control