Unknown,Transcriptomics,Genomics,Proteomics

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Nucleosome driven transcription factor binding and gene regulation [ChIP-Seq]

ABSTRACT: Elucidating the global function of a transcription factor implies the identification of its target genes and genomic binding sites. The role of chromatin in this context is unclear, but the dominant view is that factors bind preferentially to nucleosome-depleted regions, identified as DNaseI-hypersensitive sites (DHS). Here we show by chromatin-IP, MNase and DNaseI assays followed by deep sequencing that the progesterone receptor (PR) requires nucleosomes for optimal binding and function. In breast cancer cells treated with progestins we identified 25,000 PR binding sites (PRbs), the majority encompassing several copies of the hexanucleotide TGTYCY, highly abundant in the genome. We found that functional PRbs accumulate around progesterone-induced genes mainly in enhancers, are enriched in DHS but exhibit high nucleosome occupancy. Progestin stimulation results in remodeling of these nucleosomes with displacement of histones H1 and H2A/H2B dimers. Our results strongly suggest that nucleosomes are crucial for PR binding and hormonal gene regulation. T47D-MTVL human breast cancer cells were incubated with the progestin R5020 for different times between 0 to 360 minutes at 37ÂºC. ChIP-seq experiments were performed using antibodies against progesterone receptor and a single Sample each with anti-H3K4me3 and anti-H3K4me1. Mononucleosomal DNA was prepared from cells untreated or stimulated 60 min with R5020 and subjected to deep sequencing using the Solexa Genome Analyzer.

ORGANISM(S): Homo sapiens

SUBMITTER: Miguel Beato

PROVIDER: E-GEOD-41466 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Although non-genomic steroid receptor pathways have been studied over the past decade, little is known about the direct gene expression changes that take place as a consequence of their activation. Progesterone controls proliferation of rat endometrial stromal cells during the peri-implantation phase of pregnancy. We showed that picomolar concentration of progestin R5020 mimics this control in UIII endometrial stromal cells via ERK1-2 and AKT activation mediated by interaction of Progesterone Receptor (PR) with Estrogen Receptor beta (ERb) and without transcriptional activity of endogenous PR and ER. Here we identify early downstream targets of cytoplasmic PR signaling and their possible role in endometrial stromal cell proliferation. Microarray analysis of global gene expression changes in UIII cells treated for 45 min with progestin identified 97 up- and 341 down-regulated genes. The most over-represented molecular functions were transcription factors and regulatory factors associated with cell proliferation and cell cycle, a large fraction of which were repressors down-regulated by hormone. Further analysis verified that progestins regulate Ccnd1, JunD, Usf1, Gfi1, Cyr61, and Cdkn1b through PR-mediated activation of ligand-free ER, ERK1-2 or AKT, in the absence of genomic PR binding. ChIP experiments show that progestin promoted the interaction of USF1 with the proximal promoter of the Cdc2 gene, and Usf1 knockdown abrogated Cdc2 progestin-dependent transcriptional regulation providing a mechanism for direct regulation of its expression. Finally, Cdc2 knockdown blocked R5020 induced UIII cell proliferation. We conclude that progestin induced proliferation of endometrial stromal cells requires ERK1-2 and AKT mediated early regulation of USF1, that induces Cdc2. To our knowledge, this is the first description of early target genes of progestin-activated classical PR via crosstalk with protein kinases and independently of hormone receptor binding to the genomic targets.

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Nucleosome driven transcription factor binding and gene regulation [ChIP-Seq]

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