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  • Hormone-induced and DNA demethylation-induced relief of a tissue-specific and developmentally regulated block in transcriptional elongation.

Hormone-induced and DNA demethylation-induced relief of a tissue-specific and developmentally regulated block in transcriptional elongation.

The Journal of biological chemistry (2014-10-22)
Manjeet K Rao, Yuiko Matsumoto, Marcy E Richardson, Subbarayalu Panneerdoss, Anjana Bhardwaj, Jacqueline M Ward, Sreenath Shanker, Anilkumar Bettegowda, Miles F Wilkinson
ABSTRACT

Genome-wide studies have revealed that genes commonly have a high density of RNA polymerase II just downstream of the transcription start site. This has raised the possibility that genes are commonly regulated by transcriptional elongation, but this remains largely untested in vivo, particularly in vertebrates. Here, we show that the proximal promoter from the Rhox5 homeobox gene recruits polymerase II and begins elongating in all tissues and cell lines that we tested, but it only completes elongation in a tissue-specific and developmentally regulated manner. Relief of the elongation block is associated with recruitment of the elongation factor P-TEFb, the co-activator GRIP1, the chromatin remodeling factor BRG1, and specific histone modifications. We provide evidence that two mechanisms relieve the elongation block at the proximal promoter: demethylation and recruitment of androgen receptor. Together, our findings support a model in which promoter proximal pausing helps confer tissue-specific and developmental gene expression through a mechanism regulated by DNA demethylation-dependent nuclear hormone receptor recruitment.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Anti-trimethyl-Histone H3 (Lys9) Antibody, clone 6F12-H4, clone 6F12-H4, from mouse
Sigma-Aldrich
Anti-RNA polymerase II subunit B1 (phospho-CTD Ser-5) Antibody, clone 3E8, culture supernatant, clone 3E8, from rat
Sigma-Aldrich
Anti-trimethyl-Histone H3 (Lys4) Antibody, Upstate®, from rabbit
Sigma-Aldrich
Anti-dimethyl-Histone H3 (Lys9) Antibody, Upstate®, from rabbit
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Anti-RNA polymerase II subunit B1 (phospho CTD Ser-2), clone 3E10 (rat monoclonal)., culture supernatant, clone 3E10, from rat
Sigma-Aldrich
Anti-BRG1 Antibody, clone 3G4, clone 3G4, from rat
Sigma-Aldrich
Anti-GRIP-1 Antibody, CT, from rabbit, purified by affinity chromatography
Sigma-Aldrich
ChIPAb+ RNA Pol II - ChIP Validated Antibody and Primer Set, from mouse