Hormone-induced and DNA Demethylation-induced Relief of a Tissue-specific and Developmentally Regulated Block in Transcriptional Elongation

Manjeet K Rao,Yuiko Matsumoto,Marcy E Richardson,Subbarayalu Panneerdoss,Anjana Bhardwaj,Jacqueline M Ward,Sreenath Shanker,Anilkumar Bettegowda,Miles F Wilkinson

doi:10.1074/jbc.m114.615435

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.

Highlights

The regulation of transcriptional elongation in vertebrates in vivo is poorly understood
To test whether reproductive homeobox-5 (Rhox5) expression is regulated at the level of transcriptional elongation, we examined polymerase II (pol II) occupancy at the 3Ј end of the Rhox5 gene and found that pol II was only detectable at this position in the testis but not in the SV or liver (Fig. 1C, right panel)
We demonstrate that pol II is recruited to its proximal promoter and nascent transcripts begins to elongate; the progression of pol II is blocked or impeded ϳ100 nt downstream of the transcription start site in most tissues (Figs. 1–3)

Summary

Background

The regulation of transcriptional elongation in vertebrates in vivo is poorly understood. 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. Components of the general transcriptional apparatus have been shown to confer functionally specialized transcription initiation complexes that mediate the promoter selectivity required for tissue- and cell type-specific transcription [1] Another potential regulatory step is the one following transcriptional initiation—the pausing of RNA polymerase II (pol II) immediately downstream of the promoter [2, 3]. The precise mechanisms by which these factors influence Pp transcription are not known In this communication, we provide evidence that both the tissue-specific and developmentally regulated pattern of Pp expression is controlled, at least in part, at the level of transcriptional elongation. Our discovery that both DNA methylation and nuclear hormones control Rhox transcriptional elongation significantly expands our understanding of how transcriptional elongation is regulated and provides potential insights into its physiological role

EXPERIMENTAL PROCEDURES

RESULTS

Testis SV

DISCUSSION