The classical model of gene regulation posits that transcription factors recruit proteins that remodel the chromatin structure and recruit the basal transcription machinery to elicit transcription initiation. Recent studies conducted in human embryonic stem cells and Drosophila melanogaster embryos have identified transcriptional elongation as a widely used alternative control mechanism of transcription. This proposal addresses whether a nuclear hormone receptor can control transcription at the level of elongation using the androgen-regulated Rhox5 gene as a model system. Rhox5 is an X-linked homeobox gene whose expression is restricted to specific somatic cell types within reproductive tissues and whose absence causes subfertility in male mice. Rhox5 has two different promoters that are independently regulated. The subject of this proposal is the Rhox5 proximal promoter (Pp), which the Wilkinson laboratory showed depends on testosterone and androgen receptor (AR) for expression in Sertoli and caput epididymal cells. In other cell types, RNA polymerase (Pol) II is recruited to the Pp and short mRNAs are generated, but transcription does not propagate beyond ~100 nucleotides. Several lines of preliminary evidence suggest that escape from this transcriptional elongation block requires AR, a notion that will be directly tested in this proposal. Because AR is relatively widely expressed, this suggests that a mechanism exists that recruits AR to the Pp only in Sertoli and caput epididymal cells. A strong candidate to control AR recruitment is DNA methylation, as many of the AR- binding sites in the Pp are methylated in tissues and developmental stages that exhibit the elongation block. Furthermore, in vitro methylation of the Pp inhibits its transcription. Although promoter methylation has been shown to repress transcriptional elongation in Neurospora, this is the first suggestion that DNA methylation has this effect in mammals. Together, the data lead to the hypothesis that methylation of the Pp blocks AR recruitment, thereby preventing transcriptional elongation. A further hypothesis is that AR promotes transcriptional elongation by recruiting P-TEFb, which is known to interact with AR and is essential for completion of transcriptional elongation by virtue of its ability to phosphorylate the large subunit of Pol II. This proposal aims to test these hypotheses with a focus on how they explain the cell type-specific and developmentally regulated expression pattern of the Rhox5 gene. This work has the potential to alter existing paradigms for how nuclear hormone receptors and epigenetic signals regulate transcription.
The projects outlined in this application will define a paradigm shift in role of the androgen receptor in the control of transcription elongation. These projects also aim to demonstrate for the first time, that methylation can control transcriptional elongation in mammals. The control of these two phenomena will be analyzed in the developmental and tissue-specific program that allows the expression of Rhox5 Pp in the mouse testis.
|Richardson, Marcy E; Bleiziffer, Andreas; Tuttelmann, Frank et al. (2014) Epigenetic regulation of the RHOX homeobox gene cluster and its association with human male infertility. Hum Mol Genet 23:12-23|