TFIIH-XPD Co-Activator Protein in Photoreceptor and Retinal Pigment Epithelial Gene Transcription and Disease

Abstract

Purpose: : The general transcription factor complex TFIIH plays an important role in DNA repair and transcription. Mutations in XPD, a subunit of TFIIH, cause autosomal recessive syndromes with retinal and other ocular defects. Using yeast two-hybrid assays, we initially identified XPD as an interacting partner of NR2E3, a nuclear receptor required for rod differentiation. The goal of this research was to determine the role of TFIIH-XPD in photoreceptor and RPE gene transcription and associated retinal disease.

Methods: : Co-immunoprecipitation, chromatin immunoprecipitation (ChIP) and co-transfection assays investigated XPD interactions with NR2E3 and other photoreceptor transcription factors (PhTFs). Changes in retinal and RPE gene expression in Xpd^TTD mice carrying a hypomorph mutation of XPD were assessed by immunocytohistochemistry and qRT-PCR.

Results: : XPD protein is present in the nucleus of all mouse retinal and RPE cell types, including photoreceptors and their precursors. Co-immunoprecipitation showed that XPD interacts not only with NR2E3, but also the cone-rod homeobox CRX and bZIP factor NRL. Each PhTF interacts with XPD via its regulatory domain. In transfected NIH3T3 cells, recombinant XPD enhanced the activation of the rhodopsin promoter by PhTFs, while shRNA-mediated XPD knockdown blocked transcriptional activation. ChIP assays on P14 wild-type mouse retinas showed that XPD and other components of TFIIH bind to promoter and coding regions of each opsin gene along with PhTFs. XPD target binding is reduced in mutant retinas lacking each PhTF, suggesting that XPD is a PhTF co-activator. The importance of TFIIH-XPD was further revealed by photoreceptor defects in Xpd^TTD mice: Reduced amplitudes of dark and light adapted ERG "a" and "b" waves in young adults, consistent with decreased transcription of PhTF target genes as measured by qRT-PCR. Expression of several RPE-specific genes were also reduced in Xpd^TTD mice.

Conclusions: : TFIIH-XPD co-activator complex is essential for photoreceptor and RPE gene transcription and function. The cellular and molecular mechanisms by which XPD mutations cause retina/RPE defects are under investigation.

Keywords: gene/expression • transcription factors • degenerations/dystrophies