Transcription-induced CAG repeat contraction in human cells is mediated in part by transcription-coupled nucleotide excision repair

Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, Texas 77030, USA

^* To whom correspondence should be addressed. Email: jwilson{at}bcm.tmc.edu.

	Abstract

Expansions of CAG repeat tracts in the germline underlie several neurological diseases. In human patients and mouse models, CAG repeat tracts display an ongoing instability in neurons, which may exacerbate disease symptoms. It is unclear how repeats are destabilized in nondividing cells, but it cannot involve DNA replication. We showed previously that transcription through CAG repeats induces their instability. Here, we present a genetic analysis of the link between transcription-induced repeat instability and nucleotide excision repair (NER) in human cells. We show that siRNA-mediated knockdown of CSB, a component specifically required for transcription-coupled NER (TC-NER), and knockdowns of ERCC1 and XPG, which incise DNA adjacent to damage, stabilize CAG repeat tracts. These results suggest that TC-NER is involved in the pathway for transcription-induced CAG repeat instability. By contrast, knockdowns of OGG1 or APEX1, key components involved in base excision repair, did not affect repeat instability. In addition, repeats are stabilized by knockdown of transcription factor IIS, consistent with a requirement for RNA polymerase II (RNAPII) to backtrack from a transcription block. Repeats are also stabilized by knockdown of either BRCA1 or BARD1, which together function as an E3 ligase that can ubiquitinate arrested RNAPII. Treatment with the proteasome inhibitor, MG132, which stabilizes repeats, confirms proteasome involvement. We integrate these observations into a tentative pathway for transcription-induced CAG repeat instability that can account for the contractions observed here, and potentially for the contractions and expansions seen in human diseases.