Transcription promotes contraction of CAG repeat tracts in human cells

Abstract

Induced transcription through CAG repeats in human cells increases repeat contraction approximately 15-fold in both confluent and proliferating cells. Repeats are stabilized against contraction by siRNA knockdown of MSH2, MSH3 or XPA, but not by knockdown of MSH6, XPC or FEN1. These results define a pathway for CAG.CTG repeat contraction that is initiated by transcription, depends on elements of mismatch and nucleotide-excision repair and does not require DNA replication.

Topoisomerase 1 and Single-Strand Break Repair Modulate Transcription-Induced CAG Repeat Contraction in Human Cells

“…FLAH25 human cells that consists of a selection marker, the HPRT (hypoxanthine-guanine phosphoribosyl transferase) minigene, containing a (CAG) 95 repeat tract driven by an inducible promoter ( Fig. 1A) (28). Using this assay, we can detect large CAG contraction events that reduce the size of the repeat tract from 95 to 38 units or fewer.…”

“…FLAH25 cells were derived from the human fibrosarcoma HT1080 cell line. Construction of this cell line was described previously (28). FLAH25 cells carry a spontaneous nonreverting deletion of the single endogenous HPRT gene; an integrated plasmid that constitutively expresses rtTA protein, which is the reverse tetracycline repressor protein fused to the HSV VP16 activation domain (17); and a single copy of the HPRT minigene with a CAG 95 tract embedded in its single intron.…”

“…FLAH25 cells carry a spontaneous nonreverting deletion of the single endogenous HPRT gene; an integrated plasmid that constitutively expresses rtTA protein, which is the reverse tetracycline repressor protein fused to the HSV VP16 activation domain (17); and a single copy of the HPRT minigene with a CAG 95 tract embedded in its single intron. The expression of the HPRT minigene in FLAH25 cells is driven by the promoter, pCMV-mini, which is suppressed by rtTA, but can be induced 22-fold by doxycycline (28). FLAH25 cells were grown at 37°C with 5% CO 2 in Dulbecco modified Eagle medium/F-12 medium supplemented with 10% fetal bovine serum and 1% minimal essential medium nonessential amino acids.…”

“…We have previously demonstrated that transcription-coupled nucleotide excision repair (TC-NER) normally functions to increase transcription-induced CAG repeat contractions (28,32). If TC-NER were the cause of the increase in CAG contractions that arise by inhibiting the TOP1-TDP1-SSBR pathway, then we should be able to block the increase by simultaneously knocking down both pathways.…”

Androgen receptor CAG repeat length contraction in diseased and non-diseased prostatic tissues

“…Our observations are in keeping with these results, which are suggestive of an early embryonic phenomenon. In addition, recently, Lin et al 43 demonstrated that induced transcription of CAG repeats increased large repeat CAG contractions by 15-fold with no evidence of expansions. These observations may therefore help explain why AR CAG repeat contractions are limited to the prostate as the prostate, almost uniquely expresses higher levels of AR, at an early stage of development.…”