Convergent Transcription through a Long CAG Tract Destabilizes Repeats and Induces Apoptosis

Abstract

Short repetitive sequences are common in the human genome, and many fall within transcription units. We have previously shown that transcription through CAG repeat tracts destabilizes them in a way that depends on transcription-coupled nucleotide excision repair and mismatch repair. Recent observations that antisense transcription accompanies sense transcription in many human genes led us to test the effects of antisense transcription on triplet repeat instability in human cells. Here, we report that simultaneous sense and antisense transcription (convergent transcription) initiated from two inducible promoters flanking a CAG 95 tract in a nonessential gene enhances repeat instability synergistically, arrests the cell cycle, and causes massive cell death via apoptosis. Using chemical inhibitors and small interfering RNA (siRNA) knockdowns, we identified the ATR (ataxia-telangiectasia mutated [ATM] and Rad3 related) signaling pathway as a key mediator of this cellular response. RNA polymerase II, replication protein A (RPA), and components of the ATR signaling pathway accumulate at convergently transcribed repeat tracts, accompanied by phosphorylation of ATR, CHK1, and p53. Cell death depends on simultaneous sense and antisense transcription and is proportional to their relative levels, it requires the presence of the repeat tract, and it occurs in both proliferating and nonproliferating cells. Convergent transcription through a CAG repeat represents a novel mechanism for triggering a cellular stress response, one that is initiated by events at a single locus in the genome and resembles the response to DNA damage.

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

“…There is generally good agreement between results in our assay in human cells and those in mouse models (29)(30)(31). Among the 20 genes shown to affect CAG repeat contraction in human cells (6,27,28,31,32,37), six have been tested in a mouse model and also shown to modulate CAG repeat instability, including MSH2 (22,47,48), MSH3 (52), PMS2 (12), DNMT1 (6), CSB (20), and XPA (L. Hubert et al, unpublished data).…”

Environmental stress induces trinucleotide repeat mutagenesis in human cells

“…We found that induction of transcription, which efficiently destabilizes CAG repeat tracts in human cells (30,31,33,34), is not required for stress-induced production of GFP + cells. Heat, cold, hypoxic, and oxidative stress induced the same four-to fivefold increase in GFP + cells regardless of whether transcription of the GFP gene was induced by doxycycline before stress and occurred throughout the 3-d recovery period (Fig.…”

“…To establish a link between environmental stresses and CAG repeat instability, we used a GFP-based fluorescence assay to detect repeat contractions (30). As discussed previously, repeat contractions are a good indicator of repeat instability, including expansions (31). The fluorescence assay uses GFP(CAG) 89 cells, which are derived from T-REx HEK293 cells and carry a chromosomal GFP minigene (32) with a CAG 89 tract in the middle of its intron (Fig.…”

Oligodeoxynucleotide Binding to (CTG) · (CAG) Microsatellite Repeats Inhibits Replication Fork Stalling, Hairpin Formation, and Genome Instability

“…The (CTG) n · (CAG) n TNR can cause DNA polymerase stuttering and form hairpin structures in vitro (4)(5)(6)(7) and in vivo (8), which lead to TNR length instability (contraction or expansion). TNR hairpins may be formed during DNA replication (8), transcription (9), and DNA repair (10). Particularly in postmitotic cells, mismatch repair (MMR) pathways are thought to play a role in TNR instability, although the precise role of human MMR in (CTG) n · (CAG) n stability in humans remains unresolved (11,12).…”