Replication-dependent instability at (CTG) x (CAG) repeat hairpins in human cells

Abstract

Instability of (CTG) x (CAG) microsatellite trinucleotide repeat (TNR) sequences is responsible for more than a dozen neurological or neuromuscular diseases. TNR instability during DNA synthesis is thought to involve slipped-strand or hairpin structures in template or nascent DNA strands, although direct evidence for hairpin formation in human cells is lacking. We have used targeted recombination to create a series of isogenic HeLa cell lines in which (CTG) x (CAG) repeats are replicated from an ectopic copy of the Myc (also known as c-myc) replication origin. In this system, the tendency of chromosomal (CTG) x (CAG) tracts to expand or contract was affected by origin location and the leading or lagging strand replication orientation of the repeats, and instability was enhanced by prolonged cell culture, increased TNR length and replication inhibition. Hairpin cleavage by synthetic zinc finger nucleases in these cells has provided the first direct evidence for the formation of hairpin structures during replication in vivo.

Figure 1

Hairpin induced trinucleotide repeat instability. The TNR is indicated by grey lines, flanking DNA by black lines. (a) Nascent strand hairpin formation results in overreplication of a segment of the TNR in one chromatid. A second round of replication of the hairpin strand fixes the expanded allelle in the genome. (b) Template strand hairpin formation results in underreplication of a segment of the TNR in one chromatid. A second round of replication of the non-hairpin strand fixes the contracted allelle in the genome. (Adapted from 9).

Figure 2

DNA replication affects TNR instability. (a) Diagram of ectopic origin sites. Arrowheads, PCR primer positions; bracket, DUE deletion (ΔDUE). (b) PCR analysis of (CTG)₁₀₂•(CAG)₁₀₂ cells cultured for 25 population doublings. Lane 1, (CTG)₁₀₂ cells; lane 2, (CAG)₁₀₂ cells; M, molecular weight marker. The progenitor (CTG)₁₀₂•(CAG)₁₀₂ band is indicated. (c) PCR analysis of (CTG)₁₀₂•(CAG)₁₀₂ cells cultured for 250 population doublings. Lane 1, (CTG)₁₀₂ cells; lane 2, (CAG)₁₀₂ cells. (d) PCR analysis of (CTG)₁₂•(CAG)₁₂ cells cultured for 25 or 250 population doublings. Lanes 1, 3, (CTG)₁₂ cells; lanes 2, 4, (CAG)₁₂ cells. The progenitor (CTG)₁₂•(CAG)₁₂ band is indicated. (e) PCR analysis of ΔDUE-(CTG)₁₀₂•(CAG)₁₀₂ cells cultured for 25 or 250 population doublings. Lanes 1, 3, ΔDUE-(CTG)₁₀₂ cells; lanes 2, 4, ΔDUE-(CAG)₁₀₂ cells.

Figure 3

ZFN cleave specifically in vitro. (a) Binding of a ZFN_CTG and ZFN_CAG heterodimer capable of cleaving heteroduplex DNA. Fok I_CD, Fok I catalytic domain; ZFP_GCT, GCT recognition zinc finger protein; ZF_AGC, AGC recognition zinc finger protein. (b) Predicted modes of ZFN_CTG monomer binding to heteroduplex DNA (upper) or homodimeric ZFN_CTG capable of cleaving (CTG)_n hairpin DNA (lower). (c) The linear (CTG)₁₀₂•(CAG)₁₀₂ PCR product was gel purified and reamplified. Time course of cleavage of the reamplified (CTG)₁₀₂•(CAG)₁₀₂ PCR products with ZFN_CTG (10% of immunoprecipitate, lanes 1–3) or ZFN_CAG (10% of immunoprecipitate, lanes 4–6). (d) Time course of cleavage of the reamplified (CTG)₁₀₂•(CAG)₁₀₂ PCR products with a mixture of ZFN_CTG and ZFN_CAG (5% of each immunoprecipitate).

Figure 4

(CTG)₁₀₂•(CAG)₁₀₂ TNRs form hairpins in vivo. (CTG)₁₀₂•(CAG)₁₀₂ cells were cultured for 25 population doublings. (a) spPCR of DNA from (CTG)₁₀₂ cells or (CAG)₁₀₂ cells transfected, respectively, with empty vector (lanes 1–4, 9–12) or ZFN_CTG expression plasmid (lanes 5–8, 13–16). (b) spPCR of DNA from (CTG)₁₀₂ cells (lanes 1–5) or (CAG)₁₀₂ cells (lanes 6–9) transfected with ZFN_CAG expression plasmid. (c) spPCR of DNA from (CTG)₁₀₂ cells or (CAG)₁₀₂ cells transfected, respectively, with empty vector (lanes 1–4, 5–8) or a 0.5:0.5 mixture of ZFN_CTG and ZFN_CAG expression plasmids (lanes 9–12, 13–16). (d) spPCR of DNA from (CTG)₁₀₂ cells or (CAG)₁₀₂ cells transfected with ZFP_CTG (lanes 1–8) or ZFP_CAG (lanes 9–16) expression plasmids. Lanes 1–4 and 5–8 were merged from nonadjacent lanes of the same gel, as were lanes 9–12 and lanes 13–16. (See Supplementary Figure 11 for full gel images).

Figure 5

(CTG)₁₀₂•(CAG)₁₀₂ hairpin formation is suppressed by serum starvation. (CTG)₁₀₂•(CAG)₁₀₂ cells were grown for 25 population doublings and transferred to medium containing 0.5% serum for 48 hr. (a) spPCR of DNA from (CTG)₁₀₂ cells (lanes 1–8) or (CAG)₁₀₂ cells (lanes 9–16) transfected with the ZFN_CTG expression plasmid. (b) spPCR of DNA from (CTG)₁₀₂ cells (lanes 1–8) or (CAG)₁₀₂ cells (lanes 9–16) transfected with the ZFN_CAG expression plasmid. (c) spPCR of DNA from (CTG)₁₀₂ cells (lanes 1–8) or (CAG)₁₀₂ cells (lanes 9–16) transfected with the ZFN_CAG and ZFN_CAG expression plasmids.

Figure 6

(CTG)₁₂•(CAG)₁₂ TNRs are stable in vivo. (CTG)₁₂ or (CAG)₁₂ cells were cultured for 25 population doublings. (a) PCR of DNA from (CTG)₁₂ (lane 1, 3) or (CAG)₁₂ cells (lane 2, 4) treated with aphidicolin, or mock treated, as indicated. (b) PCR of DNA from (CTG)₁₂ (lane 1, 3, 5, 7) or (CAG)₁₂ cells (lane 2, 4, 6, 8) treated with emetine, Fen1 siRNA, or mock treated, as indicated. (c) spPCR of DNA from untreated (CTG)₁₂ (lane 1–8) or (CAG)₁₂ cells (lane 9–16). (d) spPCR of DNA from (CTG)₁₂ or (CAG)₁₂ cells expressing ZFN_CTG (lanes 1–8) or ZFN_CAG (lanes 9–16), as indicated. (e) spPCR of DNA from (CTG)₁₂ (Lanes 1–6) or (CAG)₁₂ cells (lanes 7–12) expressing a 0.5:0.5 mixture of ZFN_CTG and ZFN_CAG.