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. 2013 Apr;23(4):705-15.
doi: 10.1101/gr.146357.112. Epub 2012 Dec 17.

Mapping genomic hotspots of DNA damage by a single-strand-DNA-compatible and strand-specific ChIP-seq method

Zhi-Xiong Zhou  1 , Mei-Jun Zhang, Xu Peng, Yuko Takayama, Xing-Ya Xu, Ling-Zhi Huang, Li-Lin Du
Affiliations

Mapping genomic hotspots of DNA damage by a single-strand-DNA-compatible and strand-specific ChIP-seq method

Zhi-Xiong Zhou et al. Genome Res. 2013 Apr.

Abstract

Spontaneous DNA damage may occur nonrandomly in the genome, especially when genome maintenance mechanisms are undermined. We developed single-strand DNA (ssDNA)-associated protein immunoprecipitation followed by sequencing (SPI-seq) to map genomic hotspots of DNA damage. We demonstrated this method with Rad52, a homologous recombination repair protein, which binds to ssDNA formed at DNA lesions. SPI-seq faithfully detected, in fission yeast, Rad52 enrichment at artificially induced double-strand breaks (DSBs) as well as endogenously programmed DSBs for mating-type switching. Applying Rad52 SPI-seq to fission yeast mutants defective in DNA helicase Pfh1 or histone H3K56 deacetylase Hst4, led to global views of DNA lesion hotspots emerging in these mutants. We also found serendipitously that histone dosage aberration can activate retrotransposon Tf2 and cause the accumulation of a Tf2 cDNA species bound by Rad52. SPI-seq should be widely applicable for mapping sites of DNA damage and uncovering the causes of genome instability.

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Figures

Figure 1.
Figure 1.
SPI-seq assay detected Rad52 bound at artificially induced and naturally occurring double-strand breaks (DSBs). (A) An adaptor-ligation scheme specially designed for single-strand DNA (ssDNA). (B) The expected outcome of SPI-seq when applied to an HO-induced DSB. Because of our adaptor-ligation design, the chromatin immunoprecipitation (ChIP)–enriched sequencing reads should map to the strands opposite to the ones bound by Rad52. (C) SPI-seq analysis of HO-induced DSBs. The upper and middle panels show the same read density profiles generated by kernel density estimation. (Upper panel) The whole chromosome 1. The y-axis scale of the ChIP signal track is limited to 60 to allow the visualization of the weak signals at the endogenous HO sites. (Middle panel) Close-up view of arg3∷HO-site and its surrounding region. The ChIP signal track shows the full height of the ChIP signal at arg3∷HO-site. Green arrowheads point to the HO-site-flanking regions where strand-specific signal loss occurred in the input, presumably due to resection. (Lower panel) ChIP-enriched reads mapped to individual nucleotide positions at the HO cleavage site, without kernel smoothing. (D) SPI-seq signals at mating-type loci. To visualize reads mapped to the duplicated mating-type cassette, nonuniquely aligned reads were randomly assigned. The units on the y-axes in C and D are reads per 10 million.
Figure 2.
Figure 2.
Rad52 enrichment patterns in pfh1-R20. Rad52 SPI-seq read distribution along chromosome 1 in pfh1-R20 at 20°C. The units on the y-axes are reads per 10 million. The positions and naming of replication origins are as described by Hayashi et al. (2007).
Figure 3.
Figure 3.
Analysis of the narrow Rad52 peaks in pfh1-R20. (A) Rad52 peaks identified by MACS were analyzed to detect their associations with known genomic features. The numbers of observed peaks overlapping with genomic features are listed. For details, see Supplemental Methods. (B) Origins that overlap with Rad52 peaks are enriched with those close to Pol III genes. An origin is deemed associated with a Pol III gene if it is within 300 bp from a Pol III gene. P-value was determined by Fisher's exact test. (C) Heatmap representations of the SPI-seq signals at the 32 singleton peaks overlapping with Pol III genes. The peaks are aligned according to the Pol III gene transcription orientation, with the first nucleotide of the mature RNAs as the alignment point. (D) Average plots of Rad52 SPI-seq signals at the 32 singleton peaks overlapping with Pol III genes. The peaks are aligned as in C. (E) A scatter plot of the distances between Pol III genes and their neighboring origins. The distance from the 5′ end of a Pol III gene to its upstream origin was denoted as its “U” value, while the distance from the 3′ end to the downstream origin as its “D” value. Peak genes refer to the Pol III genes overlapping with the singleton peaks. To reduce errors caused by imprecise origin mapping, we did not include the genes whose distance to the closest origin is <250 bp. (F) The ratios of U/D for peak and nonpeak Pol III genes were plotted. P-value was obtained by comparing the log-transformed ratios of the two groups using Welch's t-test.
Figure 4.
Figure 4.
Replication-coupled Rad52 enrichment patterns in hst4Δ. (A) Rad52 SPI-seq read distribution along chromosome 3. (B) Averaged SPI-seq signal within inter-origin regions of different sizes. Inter-origin regions were classified into three groups according to their sizes. We divided each inter-origin region to 100 bins of equal sizes. The average read number within each bin was calculated, and a sliding window with a window size of 10 bins and a step size of five bins was applied to smooth the curves. (C) Rad52 SPI-seq analysis of hst4Δ cells synchronously released into cell cycle from a G2 arrest. The septation index (percentage of septated cells) shown on the left was measured with Calcofluor staining. The sequencing reads from inter-origin regions >50 kb were used to draw the average plots shown on the right. We first calculated the average read number at each nucleotide position within the first 35 kb and the last 15 kb of the inter-origin regions, and then applied a sliding window with a window size of 1 kb and a step size of 0.1 kb to smooth the curves. (D) Rad52 SPI-seq analysis of swi1Δ and swi3Δ. Average plots were drawn as in C. In A and D, a bandwidth of 500 bp was used for kernel density estimation, and the units on the y-axes of the browser views are reads per 10 million.
Figure 5.
Figure 5.
Rad52 enrichment on retrotransposon cDNA in histone dosage mutants. (A) Rad52 SPI-seq read distribution along chromosome 1 in Δ1Δ3. Enrichment at Tf2 retrotransposons was detected when nonuniquely aligned reads were randomly assigned and visualized. (B) Rad52 SPI-seq reads from Δ1Δ3 were aligned to a full-length Tf2 sequence (GenBank L10324.1). (Bottom panel) The unaveraged read numbers at primer-binding site (PBS). (C) ChIP-PCR detection of Rad52 enrichment on Tf2-12-neoAI. (D) Northern blotting analysis of Tf2 transcription in H3-H4 gene deletion mutants and ams2Δ. (E) Rad52 SPI-seq analysis of histone deletion mutants and mutants with higher-than-normal Tf2 expression. The tandem-linked Tf2-7 and Tf2-8 and their surrounding regions are shown. (F) Tf2 transposition frequency determined using Tf2-12-neoAI. The standard deviation was obtained from four independent cultures. The units on the y-axes of the browser views in A and E are reads per 10 million.
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References

    1. Arcangioli B, Roseaulin L, Holmes A 2007. Mating-type switching in S. pombe. In Topics in current genetics, Vol. 17, pp. 251–283. Springer, Berlin/Heidelberg
    1. Beach DH, Klar AJ 1984. Rearrangements of the transposable mating-type cassettes of fission yeast. EMBO J 3: 603–610 - PMC - PubMed
    1. Bekker-Jensen S, Lukas C, Kitagawa R, Melander F, Kastan MB, Bartek J, Lukas J 2006. Spatial organization of the mammalian genome surveillance machinery in response to DNA strand breaks. J Cell Biol 173: 195–206 - PMC - PubMed
    1. Bowen NJ, Jordan IK, Epstein JA, Wood V, Levin HL 2003. Retrotransposons and their recognition of pol II promoters: A comprehensive survey of the transposable elements from the complete genome sequence of Schizosaccharomyces pombe. Genome Res 13: 1984–1997 - PMC - PubMed
    1. Buhler C, Borde V, Lichten M 2007. Mapping meiotic single-strand DNA reveals a new landscape of DNA double-strand breaks in Saccharomyces cerevisiae. PLoS Biol 5: e324. - PMC - PubMed

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