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. 2011 May 1;25(9):959-71.
doi: 10.1101/gad.2030811.

MDC1 directs chromosome-wide silencing of the sex chromosomes in male germ cells

Yosuke Ichijima  1 , Misako Ichijima, Zhenkun Lou, André Nussenzweig, R Daniel Camerini-Otero, Junjie Chen, Paul R Andreassen, Satoshi H Namekawa
Affiliations

MDC1 directs chromosome-wide silencing of the sex chromosomes in male germ cells

Yosuke Ichijima et al. Genes Dev. .

Abstract

Chromosome-wide inactivation is an epigenetic signature of sex chromosomes. The mechanism by which the chromosome-wide domain is recognized and gene silencing is induced remains unclear. Here we identify an essential mechanism underlying the recognition of the chromosome-wide domain in the male germline. We show that mediator of DNA damage checkpoint 1 (MDC1), a binding partner of phosphorylated histone H2AX (γH2AX), defines the chromosome-wide domain, initiates meiotic sex chromosome inactivation (MSCI), and leads to XY body formation. Importantly, MSCI consists of two genetically separable steps. The first step is the MDC1-independent recognition of the unsynapsed axis by DNA damage response (DDR) factors such as ataxia telangiectasia and Rad3-related (ATR), TOPBP1, and γH2AX. The second step is the MDC1-dependent chromosome-wide spreading of DDR factors to the entire chromatin. Furthermore, we demonstrate that, in somatic cells, MDC1-dependent amplification of the γH2AX signal occurs following replicative stress and is associated with transcriptional silencing. We propose that a common DDR pathway underlies both MSCI and the response of somatic cells to replicative stress. These results establish that the DDR pathway centered on MDC1 triggers epigenetic silencing of sex chromosomes in germ cells.

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Figures

Figure 1.
Figure 1.
MDC1 directs chromosome-wide spreading of DDR factors. (A–F) Immunostaining of meiotic chromosome spreads using antibodies against DDR factors together with the anti-SCP3 antibody. SCP3 staining displays the status of chromosome synapsis and is used to distinguish meiotic stages. All images are wide-field images of pachytene spermatocytes. Areas surrounding sex chromosomes are highlighted in dotted rectangles and are magnified in the right panels. (Arrows) Sex chromosomes. (G) Pictorial representation about the role of MDC1 in MSCI. The first step is MDC1-independent recognition of the unsynapsed axis. The second step is MDC1-dependent spreading of DDR factors to the chromosome-wide domain.
Figure 2.
Figure 2.
MDC1 is required for MSCI. (A,B) Immunostaining with anti-Pol II antibody in Mdc1+/− and Mdc1−/− spermatocytes, respectively. Unsynapsed axes of sex chromosomes are detected with anti-BRCA1 antibody. Areas surrounding sex chromosomes are highlighted in dotted rectangles and are magnified in the right panels. The outline of the XY body is highlighted with a dotted oval. (Arrow) Centromeric end of the X chromosome; (arrowhead) Y-chromosome part of the sex chromosome pair. (C,D) Pictorial representation of the unsynapsed axes of sex chromosomes. (E,F) Combined immunostaining with anti-BRCA1 antibody and Cot-1 RNA FISH in Mdc1+/− and Mdc1−/− cells, respectively. Meiotic slides were prepared as described (Namekawa et al. 2006). All images are deconvolved single Z-sections.
Figure 3.
Figure 3.
Microarray analysis of Mdc1−/− testis at 16.5 d old. (A) Comparison of gene expression levels in Mdc1−/− versus Mdc1+/+ cells among autosomes or X and Y chromosomes. The central dot is the median, the boxes encompass 50% of data points, and the error bars indicate 90% of data points. P was derived from a paired t-test. (B) Average expression levels in Mdc1−/− versus Mdc1+/+ mice for each chromosome. (C) Expression levels along the location of representative autosomes (Chr1–Chr3) and the X chromosome. Locations are distances from proximal ends of chromosomes (in megabases). Arrowheads denote average expression levels in each chromosome.
Figure 4.
Figure 4.
γH2AX–MDC1 signaling is the primary step in XY body formation. (A–L) Immunostaining of meiotic chromosome spreads using the indicated antibodies together with the anti-SCP3 antibody. All pictures represent pachytene spermatocytes. (Arrows) Sex chromosomes. All images are wide-field images. (M) Conformation of sex chromosome axes detected by BRCA1 staining in pachytene Mdc1+/− and Mdc1−/− spermatocytes. To preserve relative three-dimensional conformation, meiotic slides were prepared as described (Namekawa et al. 2006). (N) Summary of linear distances between both ends of sex chromosome axes. The normalized distance is the linear distance between both ends detected with BRCA1 staining normalized to the nuclear diameter. The central dot is the median, the boxes encompass 50% of data points, and the error bars indicate 90% of data points. Meiotic stages were judged by simultaneous staining with SCP3 and H1t. P-values were calculated using an unpaired t-test. (*) P < 0.01; (**) P < 0.001.
Figure 5.
Figure 5.
Meiotic recombination in the Mdc1 mutant. Immunostaining of meiotic chromosome spreads using the indicated antibodies together with the anti-SCP3 antibody. All images are wide-field images. (Arrows) Sex chromosomes. (A,B) Chromosome synapsis occurs normally at the pachytene stage in Mdc1+/− and Mdc1−/− spermatocytes. (C) Mdc1−/− spermatocytes exhibit sex chromosome-specific synaptic defects. (D) Illegitimate associations of the X axis to autosomes in Mdc1−/− spermatocytes. Areas surrounding sex chromosomes (dotted rectangle) are magnified in the right panels, and schematics of chromosome axes are shown below. (E,F) Rad51 foci formation is normal at the zygotene stage in Mdc1+/− and Mdc1−/− spermatocytes. (G,H) Rad51 foci disappear from autosomal regions and remain only on the X-chromosome axis at the pachytene stage in Mdc1+/− and Mdc1−/− spermatocytes. (I,J) RPA foci formation is normal at the pachytene stage in Mdc1+/− and Mdc1−/− spermatocytes. (K) MLH1 marks the sites of crossover at the pachytene stage in Mdc1+/− spermatocytes. (L) MLH1 foci are abolished in Mdc1−/− at the pachytene stage. (M) MDC1 localizes on the pseudo sex body in Spo11−/− spermatocytes. (Arrowhead) Pseudo sex body. (N) The pseudo sex body is abolished in spermatocytes of the Spo11−/−Mdc1−/− double mutant.
Figure 6.
Figure 6.
MDC1 amplifies γH2AX signal after replicative stress in somatic cells. (A) Replicative stress induction by HU and the appearance of γH2AX foci during S phase. U2OS cells were treated with 10 μM EdU for 30 min prior to 1 mM HU treatment for 2 h. Images are confocal images using Optiglid. (Arrowheads) S-phase cells. Dotted circles show nuclear regions. (B) γH2AX signal amplification is hampered by the knockdown of MDC1. U2OS cells were treated with siRNAs for 48 h prior to 1 mM HU treatment for 2 h. siRNA for luciferase gene (siLuc) was used as a negative control. Images are confocal images using Optiglid. (C) Quantification of γH2AX signal intensity after MDC1 knockdown. More than 30 nuclei of three independent experiments were examined, quantified using the ImageJ software, and normalized against the value of control siRNA (siLuc). P was derived from an unpaired t-test. The error bar shows standard deviation. (D) Model showing the role of MDC1 in γH2AX amplification. The first step is MDC1-independent induction of γH2AX. The second step is MDC1-dependent amplification of γH2AX signals in a feedback loop with TOPBP1 and ATR. MDC1 directs spreading of DDR factors to adjacent nucleosomes. (E) Foci of γH2AX and Pol II are mutually exclusive in U2OS cells after the 2-h HU treatment. Serial Z-sections were deconvolved and a representative Z-section is shown. Areas in dotted rectangles are magnified in the right panels. (Arrows) Sites of γH2AX foci.
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References

    1. Ahmed EA, van der Vaart A, Barten A, Kal HB, Chen J, Lou Z, Minter-Dykhouse K, Bartkova J, Bartek J, de Boer P, et al. 2007. Differences in DNA double strand breaks repair in male germ cell types: lessons learned from a differential expression of Mdc1 and 53BP1. DNA Repair (Amst) 6: 1243–1254 - PubMed
    1. Andreassen PR, Niedernhofer LJ 2009. Fanconi anemia: a paradigm of discovering molecular pathways from patients. Mutat Res 668: 1–3 - PMC - PubMed
    1. Baarends WM, Wassenaar E, van der Laan R, Hoogerbrugge J, Sleddens-Linkels E, Hoeijmakers JH, de Boer P, Grootegoed JA 2005. Silencing of unpaired chromatin and histone H2A ubiquitination in mammalian meiosis. Mol Cell Biol 25: 1041–1053 - PMC - PubMed
    1. Baker SM, Plug AW, Prolla TA, Bronner CE, Harris AC, Yao X, Christie DM, Monell C, Arnheim N, Bradley A, et al. 1996. Involvement of mouse Mlh1 in DNA mismatch repair and meiotic crossing over. Nat Genet 13: 336–342 - PubMed
    1. Barchi M, Mahadevaiah S, Di Giacomo M, Baudat F, de Rooij DG, Burgoyne PS, Jasin M, Keeney S 2005. Surveillance of different recombination defects in mouse spermatocytes yields distinct responses despite elimination at an identical developmental stage. Mol Cell Biol 25: 7203–7215 - PMC - PubMed

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