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Extensive meiotic asynapsis in mice antagonises meiotic silencing of unsynapsed chromatin and consequently disrupts meiotic sex chromosome inactivation.

Mahadevaiah SK, Bourc'his D, de Rooij DG, Bestor TH, Turner JM, Burgoyne PS - J. Cell Biol. (2008)

Bottom Line: In contrast, three mutants (Dnmt3l, Msh5, and Dmc1) with high levels of asynapsis and numerous persistent unrepaired DSBs have a severely impaired MSUC response.We suggest that MSUC-related proteins, including the MSUC initiator BRCA1, are sequestered at unrepaired DSBs.Apoptosis does not occur in mice with a single additional asynapsed chromosome with unrepaired meiotic DSBs and no disturbance of MSCI.

View Article: PubMed Central - PubMed

Affiliation: Division of Stem Cell Biology and Developmental Genetics, Medical Research Council National Institute for Medical Research, London NW7 1AA, England, UK.

ABSTRACT
Chromosome synapsis during zygotene is a prerequisite for the timely homologous recombinational repair of meiotic DNA double-strand breaks (DSBs). Unrepaired DSBs are thought to trigger apoptosis during midpachytene of male meiosis if synapsis fails. An early pachytene response to asynapsis is meiotic silencing of unsynapsed chromatin (MSUC), which, in normal males, silences the X and Y chromosomes (meiotic sex chromosome inactivation [MSCI]). In this study, we show that MSUC occurs in Spo11- mouse spermatocytes with extensive asynapsis but lacking meiotic DSBs. In contrast, three mutants (Dnmt3l, Msh5, and Dmc1) with high levels of asynapsis and numerous persistent unrepaired DSBs have a severely impaired MSUC response. We suggest that MSUC-related proteins, including the MSUC initiator BRCA1, are sequestered at unrepaired DSBs. All four mutants fail to silence the X and Y chromosomes (MSCI failure), which is sufficient to explain the midpachytene apoptosis. Apoptosis does not occur in mice with a single additional asynapsed chromosome with unrepaired meiotic DSBs and no disturbance of MSCI.

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Increasing asynapsis may attenuate the MSUC response by sequestering BRCA1 at unrepaired DSBs. (a and b) In Dnmt3l−/− pachytene spermatocytes, increasing asynapsis is associated with increasingly focal BRCA1 staining. In the two spermatocytes shown, the Y chromosome has failed to synapse with the X chromosome. (c and d) Enlargements of the X (presumed) and Y chromosomes show that BRCA1 covers the axes of both chromosomes from the spermatocyte with only the X and Y asynapsed, but, in the spermatocyte with more extensive asynapsis, the BRCA1 has become more focal on the presumed X and is restricted on the Y chromosome to a single focus. (e) Quantitation of the axially located BRCA1 in pachytene spermatocytes shows that it does not increase with increasing asynapsis (fitted blue regression line). The projected increase in BRCA1 signal if it was in proportion to the amount of asynapsed axis is denoted by the red line. (f) The focal BRCA1 staining seen when asynapsis is extensive is largely DSB associated, as indicated by costaining for RAD51. Bars, 10 μm.
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fig4: Increasing asynapsis may attenuate the MSUC response by sequestering BRCA1 at unrepaired DSBs. (a and b) In Dnmt3l−/− pachytene spermatocytes, increasing asynapsis is associated with increasingly focal BRCA1 staining. In the two spermatocytes shown, the Y chromosome has failed to synapse with the X chromosome. (c and d) Enlargements of the X (presumed) and Y chromosomes show that BRCA1 covers the axes of both chromosomes from the spermatocyte with only the X and Y asynapsed, but, in the spermatocyte with more extensive asynapsis, the BRCA1 has become more focal on the presumed X and is restricted on the Y chromosome to a single focus. (e) Quantitation of the axially located BRCA1 in pachytene spermatocytes shows that it does not increase with increasing asynapsis (fitted blue regression line). The projected increase in BRCA1 signal if it was in proportion to the amount of asynapsed axis is denoted by the red line. (f) The focal BRCA1 staining seen when asynapsis is extensive is largely DSB associated, as indicated by costaining for RAD51. Bars, 10 μm.

Mentions: Our finding that the amount of γH2AX does not increase in response to asynapsis (Fig. 3 i), even though in spermatocytes H2AX has been shown to be abundant throughout the nucleus (Fernandez-Capetillo et al., 2003), suggests that something is limiting the phosphorylation of H2AX by ATR. This could be limited availability of ATR itself or of BRCA1, which is involved in ATR recruitment to asynapsed axes (Turner et al., 2004). We have used RNA FISH to assess Atr and Brca1 transcription (Fig. S2, a–j); Atr is robustly transcribed throughout pachytene, but Brca1 transcription is shut down in the course of pachytene. More importantly, with increasing asynapsis in Dnmt3l−/− pachytene spermatocytes, it was apparent that the BRCA1 staining associated with these asynapsed axes became weaker and more focal (Fig. 4, a and b). Quantitation of the axially located BRCA1 in pachytene spermatocytes confirmed that it does not increase concomitantly with increasing asynapsis (Fig. 4 e). Because recruitment of ATR to the axes during the MSUC response is dependent on the prior recruitment of BRCA1, this strongly implicates BRCA1 recruitment to the asynapsed axes as the limiting factor.


Extensive meiotic asynapsis in mice antagonises meiotic silencing of unsynapsed chromatin and consequently disrupts meiotic sex chromosome inactivation.

Mahadevaiah SK, Bourc'his D, de Rooij DG, Bestor TH, Turner JM, Burgoyne PS - J. Cell Biol. (2008)

Increasing asynapsis may attenuate the MSUC response by sequestering BRCA1 at unrepaired DSBs. (a and b) In Dnmt3l−/− pachytene spermatocytes, increasing asynapsis is associated with increasingly focal BRCA1 staining. In the two spermatocytes shown, the Y chromosome has failed to synapse with the X chromosome. (c and d) Enlargements of the X (presumed) and Y chromosomes show that BRCA1 covers the axes of both chromosomes from the spermatocyte with only the X and Y asynapsed, but, in the spermatocyte with more extensive asynapsis, the BRCA1 has become more focal on the presumed X and is restricted on the Y chromosome to a single focus. (e) Quantitation of the axially located BRCA1 in pachytene spermatocytes shows that it does not increase with increasing asynapsis (fitted blue regression line). The projected increase in BRCA1 signal if it was in proportion to the amount of asynapsed axis is denoted by the red line. (f) The focal BRCA1 staining seen when asynapsis is extensive is largely DSB associated, as indicated by costaining for RAD51. Bars, 10 μm.
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fig4: Increasing asynapsis may attenuate the MSUC response by sequestering BRCA1 at unrepaired DSBs. (a and b) In Dnmt3l−/− pachytene spermatocytes, increasing asynapsis is associated with increasingly focal BRCA1 staining. In the two spermatocytes shown, the Y chromosome has failed to synapse with the X chromosome. (c and d) Enlargements of the X (presumed) and Y chromosomes show that BRCA1 covers the axes of both chromosomes from the spermatocyte with only the X and Y asynapsed, but, in the spermatocyte with more extensive asynapsis, the BRCA1 has become more focal on the presumed X and is restricted on the Y chromosome to a single focus. (e) Quantitation of the axially located BRCA1 in pachytene spermatocytes shows that it does not increase with increasing asynapsis (fitted blue regression line). The projected increase in BRCA1 signal if it was in proportion to the amount of asynapsed axis is denoted by the red line. (f) The focal BRCA1 staining seen when asynapsis is extensive is largely DSB associated, as indicated by costaining for RAD51. Bars, 10 μm.
Mentions: Our finding that the amount of γH2AX does not increase in response to asynapsis (Fig. 3 i), even though in spermatocytes H2AX has been shown to be abundant throughout the nucleus (Fernandez-Capetillo et al., 2003), suggests that something is limiting the phosphorylation of H2AX by ATR. This could be limited availability of ATR itself or of BRCA1, which is involved in ATR recruitment to asynapsed axes (Turner et al., 2004). We have used RNA FISH to assess Atr and Brca1 transcription (Fig. S2, a–j); Atr is robustly transcribed throughout pachytene, but Brca1 transcription is shut down in the course of pachytene. More importantly, with increasing asynapsis in Dnmt3l−/− pachytene spermatocytes, it was apparent that the BRCA1 staining associated with these asynapsed axes became weaker and more focal (Fig. 4, a and b). Quantitation of the axially located BRCA1 in pachytene spermatocytes confirmed that it does not increase concomitantly with increasing asynapsis (Fig. 4 e). Because recruitment of ATR to the axes during the MSUC response is dependent on the prior recruitment of BRCA1, this strongly implicates BRCA1 recruitment to the asynapsed axes as the limiting factor.

Bottom Line: In contrast, three mutants (Dnmt3l, Msh5, and Dmc1) with high levels of asynapsis and numerous persistent unrepaired DSBs have a severely impaired MSUC response.We suggest that MSUC-related proteins, including the MSUC initiator BRCA1, are sequestered at unrepaired DSBs.Apoptosis does not occur in mice with a single additional asynapsed chromosome with unrepaired meiotic DSBs and no disturbance of MSCI.

View Article: PubMed Central - PubMed

Affiliation: Division of Stem Cell Biology and Developmental Genetics, Medical Research Council National Institute for Medical Research, London NW7 1AA, England, UK.

ABSTRACT
Chromosome synapsis during zygotene is a prerequisite for the timely homologous recombinational repair of meiotic DNA double-strand breaks (DSBs). Unrepaired DSBs are thought to trigger apoptosis during midpachytene of male meiosis if synapsis fails. An early pachytene response to asynapsis is meiotic silencing of unsynapsed chromatin (MSUC), which, in normal males, silences the X and Y chromosomes (meiotic sex chromosome inactivation [MSCI]). In this study, we show that MSUC occurs in Spo11- mouse spermatocytes with extensive asynapsis but lacking meiotic DSBs. In contrast, three mutants (Dnmt3l, Msh5, and Dmc1) with high levels of asynapsis and numerous persistent unrepaired DSBs have a severely impaired MSUC response. We suggest that MSUC-related proteins, including the MSUC initiator BRCA1, are sequestered at unrepaired DSBs. All four mutants fail to silence the X and Y chromosomes (MSCI failure), which is sufficient to explain the midpachytene apoptosis. Apoptosis does not occur in mice with a single additional asynapsed chromosome with unrepaired meiotic DSBs and no disturbance of MSCI.

Show MeSH
Related in: MedlinePlus