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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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The pseudo–sex body domain of Spo11- pachytene spermatocytes is transcriptionally silenced. (a and b) Cot1 RNA FISH staining detects nascent transcripts within the nucleus; the pseudo–sex body marked by γH2AX is a transcriptionally repressed domain. (c and d) In Spo11- pachytene spermatocytes, as a result of asynapsis/nonhomologous synapsis, the two autosomal Atr loci are nearly always well separated. In the cell shown, there is a single Atr RNA FISH signal (arrows); DNA FISH shows that the second non-transcribing Atr locus lies within the pseudo–sex body domain (white outline). (e and f) A pachytene spermatocyte showing transcription of the Y chromosomal gene Zfy2 when it is not located in the pseudo–sex body. (g and h) A nontranscribing Zfy2 locus lying within the pseudo–sex body. (i–n) RNA/DNA FISH for the Ddx3x/Usp9x X chromosomal BAC showing two transcribing pachytene spermatocytes with the locus outside the pseudo–sex body and one nontranscribing pachytene spermatocyte in which the locus lies within the pseudo–sex body. (a–n) White outlines indicate the extent of the γH2AX domain before DNA FISH, and arrows point to the FISH signals (either RNA FISH or DNA FISH). Bar, 5 μm.
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fig2: The pseudo–sex body domain of Spo11- pachytene spermatocytes is transcriptionally silenced. (a and b) Cot1 RNA FISH staining detects nascent transcripts within the nucleus; the pseudo–sex body marked by γH2AX is a transcriptionally repressed domain. (c and d) In Spo11- pachytene spermatocytes, as a result of asynapsis/nonhomologous synapsis, the two autosomal Atr loci are nearly always well separated. In the cell shown, there is a single Atr RNA FISH signal (arrows); DNA FISH shows that the second non-transcribing Atr locus lies within the pseudo–sex body domain (white outline). (e and f) A pachytene spermatocyte showing transcription of the Y chromosomal gene Zfy2 when it is not located in the pseudo–sex body. (g and h) A nontranscribing Zfy2 locus lying within the pseudo–sex body. (i–n) RNA/DNA FISH for the Ddx3x/Usp9x X chromosomal BAC showing two transcribing pachytene spermatocytes with the locus outside the pseudo–sex body and one nontranscribing pachytene spermatocyte in which the locus lies within the pseudo–sex body. (a–n) White outlines indicate the extent of the γH2AX domain before DNA FISH, and arrows point to the FISH signals (either RNA FISH or DNA FISH). Bar, 5 μm.

Mentions: We then asked whether the γH2AX-positive pseudo–sex body domain was transcriptionally repressed by use of a Cot1 probe to detect nascent transcripts (Turner et al., 2005) and then immunostained for γH2AX to locate the pseudo–sex body domain. This showed that the γH2AX domain was indeed transcriptionally repressed in cells that had a clear pseudo–sex body (Fig. 2, a and b). We next combined γH2AX immunostaining with RNA FISH and DNA FISH for Atr, which is autosomal and in wild-type males is robustly expressed throughout pachytene (Fig. S2, a–e; available at http://www.jcb.org/cgi/content/full/jcb.200710195/DC1). It was predicted that any pachytene cells with an Atr locus that failed to transcribe would have the Atr locus within the transcriptionally repressed γH2AX domain. 7/42 (17%) pachytene cells had one transcribed and one nontranscribed Atr locus, and, in each case, the nontranscribing locus was within the γH2AX-positive region (Fig. 2, c and d). We next performed RNA FISH analysis for the Y-located gene Zfy2 that in normal males is weakly expressed in leptotene and zygotene spermatocytes when chromatin condensation is associated with global transcriptional repression and is then shut down by MSCI/MSUC throughout pachytene (Fig. S2, k–o). This revealed that 70/74 (95%) Spo11−/− pachytene cells were inappropriately expressing Zfy2 (Fig. 2, e and f); in 69 of these cells, the Zfy2 locus was located away from the γH2AX-positive domain, and one weakly expressed locus was at the edge of the domain. In the four nonexpressing cells, the Zfy2 locus was located within the γH2AX-positive domain (Fig. 2, g and h). Finally, we performed RNA FISH analysis using an X-chromosomal bacterial artificial chromosome (BAC) probe encompassing Ddx3x and 28 kb of Usp9x. We have found that this BAC gives no detectable RNA FISH signal in normal spermatocytes (Figs. S2, p–t; and S4, a and b); however, in Brca1Δ11/Δ11 males (which have extensive MSCI failure; Turner et al., 2004), a Ddx3x/Usp9x signal becomes progressively more evident during pachytene (Figs. S3, g–i; and S4, c and d). This provides a robust pachytene-specific marker of MSCI failure but will underestimate the proportion of pachytene cells with MSCI failure because transcription does not commence immediately upon entry of cells into pachytene. In Spo11−/− males, 36/43 (84%) pachytene cells were found to be inappropriately expressing Ddx3x/Usp9x because the locus was not within the γH2AX-positive domain (Fig. 2, i–l). In four of the seven nonexpressing cells, the Ddx3x/Usp9x locus lay within the γH2AX domain (Fig. 2, m and n).


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)

The pseudo–sex body domain of Spo11- pachytene spermatocytes is transcriptionally silenced. (a and b) Cot1 RNA FISH staining detects nascent transcripts within the nucleus; the pseudo–sex body marked by γH2AX is a transcriptionally repressed domain. (c and d) In Spo11- pachytene spermatocytes, as a result of asynapsis/nonhomologous synapsis, the two autosomal Atr loci are nearly always well separated. In the cell shown, there is a single Atr RNA FISH signal (arrows); DNA FISH shows that the second non-transcribing Atr locus lies within the pseudo–sex body domain (white outline). (e and f) A pachytene spermatocyte showing transcription of the Y chromosomal gene Zfy2 when it is not located in the pseudo–sex body. (g and h) A nontranscribing Zfy2 locus lying within the pseudo–sex body. (i–n) RNA/DNA FISH for the Ddx3x/Usp9x X chromosomal BAC showing two transcribing pachytene spermatocytes with the locus outside the pseudo–sex body and one nontranscribing pachytene spermatocyte in which the locus lies within the pseudo–sex body. (a–n) White outlines indicate the extent of the γH2AX domain before DNA FISH, and arrows point to the FISH signals (either RNA FISH or DNA FISH). Bar, 5 μm.
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Related In: Results  -  Collection

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fig2: The pseudo–sex body domain of Spo11- pachytene spermatocytes is transcriptionally silenced. (a and b) Cot1 RNA FISH staining detects nascent transcripts within the nucleus; the pseudo–sex body marked by γH2AX is a transcriptionally repressed domain. (c and d) In Spo11- pachytene spermatocytes, as a result of asynapsis/nonhomologous synapsis, the two autosomal Atr loci are nearly always well separated. In the cell shown, there is a single Atr RNA FISH signal (arrows); DNA FISH shows that the second non-transcribing Atr locus lies within the pseudo–sex body domain (white outline). (e and f) A pachytene spermatocyte showing transcription of the Y chromosomal gene Zfy2 when it is not located in the pseudo–sex body. (g and h) A nontranscribing Zfy2 locus lying within the pseudo–sex body. (i–n) RNA/DNA FISH for the Ddx3x/Usp9x X chromosomal BAC showing two transcribing pachytene spermatocytes with the locus outside the pseudo–sex body and one nontranscribing pachytene spermatocyte in which the locus lies within the pseudo–sex body. (a–n) White outlines indicate the extent of the γH2AX domain before DNA FISH, and arrows point to the FISH signals (either RNA FISH or DNA FISH). Bar, 5 μm.
Mentions: We then asked whether the γH2AX-positive pseudo–sex body domain was transcriptionally repressed by use of a Cot1 probe to detect nascent transcripts (Turner et al., 2005) and then immunostained for γH2AX to locate the pseudo–sex body domain. This showed that the γH2AX domain was indeed transcriptionally repressed in cells that had a clear pseudo–sex body (Fig. 2, a and b). We next combined γH2AX immunostaining with RNA FISH and DNA FISH for Atr, which is autosomal and in wild-type males is robustly expressed throughout pachytene (Fig. S2, a–e; available at http://www.jcb.org/cgi/content/full/jcb.200710195/DC1). It was predicted that any pachytene cells with an Atr locus that failed to transcribe would have the Atr locus within the transcriptionally repressed γH2AX domain. 7/42 (17%) pachytene cells had one transcribed and one nontranscribed Atr locus, and, in each case, the nontranscribing locus was within the γH2AX-positive region (Fig. 2, c and d). We next performed RNA FISH analysis for the Y-located gene Zfy2 that in normal males is weakly expressed in leptotene and zygotene spermatocytes when chromatin condensation is associated with global transcriptional repression and is then shut down by MSCI/MSUC throughout pachytene (Fig. S2, k–o). This revealed that 70/74 (95%) Spo11−/− pachytene cells were inappropriately expressing Zfy2 (Fig. 2, e and f); in 69 of these cells, the Zfy2 locus was located away from the γH2AX-positive domain, and one weakly expressed locus was at the edge of the domain. In the four nonexpressing cells, the Zfy2 locus was located within the γH2AX-positive domain (Fig. 2, g and h). Finally, we performed RNA FISH analysis using an X-chromosomal bacterial artificial chromosome (BAC) probe encompassing Ddx3x and 28 kb of Usp9x. We have found that this BAC gives no detectable RNA FISH signal in normal spermatocytes (Figs. S2, p–t; and S4, a and b); however, in Brca1Δ11/Δ11 males (which have extensive MSCI failure; Turner et al., 2004), a Ddx3x/Usp9x signal becomes progressively more evident during pachytene (Figs. S3, g–i; and S4, c and d). This provides a robust pachytene-specific marker of MSCI failure but will underestimate the proportion of pachytene cells with MSCI failure because transcription does not commence immediately upon entry of cells into pachytene. In Spo11−/− males, 36/43 (84%) pachytene cells were found to be inappropriately expressing Ddx3x/Usp9x because the locus was not within the γH2AX-positive domain (Fig. 2, i–l). In four of the seven nonexpressing cells, the Ddx3x/Usp9x locus lay within the γH2AX domain (Fig. 2, m and n).

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