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SPO11-independent DNA repair foci and their role in meiotic silencing.

Carofiglio F, Inagaki A, de Vries S, Wassenaar E, Schoenmakers S, Vermeulen C, van Cappellen WA, Sleddens-Linkels E, Grootegoed JA, Te Riele HP, de Massy B, Baarends WM - PLoS Genet. (2013)

Bottom Line: Interestingly, we observed foci of proteins involved in the processing of DNA damage, such as RAD51, DMC1, and RPA, both in Spo11(YF/YF) and Spo11 knockout meiocytes.These foci preferentially localized to the areas that undergo MSUC and form the so-called pseudo XY body.In wild type pachytene oocytes we observed meiotic silencing in two types of pseudo XY bodies, one type containing DMC1 and RAD51 foci on unsynapsed axes, and another type containing only RAD51 foci, mainly on synapsed axes.

View Article: PubMed Central - PubMed

Affiliation: Department of Reproduction and Development, Erasmus MC - University Medical Center, Rotterdam, The Netherlands.

ABSTRACT
In mammalian meiotic prophase, the initial steps in repair of SPO11-induced DNA double-strand breaks (DSBs) are required to obtain stable homologous chromosome pairing and synapsis. The X and Y chromosomes pair and synapse only in the short pseudo-autosomal regions. The rest of the chromatin of the sex chromosomes remain unsynapsed, contains persistent meiotic DSBs, and the whole so-called XY body undergoes meiotic sex chromosome inactivation (MSCI). A more general mechanism, named meiotic silencing of unsynapsed chromatin (MSUC), is activated when autosomes fail to synapse. In the absence of SPO11, many chromosomal regions remain unsynapsed, but MSUC takes place only on part of the unsynapsed chromatin. We asked if spontaneous DSBs occur in meiocytes that lack a functional SPO11 protein, and if these might be involved in targeting the MSUC response to part of the unsynapsed chromatin. We generated mice carrying a point mutation that disrupts the predicted catalytic site of SPO11 (Spo11(YF/YF)), and blocks its DSB-inducing activity. Interestingly, we observed foci of proteins involved in the processing of DNA damage, such as RAD51, DMC1, and RPA, both in Spo11(YF/YF) and Spo11 knockout meiocytes. These foci preferentially localized to the areas that undergo MSUC and form the so-called pseudo XY body. In SPO11-deficient oocytes, the number of repair foci increased during oocyte development, indicating the induction of S phase-independent, de novo DNA damage. In wild type pachytene oocytes we observed meiotic silencing in two types of pseudo XY bodies, one type containing DMC1 and RAD51 foci on unsynapsed axes, and another type containing only RAD51 foci, mainly on synapsed axes. Taken together, our results indicate that in addition to asynapsis, persistent SPO11-induced DSBs are important for the initiation of MSCI and MSUC, and that SPO11-independent DNA repair foci contribute to the MSUC response in oocytes.

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Model for the roles of SPO11-dependent and -independent meiotic DSBs in synapsis and meiotic silencing.In spermatocytes and oocytes, SPO11 generates many meiotic DSBs which are repaired via homologous recombination (HR). This repair process requires the use of the homologous chromosome as a repair template and promotes homologous chromosome synapsis. Once the homologs are in close juxtaposition, synapsis proceeds. Subsequently, repair may occur faster, perhaps now allowing the use of both the homologous chromosome and the sister chromatid as a template for repair. In the absence of a repair template, DSBs persist, inhibiting synapsis between non-homologous partners, although some repair via the sister chromatid on chromosomes that are not synapsed is not excluded. Conversely, asynapsis also contributes to the persistence of DSBs when repair via the sister chromatid remains suppressed. The presence of persistent DSBs on unsynapsed axes, may lead to local accumulation of γH2AX and activate a positive feedback mechanism that involves HORMAD activation, followed by recruitment of ATR, which will lead to rapid spreading of a signal along the unsynapsed axes that will then induce accumulation of γH2AX on the chromatin surrounding these axes. This process always occurs on the XY pair in spermatocytes and leads to MSCI. In the absence of SPO11-induced DSBs, SPO11-independent DNA damage nucleates MSUC via the same mechanism. In spermatocytes, SPO11-independent DNA repair foci may represent remnant DSBs that have formed during the premeiotic S phase. In oocytes (both wild type and SPO11-mutant), SPO11-independent DNA repair foci form late, at a time point corresponding to early pachytene. Such de novo induced DNA repair foci, most likely caused by some form of DNA damage, together with unrepaired SPO11-induced DSBs, and frequently in combination with occasional asynapsis, result in γH2AX accumulation and activation of MSUC. Representative images of the (pseudo) XY body in male and female nuclei from wild type (wt) and Spo11YF/YF nuclei are shown. The immunostainings show SYCP3 (red), γH2AX (blue) and RAD51 (green).
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pgen-1003538-g009: Model for the roles of SPO11-dependent and -independent meiotic DSBs in synapsis and meiotic silencing.In spermatocytes and oocytes, SPO11 generates many meiotic DSBs which are repaired via homologous recombination (HR). This repair process requires the use of the homologous chromosome as a repair template and promotes homologous chromosome synapsis. Once the homologs are in close juxtaposition, synapsis proceeds. Subsequently, repair may occur faster, perhaps now allowing the use of both the homologous chromosome and the sister chromatid as a template for repair. In the absence of a repair template, DSBs persist, inhibiting synapsis between non-homologous partners, although some repair via the sister chromatid on chromosomes that are not synapsed is not excluded. Conversely, asynapsis also contributes to the persistence of DSBs when repair via the sister chromatid remains suppressed. The presence of persistent DSBs on unsynapsed axes, may lead to local accumulation of γH2AX and activate a positive feedback mechanism that involves HORMAD activation, followed by recruitment of ATR, which will lead to rapid spreading of a signal along the unsynapsed axes that will then induce accumulation of γH2AX on the chromatin surrounding these axes. This process always occurs on the XY pair in spermatocytes and leads to MSCI. In the absence of SPO11-induced DSBs, SPO11-independent DNA damage nucleates MSUC via the same mechanism. In spermatocytes, SPO11-independent DNA repair foci may represent remnant DSBs that have formed during the premeiotic S phase. In oocytes (both wild type and SPO11-mutant), SPO11-independent DNA repair foci form late, at a time point corresponding to early pachytene. Such de novo induced DNA repair foci, most likely caused by some form of DNA damage, together with unrepaired SPO11-induced DSBs, and frequently in combination with occasional asynapsis, result in γH2AX accumulation and activation of MSUC. Representative images of the (pseudo) XY body in male and female nuclei from wild type (wt) and Spo11YF/YF nuclei are shown. The immunostainings show SYCP3 (red), γH2AX (blue) and RAD51 (green).

Mentions: The percentage of cells with γH2AX accumulation in a pseudo XY body is highly reduced in Spo11−/− Hormad1−/− or Spo11−/− Hormad2−/− double mutant spermatocytes [22], [23]. This illustrates the important role of HORMAD proteins in the MSUC response. Yet the localization of HORMAD1 to all unsynapsed chromatin in Spo11 knockout spermatocytes [22], [23]), and the presence of some nuclei with a proper MSUC response in Spo11−/− Hormad1−/− spermatocytes indicate that, apart from HORMAD proteins, an additional localizing event is needed for pseudo XY body nucleation. Taken together, these and our observations support the hypothesis that both asynapsis, detected by HORMADs, and persistent SPO11-independent DNA repair foci are involved in the induction of H2AX phosphorylation and the establishment of meiotic silencing in pseudo XY bodies in Spo11YF/YF oocyte nuclei. We would like to propose that MSCI in wild type spermatocytes is then also triggered by both persistent DSBs, in this case SPO11-dependent, and the presence of unsynapsed chromatin (schematically presented in Figure 9).


SPO11-independent DNA repair foci and their role in meiotic silencing.

Carofiglio F, Inagaki A, de Vries S, Wassenaar E, Schoenmakers S, Vermeulen C, van Cappellen WA, Sleddens-Linkels E, Grootegoed JA, Te Riele HP, de Massy B, Baarends WM - PLoS Genet. (2013)

Model for the roles of SPO11-dependent and -independent meiotic DSBs in synapsis and meiotic silencing.In spermatocytes and oocytes, SPO11 generates many meiotic DSBs which are repaired via homologous recombination (HR). This repair process requires the use of the homologous chromosome as a repair template and promotes homologous chromosome synapsis. Once the homologs are in close juxtaposition, synapsis proceeds. Subsequently, repair may occur faster, perhaps now allowing the use of both the homologous chromosome and the sister chromatid as a template for repair. In the absence of a repair template, DSBs persist, inhibiting synapsis between non-homologous partners, although some repair via the sister chromatid on chromosomes that are not synapsed is not excluded. Conversely, asynapsis also contributes to the persistence of DSBs when repair via the sister chromatid remains suppressed. The presence of persistent DSBs on unsynapsed axes, may lead to local accumulation of γH2AX and activate a positive feedback mechanism that involves HORMAD activation, followed by recruitment of ATR, which will lead to rapid spreading of a signal along the unsynapsed axes that will then induce accumulation of γH2AX on the chromatin surrounding these axes. This process always occurs on the XY pair in spermatocytes and leads to MSCI. In the absence of SPO11-induced DSBs, SPO11-independent DNA damage nucleates MSUC via the same mechanism. In spermatocytes, SPO11-independent DNA repair foci may represent remnant DSBs that have formed during the premeiotic S phase. In oocytes (both wild type and SPO11-mutant), SPO11-independent DNA repair foci form late, at a time point corresponding to early pachytene. Such de novo induced DNA repair foci, most likely caused by some form of DNA damage, together with unrepaired SPO11-induced DSBs, and frequently in combination with occasional asynapsis, result in γH2AX accumulation and activation of MSUC. Representative images of the (pseudo) XY body in male and female nuclei from wild type (wt) and Spo11YF/YF nuclei are shown. The immunostainings show SYCP3 (red), γH2AX (blue) and RAD51 (green).
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3675022&req=5

pgen-1003538-g009: Model for the roles of SPO11-dependent and -independent meiotic DSBs in synapsis and meiotic silencing.In spermatocytes and oocytes, SPO11 generates many meiotic DSBs which are repaired via homologous recombination (HR). This repair process requires the use of the homologous chromosome as a repair template and promotes homologous chromosome synapsis. Once the homologs are in close juxtaposition, synapsis proceeds. Subsequently, repair may occur faster, perhaps now allowing the use of both the homologous chromosome and the sister chromatid as a template for repair. In the absence of a repair template, DSBs persist, inhibiting synapsis between non-homologous partners, although some repair via the sister chromatid on chromosomes that are not synapsed is not excluded. Conversely, asynapsis also contributes to the persistence of DSBs when repair via the sister chromatid remains suppressed. The presence of persistent DSBs on unsynapsed axes, may lead to local accumulation of γH2AX and activate a positive feedback mechanism that involves HORMAD activation, followed by recruitment of ATR, which will lead to rapid spreading of a signal along the unsynapsed axes that will then induce accumulation of γH2AX on the chromatin surrounding these axes. This process always occurs on the XY pair in spermatocytes and leads to MSCI. In the absence of SPO11-induced DSBs, SPO11-independent DNA damage nucleates MSUC via the same mechanism. In spermatocytes, SPO11-independent DNA repair foci may represent remnant DSBs that have formed during the premeiotic S phase. In oocytes (both wild type and SPO11-mutant), SPO11-independent DNA repair foci form late, at a time point corresponding to early pachytene. Such de novo induced DNA repair foci, most likely caused by some form of DNA damage, together with unrepaired SPO11-induced DSBs, and frequently in combination with occasional asynapsis, result in γH2AX accumulation and activation of MSUC. Representative images of the (pseudo) XY body in male and female nuclei from wild type (wt) and Spo11YF/YF nuclei are shown. The immunostainings show SYCP3 (red), γH2AX (blue) and RAD51 (green).
Mentions: The percentage of cells with γH2AX accumulation in a pseudo XY body is highly reduced in Spo11−/− Hormad1−/− or Spo11−/− Hormad2−/− double mutant spermatocytes [22], [23]. This illustrates the important role of HORMAD proteins in the MSUC response. Yet the localization of HORMAD1 to all unsynapsed chromatin in Spo11 knockout spermatocytes [22], [23]), and the presence of some nuclei with a proper MSUC response in Spo11−/− Hormad1−/− spermatocytes indicate that, apart from HORMAD proteins, an additional localizing event is needed for pseudo XY body nucleation. Taken together, these and our observations support the hypothesis that both asynapsis, detected by HORMADs, and persistent SPO11-independent DNA repair foci are involved in the induction of H2AX phosphorylation and the establishment of meiotic silencing in pseudo XY bodies in Spo11YF/YF oocyte nuclei. We would like to propose that MSCI in wild type spermatocytes is then also triggered by both persistent DSBs, in this case SPO11-dependent, and the presence of unsynapsed chromatin (schematically presented in Figure 9).

Bottom Line: Interestingly, we observed foci of proteins involved in the processing of DNA damage, such as RAD51, DMC1, and RPA, both in Spo11(YF/YF) and Spo11 knockout meiocytes.These foci preferentially localized to the areas that undergo MSUC and form the so-called pseudo XY body.In wild type pachytene oocytes we observed meiotic silencing in two types of pseudo XY bodies, one type containing DMC1 and RAD51 foci on unsynapsed axes, and another type containing only RAD51 foci, mainly on synapsed axes.

View Article: PubMed Central - PubMed

Affiliation: Department of Reproduction and Development, Erasmus MC - University Medical Center, Rotterdam, The Netherlands.

ABSTRACT
In mammalian meiotic prophase, the initial steps in repair of SPO11-induced DNA double-strand breaks (DSBs) are required to obtain stable homologous chromosome pairing and synapsis. The X and Y chromosomes pair and synapse only in the short pseudo-autosomal regions. The rest of the chromatin of the sex chromosomes remain unsynapsed, contains persistent meiotic DSBs, and the whole so-called XY body undergoes meiotic sex chromosome inactivation (MSCI). A more general mechanism, named meiotic silencing of unsynapsed chromatin (MSUC), is activated when autosomes fail to synapse. In the absence of SPO11, many chromosomal regions remain unsynapsed, but MSUC takes place only on part of the unsynapsed chromatin. We asked if spontaneous DSBs occur in meiocytes that lack a functional SPO11 protein, and if these might be involved in targeting the MSUC response to part of the unsynapsed chromatin. We generated mice carrying a point mutation that disrupts the predicted catalytic site of SPO11 (Spo11(YF/YF)), and blocks its DSB-inducing activity. Interestingly, we observed foci of proteins involved in the processing of DNA damage, such as RAD51, DMC1, and RPA, both in Spo11(YF/YF) and Spo11 knockout meiocytes. These foci preferentially localized to the areas that undergo MSUC and form the so-called pseudo XY body. In SPO11-deficient oocytes, the number of repair foci increased during oocyte development, indicating the induction of S phase-independent, de novo DNA damage. In wild type pachytene oocytes we observed meiotic silencing in two types of pseudo XY bodies, one type containing DMC1 and RAD51 foci on unsynapsed axes, and another type containing only RAD51 foci, mainly on synapsed axes. Taken together, our results indicate that in addition to asynapsis, persistent SPO11-induced DSBs are important for the initiation of MSCI and MSUC, and that SPO11-independent DNA repair foci contribute to the MSUC response in oocytes.

Show MeSH
Related in: MedlinePlus