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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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Enrichment of DNA repair markers in the pseudo XY bodies of Spo11+/+ oocytes.(A) Oocyte nuclei from Spo11+/+ E17.5 embryos were immunostained with anti-SYCP3 (red), anti-γH2AX (blue) and anti-RAD51 (green, upper panel) or anti-DMC1 (green, middle panel) or RPA (green, lower panel). Foci of each marker listed above are indicated with arrowheads. Numbers in the bottom left corner of every picture represent the percentage of nuclei of the respective type in the analyzed cell population. Quantification of the four fractions (defined in Figure 3A) was performed in 271, 54, and 53 oocyte nuclei, for RAD51, DMC1, and RPA protein foci, respectively. (B) The number of RAD51, DMC1, and RPA foci was counted in pachytene oocyte nuclei showing both a pseudo XY body and foci (red circle). The average total number of foci of each protein per nucleus is reported in the first column of the table. The second and the third column show the percentage of foci located within a pseudo XY body-like domain and the percentage of pseudo XY body-like domains which contained at least one focus.
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pgen-1003538-g006: Enrichment of DNA repair markers in the pseudo XY bodies of Spo11+/+ oocytes.(A) Oocyte nuclei from Spo11+/+ E17.5 embryos were immunostained with anti-SYCP3 (red), anti-γH2AX (blue) and anti-RAD51 (green, upper panel) or anti-DMC1 (green, middle panel) or RPA (green, lower panel). Foci of each marker listed above are indicated with arrowheads. Numbers in the bottom left corner of every picture represent the percentage of nuclei of the respective type in the analyzed cell population. Quantification of the four fractions (defined in Figure 3A) was performed in 271, 54, and 53 oocyte nuclei, for RAD51, DMC1, and RPA protein foci, respectively. (B) The number of RAD51, DMC1, and RPA foci was counted in pachytene oocyte nuclei showing both a pseudo XY body and foci (red circle). The average total number of foci of each protein per nucleus is reported in the first column of the table. The second and the third column show the percentage of foci located within a pseudo XY body-like domain and the percentage of pseudo XY body-like domains which contained at least one focus.

Mentions: To analyse this further, we studied the localisation of other proteins involved in homologous recombination (DMC1 and RPA) in relation to the formation of a γH2AX domain. Again we divided the oocyte population in four subgroups, based on the detection of γH2AX and the three DNA repair markers. As expected, the majority of pachytene oocytes showed complete synapsis of all chromosomes and no clear γH2AX-positive domain. Around 20–30% of nuclei showed pseudo XY bodies, as defined by the presence of one or a few distinct γH2AX-positive domains (Figure 6A). Approximately half of the pachytene nuclei lacked both γH2AX domains and RAD51 or DMC1 foci, whereas no nuclei were found without RPA foci (Figure 6A, B). We did not observe any pseudo XY body in nuclei without RAD51 foci, but 13% of the nuclei contained a γH2AX domain but no DMC1 foci (Figure 6A). RPA is known to mark DSB repair spots after RAD51-mediated strand invasion and during homologous recombination, to protect the ssDNA regions generated during this process [55]. This explains the fact that RPA foci are always present in E17.5 oocyte nuclei which are at a mid-meiotic stage and have not yet completed the homologous recombination process at all DSB repair sites. Also, since RPA is engaged in completing recombination at synapsed autosomal sites, a relatively small fraction of the RPA foci colocalizes with pseudo XY bodies. In contrast, most DMC1 and RAD51 foci localize to γH2AX domains, similar to what was found for Spo11YF/YF oocyte nuclei (Figure 6B), although DMC1 foci are found more frequently and in higher numbers in pseudo XY bodies in Spo11+/+ compared to Spo11YF/YF oocytes. DMC1 foci colocalized with RAD51 foci when both were present in the pseudo XY body (Figure S6C).


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)

Enrichment of DNA repair markers in the pseudo XY bodies of Spo11+/+ oocytes.(A) Oocyte nuclei from Spo11+/+ E17.5 embryos were immunostained with anti-SYCP3 (red), anti-γH2AX (blue) and anti-RAD51 (green, upper panel) or anti-DMC1 (green, middle panel) or RPA (green, lower panel). Foci of each marker listed above are indicated with arrowheads. Numbers in the bottom left corner of every picture represent the percentage of nuclei of the respective type in the analyzed cell population. Quantification of the four fractions (defined in Figure 3A) was performed in 271, 54, and 53 oocyte nuclei, for RAD51, DMC1, and RPA protein foci, respectively. (B) The number of RAD51, DMC1, and RPA foci was counted in pachytene oocyte nuclei showing both a pseudo XY body and foci (red circle). The average total number of foci of each protein per nucleus is reported in the first column of the table. The second and the third column show the percentage of foci located within a pseudo XY body-like domain and the percentage of pseudo XY body-like domains which contained at least one focus.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1003538-g006: Enrichment of DNA repair markers in the pseudo XY bodies of Spo11+/+ oocytes.(A) Oocyte nuclei from Spo11+/+ E17.5 embryos were immunostained with anti-SYCP3 (red), anti-γH2AX (blue) and anti-RAD51 (green, upper panel) or anti-DMC1 (green, middle panel) or RPA (green, lower panel). Foci of each marker listed above are indicated with arrowheads. Numbers in the bottom left corner of every picture represent the percentage of nuclei of the respective type in the analyzed cell population. Quantification of the four fractions (defined in Figure 3A) was performed in 271, 54, and 53 oocyte nuclei, for RAD51, DMC1, and RPA protein foci, respectively. (B) The number of RAD51, DMC1, and RPA foci was counted in pachytene oocyte nuclei showing both a pseudo XY body and foci (red circle). The average total number of foci of each protein per nucleus is reported in the first column of the table. The second and the third column show the percentage of foci located within a pseudo XY body-like domain and the percentage of pseudo XY body-like domains which contained at least one focus.
Mentions: To analyse this further, we studied the localisation of other proteins involved in homologous recombination (DMC1 and RPA) in relation to the formation of a γH2AX domain. Again we divided the oocyte population in four subgroups, based on the detection of γH2AX and the three DNA repair markers. As expected, the majority of pachytene oocytes showed complete synapsis of all chromosomes and no clear γH2AX-positive domain. Around 20–30% of nuclei showed pseudo XY bodies, as defined by the presence of one or a few distinct γH2AX-positive domains (Figure 6A). Approximately half of the pachytene nuclei lacked both γH2AX domains and RAD51 or DMC1 foci, whereas no nuclei were found without RPA foci (Figure 6A, B). We did not observe any pseudo XY body in nuclei without RAD51 foci, but 13% of the nuclei contained a γH2AX domain but no DMC1 foci (Figure 6A). RPA is known to mark DSB repair spots after RAD51-mediated strand invasion and during homologous recombination, to protect the ssDNA regions generated during this process [55]. This explains the fact that RPA foci are always present in E17.5 oocyte nuclei which are at a mid-meiotic stage and have not yet completed the homologous recombination process at all DSB repair sites. Also, since RPA is engaged in completing recombination at synapsed autosomal sites, a relatively small fraction of the RPA foci colocalizes with pseudo XY bodies. In contrast, most DMC1 and RAD51 foci localize to γH2AX domains, similar to what was found for Spo11YF/YF oocyte nuclei (Figure 6B), although DMC1 foci are found more frequently and in higher numbers in pseudo XY bodies in Spo11+/+ compared to Spo11YF/YF oocytes. DMC1 foci colocalized with RAD51 foci when both were present in the pseudo XY body (Figure S6C).

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