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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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Transcriptional silencing of the pseudo XY body in spermatocytes and oocytes.(A–B) Double immunostaining with anti-γH2AX and anti-RNA polymerase II of spermatocyte (A) and oocyte (B) nuclei from Spo11−/− (A, upper panel), Spo11YF/YF (A–B, lower panels), and Spo11+/+ (B, upper panel) animals. Nuclear domains enriched in γH2AX are marked by a dashed circle. (C) Scatter plots of the relative amount of RNA polII in a γH2AX domain normalized to the RNA polII level in a non-heterochromatic area of the same nucleus. Every dot represents a nucleus. RNA polII levels are compared between γH2AX domains (pseudo XY body) of Spo11+/+ and Spo11YF/YF E17.5 oocytes, and the proper sex body in Spo11+/+ mid-pachytene spermatocytes. Grey lines indicate the average. No significant difference between the wild type pachytene spermatocyte nuclei and either E17.5 oocyte nuclei group was observed (Mann-Whitney, confidence interval p<0.01).
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pgen-1003538-g002: Transcriptional silencing of the pseudo XY body in spermatocytes and oocytes.(A–B) Double immunostaining with anti-γH2AX and anti-RNA polymerase II of spermatocyte (A) and oocyte (B) nuclei from Spo11−/− (A, upper panel), Spo11YF/YF (A–B, lower panels), and Spo11+/+ (B, upper panel) animals. Nuclear domains enriched in γH2AX are marked by a dashed circle. (C) Scatter plots of the relative amount of RNA polII in a γH2AX domain normalized to the RNA polII level in a non-heterochromatic area of the same nucleus. Every dot represents a nucleus. RNA polII levels are compared between γH2AX domains (pseudo XY body) of Spo11+/+ and Spo11YF/YF E17.5 oocytes, and the proper sex body in Spo11+/+ mid-pachytene spermatocytes. Grey lines indicate the average. No significant difference between the wild type pachytene spermatocyte nuclei and either E17.5 oocyte nuclei group was observed (Mann-Whitney, confidence interval p<0.01).

Mentions: The transcriptional silencing in the XY body can be immunocytochemically visualized as an area that is relatively depleted of RNA polymerase II [17]. To verify that the γH2AX domain detected in SPO11-deficient spermatocytes and oocytes is a transcriptionally silenced region, we performed RNA polymerase II (RNA pol II) staining and indeed observed a depletion of this enzyme from the areas enriched for γH2AX in Spo11−/− and Spo11YF/YFspermatocytes and oocytes (Figure 2A and B). To verify the results, we quantified the relative average intensity of RNA pol II staining in the γH2AX domain in oocytes, and compared it to the relative intensity in the true XY body of wild type pachytene spermatocytes (Figure 2C). Despite the fact that we observed variable depletion levels within each of the three analysed categories, the relative average level of RNA pol II in γH2AX domains of wild type (0.77±0.16, n = 30) and Spo11YF/YF (0.76±0.18, n = 30) oocytes is similar, and also comparable to what is observed for the XY body in male wild type spermatocytes (0.69±0.14, n = 30) (Mann-Whitney, confidence interval p<0.001), indicating a significant transcriptional silencing.


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)

Transcriptional silencing of the pseudo XY body in spermatocytes and oocytes.(A–B) Double immunostaining with anti-γH2AX and anti-RNA polymerase II of spermatocyte (A) and oocyte (B) nuclei from Spo11−/− (A, upper panel), Spo11YF/YF (A–B, lower panels), and Spo11+/+ (B, upper panel) animals. Nuclear domains enriched in γH2AX are marked by a dashed circle. (C) Scatter plots of the relative amount of RNA polII in a γH2AX domain normalized to the RNA polII level in a non-heterochromatic area of the same nucleus. Every dot represents a nucleus. RNA polII levels are compared between γH2AX domains (pseudo XY body) of Spo11+/+ and Spo11YF/YF E17.5 oocytes, and the proper sex body in Spo11+/+ mid-pachytene spermatocytes. Grey lines indicate the average. No significant difference between the wild type pachytene spermatocyte nuclei and either E17.5 oocyte nuclei group was observed (Mann-Whitney, confidence interval p<0.01).
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1003538-g002: Transcriptional silencing of the pseudo XY body in spermatocytes and oocytes.(A–B) Double immunostaining with anti-γH2AX and anti-RNA polymerase II of spermatocyte (A) and oocyte (B) nuclei from Spo11−/− (A, upper panel), Spo11YF/YF (A–B, lower panels), and Spo11+/+ (B, upper panel) animals. Nuclear domains enriched in γH2AX are marked by a dashed circle. (C) Scatter plots of the relative amount of RNA polII in a γH2AX domain normalized to the RNA polII level in a non-heterochromatic area of the same nucleus. Every dot represents a nucleus. RNA polII levels are compared between γH2AX domains (pseudo XY body) of Spo11+/+ and Spo11YF/YF E17.5 oocytes, and the proper sex body in Spo11+/+ mid-pachytene spermatocytes. Grey lines indicate the average. No significant difference between the wild type pachytene spermatocyte nuclei and either E17.5 oocyte nuclei group was observed (Mann-Whitney, confidence interval p<0.01).
Mentions: The transcriptional silencing in the XY body can be immunocytochemically visualized as an area that is relatively depleted of RNA polymerase II [17]. To verify that the γH2AX domain detected in SPO11-deficient spermatocytes and oocytes is a transcriptionally silenced region, we performed RNA polymerase II (RNA pol II) staining and indeed observed a depletion of this enzyme from the areas enriched for γH2AX in Spo11−/− and Spo11YF/YFspermatocytes and oocytes (Figure 2A and B). To verify the results, we quantified the relative average intensity of RNA pol II staining in the γH2AX domain in oocytes, and compared it to the relative intensity in the true XY body of wild type pachytene spermatocytes (Figure 2C). Despite the fact that we observed variable depletion levels within each of the three analysed categories, the relative average level of RNA pol II in γH2AX domains of wild type (0.77±0.16, n = 30) and Spo11YF/YF (0.76±0.18, n = 30) oocytes is similar, and also comparable to what is observed for the XY body in male wild type spermatocytes (0.69±0.14, n = 30) (Mann-Whitney, confidence interval p<0.001), indicating a significant transcriptional silencing.

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