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Conditional inactivation of the DNA damage response gene Hus1 in mouse testis reveals separable roles for components of the RAD9-RAD1-HUS1 complex in meiotic chromosome maintenance.

Lyndaker AM, Lim PX, Mleczko JM, Diggins CE, Holloway JK, Holmes RJ, Kan R, Schlafer DH, Freire R, Cohen PE, Weiss RS - PLoS Genet. (2013)

Bottom Line: Hus1 loss in testicular germ cells resulted in meiotic defects, germ cell depletion, and severely compromised fertility.Interestingly, RAD1 had a broader distribution that only partially overlapped with RAD9, and localization of both RAD1 and the ATR activator TOPBP1 to the XY body and to unsynapsed autosomes was intact in Hus1 conditional knockouts.We conclude that mammalian HUS1 acts as a component of the canonical 9-1-1 complex during meiotic prophase I to promote DSB repair and further propose that RAD1 and TOPBP1 respond to unsynapsed chromatin through an alternative mechanism that does not require RAD9 or HUS1.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Sciences, Cornell University, Ithaca, New York, USA.

ABSTRACT
The RAD9-RAD1-HUS1 (9-1-1) complex is a heterotrimeric PCNA-like clamp that responds to DNA damage in somatic cells by promoting DNA repair as well as ATR-dependent DNA damage checkpoint signaling. In yeast, worms, and flies, the 9-1-1 complex is also required for meiotic checkpoint function and efficient completion of meiotic recombination; however, since Rad9, Rad1, and Hus1 are essential genes in mammals, little is known about their functions in mammalian germ cells. In this study, we assessed the meiotic functions of 9-1-1 by analyzing mice with germ cell-specific deletion of Hus1 as well as by examining the localization of RAD9 and RAD1 on meiotic chromosomes during prophase I. Hus1 loss in testicular germ cells resulted in meiotic defects, germ cell depletion, and severely compromised fertility. Hus1-deficient primary spermatocytes exhibited persistent autosomal γH2AX and RAD51 staining indicative of unrepaired meiotic DSBs, synapsis defects, an extended XY body domain often encompassing partial or whole autosomes, and an increase in structural chromosome abnormalities such as end-to-end X chromosome-autosome fusions and ruptures in the synaptonemal complex. Most of these aberrations persisted in diplotene-stage spermatocytes. Consistent with a role for the 9-1-1 complex in meiotic DSB repair, RAD9 localized to punctate, RAD51-containing foci on meiotic chromosomes in a Hus1-dependent manner. Interestingly, RAD1 had a broader distribution that only partially overlapped with RAD9, and localization of both RAD1 and the ATR activator TOPBP1 to the XY body and to unsynapsed autosomes was intact in Hus1 conditional knockouts. We conclude that mammalian HUS1 acts as a component of the canonical 9-1-1 complex during meiotic prophase I to promote DSB repair and further propose that RAD1 and TOPBP1 respond to unsynapsed chromatin through an alternative mechanism that does not require RAD9 or HUS1.

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RAD1 and RAD9 localization to meiotic chromosomes is differentially affected by Hus1 loss.Meiotic chromosome spreads from Stra8-Cre Hus1 CKO mice were stained for SYCP3 and RAD1 (A, C, D) or RAD9 (B, E). A,C,D. RAD1 continued to localize to sex chromosomes and aberrant meiotic chromosomes in the absence of Hus1. Most notably, RAD1 localization to asynapsed autosomes and chromosomes with the sex body domain persisted in cells from Hus1 CKO mice. By contrast, RAD9 localization to both normal and aberrant chromosome structures was abolished following Hus1 loss (B,E).
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pgen-1003320-g009: RAD1 and RAD9 localization to meiotic chromosomes is differentially affected by Hus1 loss.Meiotic chromosome spreads from Stra8-Cre Hus1 CKO mice were stained for SYCP3 and RAD1 (A, C, D) or RAD9 (B, E). A,C,D. RAD1 continued to localize to sex chromosomes and aberrant meiotic chromosomes in the absence of Hus1. Most notably, RAD1 localization to asynapsed autosomes and chromosomes with the sex body domain persisted in cells from Hus1 CKO mice. By contrast, RAD9 localization to both normal and aberrant chromosome structures was abolished following Hus1 loss (B,E).

Mentions: Prompted by the differences in RAD1 and RAD9 localization described above, we next assessed localization of these 9-1-1 subunits in Hus1 CKO spermatocytes. Since the chromatin association and nuclear localization of the 9-1-1 complex in somatic cells requires all three subunits [48], we expected RAD9 and RAD1 to be depleted from chromosomes in the absence of HUS1. However, the more extensive RAD1 staining observed in normal and aberrant spermatocytes suggested a possible separation of functions for the individual subunits. Remarkably, bright RAD1 staining of XY cores as well as punctate staining of autosomes persisted upon Hus1 deletion (Figure 9A). By contrast, RAD9 foci were undetectable in nuclei from Hus1 CKO mice (Figure 9B). Whereas RAD9 staining was readily detected in all control nuclei, approximately 99% of Stra8-Cre Hus1 CKO pachytene nuclei lacked RAD9 foci. The continued presence of RAD1 on meiotic chromosomes following Hus1 loss despite the significant reduction in total RAD1 levels (Figure S3) may indicate that a fraction of RAD1 protein participates in chromatin-associated complexes that remain stable independently of HUS1, while the remaining pool of RAD1 is unstable in the absence of HUS1.


Conditional inactivation of the DNA damage response gene Hus1 in mouse testis reveals separable roles for components of the RAD9-RAD1-HUS1 complex in meiotic chromosome maintenance.

Lyndaker AM, Lim PX, Mleczko JM, Diggins CE, Holloway JK, Holmes RJ, Kan R, Schlafer DH, Freire R, Cohen PE, Weiss RS - PLoS Genet. (2013)

RAD1 and RAD9 localization to meiotic chromosomes is differentially affected by Hus1 loss.Meiotic chromosome spreads from Stra8-Cre Hus1 CKO mice were stained for SYCP3 and RAD1 (A, C, D) or RAD9 (B, E). A,C,D. RAD1 continued to localize to sex chromosomes and aberrant meiotic chromosomes in the absence of Hus1. Most notably, RAD1 localization to asynapsed autosomes and chromosomes with the sex body domain persisted in cells from Hus1 CKO mice. By contrast, RAD9 localization to both normal and aberrant chromosome structures was abolished following Hus1 loss (B,E).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3585019&req=5

pgen-1003320-g009: RAD1 and RAD9 localization to meiotic chromosomes is differentially affected by Hus1 loss.Meiotic chromosome spreads from Stra8-Cre Hus1 CKO mice were stained for SYCP3 and RAD1 (A, C, D) or RAD9 (B, E). A,C,D. RAD1 continued to localize to sex chromosomes and aberrant meiotic chromosomes in the absence of Hus1. Most notably, RAD1 localization to asynapsed autosomes and chromosomes with the sex body domain persisted in cells from Hus1 CKO mice. By contrast, RAD9 localization to both normal and aberrant chromosome structures was abolished following Hus1 loss (B,E).
Mentions: Prompted by the differences in RAD1 and RAD9 localization described above, we next assessed localization of these 9-1-1 subunits in Hus1 CKO spermatocytes. Since the chromatin association and nuclear localization of the 9-1-1 complex in somatic cells requires all three subunits [48], we expected RAD9 and RAD1 to be depleted from chromosomes in the absence of HUS1. However, the more extensive RAD1 staining observed in normal and aberrant spermatocytes suggested a possible separation of functions for the individual subunits. Remarkably, bright RAD1 staining of XY cores as well as punctate staining of autosomes persisted upon Hus1 deletion (Figure 9A). By contrast, RAD9 foci were undetectable in nuclei from Hus1 CKO mice (Figure 9B). Whereas RAD9 staining was readily detected in all control nuclei, approximately 99% of Stra8-Cre Hus1 CKO pachytene nuclei lacked RAD9 foci. The continued presence of RAD1 on meiotic chromosomes following Hus1 loss despite the significant reduction in total RAD1 levels (Figure S3) may indicate that a fraction of RAD1 protein participates in chromatin-associated complexes that remain stable independently of HUS1, while the remaining pool of RAD1 is unstable in the absence of HUS1.

Bottom Line: Hus1 loss in testicular germ cells resulted in meiotic defects, germ cell depletion, and severely compromised fertility.Interestingly, RAD1 had a broader distribution that only partially overlapped with RAD9, and localization of both RAD1 and the ATR activator TOPBP1 to the XY body and to unsynapsed autosomes was intact in Hus1 conditional knockouts.We conclude that mammalian HUS1 acts as a component of the canonical 9-1-1 complex during meiotic prophase I to promote DSB repair and further propose that RAD1 and TOPBP1 respond to unsynapsed chromatin through an alternative mechanism that does not require RAD9 or HUS1.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Sciences, Cornell University, Ithaca, New York, USA.

ABSTRACT
The RAD9-RAD1-HUS1 (9-1-1) complex is a heterotrimeric PCNA-like clamp that responds to DNA damage in somatic cells by promoting DNA repair as well as ATR-dependent DNA damage checkpoint signaling. In yeast, worms, and flies, the 9-1-1 complex is also required for meiotic checkpoint function and efficient completion of meiotic recombination; however, since Rad9, Rad1, and Hus1 are essential genes in mammals, little is known about their functions in mammalian germ cells. In this study, we assessed the meiotic functions of 9-1-1 by analyzing mice with germ cell-specific deletion of Hus1 as well as by examining the localization of RAD9 and RAD1 on meiotic chromosomes during prophase I. Hus1 loss in testicular germ cells resulted in meiotic defects, germ cell depletion, and severely compromised fertility. Hus1-deficient primary spermatocytes exhibited persistent autosomal γH2AX and RAD51 staining indicative of unrepaired meiotic DSBs, synapsis defects, an extended XY body domain often encompassing partial or whole autosomes, and an increase in structural chromosome abnormalities such as end-to-end X chromosome-autosome fusions and ruptures in the synaptonemal complex. Most of these aberrations persisted in diplotene-stage spermatocytes. Consistent with a role for the 9-1-1 complex in meiotic DSB repair, RAD9 localized to punctate, RAD51-containing foci on meiotic chromosomes in a Hus1-dependent manner. Interestingly, RAD1 had a broader distribution that only partially overlapped with RAD9, and localization of both RAD1 and the ATR activator TOPBP1 to the XY body and to unsynapsed autosomes was intact in Hus1 conditional knockouts. We conclude that mammalian HUS1 acts as a component of the canonical 9-1-1 complex during meiotic prophase I to promote DSB repair and further propose that RAD1 and TOPBP1 respond to unsynapsed chromatin through an alternative mechanism that does not require RAD9 or HUS1.

Show MeSH
Related in: MedlinePlus