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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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Conditional Hus1 inactivation in the mouse testis results in reduced testis size and sperm count.A. Western blot analysis of HUS1 protein in adult (12-week old) control and Hus1 CKO testes. B, E. Photographs of testes from 12-week old Hus1 CKO and control mice from Stra8-Cre and Spo11-Cre crosses, respectively. In these representative images, the Stra8-Cre control is Cre-positive Hus1+/flox, whereas the Spo11-Cre control is Cre-negative Hus1flox/Δ1. C, F. Testis weights of 17-day and 12-week old adult Hus1 CKO and control mice from both Stra8-Cre and Spo11-Cre crosses, shown as the mean testis weight relative to body weight ± SEM. D, G. Epididymal sperm counts from Stra8-Cre and Spo11-Cre Hus1 CKO 12-week old males, shown as the mean ± SEM. Statistically significant differences between Hus1 CKO and the respective control as determined by Student's t-test are indicated (* at p<0.001; ‡ at p<0.01).
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pgen-1003320-g001: Conditional Hus1 inactivation in the mouse testis results in reduced testis size and sperm count.A. Western blot analysis of HUS1 protein in adult (12-week old) control and Hus1 CKO testes. B, E. Photographs of testes from 12-week old Hus1 CKO and control mice from Stra8-Cre and Spo11-Cre crosses, respectively. In these representative images, the Stra8-Cre control is Cre-positive Hus1+/flox, whereas the Spo11-Cre control is Cre-negative Hus1flox/Δ1. C, F. Testis weights of 17-day and 12-week old adult Hus1 CKO and control mice from both Stra8-Cre and Spo11-Cre crosses, shown as the mean testis weight relative to body weight ± SEM. D, G. Epididymal sperm counts from Stra8-Cre and Spo11-Cre Hus1 CKO 12-week old males, shown as the mean ± SEM. Statistically significant differences between Hus1 CKO and the respective control as determined by Student's t-test are indicated (* at p<0.001; ‡ at p<0.01).

Mentions: Several lines of evidence from non-vertebrate organisms suggest that the mammalian 9-1-1 complex, the components of which are highly expressed in mouse and human testis, is likely to be critical for normal germ cell development and completion of meiosis. In order to assess the meiotic functions of the 9-1-1 complex, we generated mice in which the Hus1 gene was conditionally inactivated specifically in testicular germ cells. We combined a conditional floxed Hus1 allele (Figure S1A; [46]) with two Cre-expressing lines, Stra8-Cre and Spo11-Cre. Stra8-Cre mice begin to express the CRE recombinase in spermatogonia [47], which undergo several rounds of mitotic division prior to meiosis, whereas CRE expression in Spo11-Cre mice begins in spermatocytes that have initiated meiosis (Figures S1 and S2; Text S1). We focused our analysis on mice made with Stra8-Cre and used the Spo11-Cre line to confirm our findings and to assess whether defects we observed originated during pre-meiotic or meiotic processes. Hus1 deletion in the testis was confirmed by Southern blot detection of Hus1Δ2,3, the allele produced by CRE-mediated recombination (Figures S1C, S1D and S2D). As shown in Figure 1A and Figure S3B, Western blotting of whole testis lysates further confirmed that Hus1 gene deletion using Stra8-Cre resulted in a drastic reduction in HUS1 protein level in the testis. Despite high genomic Hus1 deletion efficiency, the reduction in HUS1 protein levels was more subtle in Spo11-Cre Hus1 conditional knockout (CKO) mice, consistent with expectation that CRE expression from the Spo11 promoter would result in HUS1 loss in a more restricted subset of cells and with delayed kinetics relative to that in mice with Stra8-Cre (Figure S3C). Hus1 loss also led to a significant reduction in total RAD9 and RAD1 protein levels in testes from Stra8-Cre Hus1 CKO animals (Figure S3A, S3B), indicating that the entire 9-1-1 complex was destabilized 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)

Conditional Hus1 inactivation in the mouse testis results in reduced testis size and sperm count.A. Western blot analysis of HUS1 protein in adult (12-week old) control and Hus1 CKO testes. B, E. Photographs of testes from 12-week old Hus1 CKO and control mice from Stra8-Cre and Spo11-Cre crosses, respectively. In these representative images, the Stra8-Cre control is Cre-positive Hus1+/flox, whereas the Spo11-Cre control is Cre-negative Hus1flox/Δ1. C, F. Testis weights of 17-day and 12-week old adult Hus1 CKO and control mice from both Stra8-Cre and Spo11-Cre crosses, shown as the mean testis weight relative to body weight ± SEM. D, G. Epididymal sperm counts from Stra8-Cre and Spo11-Cre Hus1 CKO 12-week old males, shown as the mean ± SEM. Statistically significant differences between Hus1 CKO and the respective control as determined by Student's t-test are indicated (* at p<0.001; ‡ at p<0.01).
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3585019&req=5

pgen-1003320-g001: Conditional Hus1 inactivation in the mouse testis results in reduced testis size and sperm count.A. Western blot analysis of HUS1 protein in adult (12-week old) control and Hus1 CKO testes. B, E. Photographs of testes from 12-week old Hus1 CKO and control mice from Stra8-Cre and Spo11-Cre crosses, respectively. In these representative images, the Stra8-Cre control is Cre-positive Hus1+/flox, whereas the Spo11-Cre control is Cre-negative Hus1flox/Δ1. C, F. Testis weights of 17-day and 12-week old adult Hus1 CKO and control mice from both Stra8-Cre and Spo11-Cre crosses, shown as the mean testis weight relative to body weight ± SEM. D, G. Epididymal sperm counts from Stra8-Cre and Spo11-Cre Hus1 CKO 12-week old males, shown as the mean ± SEM. Statistically significant differences between Hus1 CKO and the respective control as determined by Student's t-test are indicated (* at p<0.001; ‡ at p<0.01).
Mentions: Several lines of evidence from non-vertebrate organisms suggest that the mammalian 9-1-1 complex, the components of which are highly expressed in mouse and human testis, is likely to be critical for normal germ cell development and completion of meiosis. In order to assess the meiotic functions of the 9-1-1 complex, we generated mice in which the Hus1 gene was conditionally inactivated specifically in testicular germ cells. We combined a conditional floxed Hus1 allele (Figure S1A; [46]) with two Cre-expressing lines, Stra8-Cre and Spo11-Cre. Stra8-Cre mice begin to express the CRE recombinase in spermatogonia [47], which undergo several rounds of mitotic division prior to meiosis, whereas CRE expression in Spo11-Cre mice begins in spermatocytes that have initiated meiosis (Figures S1 and S2; Text S1). We focused our analysis on mice made with Stra8-Cre and used the Spo11-Cre line to confirm our findings and to assess whether defects we observed originated during pre-meiotic or meiotic processes. Hus1 deletion in the testis was confirmed by Southern blot detection of Hus1Δ2,3, the allele produced by CRE-mediated recombination (Figures S1C, S1D and S2D). As shown in Figure 1A and Figure S3B, Western blotting of whole testis lysates further confirmed that Hus1 gene deletion using Stra8-Cre resulted in a drastic reduction in HUS1 protein level in the testis. Despite high genomic Hus1 deletion efficiency, the reduction in HUS1 protein levels was more subtle in Spo11-Cre Hus1 conditional knockout (CKO) mice, consistent with expectation that CRE expression from the Spo11 promoter would result in HUS1 loss in a more restricted subset of cells and with delayed kinetics relative to that in mice with Stra8-Cre (Figure S3C). Hus1 loss also led to a significant reduction in total RAD9 and RAD1 protein levels in testes from Stra8-Cre Hus1 CKO animals (Figure S3A, S3B), indicating that the entire 9-1-1 complex was destabilized 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