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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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Hus1 inactivation results in abnormal accumulation of γH2AX on autosomes, an extended sex body domain, inclusion of autosomes within the sex body, and X-autosome fusions.A–F. Immunofluorescence staining for γH2AX and SYCP3. γH2AX staining persisted in a perpendicular pattern on synapsed autosomes in pachytene (B) and diplotene stage (C), and in clouds surrounding unsynapsed chromosomal regions (D). The sex body domain marked by γH2AX was extended in Stra8-Cre Hus1 CKOs (B,F), and often contained whole or partial autosomes (B,F). Some Stra8-Cre Hus1 CKO nuclei exhibited apparent X chromosome-autosome end-to-end fusions (E). G,H. Western blot analysis of CHK1 (G) and CHK2 (H) in total testis extracts from mice of the indicated genotypes. In the CHK2 immunoblot, pre-B cells that were untreated (U) or treated with 5Gy ionizing radiation (IR) are included as controls. β-actin is shown as a loading control. In Hus1 CKO samples, total CHK1 levels were increased on average 2.9-fold over controls, and phosphorylated CHK1 (S345) levels were increased 4.1-fold. CHK2 phosphorylation was not detected in the absence of Hus1.
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pgen-1003320-g003: Hus1 inactivation results in abnormal accumulation of γH2AX on autosomes, an extended sex body domain, inclusion of autosomes within the sex body, and X-autosome fusions.A–F. Immunofluorescence staining for γH2AX and SYCP3. γH2AX staining persisted in a perpendicular pattern on synapsed autosomes in pachytene (B) and diplotene stage (C), and in clouds surrounding unsynapsed chromosomal regions (D). The sex body domain marked by γH2AX was extended in Stra8-Cre Hus1 CKOs (B,F), and often contained whole or partial autosomes (B,F). Some Stra8-Cre Hus1 CKO nuclei exhibited apparent X chromosome-autosome end-to-end fusions (E). G,H. Western blot analysis of CHK1 (G) and CHK2 (H) in total testis extracts from mice of the indicated genotypes. In the CHK2 immunoblot, pre-B cells that were untreated (U) or treated with 5Gy ionizing radiation (IR) are included as controls. β-actin is shown as a loading control. In Hus1 CKO samples, total CHK1 levels were increased on average 2.9-fold over controls, and phosphorylated CHK1 (S345) levels were increased 4.1-fold. CHK2 phosphorylation was not detected in the absence of Hus1.

Mentions: Because the 9-1-1 complex protects chromosomal integrity in somatic cells, we hypothesized that germ cell loss in Hus1 CKOs might be due to DNA damage accumulation. We prepared meiotic chromosome spreads and assessed the localization of γH2AX, the phosphorylated form of the histone variant H2AX, by indirect immunofluorescence. γH2AX marks DSBs as well as unsynapsed chromatin, and is phosphorylated by the ATM and ATR kinases [26], [29]. We found that γH2AX staining during leptotene and zygotene, when meiotic DSBs are generated and processed, was similar in Hus1 CKOs and controls. Furthermore, γH2AX localized normally to the sex body domain in pachytene Hus1 CKO spermatocytes (Figure 3), suggesting that ATR-dependent H2AX phosphorylation is independent of HUS1 as is also true during responses to replication stress [51]. However, in Hus1 mutant spermatocytes, the sex body domain marked by γH2AX was enlarged and extended, and often encompassed parts of or entire autosomes (Figure 3B, 3C, 3F). 56% of mutant pachytene spermatocytes showed some type of sex body abnormality (compared to 13% of control nuclei), with 39% of Stra8-Cre Hus1 CKO nuclei having sex body extensions or protrusions and 33% with autosome inclusion (compared to 4% and 9%, respectively, in control spermatocytes; Table 2). Similar phenotypes were also observed in Spo11-Cre Hus1 CKO spermatocytes (Table 2). Autosomes included in the sex body domain often appeared synapsed (based on chromosome number and SYCP3 staining intensity), though asynapsed chromosomes and parts of chromosomes were also included in the sex body, as shown in Figure 3F. Additionally, a substantial number of meiotic nuclei harbored apparent end-to-end fusions between the X chromosome and an autosome (Figure 3E). In such cases, the X and Y chromosomes were synapsed at the pseudoautosomal region (PAR) and were contained within the sex body domain containing γH2AX, while the autosome remained outside of the sex body domain and was devoid of γH2AX. The frequency of X-autosome fusions was significantly increased in Stra8-Cre Hus1 CKO pachytene spermatocytes, from 2% in controls to 12% in CKO mice (p<0.001; Table 2).


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)

Hus1 inactivation results in abnormal accumulation of γH2AX on autosomes, an extended sex body domain, inclusion of autosomes within the sex body, and X-autosome fusions.A–F. Immunofluorescence staining for γH2AX and SYCP3. γH2AX staining persisted in a perpendicular pattern on synapsed autosomes in pachytene (B) and diplotene stage (C), and in clouds surrounding unsynapsed chromosomal regions (D). The sex body domain marked by γH2AX was extended in Stra8-Cre Hus1 CKOs (B,F), and often contained whole or partial autosomes (B,F). Some Stra8-Cre Hus1 CKO nuclei exhibited apparent X chromosome-autosome end-to-end fusions (E). G,H. Western blot analysis of CHK1 (G) and CHK2 (H) in total testis extracts from mice of the indicated genotypes. In the CHK2 immunoblot, pre-B cells that were untreated (U) or treated with 5Gy ionizing radiation (IR) are included as controls. β-actin is shown as a loading control. In Hus1 CKO samples, total CHK1 levels were increased on average 2.9-fold over controls, and phosphorylated CHK1 (S345) levels were increased 4.1-fold. CHK2 phosphorylation was not detected in the absence of Hus1.
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Related In: Results  -  Collection

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pgen-1003320-g003: Hus1 inactivation results in abnormal accumulation of γH2AX on autosomes, an extended sex body domain, inclusion of autosomes within the sex body, and X-autosome fusions.A–F. Immunofluorescence staining for γH2AX and SYCP3. γH2AX staining persisted in a perpendicular pattern on synapsed autosomes in pachytene (B) and diplotene stage (C), and in clouds surrounding unsynapsed chromosomal regions (D). The sex body domain marked by γH2AX was extended in Stra8-Cre Hus1 CKOs (B,F), and often contained whole or partial autosomes (B,F). Some Stra8-Cre Hus1 CKO nuclei exhibited apparent X chromosome-autosome end-to-end fusions (E). G,H. Western blot analysis of CHK1 (G) and CHK2 (H) in total testis extracts from mice of the indicated genotypes. In the CHK2 immunoblot, pre-B cells that were untreated (U) or treated with 5Gy ionizing radiation (IR) are included as controls. β-actin is shown as a loading control. In Hus1 CKO samples, total CHK1 levels were increased on average 2.9-fold over controls, and phosphorylated CHK1 (S345) levels were increased 4.1-fold. CHK2 phosphorylation was not detected in the absence of Hus1.
Mentions: Because the 9-1-1 complex protects chromosomal integrity in somatic cells, we hypothesized that germ cell loss in Hus1 CKOs might be due to DNA damage accumulation. We prepared meiotic chromosome spreads and assessed the localization of γH2AX, the phosphorylated form of the histone variant H2AX, by indirect immunofluorescence. γH2AX marks DSBs as well as unsynapsed chromatin, and is phosphorylated by the ATM and ATR kinases [26], [29]. We found that γH2AX staining during leptotene and zygotene, when meiotic DSBs are generated and processed, was similar in Hus1 CKOs and controls. Furthermore, γH2AX localized normally to the sex body domain in pachytene Hus1 CKO spermatocytes (Figure 3), suggesting that ATR-dependent H2AX phosphorylation is independent of HUS1 as is also true during responses to replication stress [51]. However, in Hus1 mutant spermatocytes, the sex body domain marked by γH2AX was enlarged and extended, and often encompassed parts of or entire autosomes (Figure 3B, 3C, 3F). 56% of mutant pachytene spermatocytes showed some type of sex body abnormality (compared to 13% of control nuclei), with 39% of Stra8-Cre Hus1 CKO nuclei having sex body extensions or protrusions and 33% with autosome inclusion (compared to 4% and 9%, respectively, in control spermatocytes; Table 2). Similar phenotypes were also observed in Spo11-Cre Hus1 CKO spermatocytes (Table 2). Autosomes included in the sex body domain often appeared synapsed (based on chromosome number and SYCP3 staining intensity), though asynapsed chromosomes and parts of chromosomes were also included in the sex body, as shown in Figure 3F. Additionally, a substantial number of meiotic nuclei harbored apparent end-to-end fusions between the X chromosome and an autosome (Figure 3E). In such cases, the X and Y chromosomes were synapsed at the pseudoautosomal region (PAR) and were contained within the sex body domain containing γH2AX, while the autosome remained outside of the sex body domain and was devoid of γH2AX. The frequency of X-autosome fusions was significantly increased in Stra8-Cre Hus1 CKO pachytene spermatocytes, from 2% in controls to 12% in CKO mice (p<0.001; Table 2).

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