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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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TOPBP1 localization to and RNA Pol II exclusion from the sex body domain remain unperturbed following Hus1 inactivation.A–E. Immunofluorescence staining for TOPBP1 (A–C) and RNA Polymerase II (D–E) in control (Cre-negative Hus1flox/Δ1) and Stra8-Cre Hus1 CKO pachytene spermatocytes. The asterisk in B indicates an autosome partially included within the sex body domain and coated with TOPBP1. Arrows in C indicate abnormal TOPBP1 staining in a perpendicular pattern on autosomes in pachytene Hus1 CKOs.
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pgen-1003320-g006: TOPBP1 localization to and RNA Pol II exclusion from the sex body domain remain unperturbed following Hus1 inactivation.A–E. Immunofluorescence staining for TOPBP1 (A–C) and RNA Polymerase II (D–E) in control (Cre-negative Hus1flox/Δ1) and Stra8-Cre Hus1 CKO pachytene spermatocytes. The asterisk in B indicates an autosome partially included within the sex body domain and coated with TOPBP1. Arrows in C indicate abnormal TOPBP1 staining in a perpendicular pattern on autosomes in pachytene Hus1 CKOs.

Mentions: During meiosis, TOPBP1 normally colocalizes with ATR at sites of unsynapsed chromatin, including unsynapsed autosomes in early spermatocytes as well as the XY chromatin throughout prophase I [24], [60]. In mammalian somatic cells, the best characterized function of 9-1-1 is to recruit TOPBP1 to sites of DNA damage, where it physically interacts with and stimulates the kinase activity of ATR [61]–[63]. Therefore, we next tested whether Hus1 loss affected the meiotic localization of TOPBP1. In contrast to the 9-1-1-dependent mechanism elucidated in somatic cells, TOPBP1 localization to the asynapsed sex chromatin was unperturbed in Hus1 CKO mice (Figure 6). Additionally, TOPBP1 localized to autosomes that resided within the sex body domain (Figure 6B, asterisk), and to perpendicular foci on some autosomes in Stra8-Cre Hus1 CKOs (Figure 6C) similar to the γH2AX eruptions described above, indicating that HUS1 loss does not preclude assembly of TOPBP1 on chromatin and results in TOPBP1 localization to abnormal autosomes as well as to unsynapsed sex chromatin.


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)

TOPBP1 localization to and RNA Pol II exclusion from the sex body domain remain unperturbed following Hus1 inactivation.A–E. Immunofluorescence staining for TOPBP1 (A–C) and RNA Polymerase II (D–E) in control (Cre-negative Hus1flox/Δ1) and Stra8-Cre Hus1 CKO pachytene spermatocytes. The asterisk in B indicates an autosome partially included within the sex body domain and coated with TOPBP1. Arrows in C indicate abnormal TOPBP1 staining in a perpendicular pattern on autosomes in pachytene Hus1 CKOs.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1003320-g006: TOPBP1 localization to and RNA Pol II exclusion from the sex body domain remain unperturbed following Hus1 inactivation.A–E. Immunofluorescence staining for TOPBP1 (A–C) and RNA Polymerase II (D–E) in control (Cre-negative Hus1flox/Δ1) and Stra8-Cre Hus1 CKO pachytene spermatocytes. The asterisk in B indicates an autosome partially included within the sex body domain and coated with TOPBP1. Arrows in C indicate abnormal TOPBP1 staining in a perpendicular pattern on autosomes in pachytene Hus1 CKOs.
Mentions: During meiosis, TOPBP1 normally colocalizes with ATR at sites of unsynapsed chromatin, including unsynapsed autosomes in early spermatocytes as well as the XY chromatin throughout prophase I [24], [60]. In mammalian somatic cells, the best characterized function of 9-1-1 is to recruit TOPBP1 to sites of DNA damage, where it physically interacts with and stimulates the kinase activity of ATR [61]–[63]. Therefore, we next tested whether Hus1 loss affected the meiotic localization of TOPBP1. In contrast to the 9-1-1-dependent mechanism elucidated in somatic cells, TOPBP1 localization to the asynapsed sex chromatin was unperturbed in Hus1 CKO mice (Figure 6). Additionally, TOPBP1 localized to autosomes that resided within the sex body domain (Figure 6B, asterisk), and to perpendicular foci on some autosomes in Stra8-Cre Hus1 CKOs (Figure 6C) similar to the γH2AX eruptions described above, indicating that HUS1 loss does not preclude assembly of TOPBP1 on chromatin and results in TOPBP1 localization to abnormal autosomes as well as to unsynapsed sex chromatin.

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