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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 knockout meiotic chromosomes display synapsis defects and ruptures in the synaptonemal complex.Meiotic chromosome spreads from control (Cre-negative Hus1flox/Δ1) and Stra8-Cre Hus1 CKO mice were stained for SYCP1 and SYCP3, as well as with DAPI. A. Normal synapsis of pachytene chromosomes in control males, as indicated by SYCP1 and SYCP3 immunofluorescence. B. Chromosomal asynapsis in pachytene-stage Stra8-Cre Hus1 CKO nuclei, with unsynapsed chromosomal regions devoid of SYCP1. C. Diplotene Stra8-Cre Hus1 CKO chromosomes with ruptures in the SC lateral elements, as indicated by arrows.
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pgen-1003320-g005: Conditional Hus1 knockout meiotic chromosomes display synapsis defects and ruptures in the synaptonemal complex.Meiotic chromosome spreads from control (Cre-negative Hus1flox/Δ1) and Stra8-Cre Hus1 CKO mice were stained for SYCP1 and SYCP3, as well as with DAPI. A. Normal synapsis of pachytene chromosomes in control males, as indicated by SYCP1 and SYCP3 immunofluorescence. B. Chromosomal asynapsis in pachytene-stage Stra8-Cre Hus1 CKO nuclei, with unsynapsed chromosomal regions devoid of SYCP1. C. Diplotene Stra8-Cre Hus1 CKO chromosomes with ruptures in the SC lateral elements, as indicated by arrows.

Mentions: To investigate additional causes for germ cell loss in Hus1 CKOs, we assessed the localization of various meiotic markers on chromosome spreads from control and Stra8-Cre Hus1 CKO males. Meiotic chromosome synapsis appeared normal in most Hus1 mutant nuclei, with colocalization of the synaptonemal complex proteins SYCP1 and SYCP3 in pachytene spermatocytes and the presence of 19 pairs of fully synapsed homologs in addition to the X and Y chromosomes paired at the pseudoautosomal region. However, as shown in Figure 5B, a significant number of Stra8-Cre Hus1 CKO nuclei exhibited synapsis defects, usually involving the X chromosome (14% of Hus1 CKOs versus 3% of controls; N = 161 and 185, respectively; p<0.001). Notably, Hus1 CKOs also frequently displayed ruptures in the synaptonemal complex during diplotene (36% of Stra8-Cre Hus1 CKOs and 33% of Spo11-Cre Hus1 CKOs compared to 2–6% of controls; Table 2). In these cases, there were clear discontinuities in synaptonemal complex protein staining and the ends of the broken SC were spatially separated (Figure 5C, arrows), indicating either a defect in SC integrity or breakage of chromosomal DNA 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 knockout meiotic chromosomes display synapsis defects and ruptures in the synaptonemal complex.Meiotic chromosome spreads from control (Cre-negative Hus1flox/Δ1) and Stra8-Cre Hus1 CKO mice were stained for SYCP1 and SYCP3, as well as with DAPI. A. Normal synapsis of pachytene chromosomes in control males, as indicated by SYCP1 and SYCP3 immunofluorescence. B. Chromosomal asynapsis in pachytene-stage Stra8-Cre Hus1 CKO nuclei, with unsynapsed chromosomal regions devoid of SYCP1. C. Diplotene Stra8-Cre Hus1 CKO chromosomes with ruptures in the SC lateral elements, as indicated by arrows.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1003320-g005: Conditional Hus1 knockout meiotic chromosomes display synapsis defects and ruptures in the synaptonemal complex.Meiotic chromosome spreads from control (Cre-negative Hus1flox/Δ1) and Stra8-Cre Hus1 CKO mice were stained for SYCP1 and SYCP3, as well as with DAPI. A. Normal synapsis of pachytene chromosomes in control males, as indicated by SYCP1 and SYCP3 immunofluorescence. B. Chromosomal asynapsis in pachytene-stage Stra8-Cre Hus1 CKO nuclei, with unsynapsed chromosomal regions devoid of SYCP1. C. Diplotene Stra8-Cre Hus1 CKO chromosomes with ruptures in the SC lateral elements, as indicated by arrows.
Mentions: To investigate additional causes for germ cell loss in Hus1 CKOs, we assessed the localization of various meiotic markers on chromosome spreads from control and Stra8-Cre Hus1 CKO males. Meiotic chromosome synapsis appeared normal in most Hus1 mutant nuclei, with colocalization of the synaptonemal complex proteins SYCP1 and SYCP3 in pachytene spermatocytes and the presence of 19 pairs of fully synapsed homologs in addition to the X and Y chromosomes paired at the pseudoautosomal region. However, as shown in Figure 5B, a significant number of Stra8-Cre Hus1 CKO nuclei exhibited synapsis defects, usually involving the X chromosome (14% of Hus1 CKOs versus 3% of controls; N = 161 and 185, respectively; p<0.001). Notably, Hus1 CKOs also frequently displayed ruptures in the synaptonemal complex during diplotene (36% of Stra8-Cre Hus1 CKOs and 33% of Spo11-Cre Hus1 CKOs compared to 2–6% of controls; Table 2). In these cases, there were clear discontinuities in synaptonemal complex protein staining and the ends of the broken SC were spatially separated (Figure 5C, arrows), indicating either a defect in SC integrity or breakage of chromosomal DNA 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