Limits...
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.

Show MeSH

Related in: MedlinePlus

RAD9 and RAD1 localize in overlapping yet distinct patterns on meiotic chromosomes.A. Meiotic chromosome spreads from control animals were stained for RAD9 and RAD1. RAD9 colocalized with a subset of RAD1 foci in control spermatocytes. A region containing one X-Y pair is shown at higher magnification. B–E. Meiotic chromosome spreads from Slx4mut/mut (B, C) or Msh4−/− (D, E) mice were stained for SYCP3 and RAD1 (B, D) or RAD9 (C, E). RAD1 localized more continuously along sex chromosome cores and autosomes and to asynaptic sites in Slx4 and Msh4 mutants, whereas RAD9 formed fewer, more punctate foci along asynapsed chromosome cores. Chromosome spreads in panels B–E were additionally stained using human CREST serum, which marks centromeric regions. CREST signal is detected in the red channel (middle column) and in the merged images.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3585019&req=5

pgen-1003320-g008: RAD9 and RAD1 localize in overlapping yet distinct patterns on meiotic chromosomes.A. Meiotic chromosome spreads from control animals were stained for RAD9 and RAD1. RAD9 colocalized with a subset of RAD1 foci in control spermatocytes. A region containing one X-Y pair is shown at higher magnification. B–E. Meiotic chromosome spreads from Slx4mut/mut (B, C) or Msh4−/− (D, E) mice were stained for SYCP3 and RAD1 (B, D) or RAD9 (C, E). RAD1 localized more continuously along sex chromosome cores and autosomes and to asynaptic sites in Slx4 and Msh4 mutants, whereas RAD9 formed fewer, more punctate foci along asynapsed chromosome cores. Chromosome spreads in panels B–E were additionally stained using human CREST serum, which marks centromeric regions. CREST signal is detected in the red channel (middle column) and in the merged images.

Mentions: The RAD1 subunit of 9-1-1 was previously determined to localize to meiotic chromosomes, where RAD1 foci outnumber RAD51 foci and nearly continuously coat pachytene chromosome cores, especially along the unsynapsed X and Y [41]. To reconcile this staining with our observations of fewer, more punctate RAD9 foci, we assessed the colocalization of RAD1 and RAD9 subunits in control nuclei. These experiments revealed that RAD1 had a broader distribution than RAD9, with RAD1 foci present along the entire unsynapsed regions of the X and Y while RAD9 was detected primarily in bright foci along the X. Overall, only 23% of RAD1 foci colocalized with RAD9 (16% of autosomal and 46% of XY RAD1 foci colocalized with RAD9). A larger percentage (64%) of RAD9 foci colocalized with RAD1, particularly on the sex chromosomes (52% of autosomal and 86% of XY RAD9 foci colocalized with RAD1). We next assessed both RAD1 and RAD9 localization on abnormal chromosomes in Slx4mut/mut and Msh4−/− mutant spermatocytes, which exhibit asynapsis as well as inclusion of autosomes within the sex body domain [55], [66]. Consistent with the co-staining results, RAD1 localized along synapsed autosomal cores and to a greater extent along asynapsed autosomal and XY cores (Figure 8B, 8D). RAD9 on the other hand localized in a more punctate pattern along chromosome cores in the sex body domain and on asynapsed autosomes (Figure 8C, 8E). Together, these results indicate that RAD1 and RAD9 partially colocalize along abnormal chromosomes and the sex chromosome cores, and that a subset of RAD1-containing sites lack RAD9.


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)

RAD9 and RAD1 localize in overlapping yet distinct patterns on meiotic chromosomes.A. Meiotic chromosome spreads from control animals were stained for RAD9 and RAD1. RAD9 colocalized with a subset of RAD1 foci in control spermatocytes. A region containing one X-Y pair is shown at higher magnification. B–E. Meiotic chromosome spreads from Slx4mut/mut (B, C) or Msh4−/− (D, E) mice were stained for SYCP3 and RAD1 (B, D) or RAD9 (C, E). RAD1 localized more continuously along sex chromosome cores and autosomes and to asynaptic sites in Slx4 and Msh4 mutants, whereas RAD9 formed fewer, more punctate foci along asynapsed chromosome cores. Chromosome spreads in panels B–E were additionally stained using human CREST serum, which marks centromeric regions. CREST signal is detected in the red channel (middle column) and in the merged images.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1003320-g008: RAD9 and RAD1 localize in overlapping yet distinct patterns on meiotic chromosomes.A. Meiotic chromosome spreads from control animals were stained for RAD9 and RAD1. RAD9 colocalized with a subset of RAD1 foci in control spermatocytes. A region containing one X-Y pair is shown at higher magnification. B–E. Meiotic chromosome spreads from Slx4mut/mut (B, C) or Msh4−/− (D, E) mice were stained for SYCP3 and RAD1 (B, D) or RAD9 (C, E). RAD1 localized more continuously along sex chromosome cores and autosomes and to asynaptic sites in Slx4 and Msh4 mutants, whereas RAD9 formed fewer, more punctate foci along asynapsed chromosome cores. Chromosome spreads in panels B–E were additionally stained using human CREST serum, which marks centromeric regions. CREST signal is detected in the red channel (middle column) and in the merged images.
Mentions: The RAD1 subunit of 9-1-1 was previously determined to localize to meiotic chromosomes, where RAD1 foci outnumber RAD51 foci and nearly continuously coat pachytene chromosome cores, especially along the unsynapsed X and Y [41]. To reconcile this staining with our observations of fewer, more punctate RAD9 foci, we assessed the colocalization of RAD1 and RAD9 subunits in control nuclei. These experiments revealed that RAD1 had a broader distribution than RAD9, with RAD1 foci present along the entire unsynapsed regions of the X and Y while RAD9 was detected primarily in bright foci along the X. Overall, only 23% of RAD1 foci colocalized with RAD9 (16% of autosomal and 46% of XY RAD1 foci colocalized with RAD9). A larger percentage (64%) of RAD9 foci colocalized with RAD1, particularly on the sex chromosomes (52% of autosomal and 86% of XY RAD9 foci colocalized with RAD1). We next assessed both RAD1 and RAD9 localization on abnormal chromosomes in Slx4mut/mut and Msh4−/− mutant spermatocytes, which exhibit asynapsis as well as inclusion of autosomes within the sex body domain [55], [66]. Consistent with the co-staining results, RAD1 localized along synapsed autosomal cores and to a greater extent along asynapsed autosomal and XY cores (Figure 8B, 8D). RAD9 on the other hand localized in a more punctate pattern along chromosome cores in the sex body domain and on asynapsed autosomes (Figure 8C, 8E). Together, these results indicate that RAD1 and RAD9 partially colocalize along abnormal chromosomes and the sex chromosome cores, and that a subset of RAD1-containing sites lack RAD9.

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