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

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.

Clamping down on mammalian meiosis

“…Consistent with the possibility that the mammalian 9-1-1 complex plays critical roles during gametogenesis, 9-1-1 subunits are highly expressed in the mouse testis, 14,15,33,[65][66][67] and both RAD1 and RAD9A localize to meiotic chromosomes. 15,33 Furthermore, Hus1 gene expression is reduced in the mouse testis in the absence of SPO11, 68 although it remains possible that this indicates that HUS1 function requires progression further into meiotic prophase I rather than a role specifically in meiotic recombination. In order to elucidate the germline functions of murine 9-1-1, conditional mutations were generated in Rad9a 14 and Hus1 15 using mice that express CRE recombinase in undifferentiated spermatogonia under control of the Stra8 promoter.…”

“…15 Testis size was drastically reduced in both models, and seminiferous tubules exhibited severely decreased cellularity, vacuolization, large multinucleate cells, and increased apoptosis. 14,15 Despite expected defects in mitotically dividing spermatogonia concurrent with depletion of RAD9A and HUS1, no overt pre-meiotic abnormalities were observed in Rad9a CKOs (A Vasileva, DJ Wolgemuth, HB Lieberman, unpublished results).…”

“…[33][34][35] However, many details remain to be resolved regarding the mode of ATR activation in germ cells, and to the best of our knowledge, the Rad9a 14 and Hus1 studies are the first to suggest that meiotic ATR activation is, at least in part, 9-1-1-independent. 15 A variety of studies in somatic cells indicate that the 9-1-1 complex not only contributes to ATR activation but also participates directly in DNA repair.…”

“…15 In both models, the majority of germ cells underwent apoptosis during the pachytene stage of meiosis, although there were subtle yet distinct differences in the timing. Rad9a CKO spermatocytes underwent apoptosis by early to midpachynema, leaving few late pachytene or diplotene cells.…”

“…Studies in lower eukaryotes suggest that the 9-1-1 complex may have critical roles in meiotic recombination and homologous chromosome synapsis as well as meiotic checkpoint signaling. [4][5][6][7][8][9][10][11][12][13] The germline functions of mammalian 9-1-1 were investigated in 2 recent studies in which conditional mutations in Rad9a 14 and Hus1 15 were targeted to undifferentiated spermatogonia using mice that express CRE recombinase under the control of the Stra8 promoter.…”