Key mediators of somatic ATR signaling localize to unpaired chromosomes in spermatocytes

Abstract

Meiotic silencing of unpaired chromatin (MSUC) occurs during the first meiotic prophase, as chromosomes that fail to pair are sequestered into a transcriptionally repressive nuclear domain. This phenomenon is exemplified by the heterologous sex chromosomes of male mammals, where the ATR DNA damage response kinase is crucial for this silencing event. However, the mechanisms underlying the initiation of MSUC remain unknown. Here, we show that essential components of ATR signaling in murine somatic cells are spatially confined to unpaired chromosomes in spermatocytes, including the ATR-dependent phosphorylation of the single-stranded DNA (ssDNA)-binding complex replication protein A (RPA) and the checkpoint kinase CHK1. These observations support a model in which ssDNA plays a central role in the recruitment of ATR during MSUC, and provide a link to meiotic progression through activation of CHK1.

Keywords: CHEK1; Meiosis; Mouse; Spermatogenesis; Transcriptional regulation.

Fig. 1.

Phosphorylated RPA is a mark of asynapsed chromosomes in mouse primary spermatocytes. (A) RPA32 phosphorylated at serine 33 (pRPA) first colocalizes with the γH2AFX -coated sex chromosomes of wild-type (WT) late zygotene spermatocytes (arrows). (B) Higher magnification of the boxed region in A. (C) The sex chromosomes of all pachytene spermatocytes are enriched for pRPA. (D) Higher magnification view of XY body from C. pRPA staining is highest near axial elements (B,D, arrows). (E) An unpaired autosome in the XY body of Ts65(16:17)Dn spermatocytes, marked by BRCA1 and γH2AFX, is also enriched for pRPA. The arrow indicates the autosomal translocation.

Fig. 2.

Specificity of pRPA antibody. WT spermatocytes were incubated with a molar excess of specific or non-specific phosphopeptides, along with antibodies against pRPA. The pRPA signal associated with the XY body (A) was completely abolished in the presence of a peptide specific for this antibody (pS33) (B). By contrast, even a 1000-fold molar excess of a peptide corresponding to SMC3, a known component of the XY body, had no effect on the association of the pRPA antibody with the sex chromosomes (C).

Fig. 3.

Phosphorylation of RPA on the meiotic sex chromosomes is dependent on ATR. ATR protein levels in spermatocytes from control (A) and Atr cKO (B) males. pRPA enrichment on the XY body in control (C) and Atr cKO (D) spermatocytes. Immunostaining with antibodies against RPA32, a component of the RPA complex, shows similar focal patterns in control pachytene spermatocytes (E) and spermatocytes from Atr cKO males with undetectable ATR protein (F). In control spermatocytes, the NBS1 nuclease is associated with the XY body (G). By contrast, Atr cKO spermatocytes with low levels of ATR had undetectable NBS1 enrichment on these chromosomes (H). Arrows indicate the sex chromosomes.

Fig. 4.

Phosphorylation of RPA is independent of Spo11. (A) pRPA is associated with the XY chromosomes in 100% of WT spermatocytes. (B) 70% of Spo11^−/− spermatocytes show colocalization of pRPA with γH2AFX-positive domains. (C) Broad, nuclear pRPA distribution is evident in 30% of mutant spermatocytes. Bright pRPA foci are common in both classes of Spo11^−/− spermatocytes. Arrows indicate regions undergoing MSUC.

Fig. 5.

ATR-dependent localization and phosphorylation of CHK1 on the XY body. The asynapsed sex chromosomes show enrichment of the checkpoint protein CHK1 in WT spermatocytes (A). Furthermore, the XY body of WT spermatocytes is associated with phosphorylated forms of CHK1: at serine 317 (S317; B) and serine 345 (S345; C), both of which are target residues of the ATR kinase. The enrichment of pCHK1 (S317) and pCHK1 (S345) is undetectable in spermatocytes from Atr cKO males (D,E). Arrows indicate the position of the XY chromosomes.

Fig. 6.

Spo11 deficiency severely abrogates the association of pCHK1 with asynapsed chromatin. Localization of pCHK1 (S317) (A) and pCHK1 (S345) (B) to the sex chromosomes and regions undergoing MSUC in WT and Spo11^−/− spermatocytes. Arrows indicate regions undergoing MSUC.