ATR acts stage specifically to regulate multiple aspects of mammalian meiotic silencing

Hélène Royo, Haydn Prosser, Yaroslava Ruzankina, et al

Genes Dev. 2013 27: 1484-1494

Access the most recent version at doi:10.1101/gad.219477.113

Abstract

In mammals, homologs that fail to synapse during meiosis are transcriptionally inactivated. This process, meiotic silencing, drives inactivation of the heterologous XY bivalent in male germ cells (meiotic sex chromosome inactivation [MSCI]) and is thought to act as a meiotic surveillance mechanism. The checkpoint protein ATM and Rad3-related (ATR) localizes to unsynapsed chromosomes, but its role in the initiation and maintenance of meiotic silencing is unknown. Here we show that ATR has multiple roles in silencing. ATR first regulates HORMA (Hop1, Rev7, and Mad2) domain protein HORMAD1/2 phosphorylation and localization of breast cancer I (BRCA1) and ATR cofactors ATR-interacting peptide (ATRIP)/topoisomerase 2-binding protein 1 (TOPBP1) at unsynapsed axes. Later, it acts as an adaptor, transducing signaling at unsynapsed axes into surrounding chromatin in a manner that requires interdependence with mediator of DNA damage checkpoint 1 (MDC1) and H2AFX. Finally, ATR catalyzes histone H2AFX phosphorylation, the epigenetic event leading to gene inactivation. Using a novel genetic strategy in which MSCI is used to silence a chosen gene in pachytene, we show that ATR depletion does not disrupt the maintenance of silencing and that silencing comprises two phases: The first is dynamic and reversible, and the second is stable and irreversible. Our work identifies a role for ATR in the epigenetic regulation of gene expression and presents a new technique for ablating gene function in the germline.

Results

☆ATR ablation causes meiotic arrest and defective H2AFX phosphorylation

☆ATR also regulates localization of silencing factors at unsynapsed AEs

☆A positive feedback loop between ATR, MDC1, and H2AFX amplifies the silencing response

☆Atr is dispensable for the maintenance of meiotic silencing

“In this study, we also present a novel genetic strategy whereby the expression of a gene of interest can be silenced in the germline using MSCI. We used this to demonstrate that loss of ATR after the initiation of XY inactivation does not impair H2AFX phosphorylation or the maintenance of silencing. Our data suggest that H2AFX phosphorylation is not an ongoing process but is restricted to early in pachytene, a property that contrasts with the more dynamic behavior of other histone modifications (e.g., H3K9me3) during the maintenance phase of MSCI (van derHeijden et al. 2007).Notably, the X-targeting strategy described here leads to dramatic and sustained depletion of gene expression. It may therefore prove superior to Cre recombinase-based approaches, which exhibit mosaic Cre-expression and varying excision efficiencies, in interrogating the functions of genes expressed during late prophase or spermiogenesis.”