MAMERR interacts with HSF2BP and BRCA2 to regulate the ubiquitination of mouse pachytene spermatocytes

Abstract

Meiosis is a distinct cell division process that produces haploid gametes from diploid progenitor cells. Previous studies indicate that the male meiosis recombination regulator (MAMERR) regulates homologous recombination during mouse meiotic prophase I. Deletion of MAMERR in mice causes mild impairments in DNA meiotic recombinase 1 and radiation-sensitive protein 51 recruitment. Given the widespread distribution of MAMERR on chromosomes at pachynema, we speculate that MAMERR may have broader functions in mouse meiotic prophase I.

In this thesis, we firstly identified MAMERR's precise localization and expression in mouse testis. Histological analysis demonstrated that MAMERR is essential for meiotic prophase I in Mamerr-/- mice, and apoptosis occurred at the early pachynema stage in these mice. RNA sequencing showed that meiotic sex chromosome inactivation (MSCI) failed to be established in Mamerr-/- pachytene spermatocytes. Moreover, transcription was also globally dysregulated on autosomes. These results indicated that MAMERR regulates transcription in mouse pachytene spermatocytes.

MAMERR also participated in regulating ubiquitination in mouse pachytene spermatocytes, and analysis of ubiquitination revealed increased ubiquitination in Mamerr-/- pachytene spermatocytes. Subsequent analysis using ubiquitination modification mass spectrometry (Ub-MS) showed a substantial increase in ubiquitinated protein sites, including numerous ubiquitinated sites of ubiquitin-proteasome system-associated proteins and meiosis-associated proteins, in both Mamerr-/- mouse testes and Mamerr-/- pachytene spermatocytes. Notably, ubiquitinated sites of meiosis-associated proteins, such as ataxia telangiectasia and RAD3 related and senataxin, were significantly increased in Mamerr-/- pachytene spermatocytes, which may be associated with the failure of MSCI establishment. Moreover, we generated and verified a mouse model overexpressing MAMERR and found that overexpression of MAMERR could significantly suppress ubiquitination in mouse pachytene spermatocytes. Through further mechanistic studies of how MAMERR might regulate ubiquitination, we found that overexpression of MAMERR also suppressed ubiquitination in HEK293T cells. However, in vitro deubiquitinase assays with purified recombinant MAMERR protein revealed that MAMERR did not exhibit deubiquitinase activity.

Considering the interconnected structural and functional interplay among MAMERR, heat shock factor 2 binding protein (HSF2BP), and breast cancer type 2 susceptibility protein (BRCA2), we hypothesized that HSF2BP and BRCA2 are involved in the ubiquitination regulation process of MAMERR. Remarkably, our results showed that overexpression of HSF2BP or different parts of BRCA2 significantly enhanced ubiquitination in HEK293T cells. Intriguingly, this increased ubiquitination was mitigated upon co-transfection with MAMERR, suggesting that MAMERR might interact with HSF2BP and BRCA2 to regulate ubiquitination in HEK293T cells.

In conclusion, in the work for this thesis we identified new functions of MAMERR in mouse meiotic prophase I. It is not only a regulator of homologous recombination for mouse meiotic prophase I, but it is also involved in regulating transcription and ubiquitination in mouse pachytene spermatocytes. Further research in HEK293T cells indicated that MAMERR might interact with HSF2BP and BRCA2 to regulate ubiquitination. MAMERR plays a crucial regulatory role in mouse meiotic prophase I, and research on MAMERR contributes to a deeper understanding of the mechanisms underlying transcription regulation and ubiquitination modification in mouse pachytene spermatocytes. Also, it has significant clinical implications for elucidating the pathogenesis of related infertility disorders, identifying therapeutic targets, and improving treatment effectiveness.