Characterization of TP2 interactome

Spermiogenesis is marked by an almost genome-wide histone-to-protamine transition. Protamines are sperm-specific, small and highly positively charged proteins mainly responsible for genome compaction at this step. According to previous investigations from our lab and other groups, it is known that just before their removal, histones become hyperacetylated, which creates binding sites for the first bromodomain of a testis-specific member of the BET double bromodomain protein family, Brdt. Brdt’s first bromodomain is necessary for transition proteins (TPs) and PRMs incorporation and subsequent histone removal. Histone hyperacetylation, specifically on H4 lysine residues 5 and 8 which are recognized by Brdt, is controlled by a post-meiotic enhancer of CBP/p300 acetyltransferase activity, known as Nut. The process of TP assembly itself is dependent on a prior incorporation of a histone variant known as H2A.L.2, expressed in the same cells and at the same time as TPs. To characterize the proteins associating with TP2, we performed a mass spectrometry-based proteomics analysis of TP2 co-immunoprecipitations elutes from condensing spermatids extract. The immunoprecipitation was performed using extract from stages 12-16 spermatids from wild-type and H2A.L.2-KO mice. In parallel, immunopurifications were performed using a non-specific antibody (pre-immune goat serum) to allow the identification of proteins specifically purified with TP2 as opposed to proteins non-specifically precipitated.