BET Protein BRDT Complexes With HDAC1, PRMT5, and TRIM28 and Functions in Transcriptional Repression During Spermatogenesis.

The expression of BRDT, a member of the BET sub-family of double bromodomain-containing proteins, is restricted to the male germ line, specifically to pachytene-diplotene spermatocytes and early spermatids. We previously showed that loss of the first bromodomain of BRDT by targeted mutagenesis (Brdt(ΔBD1) ) resulted in sterility and abnormalities in spermiogenesis, but little is known about BRDT's function at the molecular level. As part of studies designed to identify BRDT-interacting proteins we stably introduced a FLAG-tagged BRDT cDNA into 293T cells, which do not normally express BRDT. Affinity-purification of FLAG-tagged BRDT complexes indicated that BRDT has novel interactions with the histone deacetylase HDAC1, the arginine-specific histone methyltransferase 5 PRMT5, and the Tripartite motif-containing 28 protein TRIM28. Immunofluorescent microscopy revealed that BRDT co-localized with each of these proteins in round spermatids and co-immunoprecipitation of testicular extracts showed that these proteins interact with BRDT. Furthermore, they bind the promoter of H1t, a putative target of BRDT-containing complexes. This binding of H1t was lost in mice expressing the Brdt(ΔBD1) mutant protein and concomitantly, H1t expression was elevated in round spermatids. Our study reveals a role for BRDT-containing complexes in the repression of gene expression in vivo that correlates with dramatic effects on chromatin remodeling and the progression of spermiogenesis.