The bromodomain binds acetylated lysines in histones and other proteins and is found in many chromatin- associated proteins, transcription factors, and in nearly all known histone acetyltransferases. The BET sub- family is unique in that its members contain two bromodomains (BD1 and BD2) and an extra terminal (ET) domain. We have generated a mutation in the mouse BET family gene Brdt, designated Brdt?BD1, which yields an N-terminal truncated BRDT protein lacking BD1. Homozygous Brdt?BD1 male progeny are sterile, with strikingly aberrant elongating spermatids. We have identified abnormalities in round and elongating spermatids, specifically the presence of multiple chromocenters and abnormal chromatin in elongating spermatids. In addition, we have generated complete loss of BRDT function mutants (designated Brdt-/-), which, in concordance with our original hypothesis, exhibit a phenotype distinct from that of the Brdt?BD1 mutants: Brdt-/- male mice are also sterile but spermatogenesis is arrested in late meiotic prophase spermatocytes and spermatids do not form. We will extend these novel studies by examining BRDT's essential function in regulating gene expression during spermatogenesis at several distinct but not mutually exclusive levels, taking advantage of the distinct mouse models we have generated which exhibit dramatically distinct phenotypes at distinct stages of germ cell differentiation.
Aim 1 will test the hypothesis that BRDT forms functional transcriptional repression complexes, specifically with 3 proteins we have identified as interacting with BRDT and known to have repressor function-PRMT5, HDAC1, and TRIM28. We will determine the functional domains of BRDT responsible for forming these complexes, identify other components of the complexes in vivo and identify specific gene sets whose expression would be up-regulated due to partial or complete loss of BRDT function and whose mis-expression could contribute to the spermatogenic defects observed in our two mutant models.
Aim 2 will follow up on increasing evidence that BRDT is also important for transcriptional activation during spermatogenesis by continuing to mine, validate and study the expression of candidate genes identified in our microarray and ChIP-Seq data and pursue candidate activating transcription factors identified by our preliminary ChIP-Seq analysis, establishing the """"""""full transcriptomes"""""""" in the two mutant models by RNA-Seq, and identifying in vivo interacting proteins by immunoprecipitation followed by mass spectrometry.
Aim 3 will explore the function of BRDT in establishing higher order chromatin/chromosome organization in spermatocytes and spermatids, specifically examining changes in chromatin condensation, chromocenter formation, chromosome dynamics and nuclear architecture in spermatocytes and spermatids expressing a truncated BRDT protein lacking BD1 (Brdt?BD1) and in spermatocytes that lack BRDT protein altogether (Brdt-/-). These studies will provide critical new insight into the functions of BRDT during spermatogenesis and the function of the BET proteins in general during differentiation.
BRDT is a testis-specific member of the BET sub-family of double bromodomain- containing proteins which can read epigenetic marks (acetylated lysines) on histones and other proteins and which have been shown to have essential functions in diverse basic cellular processes from DNA replication to transcription to chromatin remodeling. Our targeted mutational analysis has shown that deletion of the first bromodomain in BRDT and complete loss of BRDT function in the mouse model both lead to male sterility but with strikingly different outcomes-abnormal differentiation of haploid spermatids in the former and arrest in meiosis in the latter. Our studies will provide important insight into the potential dysfunction of BRDT in cases of unexplained (or idiopathic) infertility in men and, given very recent studies showing that BET proteins are 'druggable', may provide a new and novel target for male contraception.
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