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.

Public Health Relevance

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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
4R01GM081767-08
Application #
9113618
Study Section
Cellular, Molecular and Integrative Reproduction Study Section (CMIR)
Program Officer
Haynes, Susan R
Project Start
2008-09-15
Project End
2017-07-31
Budget Start
2016-08-01
Budget End
2017-07-31
Support Year
8
Fiscal Year
2016
Total Cost
Indirect Cost
Name
Columbia University (N.Y.)
Department
Genetics
Type
Schools of Medicine
DUNS #
621889815
City
New York
State
NY
Country
United States
Zip Code
10032
Manterola, Marcia; Brown, Taylor M; Oh, Min Young et al. (2018) BRDT is an essential epigenetic regulator for proper chromatin organization, silencing of sex chromosomes and crossover formation in male meiosis. PLoS Genet 14:e1007209
Wang, Li; Wolgemuth, Debra J (2016) BET Protein BRDT Complexes With HDAC1, PRMT5, and TRIM28 and Functions in Transcriptional Repression During Spermatogenesis. J Cell Biochem 117:1429-38
Garcia-Gutierrez, Pablo; Juarez-Vicente, Francisco; Wolgemuth, Debra J et al. (2014) Pleiotrophin antagonizes Brd2 during neuronal differentiation. J Cell Sci 127:2554-64
Chachua, T; Goletiani, C; Maglakelidze, G et al. (2014) Sex-specific behavioral traits in the Brd2 mouse model of juvenile myoclonic epilepsy. Genes Brain Behav 13:702-12
Berkovits, Binyamin D; Wolgemuth, Debra J (2013) The role of the double bromodomain-containing BET genes during mammalian spermatogenesis. Curr Top Dev Biol 102:293-326
Gaucher, Jonathan; Boussouar, Faycal; Montellier, Emilie et al. (2012) Bromodomain-dependent stage-specific male genome programming by Brdt. EMBO J 31:3809-20
Berkovits, Binyamin D; Wang, Li; Guarnieri, Paolo et al. (2012) The testis-specific double bromodomain-containing protein BRDT forms a complex with multiple spliceosome components and is required for mRNA splicing and 3'-UTR truncation in round spermatids. Nucleic Acids Res 40:7162-75
Shang, Enyuan; Cui, Qingping; Wang, Xiangyuan et al. (2011) The bromodomain-containing gene BRD2 is regulated at transcription, splicing, and translation levels. J Cell Biochem 112:2784-93
Velisek, Libor; Shang, Enyuan; Veliskova, Jana et al. (2011) GABAergic neuron deficit as an idiopathic generalized epilepsy mechanism: the role of BRD2 haploinsufficiency in juvenile myoclonic epilepsy. PLoS One 6:e23656
Berkovits, Binyamin D; Wolgemuth, Debra J (2011) The first bromodomain of the testis-specific double bromodomain protein Brdt is required for chromocenter organization that is modulated by genetic background. Dev Biol 360:358-68

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