The bromodomain is an evolutionarily conserved motif that binds acetylated lysines in histones and other proteins. The BET sub-class of mammalian bromodomain-containing proteins is unique in that its members contain two bromodomains (BD1 and BD2) and an extra terminal (ET) domain. There are four BET family members in mouse (and human) - Brd2, Brd3, Brd4, and Brdt - and they are expressed in a striking and dynamic pattern in the male germ line. We have generated a mutation in the mouse Brdt gene, designated Brdt BD1, that yields a truncated protein lacking the first of the two bromodomains. Homozygous Brdt BD1 progeny are viable but the males are sterile, producing fewer sperm that are morphologically abnormal.
Aim 1 will test the hypotheses that i) Brdt functions as part of a transcription complex that regulates a set of genes whose expression is essential for spermatogenesis, and ii) that the BD1 of Brdt is required for this regulation. We will identify genes whose expression is changed in the absence of BD1 by microarray analysis and concomitantly, examine the chromatin modification status of the H1t promoter as a model for Brdt-complex binding regions.
Aim 2 will test the alternative, but not mutually exclusive, hypothesis that Brdt-containing complexes function to mark regions of the spermatid genome for subsequent recognition by complexes that are involved in the unique changes in chromatin structure during spermiogenesis. ChIP with anti-Brdt antibodies followed by genome-wide sequencing using the Solexa/Illumina 1 G technology will be used.
Aim 3 will test the hypotheses that first, the two bromodomains of Brdt have distinct functions in modulating transcription and/or chromatin re-modeling during spermiogenesis;second, that Brdt may function in several stages and processes of spermatogenesis, in addition to spermiogenesis;and third, that the Brdt BD1-mutant allele is a hypomorphic allele. We will generate a mutant allele producing Brdt protein containing BD1 but lacking BD2 (Aim 3a) and a mutant Brdt allele completely lacking functional protein (Aim 3b). We predict that the resulting phenotypes will overlap in part but will be distinct from the BD1-deficient mutant. Understanding the function of Brdt during spermatogenesis will provide a powerful developmental model system for elucidating the role of the BET genes during normal differentiation. Importantly, spermatogenesis is also a physiologically relevant system in which histone acetylation is clearly linked to chromatin remodeling.

Public Health Relevance

Brdt is a member of a sub-family of bromodomain-containing proteins which have recently been shown to have essential functions in diverse basic cellular functions from DNA replication to transcription to chromatin remodeling. Our targeted mutational analysis has shown that deletion of the first of the two bromodomains in Brdt in the mouse model leads to male sterility, but the animals are otherwise viable and the females are fertile. Our studies will provide important insight into the potential mis- function of human BRDT in cases of unexplained (or idiopathic) infertility in men and 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 #
5R01GM081767-04
Application #
8116439
Study Section
Cellular, Molecular and Integrative Reproduction Study Section (CMIR)
Program Officer
Haynes, Susan R
Project Start
2008-09-15
Project End
2013-07-31
Budget Start
2011-08-01
Budget End
2013-07-31
Support Year
4
Fiscal Year
2011
Total Cost
$326,789
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
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