The first bromodomain of the testis-specific double bromodomain protein Brdt is required for chromocenter organization that is modulated by genetic background

Mice homozygous for a mutation (Brdt∆BD1/∆BD1) lacking the first bromodomain of Brdt, a testis-specific member of the BET family of double-bromodomain containing proteins, are sterile and exhibit profound defects in chromatin remodeling during spermiogenesis. We have now observed that a prominent feature of the aberrant spermatid nuclei is a fragmented chromocenter, a structure comprised of peri-centromeric heterochromatin. There was a concomitant increase in the levels of heterochromatin protein 1 alpha (Hp1α), suggesting that the presence of multiple chromocenters was correlated with a spread of heterochromatin beyond the normal centromeric region. Brdt protein was normally present throughout the nucleus but was excluded from the chromocenter. A more densely staining region of Brdt protein appeared to separate sirtuin 1 (Sirt1) protein from contact with the chromocenter. Although still nuclear, this unique localization of Brdt protein was lost in Brdt∆BD1/∆BD1 mutant spermatids and Brdt and Sirt1 overlapped around the chromocenters. There was also ectopic localization of the H1 histone family, member N, testis-specific (H1fnt) protein in Brdt∆BD1/∆BD1 round spermatids, which may be linked to the previously reported loss of polarized localization of peri-nuclear heterochromatin foci. The extent of chromocenter fragmentation was more severe and penetrant in mutant testes on a pure 129Sv/Ev as compared to a pure C57Bl/6 background. Indeed, all aspects of the mutant phenotype were more severe on the 129 Sv/Ev background. Contrary to previous studies in genetic models where fragmented chromocenters were observed in spermatids, the Brdt∆BD1/∆BD1 mutant spermatids do not undergo apoptosis (on either background). These observations suggest that the first bromodomain of Brdt is critical in the formation and/or maintenance of an intact chromocenter and implicate this structure in proper remodeling of the chromatin architecture of the sperm head.

► Brdt protein is localized to nuclei in round spermatids, but is excluded from the chromocenter. ► An increased localization of Brdt surrounds the chromocenter, separating it from Sirt1 protein. ► This unique Brdt localization is lost in Brdt∆BD1/∆BD1 mutant spermatids. ► The Brdt∆BD1/∆BD1 mutant testis has increased levels of heterochromatin. ► Multiple chromocenters are a prominent feature of the Brdt∆BD1/∆BD1 mutant phenotype.