TBRD-1 selectively controls gene activity in the Drosophila testis and interacts with two new members of the bromodomain and extra-terminal (BET) family.

Ina Theofel,Katja Leser,Thomas Boettger,Christina Rathke,Stephan Awe,Marek Bartkuhn,Tim Hundertmark,Michael Schipper,Stefanie M K Gärtner,Renate Renkawitz-Pohl,Helen White-Cooper

doi:10.1371/journal.pone.0108267

Abstract

Multicellular organisms have evolved specialized mechanisms to control transcription in a spatial and temporal manner. Gene activation is tightly linked to histone acetylation on lysine residues that can be recognized by bromodomains. Previously, the testis-specifically expressed bromodomain protein tBRD-1 was identified in Drosophila. Expression of tBRD-1 is restricted to highly transcriptionally active primary spermatocytes. tBRD-1 is essential for male fertility and proposed to act as a co-factor of testis-specific TATA box binding protein-associated factors (tTAFs) for testis-specific transcription. Here, we performed microarray analyses to compare the transcriptomes of tbrd-1 mutant testes and wild-type testes. Our data confirmed that tBRD-1 controls gene activity in male germ cells. Additionally, comparing the transcriptomes of tbrd-1 and tTAF mutant testes revealed a subset of common target genes. We also characterized two new members of the bromodomain and extra-terminal (BET) family, tBRD-2 and tBRD-3. In contrast to other members of the BET family in animals, both possess only a single bromodomain, a characteristic feature of plant BET family members. Immunohistology techniques not only revealed that tBRD-2 and tBRD-3 partially co-localize with tBRD-1 and tTAFs in primary spermatocytes, but also that their proper subcellular distribution was impaired in tbrd-1 and tTAF mutant testes. Treating cultured male germ cells with inhibitors showed that localization of tBRD-2 and tBRD-3 depends on the acetylation status within primary spermatocytes. Yeast two-hybrid assays and co-immunoprecipitations using fly testes protein extracts demonstrated that tBRD-1 is able to form homodimers as well as heterodimers with tBRD-2, tBRD-3, and tTAFs. These data reveal for the first time the existence of single bromodomain BET proteins in animals, as well as evidence for a complex containing tBRDs and tTAFs that regulates transcription of a subset of genes with relevance for spermiogenesis.

Highlights

During spermatogenesis male germ cells must pass through a highly organized differentiation process to produce haploid sperm
Results tBRD-1 is required for gene expression in the testis Previously, we showed that tBRD-1 partially co-localizes with testis-specific TATA box binding protein-associated factors (TAFs) (tTAFs) in primary spermatocytes
We proposed that tBRD-1 acts together with tTAFs to initiate transcription of a special subset of genes with relevance for spermiogenesis [19]

Summary

Introduction

During spermatogenesis male germ cells must pass through a highly organized differentiation process to produce haploid sperm. Since most transcription ceases with entry into the meiotic divisions, spermiogenesis is mainly based on translationally repressed and stored mRNAs (e.g. mRNAs that encode protamines) transcribed in a prolonged meiotic prophase in primary spermatocytes [3,4,5]. Primary spermatocytes are highly transcriptionally active cells that produce two different types of mRNAs: those required for the primary spermatocytes themselves and those that encode proteins necessary for spermiogenesis [2]. Proteins of the aly-class exhibit a broader range of target genes and are part of the testis-specific meiotic arrest complex (tMAC) [7]. The can-class comprises proteins expressed in the testis and homologous to the TATA box binding protein-associated factors (TAFs).

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