Theofel Ina, Bartkuhn Marek, Hundertmark Tim, Boettger Thomas, Gärtner Stefanie M K, Leser Katja, Awe Stephan, Schipper Michael, Renkawitz-Pohl Renate, Rathke Christina
Philipps-University Marburg, Department of Biology, Marburg, Germany.
Institute for Genetics, Justus-Liebig-University, Giessen, Germany.
PLoS One. 2014 Sep 24;9(9):e108267. doi: 10.1371/journal.pone.0108267. eCollection 2014.
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.
多细胞生物已经进化出专门的机制来以空间和时间方式控制转录。基因激活与赖氨酸残基上的组蛋白乙酰化紧密相关，赖氨酸残基可被溴结构域识别。此前，在果蝇中鉴定出睾丸特异性表达的溴结构域蛋白tBRD-1。tBRD-1的表达仅限于高度转录活跃的初级精母细胞。tBRD-1对雄性生育力至关重要，并被认为作为睾丸特异性TATA盒结合蛋白相关因子（tTAFs）的辅助因子参与睾丸特异性转录。在此，我们进行了微阵列分析以比较tbrd-1突变体睾丸和野生型睾丸的转录组。我们的数据证实tBRD-1控制雄性生殖细胞中的基因活性。此外，比较tbrd-1和tTAF突变体睾丸的转录组揭示了一组共同的靶基因。我们还对溴结构域和额外末端（BET）家族的两个新成员tBRD-2和tBRD-3进行了表征。与动物中BET家族的其他成员不同，它们都只拥有一个溴结构域，这是植物BET家族成员的一个特征。免疫组织学技术不仅揭示了tBRD-2和tBRD-3在初级精母细胞中与tBRD-1和tTAFs部分共定位，还揭示了它们在tbrd-1和tTAF突变体睾丸中的正确亚细胞分布受到损害。用抑制剂处理培养的雄性生殖细胞表明tBRD-2和tBRD-3的定位取决于初级精母细胞内的乙酰化状态。使用果蝇睾丸蛋白提取物进行的酵母双杂交试验和免疫共沉淀表明，tBRD-1能够与tBRD-2、tBRD-3和tTAFs形成同二聚体以及异二聚体。这些数据首次揭示了动物中存在单溴结构域BET蛋白，以及存在一个包含tBRDs和tTAFs的复合物的证据，该复合物调节与精子发生相关的一组基因的转录。
