The role of bromodomain proteins for transcription of translationally repressed mRNAs with relevance for spermiogenesis and the identification of direct and indirect target genes of bromodomain proteins.
Developmental Biology
Final Report Abstract
Male germ cell differentiation proceeds to a large extent in the absence of active gene transcription. In Drosophila, hundreds of genes whose proteins are required during postmeiotic spermatid differentiation (spermiogenesis) are transcribed in primary spermatocytes. Transcription of these spermiogenesis-relevant genes depends on the sequential action of the testis meiotic arrest complex (tMAC), Mediator complex and testis-specific TFIID (tTFIID) complex. The testis-specifically expressed bromodomain proteins tBRD-1 and tBRD-2 are expressed in highly transcriptionally active primary spermatocytes. Loss of function of tBRD- 1 leads to male sterility and a down-regulation of about 420 genes. Moreover, tBRD-1 colocalises and interacts with tBRD-2 and the testis-specific TATA box binding proteinassociated factor (tTAF) Spermatocyte arrest (Sa). Here, we validated our previous microarray analyses and show that tBRD-1 is directly or indirectly involved in gene expression within the testis. In addition, we demonstrate that tBRD-2 function is essential for male fertility. The knockdown of tbrd-2 leads to altered gene expression in the testis and to defects in post-meiotic male germ cells. Our comparison of different microarray data sets showed that in primary spermatocytes, the expression of a defined number of genes depend on the function of the bromodomain proteins tBRD-1 and tBRD-2, the tMAC component Aly, the Mediator component Med22 and the tTAF Sa. Performing protein-protein interaction experiments we observed that tBRD-1 was able to form homodimers as well as heterodimers with tBRD-2 and the tTAFs Sa, Rye and Can. Heterodimer formation was detectable between tBRD-2 and Rye. Among the tTAFs homodimerisation was only demonstrated for Rye. Further, we show that also in vivo tBRD-1 forms homodimers as well as heterodimers with tBRD-2. Finally, we demonstrate that the spacer region between the two bromodomains of tBRD-1 is essential for homodimer formation and that tBRD-1 and tBRD-2 interact via the C-terminus of tBRD-1 and the region between the NET and the SEED domains of tBRD-2. Here, we clearly demonstrated that tBRD-1 and tBRD-2 regulate expression of genes necessary for spermatid differentiation. We hypothesise that both proteins act at the end of a gene regulatory cascade involving tMAC, Mediator and tTAF functions to regulate expression of spermiogenesis-relevant genes.
Publications
- (2014) tBRD-1 selectively controls gene activity in the Drosophila testis and interacts with two new members of the bromodomain and extraterminal (BET) family. PLOS One 9: e108267
Theofel I, Bartkuhn M, Hundertmark T, Boettger T, Gärtner SMK, Leser K, Awe S, Schipper M, Renkawitz-Pohl R, Rathke C
(See online at https://doi.org/10.1371/journal.pone.0108267) - (2016) The bromodomain protein tBRD-1 interacts with the BET family members tBRD-2 and tBRD-3 and is required for gene activation in Drosophila male germ cells. Dissertation
Ina Theofel
- (2017) Analysis of chromatin dynamics during Drosophila spermatogenesis. In: Methods in Molecular Biology 1471:289-303 (ISBN 978-1-4939-6338-6)
Hundertmark T, Theofel I, Eren-Ghiani Z, Miller D, Rathke C
- (2017) tBRD-1 and tBRD-2 regulate expression of genes necessary for spermatid differentiation. Biology Open 6: 439-448
Theofel I, Bartkuhn M, Boettger T, Gärtner SMK, Kreher J, Brehm A, Rathke C
(See online at https://doi.org/10.1242/bio.022467)
