Research Article: Drosophila melanogaster tPlus3a and tPlus3b ensure full male fertility by regulating transcription of Y-chromosomal, seminal fluid, and heat shock genes

Date Published: March 7, 2019

Publisher: Public Library of Science

Author(s): Tim Hundertmark, Sabrina Kreutz, Nastasja Merle, Andrea Nist, Boris Lamp, Thorsten Stiewe, Alexander Brehm, Renate Renkawitz-Pohl, Christina Rathke, Shree Ram Singh.

http://doi.org/10.1371/journal.pone.0213177

Abstract

Spermatogenesis in Drosophila melanogaster is characterized by a specific transcriptional program during the spermatocyte stage. Transcription of thousands of genes is regulated by the interaction of several proteins or complexes, including a tTAF-containing TFIID variant, tMAC, Mediator, and chromatin interactors, e.g., bromodomain proteins. We addressed how distinct subsets of target genes are selected. We characterized the highly similar proteins tPlus3a and tPlus3b, which contain a Plus3 domain and are enriched in the testis, mainly in spermatocytes. In tPlus3a and tplus3b deletion mutants generated using the CRISPR/Cas9 system, fertility was severely reduced and sperm showed defects during individualization. tPlus3a and tPlus3b heterodimerized with the bromodomain protein tBRD-1. To elucidate the role of the tPlus3a and tPlus3b proteins in transcriptional regulation, we determined the transcriptomes of tplus3a-tplus3b and tbrd-1 deletion mutants using next-generation sequencing (RNA-seq) and compared them to that of the wild-type. tPlus3a and tPlus3b positively or negatively regulated the expression of nearly 400 genes; tBRD-1 regulated 1,500 genes. Nearly 200 genes were regulated by both tPlus3a and tPlus3b and tBRD-1. tPlus3a and tPlus3b activated the Y-chromosomal genes kl-3 and kl-5, which indicates that tPlus3a and tPlus3b proteins are required for the function of distinct classes of genes. tPlus3a and tPlus3b and tBRD-1 repress genes relevant for seminal fluid and heat shock. We hypothesize that tPlus3a and tPlus3b proteins are required to specify the general transcriptional program in spermatocytes.

Partial Text

In mammals and in Drosophila melanogaster, regulation of transcription during spermatogenesis is complex. The germ cell transcriptional program covers various processes, including meiosis, post-meiotic formation of flagella, nuclear shaping, and chromatin reorganization. In flies, the majority of transcripts needed in spermatogenesis are produced in the prolonged meiotic prophase of the spermatocyte stage. Therefore, most transcripts required for post-meiotic sperm development (spermiogenesis) are translationally repressed until they are needed in later stages [1–4]. About 50% of the predicted genes in the D. melanogaster genome are expressed in the testis [5], and a large portion of the transcripts are testis enriched or even testis-specific.

D. melanogaster spermatogenesis is characterized by a unique transcriptional program in spermatocytes. Thousands of transcripts are synthesized in this germ cell stage, but most are only needed much later in post-meiotic development [1–3]. Jiang et al. [33] reported that one-third of the tTAF-dependent genes is regulated by Modulo or Acj6. In our previous studies, we described a possible interplay between tBRDs and the testis transcription machinery and a role of tBRDs in guiding the complexes Mediator, tMAC, and TFIID to specific subsets of target genes [11–12]. We hypothesize that in addition to tBRD-1 and tBRD-2, other factors are required for fine-tuning gene expression during spermatogenesis. In the current study, we characterized synthesis and function of tPlus3a and tPlus3b. We show that tPlus3a and tPlus3b and the bromodomain protein tBRD-1 co-regulate distinct groups of target genes, which underscores their potential for fine-tuning the transcriptional regulatory network in spermatocytes. tplus3a-tplus3b are target genes of Aly and MED22 [8, 34], thus probably of the tMAC and Mediator complexes and are also slightly down-regulated in tbrd-11 mutants, which indicates that regulators of transcription are interdependent.

 

Source:

http://doi.org/10.1371/journal.pone.0213177

 

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