The formation of matured and individual sperm involves a series of molecular and spectacular morphological changes of the developing cysts in Drosophila melanogaster testis. Recent advances in RNA Sequencing (RNA-Seq) technology help us to understand the complexity of eukaryotic transcriptomes by dissecting different tissues and developmental stages of organisms. To gain a better understanding of cellular differentiation of spermatogenesis, we applied RNA-Seq to analyse the testis-specific transcriptome, including coding and non-coding genes. We isolated three different parts of the wild-type testis by dissecting and cutting the different regions: 1.) the apical region, which contains stem cells and developing spermatocytes 2.) the middle region, with enrichment of meiotic cysts 3.) the basal region, which contains elongated post-meiotic cysts with spermatids. Total RNA was isolated from each region and analysed by next-generation sequencing. We collected data from the annotated 17412 Drosophila genes and identified 5381 genes with significant transcript accumulation differences between the regions, representing the main stages of spermatogenesis. We demonstrated for the first time the presence and region specific distribution of 2061 lncRNAs in testis, with 203 significant differences. Using the available modENCODE RNA-Seq data, we determined the tissue specificity indices of Drosophila genes. Combining the indices with our results, we identified genes with region-specific enrichment in testis. By multiple analyses of our results and integrating existing knowledge about Drosophila melanogaster spermatogenesis to our dataset, we were able to describe transcript composition of different regions of Drosophila testis, including several stage-specific transcripts. We present searchable visualizations that can facilitate the identification of new components that play role in the organisation and composition of different stages of spermatogenesis, including the less known, but complex regulation of post-meiotic stages.

In Drosophila and several other metazoan organisms, there are two genes that encode related but distinct homologs of ADA2-type transcriptional adaptors. Here we describe mutations of the two Ada2 genes of Drosophila melanogaster. By using mutant Drosophila lines, which allow the functional study of individual ADA2s, we demonstrate that both Drosophila Ada2 genes are essential. Ada2a and Ada2b null homozygotes are late-larva and late-pupa lethal, respectively. Double mutants have a phenotype identical to that of the Ada2a mutant. The overproduction of ADA2a protein from transgenes cannot rescue the defects resulting from the loss of Ada2b, nor does complementation work vice versa, indicating that the two Ada2 genes of Drosophila have different functions. An analysis of germ line mosaics generated by pole-cell transplantation revealed that the Ada2a function (similar to that reported for Ada2b) is required in the female germ line. A loss of the function of either of the Ada2 genes interferes with cell proliferation. Interestingly, the Ada2b null mutation reduces histone H3 K14 and H3 K9 acetylation and changes TAF10 localization, while the Ada2a null mutation does not. Moreover, the two ADA2s are differently required for the expression of the rosy gene, involved in eye pigment production, and for Dmp53-mediated apoptosis. The data presented here demonstrate that the two genes encoding homologous transcriptional adaptor ADA2 proteins in Drosophila are both essential but are functionally distinct.

The histone acetyltransferase (HAT) Gcn5 plays a role in chromatin structure and gene expression regulation as a catalytic component of multiprotein complexes, some of which also contain Ada2-type transcriptional coactivators. Data obtained mostly from studies on yeast (Saccharomyces cerevisiae) suggest that Ada2 potentiates Gcn5 activity and substrate recognition. dAda2b, one of two related Ada2 proteins of Drosophila melanogaster, was recently found to play a role in complexes acetylating histone 3 (H3). Evidence of an in vivo functional link between the related coactivator dAda2a and dGcn5, however, is lacking. Here we present data on the genetic interaction of dGcn5 and dAda2a. The loss of either dGcn5 or dAda2a function results in similar chromosome structural and developmental defects. In dAda2a mutants, the nucleosomal H4 acetylation at lysines 12 and 5 is significantly reduced, while the acetylation established by dAda2b-containing Gcn5 complexes at H3 lysines 9 and 14 is unaffected. The data presented here, together with our earlier data on the function of dAda2b, provide evidence that related Ada2 proteins of Drosophila, together with Gcn5 HAT, are involved in the acetylation of specific lysine residues in the N-terminal tails of nucleosomal H3 and H4. Our data suggest dAda2a involvement in both uniformly distributed H4 acetylation and gene-specific transcription regulation.

Uncovering mechanisms that regulate ecdysone production is an important step toward understanding the regulation of insect metamorphosis and processes in steroid-related pathologies. We report here the transcriptome analysis of Drosophila melanogaster dAda2a and dAda3 mutants, in which subunits of the ATAC acetyltransferase complex are affected. In agreement with the fact that these mutations lead to lethality at the start of metamorphosis, both the ecdysone levels and the ecdysone receptor binding to polytene chromosomes are reduced in these flies. The cytochrome genes (spookier, phantom, disembodied, and shadow) involved in steroid conversion in the ring gland are downregulated, while the gene shade, which is involved in converting ecdysone into its active form in the periphery, is upregulated in these dATAC subunit mutants. Moreover, driven expression of dAda3 at the site of ecdysone synthesis partially rescues dAda3 mutants. Mutants of dAda2b, a subunit of the dSAGA histone acetyltransferase complex, do not share phenotype characteristics and RNA profile alterations with dAda2a mutants, indicating that the ecdysone biosynthesis genes are regulated by dATAC, but not by dSAGA. Thus, we provide one of the first examples of the coordinated regulation of a functionally linked set of genes by the metazoan-specific ATAC complex.

Post-translational modifications (PTMs) of nucleosomal core histones play roles in basic biological processes via altering chromatin structure and creating target sites for proteins acting on chromatin. Several features make Drosophila a uniquely effective model for studying PTMs. Position effect variegation, polycomb repression, dosage compensation and several other processes extensively studied by the powerful tools of Drosophila genetics as well as polytene chromosome cytology reveal information on the dynamic changes of histone PTMs and factors that deposit, remove and recognize these. Recent determination of the genome-wide distribution of more than 20 different histone PTM types has resulted in a highly detailed view of chromatin landscape. This review samples from the wealth of data these analyses have provided together with data resulting from gene-targeted studies on the distribution and role of specific histone modifications and modifiers. As an example of the complex interactions among PTMs, we will also discuss crosstalk involving specific phosphorylated and acetylated histone forms.

We describe a novel Drosophila gene, dtl (Drosophila Tat-like), which encodes a 60-kDa protein with RNA binding activity and a methyltransferase (MTase) domain. Dtl has an essential role in Drosophila development. The homologs of DTL recently described include PIMT (peroxisome proliferator-activated receptor-interacting protein with a methyltransferase domain), an RNA-binding protein that interacts with and enhances the nuclear receptor coactivator function, and TGS1, the methyltransferase involved in the formation of the 2,2,7-trimethylguanosine (m₃G) cap of non-coding small RNAs. DTL is expressed throughout all of the developmental stages of Drosophila. The dtl mRNA has two ORFs (uORF and dORF). The product of dORF is the 60-kDa PIMT/TGS1 homolog protein that is translated from an internal AUG located 538 bp downstream from the 5′ end of the message. This product of dtl is responsible for the formation of the m₃G cap of small RNAs of Drosophila. Trimethylguanosine synthase activity is essential in Drosophila. The deletion in the dORF or point mutation in the putative MTase active site results in a reduced pool of m₃G cap-containing RNAs and lethality in the early pupa stage. The 5′ region of the dtl message also has the coding capacity (uORF) for a 178 amino acid protein. For complete rescue of the lethal phenotype of dtl mutants, the presence of the entire dtl transcription unit is required. Transgenes that carry mutations within the uORF restore the MTase activity but result in only partial rescue of the lethal phenotype. Interestingly, two transgenes bearing a mutation in uORF or dORF in trans can result in complete rescue.