The Drosophila histone acetyltransferase Gcn5 and transcriptional adaptor Ada2a are involved in nucleosomal histone H4 acetylation

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

The Homologous Drosophila Transcriptional Adaptors ADA2a and ADA2b Are both Required for Normal Development but Have Different Functions

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 Drosophila NURF remodelling and the ATAC histone acetylase complexes functionally interact and are required for global chromosome organization.

International audienceDrosophila Gcn5 is the catalytic subunit of the SAGA and ATAC histone acetylase complexes. Here, we show that mutations in Gcn5 and the ATAC component Ada2a induce a decondensation of the male X chromosome, similar to that induced by mutations in the Iswi and Nurf301 subunits of the NURF nucleosome remodelling complex. Genetic studies as well as transcript profiling analysis indicate that ATAC and NURF regulate overlapping sets of target genes during development. In addition, we find that Ada2a chromosome binding and histone H4-Lys12 acetylation are compromised in Iswi and Nurf301 mutants. Our results strongly suggest that NURF is required for ATAC to access the chromatin and to regulate global chromosome organization

dPNUTS is a nuclear protein that colocalises with transcriptionally active RNAPII on salivary gland polytene chromosomes.

<p>A) Distribution of <i>dPNUTS</i> transcripts detected by RNA <i>in situ</i> hybridization; <i>dPNUTS</i> transcripts are maternally provided (top left) and are ubiquitously distributed in embryos at cellularisation (top right). At gastrulation, d<i>PNUTS</i> mRNA levels are enriched in the germband and in the fore- and hind-gut (fg and hg, respectively). Later, d<i>PNUTS</i> is highly expressed in the brain (br) and ventral nerve cord (vnc). Embryonic stage and approximate age, hours post fertilization (hpf), are indicated. B) 3^rd instar wing discs stained to reveal the distribution of ectopically expressed Myc-tagged dPNUTS (green in merge), Histone H3S10ph (red in merge, marking mitotic nuclei) and DNA. C) Images of whole mount salivary gland and magnified images of an individual nucleus (below), stained to show the localization of Myc-tagged dPNUTS (green in merge) and DNA (magenta in merge). D) Line scans of images in C) reveal that Myc-tagged dPNUTS is localised to interbands that stain weakly for DNA. Fluorescence intensity of anti-Myc antibody and TOPRO-3 staining was measured along a line through the indicated chromosomal region in the images shown. The profile plot below shows that the peaks of Myc-PNUTS and DNA of staining do not overlap. E) Polytene chromosomes from salivary gland squashes showing that dPNUTS localises to a number of discrete bands that are broadly distributed. F) Merging of the green signal representing dPNUTS with the red signal representing RNAPII Ser2-P (H5) identifies sites where these two proteins co-localize (example indicated with arrow). The relative signals of dPNUTS and RNAPII Ser2-P vary between sites, but the majority dPNUTS loci colocalize with RNAPII Ser2-P staining (star indicates example where only dPNUTS staining is visible).</p

PP1 localisation is regulated by dPNUTS and binding to PP1 is important for dPNUTS function.

<p>A) Images of polytene chromosome squashes from salivary glands expressing either <i>dPNUTS^WT</i> or <i>dPNUTS^W726A</i> stained with PP1 (in green). Inset are enlarged views of the distal end of the X chromosome. Arrows indicate approximate lines along which quantitation of fluorescence (see B) was performed. B) Plots of line scans through the chromosomal region indicated in A, showing levels of PP1 staining in salivary glands expressing either <i>dPNUTS^WT</i> or <i>dPNUTS^W726A</i>. Bar graphs represent the average fluorescence in this region from 6 independent images/genotype. Genotypes are indicated by the colour key. C) Levels and distribution of Myc-dPNUTS on polytene chromosome squashes from salivary glands expressing either <i>dPNUTS^WT</i> or <i>dPNUTS^W726A</i>, as revealed by anti-Myc staining (green). D) Western blots showing levels of PP1 and Myc-dPNUTS, relative to Actin, in extracts from animals ectopically expressing <i>dPNUTS^WT</i> or <i>dPNUTS^W726A</i> under the control of <i>da-GAL4</i> (da><i>dPNUTS^WT</i> and da><i>dPNUTS^W726A</i>, respectively) compared to <i>w¹¹¹⁸</i> control (−). E) Images of adult female eyes showing that the severely reduced eye phenotype of homozygous <i>dPNUTS</i> mutant eyes is fully rescued by ectopic expression of <i>dPNUTS^WT</i> but not <i>dPNUTS^W726A</i>. F) Homozygous <i>dPNUTS^KG572</i> mutant eyes show a weaker phenotype than either <i>dPNUTS^9B</i> or <i>dPNUTS^13B</i>, and this can be enhanced by loss of one copy of <i>PP187B</i>.</p