PNUTS/PP1 regulates RNAPII-mediated gene expression and is necessary for developmental growth.

In multicellular organisms, tight regulation of gene expression ensures appropriate tissue and organismal growth throughout development. Reversible phosphorylation of the RNA Polymerase II (RNAPII) C-terminal domain (CTD) is critical for the regulation of gene expression states, but how phosphorylation is actively modified in a developmental context remains poorly understood. Protein phosphatase 1 (PP1) is one of several enzymes that has been reported to dephosphorylate the RNAPII CTD. However, PP1's contribution to transcriptional regulation during animal development and the mechanisms by which its activity is targeted to RNAPII have not been fully elucidated. Here we show that the Drosophila orthologue of the PP1 Nuclear Targeting Subunit (dPNUTS) is essential for organismal development and is cell autonomously required for growth of developing tissues. The function of dPNUTS in tissue development depends on its binding to PP1, which we show is targeted by dPNUTS to RNAPII at many active sites of transcription on chromosomes. Loss of dPNUTS function or specific disruption of its ability to bind PP1 results in hyperphosphorylation of the RNAPII CTD in whole animal extracts and on chromosomes. Consistent with dPNUTS being a global transcriptional regulator, we find that loss of dPNUTS function affects the expression of the majority of genes in developing 1(st) instar larvae, including those that promote proliferative growth. Together, these findings shed light on the in vivo role of the PNUTS-PP1 holoenzyme and its contribution to the control of gene expression during early Drosophila development.

GAL4 induces transcriptionally active puff in the absence of dSAGA- and ATAC-specific chromatin acetylation in the Drosophila melanogaster polytene chromosome.

Puffs in the polytene chromosome of Drosophila melanogaster are characteristic of sites of high-level active transcription which can be observed directly under the microscope. We studied the dependence of puff formation on chromatin modifications at a site where a GAL4-inducible transgene is located in the 61C7 cytological region. Immunostaining of salivary gland polytene chromosomes indicated no increase of either dSAGA-specific histone H3 lysine 14, or ATAC-specific histone H4 lysine 5 and 12 acetylation in the puffed region. Nor did we observe increased levels of H4K8ac, H3K18ac, or H4K16ac in the puff. In accordance with the above, puff formation as well as localization of Pol II and GAL4 was detectable at the 61C region in dAda2b and dAda2a null homozygotes, which are dSAGA- and ATAC-specific mutants, respectively. Moreover, the reduced level of JIL-1-specific H3 serine 10 phosphorylation did not abolish puff formation in ATAC mutants. Surprisingly, in wild-type animals dADA3 and GCN5 shared constituents of dSAGA and ATAC, as well as JIL-1 localized specifically to the puff, where the JIL-1-phosphorylated H3S10ph level was also high. Altogether these data strongly suggest that the GAL4 activator can induce transcription and chromatin reorganization seen as a puff without dSAGA- and ATAC-specific histone acetylation and JIL-1-specific histone H3 phosphorylation.

Loss of ATAC-specific acetylation of histone H4 at Lys12 reduces binding of JIL-1 to chromatin and phosphorylation of histone H3 at Ser10.

Various combinations of post-translational modifications of the N-terminal tails of nucleosomal histones serve as signals to govern chromatin-related processes. The relationship, however, among different types of histone modifications - most frequently acetylation, phosphorylation and methylation - and the order of their establishment has been explored only in a few cases. Here we show that a reduced level of histone H4 acetylated at Lys12 by the ATAC-HAT complex leads to a decrease in the histone H3 phosphorylation at Ser10 by the kinase JIL-1. As JIL-1 activity antagonizes histone H3 dimethylation at Lys9 by SU(VAR)3-9, our observations demonstrate the interdependent actions of an acetyltransferase, a kinase and a methyltransferase. We demonstrate that, in accord with the steps of modifications, mutations that affect ATAC subunits (such as dGcn5, dAda2a and dAda3) (1) decrease the level histone H3 phosphorylation at Ser10, (2) can be rescued partially by JIL-1 overproduction, (3) enhance the spread of histone H3 dimethylation at Lys9 and (4) are suppressed by mutations of Su(var)3-9. We propose that a reduced level of histone H4 acetylated at Lys12 by ATAC attenuates histone H3 phosphorylation at Ser10 by JIL-1 owing to reduced binding of JIL-1 to hypoacetylated chromatin.

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

Drosophila 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.

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.