In metazoans, covalent modifications of histones act in a combinatorial manner to regulate developmental gene expression (Allis and Jenuwein, 2016; Zhao and Shilatifard, 2019). Among these modifications, histone H3 lysine 27 acetylation (H3K27ac) is considered a hallmark of trithorax group (trxG) mediated gene activation that antagonizes repression by Polycomb group (PcG) proteins (Tie et al., 2009). Although the anti-silencing effect of H3K27ac and its catalyzing enzyme CREB binding protein (CBP) is widely documented in flies and mammals, how this CBP mediated H3K27ac is maintained on actively transcribed genes remains elusive (Tie et al., 2009; Pasini et al., 2010). In Drosophila, presence of only one homolog of CBP known as nejire, makes it suitable to understand its function. Importantly, chromatin immunoprecipitation sequencing (ChIP-seq) data from previously published reports revealed the presence of CBP on a majority of actively transcribed genes in Drosophila S2 cells (Philip et al., 2015). Our lab recently showed that Drosophila Ballchen (BALL), a known serine-threonine kinase that mediates histone H2AT119 phosphorylation (Aihara et al., 2004), associates with trxG target genes enriched with H3K27ac (Khan et al., 2021). In this report, we have further analyzed the link between BALL, H3K27ac and CBP. We have discovered that BALL not only binds to chromatin enriched with H3K27ac but also shares more than 77% genomic binding sites with CBP. Both BALL and CBP primarily associate with transcription start sites (TSS) and regions up to 1 Kb upstream of TSS. Four of top five DNA motifs in chromatin that CBP binds are also bound by BALL. Importantly, BALL biochemically interacts with CBP and positively contributes to maintenance of H3K27ac by CBP since depletion of BALL leads to diminished H3K27ac.

Recently we reported that BALL exhibits trxG like behavior by contributing to the maintenance of gene activation in flies (Khan et al., 2021). Since BALL was shown to co-localize with Trithorax (TRX) at genes enriched with H3K27ac, we investigated if BALL binds to chromatin together with CBP. To this end, we analyzed ChIP-seq data of BALL (Khan et al., 2021) and CBP (Philip et al., 2015) from Drosophila S2 cells. Comparison of BALL and CBP binding to chromatin revealed their co-occupancy at 4577 genes (Figure 1A and Supplementary Table 1). Further analysis revealed that both BALL and CBP shared binding sites in genomic regions up to 1 Kb upstream of the transcription start sites (TSS), however, CBP is also found at the regions that are further upstream of TSS (Figures 1B,C). This finding is in line with the reported binding of CBP in promoter as well as enhancer regions (Philip et al., 2015; Boija et al., 2017). Analysis of the genomic distribution of CBP and BALL in terms of their binding at promoter regions, gene body, introns and intergenic regions also revealed similar chromatin binding patterns (Figure 1D). Of 6,553 overlapping binding sites for both BALL and CBP, 5,865 were mainly clustered on promoter regions (Supplementary Figure 1 and Supplementary Table 2). Importantly, promoters of different genes have variable number of peaks, i.e., some promoters have multiple binding sites while others have a single binding site. Binding profile of BALL closely mirrored that of CBP and H3K27ac at a majority of genomic regions. Additionally, most of these shared loci were also expressed in S2 cells, as highlighted by analysis of RNA-seq data (Gan et al., 2010; Figure 1E). Of 4577 genes where both BALL and CBP are present, 3114 (68%) were indeed found to be actively transcribed in S2 cells (Figure 1F and Supplementary Table 1). Comparison between top scoring DNA motifs present within chromatin regions where both BALL and CBP preferentially bind revealed a distinct similarity (Figure 1G). Collectively, our data highlights that BALL binding pattern substantially overlaps the binding of CBP across the whole genome. These findings prompted us to investigate whether BALL and CBP biochemically interact with each other. To this end, co-immunoprecipitation (co-IP) experiments were performed using S2 cells that stably expressed FLAG-tagged BALL. Immunoprecipitation of BALL-FLAG resulted in precipitation of endogenous CBP as compared to empty vector control (Figure 1H). To confirm this interaction, a reciprocal co-IP was performed using anti-CBP antibody, which resulted in immunoprecipitation of BALL-FLAG (Figure 1I) as compared to mock IP. These results illustrate a hitherto unknown interaction between BALL and CBP, which may explain their co-occupancy at actively transcribed regions.

Next, we investigated if depletion of BALL affects CBP mediated H3K27ac levels globally. Western blot analysis of cells where ball was knocked down revealed drastic reduction in H3K27ac levels as compared to control cells treated with dsRNA against LacZ (Figure 2A). The global levels of histone H3 remained unchanged which were used as normalization control to quantify reduction in levels of H3K27ac (Supplementary Figure 2). To validate this finding in vivo, we generated homozygous ball² mitotic clones using the flp/FRT system. Immunostaining of imaginal discs with ball² mutant clones showed a drastic reduction in H3K27ac (Figures 2B–E and Supplementary Figure 3). Notably, not all homozygous ball² mitotic clones show diminished H3K27ac which reiterates similar effects observed in trx mutant clones on expression of actively transcribed genes. It could be explained by the complex interplay of different cell signaling pathways in imaginal discs (Klymenko and Müller, 2004).

Together, these results demonstrate that BALL positively contributes to CBP mediated H3K27ac but the molecular nature of this interaction between BALL and CBP remains unclear. Whether they are part of a single multi-protein complex and if they directly interact with each other or it involves an indirect interaction remains elusive. Since BALL is a histone kinase, it is imperative to investigate whether the impact that BALL exerts on H3K27ac is through the phosphorylation of neighboring H3 residues or due to the direct phosphorylation of CBP or its interacting proteins. Interestingly, VRK1 (Vaccinia related kinase 1), the mammalian homolog of BALL, was shown to phosphorylate CREB at serine 133 (Kang et al., 2008). This phosphorylation is known to enhance CREB interaction with CBP, leading to increased transcription of CREB target genes (Radhakrishnan et al., 1998). The VRK1, CBP and CREB nexus may explain the regulation of CBP mediated gene activation by BALL in flies, which warrants further investigation.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary Material.

AS and MHFK performed the research. MHFK, AS, ZU, HA, and MT wrote the manuscript. All authors contributed to the article and approved the submitted version.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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