Kinome-Wide RNAi Screen Uncovers Role of Ballchen in Maintenance of Gene Activation by Trithorax Group in Drosophila

Abstract

Polycomb group (PcG) and trithorax group (trxG) proteins are evolutionary conserved factors that contribute to cell fate determination and maintenance of cellular identities during development of multicellular organisms. The PcG maintains heritable patterns of gene silencing while trxG acts as anti-silencing factors by conserving activation of cell type specific genes. Genetic and molecular analysis has revealed extensive details about how different PcG and trxG complexes antagonize each other to maintain cell fates, however, the cellular signaling components that contribute to the preservation of gene expression by PcG/trxG remain elusive. Here, we report an ex vivo kinome-wide RNAi screen in Drosophila aimed at identifying cell signaling genes that facilitate trxG in counteracting PcG mediated repression. From the list of trxG candidates, Ballchen (BALL), a histone kinase known to phosphorylate histone H2A at threonine 119 (H2AT119p), was characterized as a trxG regulator. The ball mutant exhibits strong genetic interactions with Polycomb (Pc) and trithorax (trx) mutants and loss of BALL affects expression of trxG target genes. BALL co-localizes with Trithorax on chromatin and depletion of BALL results in increased H2AK118 ubiquitination, a histone mark central to PcG mediated gene silencing. Moreover, BALL was found to substantially associate with known TRX binding sites across the genome. Genome wide distribution of BALL also overlaps with H3K4me3 and H3K27ac at actively transcribed genes. We propose that BALL mediated signaling positively contributes to the maintenance of gene activation by trxG in counteracting the repressive effect of PcG.

Interplay Between BALL and CREB Binding Protein Maintains H3K27 Acetylation on Active Genes in Drosophila

“…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.…”

“…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 ).…”

“…Chromatin immunoprecipitation sequencing (ChIP-seq) analysis was done as described previously ( Khan et al, 2021 ). Briefly, the sequencing datasets were uploaded to the Galaxy web public server ( Jalili et al, 2020 ).…”

“…MEME-ChIP - motif discovery, enrichment analysis and clustering on large nucleotide datasets (Galaxy Version 4.11.2 + galaxy1) was utilized to generate DREME ( D iscriminative R egular E xpression M otif E licitation) output of BALL and CBP ChIP-seq data for de novo motif discovery ( Jalili et al, 2020 ). ChIP-seq data of CBP, BALL, and H3K27ac were taken from GSE64464 ( Philip et al, 2015 ), GSE165685 ( Khan et al, 2021 ) and GSE81795 ( Rickels et al, 2016 ), respectively. RNA-seq data of Drosophila S2 cells (GSM480160) ( Gan et al, 2010 ) was mapped using TopHat Gapped-read mapper for RNA-seq data (Galaxy Version 2.1.1).…”

“…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.…”