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.
Highlights
In metazoans, specialization of cell types that make up an organism is linked to cell type specific gene expression patterns established during early development
Drosophila cells treated with dsRNAs were co-transfected with PRE-F.Luc reporter and actin promoter-driven Renilla luciferase (R.Luc) which was used as a normalization control (Figure 1A and Supplementary Figure 1)
The depletion of BALL resulted in decreased expression of pnt and pnr as compared to cells treated with dsRNA against LacZ (Figure 3I). These results suggest that BALL is required by trithorax group (trxG) to maintain gene activation and it may counteract Polycomb group (PcG) by inhibiting H2AK118ub1, a histone modification catalyzed by PRC1 subunit dRING, and in turn shift the balance in favor of trxG
Summary
Specialization of cell types that make up an organism is linked to cell type specific gene expression patterns established during early development. Molecular analysis revealed that proteins encoded by the PcG and trxG act in large multi-protein complexes, and modify the local properties of chromatin to maintain expression patterns of their target genes. Both groups exert their functions by binding to chromosomal elements known as PREs (polycomb response elements) and by interacting with histones and transcription machinery (Kassis et al, 2017; Cavalli and Heard, 2019). In contrast to PcG, trxG is more heterogeneous and comprises of proteins that activate transcription by modifying histone tails or remodeling chromatin (Schuettengruber et al, 2017) Despite their diversity, one cellular function that unifies trxG proteins is their role in counteracting PcG mediated gene silencing
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