Kinome-wide RNAi screen uncovers role of Ballchen in maintenance of gene activation by trithorax group in Drosophila
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ABSTRACT:
PROVIDER: PRJNA695452 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR13570882.fastq.gz | Fastqsanger.gz | |||
| SRR13570883.fastq.gz | Fastqsanger.gz | |||
| SRR13570884.fastq.gz | Fastqsanger.gz | |||
| SRR13570885.fastq.gz | Fastqsanger.gz |
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Project description:We report an ex vivo kinome-wide RNAi screen in Drosophila aimed to identify cell signaling genes that facilitate trxG to counteract 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. 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, analysis of genome-wide binding profile of BALL shows an overlap with 85% known binding sites of TRX across the genome. Both BALL and TRX are highly enriched at actively transcribed genes, which also correlate with presence of H3K4me3 and H3K27ac. We propose that BALL mediated signal positively contributes to the maintenance of gene activation by trxG by counteracting the repressive effect of PcG.
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Project description: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.
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Project description:BACKGROUND:Polycomb group (PcG) and trithorax group (trxG) proteins contribute to the specialization of cell types by maintaining differential gene expression patterns. Initially discovered as positive regulators of HOX genes in forward genetic screens, trxG counteracts PcG-mediated repression of cell type-specific genes. Despite decades of extensive analysis, molecular understanding of trxG action and regulation are still punctuated by many unknowns. This study aimed at discovering novel factors that elicit an anti-silencing effect to facilitate trxG-mediated gene activation. RESULTS:We have developed a cell-based reporter system and performed a genome-wide RNAi screen to discover novel factors involved in trxG-mediated gene regulation in Drosophila. We identified more than 200 genes affecting the reporter in a manner similar to trxG genes. From the list of top candidates, we have characterized Enoki mushroom (Enok), a known histone acetyltransferase, as an important regulator of trxG in Drosophila. Mutants of enok strongly suppressed extra sex comb phenotype of Pc mutants and enhanced homeotic transformations associated with trx mutations. Enok colocalizes with both TRX and PC at chromatin. Moreover, depletion of Enok specifically resulted in an increased enrichment of PC and consequently silencing of trxG targets. This downregulation of trxG targets was also accompanied by a decreased occupancy of RNA-Pol-II in the gene body, correlating with an increased stalling at the transcription start sites of these genes. We propose that Enok facilitates trxG-mediated maintenance of gene activation by specifically counteracting PcG-mediated repression. CONCLUSION:Our ex vivo approach led to identification of new trxG candidate genes that warrant further investigation. Presence of chromatin modifiers as well as known members of trxG and their interactors in the genome-wide RNAi screen validated our reverse genetics approach. Genetic and molecular characterization of Enok revealed a hitherto unknown interplay between Enok and PcG/trxG system. We conclude that histone acetylation by Enok positively impacts the maintenance of trxG-regulated gene activation by inhibiting PRC1-mediated transcriptional repression.
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