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. 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. 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.

中文翻译：

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果蝇全基因组的RNAi筛选揭示了Enok作为新型的胸廓调节剂。

Polycomb组（PcG）和trithorax组（trxG）蛋白通过维持差异基因表达模式来促进细胞类型的专业化。最初被发现为正向遗传筛选中HOX基因的正调节剂，trxG抵消了PcG介导的细胞类型特异性基因的抑制。尽管进行了数十年的广泛分析，但许多未知因素仍使人们对trxG的作用和调节的分子理解仍然存在。这项研究旨在发现引发抗沉默作用以促进trxG介导的基因激活的新因素。我们已经开发了一个基于细胞的报告系统，并进行了全基因组的RNAi筛选，以发现果蝇中参与trxG介导的基因调控的新因子。我们以与trxG基因相似的方式鉴定了200多个影响报告基因的基因。从最有希望的候选人名单中，我们将著名的组蛋白乙酰转移酶-香菇（Enok）表征为果蝇中trxG的重要调节剂。enok的突变体强烈抑制了Pc突变体的额外性梳表型，并增强了与trx突变相关的同种异体转化。Enok在染色质上与TRX和PC共定位。此外，Enok的耗竭特别导致PC的富集增加，因此trxG靶位沉默。trxG靶的这种下调还伴随着基因体中RNA-Pol-II的占用减少，与这些基因的转录起始位点处的停滞增加有关。我们建议Enok通过专门抵消PcG介导的阻遏促进trxG介导的基因激活的维持。我们的离体方法导致鉴定新的trxG候选基因，值得进一步研究。染色质修饰剂以及已知的trxG成员及其在全基因组RNAi筛选中的相互作用因子的存在验证了我们的反向遗传学方法。Enok的遗传和分子表征揭示了Enok与PcG / trxG系统之间迄今未知的相互作用。我们得出的结论是，Enok通过抑制PRC1介导的转录阻抑作用，对组蛋白乙酰化产生积极影响，维持trxG调控的基因活化。Enok的遗传和分子表征揭示了Enok与PcG / trxG系统之间迄今未知的相互作用。我们得出的结论是，Enok通过抑制PRC1介导的转录阻抑作用，对组蛋白乙酰化产生积极影响，维持trxG调控的基因活化。Enok的遗传和分子表征揭示了Enok与PcG / trxG系统之间迄今未知的相互作用。我们得出的结论是，Enok通过抑制PRC1介导的转录阻抑作用，对组蛋白乙酰化产生积极影响，维持trxG调控的基因活化。