Acetyltransferase Enok regulates transposon silencing by promoting transcription at piRNA clusters and genes involved in piRNA biosynthesis [H3 and H3K23 ChIP-seq]. Acetyltransferase Enok regulates transposon silencing by promoting transcription at piRNA clusters and genes involved in piRNA biosynthesis [H3 and H3K23 ChIP-seq]

The PIWI-interacting RNA (piRNA) pathway is an important mechanism to suppress transposon activation in the germline that is highly conserved between Drosophila and mammals. This pathway starts from transcribing piRNA clusters to generate long piRNA precursors. The majority of piRNA clusters lacks a conventional promoter, and therefore their transcription is considered to utilize a noncanonical mechanism. However, information regarding transcriptional regulation of piRNA clusters is limited. Here, we report that the fly KAT6 acetyltransferase, Enok, is important for transposon silencing in the germline. Further small RNA-seq analysis revealed that Enok is critical for piRNA production from a specific subset of piRNA clusters including 42AB, one of the two major sources of piRNAs. Enok is the major enzyme for establishing the H3K23ac mark, and was shown to promote gene expression by acetylating H3K23. Surprisingly, our RNA-seq and ChIP-seq/ChIP-qPCR results suggest that Enok regulates expression of genes involved in the piRNA production by H3K23ac-dependent mechanisms, and also promotes transcription at piRNA clusters through H3K23ac-independent pathways. Our co-immunoprecipitation assay further suggests that Enok interacts with RNA polymerase II to facilitate transcription of piRNA clusters. Taken together, our study provides novel insights into the regulation of noncanonical transcription at piRNA clusters and transposon silencing. Overall design: Investigation of the genome-wide distribution of Histone H3 and Histone H3K23 in Enok knock-down ovaries by ChIP-seq.

PIWI互作RNA（PIWI-interacting RNA, piRNA）通路是抑制生殖系中转座子激活的重要机制，在果蝇与哺乳动物间高度保守。该通路起始于piRNA基因簇的转录，以产生长链piRNA前体。绝大多数piRNA基因簇不具备经典启动子，因此其转录被认为采用非经典机制。然而，目前关于piRNA基因簇转录调控的相关信息仍较为有限。本研究报道，果蝇KAT6乙酰转移酶Enok对于生殖系中的转座子沉默至关重要。进一步的小RNA测序（small RNA-seq）分析显示，Enok对特定亚类piRNA基因簇的piRNA生成具有关键作用，其中包括两大主要piRNA来源之一的42AB基因簇。Enok是介导组蛋白H3K23乙酰化（H3K23ac）修饰的核心酶，既往研究表明其可通过乙酰化H3K23来促进基因表达。令人意外的是，本研究的RNA测序（RNA-seq）以及染色质免疫共沉淀测序（ChIP-seq）/染色质免疫共沉淀定量PCR（ChIP-qPCR）结果显示，Enok可通过依赖H3K23ac的机制调控参与piRNA生成的基因的表达，同时还可通过不依赖H3K23ac的通路促进piRNA基因簇的转录。本研究的免疫共沉淀（co-immunoprecipitation）实验进一步表明，Enok可与RNA聚合酶II（RNA polymerase II）相互作用，以促进piRNA基因簇的转录。综上，本研究为piRNA基因簇的非经典转录调控以及转座子沉默机制提供了全新的认识。整体实验设计：通过染色质免疫共沉淀测序（ChIP-seq）分析Enok敲低卵巢中组蛋白H3与组蛋白H3K23的全基因组分布情况。