The Drosophila ZAD zinc finger protein Kipferl guides Rhino to piRNA clusters (RNA-Seq)

RNA interference systems depend on the synthesis of small RNA precursors whose sequences define the target spectrum of these silencing pathways. The Drosophila Heterochromatin Protein 1 (HP1) variant Rhino permits transcription of PIWI-interacting RNA (piRNA) precursors within transposon-rich heterochromatic loci in germline cells. Current models propose that Rhino’s specific chromatin occupancy at piRNA source loci is determined by histone marks and maternally inherited piRNAs, but also imply the existence of other, undiscovered specificity cues. Here, we identify a member of the diverse family of zinc finger associated domain (ZAD)-C2H2 proteins, Kipferl, as critical Rhino cofactor in ovaries. By binding to guanosine-rich DNA motifs and interacting with the Rhino chromodomain, Kipferl recruits Rhino to specific loci and stabilizes it on chromatin. In kipferl mutant flies, Rhino is lost from most of its target chromatin loci and instead accumulates on pericentromeric satellite arrays, resulting in decreased levels of transposon targeting piRNAs and impaired fertility. Our findings reveal that DNA sequence, in addition to the H3K9me3 mark, determines the identity of piRNA source loci and provide insight into how Rhino might be caught in the crossfire of genetic conflicts. Comparative gene expression profiling analysis of RNA-seq data for MTD-Gal4 knock-downs of control, rhino and CG2678.

RNA干扰（RNA interference）系统依赖于小RNA前体的合成，此类前体的序列决定了这类沉默通路的靶标谱。果蝇（Drosophila）的异染色质蛋白1（Heterochromatin Protein 1，HP1）变体Rhino，可使生殖细胞内富含转座子的异染色质位点中PIWI互作RNA（PIWI-interacting RNA，piRNA）前体得以转录。现有模型提出，Rhino在piRNA来源位点的特异性染色质定位由组蛋白修饰与母源piRNAs共同决定，但同时也暗示存在其他尚未被发现的特异性识别信号。本研究鉴定出锌指关联结构域（zinc finger associated domain，ZAD）-C2H2蛋白家族的一员Kipferl，是卵巢中关键的Rhino辅助因子。Kipferl通过结合富鸟嘌呤DNA基序并与Rhino的染色质结构域（chromodomain）相互作用，将Rhino招募至特定染色质位点并使其在染色质上稳定留存。在kipferl突变果蝇中，Rhino从绝大多数靶染色质位点流失，转而在着丝粒旁侧卫星序列阵列上异常聚集，这导致转座子靶向piRNAs的水平显著降低，并引发果蝇生育力受损。我们的研究发现，除H3K9me3修饰外，DNA序列同样决定piRNA来源位点的特异性，并为理解Rhino如何陷入遗传冲突的交叉火力提供了新的认知视角。本研究针对对照组、rhino基因敲低组与CG2678基因敲低组的MTD-Gal4驱动样本，完成了RNA测序（RNA-seq）数据的比较基因表达谱分析。