PhpCNF-Y transcription factor infiltrates heterochromatin to generate cryptic intron-containing transcripts crucial for small RNA production [ChIP-Seq]

The assembly of repressive heterochromatin in eukaryotic genomes is crucial for silencing lineage-inappropriate genes and repetitive DNA elements. Paradoxically, transcription of repetitive elements within constitutive heterochromatin domains is required for RNA-based mechanisms, such as the RNAi pathway, to target heterochromatin assembly proteins. However, the mechanism by which heterochromatic repeats are transcribed has been unclear. Using fission yeast, we show that the conserved trimeric transcription factor (TF) PhpCNF-Y complex can infiltrate constitutive heterochromatin via its histone-fold domains to transcribe repeat elements. PhpCNF-Y collaborates with a Zn-finger containing TF to bind repeat promoter regions with CCAAT boxes. Mutating either the TFs or the CCAAT binding site disrupts the transcription of heterochromatic repeats. Although repeat elements are transcribed from both strands, PhpCNF-Y-dependent transcripts originate from only one strand. These TF-driven transcripts contain multiple cryptic introns whose predicted splice sites are required for the generation of small interfering RNAs (siRNAs) via a mechanism requiring the spliceosome. Our analyses show that siRNA production by this TF-mediated transcription pathway is critical for heterochromatin nucleation at target repeat loci. This study reveals a mechanism by which heterochromatic repeats are transcribed, initiating their own silencing by triggering a primary cascade that produces siRNAs necessary for heterochromatin nucleation. Overall design: Chromatin immunoprecipitation DNA-sequencing (ChIP-seq) in fission yeast. Input control and immunoprecipitation samples are provided for each experiment.

真核生物基因组中抑制型异染色质（repressive heterochromatin）的组装，对于沉默与细胞谱系不相适配的基因及重复DNA元件而言至关重要。矛盾的是，组成型异染色质（constitutive heterochromatin）结构域内的重复元件的转录，对于以RNA为基础的通路（例如RNA干扰（RNA interference, RNAi）通路）招募异染色质组装蛋白的过程而言是必需的。然而，异染色质重复序列的转录机制至今仍未阐明。本研究以裂殖酵母（fission yeast）为模型，证实保守的三聚体转录因子（transcription factor, TF）PhpCNF-Y复合物可通过其组蛋白折叠结构域（histone-fold domains）侵入组成型异染色质，进而介导重复元件的转录。PhpCNF-Y与一类含有锌指结构（Zn-finger）的转录因子协同结合带有CCAAT盒的重复启动子区域。对该类转录因子或CCAAT结合位点进行突变，均会破坏异染色质重复序列的转录。尽管重复元件可从双链进行转录，但PhpCNF-Y依赖型转录本仅起源于单链。此类由转录因子驱动的转录本包含多个隐蔽内含子，其预测的剪接位点对于通过依赖剪接体的机制生成小干扰RNA（small interfering RNAs, siRNAs）而言是必需的。我们的分析表明，通过该转录因子介导的转录通路产生的siRNA，对于靶标重复位点的异染色质成核过程至关重要。本研究揭示了异染色质重复序列的转录机制：该机制通过触发一条可产生异染色质成核所需siRNA的初级级联反应，启动重复序列自身的沉默过程。总体实验设计：裂殖酵母中的染色质免疫沉淀DNA测序（chromatin immunoprecipitation DNA-sequencing, ChIP-seq）。本研究提供了每项实验的输入对照样本与免疫沉淀样本。