Laboratory of Cellular and Developmental Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA.
Genes Dev. 2011 Aug 15;25(16):1686-701. doi: 10.1101/gad.16651211.
A major role of the RNAi pathway in Schizosaccharomyces pombe is to nucleate heterochromatin, but it remains unclear whether this mechanism is conserved. To address this question in Drosophila, we performed genome-wide localization of Argonaute2 (AGO2) by chromatin immunoprecipitation (ChIP)-seq in two different embryonic cell lines and found that AGO2 localizes to euchromatin but not heterochromatin. This localization pattern is further supported by immunofluorescence staining of polytene chromosomes and cell lines, and these studies also indicate that a substantial fraction of AGO2 resides in the nucleus. Intriguingly, AGO2 colocalizes extensively with CTCF/CP190 chromatin insulators but not with genomic regions corresponding to endogenous siRNA production. Moreover, AGO2, but not its catalytic activity or Dicer-2, is required for CTCF/CP190-dependent Fab-8 insulator function. AGO2 interacts physically with CTCF and CP190, and depletion of either CTCF or CP190 results in genome-wide loss of AGO2 chromatin association. Finally, mutation of CTCF, CP190, or AGO2 leads to reduction of chromosomal looping interactions, thereby altering gene expression. We propose that RNAi-independent recruitment of AGO2 to chromatin by insulator proteins promotes the definition of transcriptional domains throughout the genome.
RNAi 途径在酿酒酵母中的一个主要作用是核化异染色质，但尚不清楚这种机制是否保守。为了在果蝇中解决这个问题，我们通过染色质免疫沉淀（ChIP）-seq 在两种不同的胚胎细胞系中进行了 Argonaute2（AGO2）的全基因组定位，发现 AGO2 定位于常染色质而不是异染色质。这种定位模式进一步得到多线染色体和细胞系免疫荧光染色的支持，这些研究还表明 AGO2 的很大一部分存在于核内。有趣的是，AGO2 与 CTCF/CP190 染色质绝缘子广泛共定位，但与内源性 siRNA 产生的基因组区域没有共定位。此外，AGO2（而不是其催化活性或 Dicer-2）是 CTCF/CP190 依赖性 Fab-8 绝缘子功能所必需的。AGO2 与 CTCF 和 CP190 物理相互作用，CTCF 或 CP190 的耗尽都会导致 AGO2 染色质关联的全基因组丢失。最后，CTCF、CP190 或 AGO2 的突变会导致染色体环相互作用减少，从而改变基因表达。我们提出，绝缘子蛋白对染色质的 RNAi 独立的 AGO2 募集促进了整个基因组转录域的定义。
