Chromatin Insulator Function and Nuclear Organization

染色质绝缘体功能和核组织

• 批准号: 8349657
• 负责人: Elissa Lei
• 金额: $ 131.09万
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8349657
• 关键词: Abdomen; Address; Affect; Biological Models; Cell Line; Cell Nucleus; Chromatin; Chromatin Loop; Chromosomes; Communication; Complex; DNA; DNA Packaging; DNA Sequence; DNA-Binding Proteins; Development; Distant; Drosophila genus; Enhancers; Euchromatin; Fission Yeast; Gene Expression; Genetic Transcription; Genome; Genomics; Gypsies; Heterochromatin; Homeobox Genes; Immunofluorescence Immunologic; Macromolecular Complexes; Mutation; Nuclear; Nuclear Matrix; Nucleosomes; Pancreatic ribonuclease; Pathway interactions; Pattern; Play; Polycomb; Production; Property; Protein Binding; Proteins; RNA; RNA Helicase; RNA Interference; Repression; Research; Role; Site; Small Interfering RNA; Specific qualifier value; Staining method; Stains; Testing; Transcriptional Regulation; Work; Zinc Fingers; blastomere structure; genome-wide; human DICER1 protein; mutant; prevent; promoter; protein complex

A major role of the RNAi pathway in S. 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 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. Fab-8 insulator activity is critical for proper spatial homeotic gene expression and abdominal development. 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S. pombe中的主要作用是成核异染色质，但尚不清楚该机制是否是保守的。 为了在果蝇中解决这个问题，我们在两个不同的胚胎细胞系中通过chip-seq对Argonaute2（AGO2）进行了全基因组定位，发现AGO2定位于构象素，而不是异染色质。 这种定位模式进一步支持了多烯染色体和细胞系的免疫荧光染色，这些研究还表明，AGO2的大部分位于细胞核中。 有趣的是，AGO2与CTCF/CP190染色质绝缘子广泛共定位，但与与内源性siRNA产生相对应的基因组区域无关。 此外，AGO2（而不是其催化活性或DICER-2）是CTCF/CP190依赖性FAB-8绝缘子功能所需的。 FAB-8绝缘子活性对于适当的空间同源基因表达和腹部发育至关重要。 AGO2与CTCF和CP190的物理相互作用，CTCF或CP190的耗竭会导致AGO2染色质关联的全基因组损失。 最后，CTCF，CP190或AGO2的突变导致染色体循环相互作用的减少，从而改变基因表达。 我们建议通过绝缘子蛋白对AGO2无关募集到染色质，从而促进了整个基因组中转录结构域的定义。