Chromatin-seq and RNA-seq of dREAM subunit deficient Drosophila S2R+ cells. Drosophila melanogaster

dREAM is a multi-functional complex which activates and represses genes involved in the G2/M transition and is involved in maintaining chromatin boundaries through interactions with CTCF and BEAF-32, however the mechanisms by which it carries out gene and chromatin regulation is yet to be uncovered. Applying a novel Mnase-seq technique, RNAi and RNA-seq we report links between transcriptional changes in S2R+ cells deficient for dREAM subunits and changes in chromatin structure. The most distinct chromatin phenotype of these cells are at CTCF binding sites, several of which are located between divergent gene pairs which show impaired regulation in cells deficient for a dREAM component. Overall design: S2R+ cells treated with dsRNA against GFP (control), mip40, E2F2, mip120 or mip130 where harvested for chromatin digestion by Micrococcal nuclease, or for total RNA purification. Purified DNA or RNA was then sequenced using an Illumina HiSeq 2000 machine. All samples were carried out in duplicate except E2F2, of which there is a single sample.

dREAM是一种多功能复合物，可激活并抑制参与G2/M期转换的基因，还能通过与CCCTC结合因子（CTCF）及边界元件结合因子32（BEAF-32）相互作用维持染色质边界，但其执行基因与染色质调控的具体机制仍有待揭示。本研究借助新型微球菌核酸酶测序（Mnase-seq）技术、RNA干扰（RNAi）及RNA测序（RNA-seq），报道了dREAM亚基缺陷的S2R+细胞中的转录变化与染色质结构改变之间的关联。这类细胞最显著的染色质表型出现在CTCF结合位点，其中多个位点位于双向基因对之间，此类基因对在dREAM组分缺陷的细胞中呈现调控异常。实验整体设计：将S2R+细胞用靶向GFP（对照组）、mip40、E2F2、mip120或mip130的双链RNA（dsRNA）进行处理，随后收集细胞，分别用于微球菌核酸酶介导的染色质消化，以及总RNA的提取纯化。将纯化得到的DNA或RNA使用Illumina HiSeq 2000测序仪进行测序。所有样本均设置生物学重复，仅E2F2组仅设置单个样本。