ChIP-on-chip with anti-RBPj antibody from embryonic AGM E11.5

Hematopoietic Stem Cells (HSC) are originated during embryonic development from endothelial-like cells located in the ventral side of the dorsal aorta around day E10-12 of murine development. This region is called AGM for Aorta/Gonad/Mesonephros refering to the tissues around the hemogenic aorta. Hematopoiesis depends on the Notch pathway and the identification of Notch-targets is important for the understanding of blood origin. Hematopoietic Stem Cells (HSCs) specification occurs in the embryonic aorta and requires Notch activation, however which are the elements regulated by Notch that control this process are mainly unknown. Here, we took a genome-wide approach to identify putative direct Notch targets by precipitating the chromatin that binds to the Notch partner RBPj in the Aorta-Gonad-Mesonephros (AGM) tissue from E11.5 mouse embryos. This assay revealed 701 gene promoter regions as candidates to be regulated by Notch in the AGM. Chromatin was obtained from a pool of 40 dissected AGMs at E11.5. Chromatin immunoprecipitation (ChIP) was performed as previously described (Aguilera et al, PNAS 2004) with minor modifications. In brief, cross-linked chromatin was sonicated for 10 minutes, medium-power, 0.5-interval; with a Bioruptor (Diagenode) and precipitated with anti-RBPJ (Chu and Bresnick, 2004). After crosslinkage reversal, DNA was used as a template for PCR or for array hybridization. Mouse promoter chip on chip microarray SET (Agilent) was used to identify RBPj targets. It covers 70,000 best identified gene regions with a-5.5 kb to + 2.5 kb range, and has on average 25 probes per gene with an average probe to probe distance of 200 bp. The ChIP-on-chip was performed with dye swaps and one IgG control was brought along. Enrichment analysis was done by comparing the precipitation normalized dye swap signal with input control signal.

造血干细胞（Hematopoietic Stem Cells，HSC）起源于胚胎发育过程中：在小鼠发育第E10-12天左右，背主动脉腹侧的内皮样细胞可分化为造血干细胞。该区域被称为主动脉-性腺-中肾区（Aorta/Gonad/Mesonephros，AGM），指代生血主动脉周围的组织。造血作用依赖于Notch信号通路，鉴定Notch靶基因对于阐明血液起源的机制具有重要意义。造血干细胞的特化过程发生于胚胎主动脉中，且需要Notch信号激活，但目前对于调控该过程的Notch下游调控元件仍知之甚少。本研究采用全基因组分析策略，通过免疫沉淀结合于Notch共因子RBPj的染色质，从E11.5天小鼠胚胎的主动脉-性腺-中肾区（AGM）组织中鉴定潜在的Notch直接靶基因。该实验共鉴定出701个基因启动子区域，可作为AGM组织中Notch调控的候选靶标。染色质样本取自40枚解剖分离的E11.5天小鼠胚胎AGM组织混合样本。染色质免疫沉淀（Chromatin Immunoprecipitation，ChIP）实验参照已发表方案（Aguilera等人，《美国国家科学院院刊》，2004年）并稍作修改。简言之，采用Diagenode公司的Bioruptor超声破碎仪，以中等功率、0.5秒间隔超声处理交联染色质10分钟，随后使用抗RBPJ抗体（Chu与Bresnick，2004年）进行免疫沉淀。完成交联逆转后，提取的DNA可作为聚合酶链式反应（Polymerase Chain Reaction，PCR）或芯片杂交的模板。本研究使用安捷伦（Agilent）公司的小鼠启动子ChIP-chip微阵列试剂盒鉴定RBPJ靶基因，该试剂盒覆盖70000个经过充分注释的基因区域，覆盖范围为转录起始位点上游5.5 kb至下游2.5 kb，平均每个基因配备25个探针，探针间平均间距为200 bp。本实验采用染料互换策略进行ChIP-chip实验，并设置IgG阴性对照。富集分析通过比较沉淀样本经标准化处理的染料互换信号与输入对照信号完成。