Identification of Myc and Max genomic targets in mouse embryonic stem cells.. Mus musculus

Myc is a master transcription factor that has been demonstrated to be required for embryonic stem cell (ESC) pluripotency, self-renewal, and inhibition of differentiation. Although recent works identified several Myc-targets in ESC the list of Myc binding sites is largely incomplete due to the low sensitivity and specificity of the antibodies available so far. To systematically identify Myc binding sites in mouse ESCs here we used a stringent streptavidin based genome-wide chromatin immunoprecipitation (ChIP-Seq) of a biotin-tagged Myc (Bio-Myc) as well as a ChIP-Seq of the Myc partner Max. This analysis identified 4273 Myc binding sites of which more than 85% co-occupied by Max, overlap with H3K4me3 positive promoters and active enhancers of transcriptional regulators, chromatin modifiers, and genes involved in stem cell self-renewing. The new sites identified were validated experimentally. This study provides a new Myc and Max binding reference in mouse ESCs. Overall design: ChIP-seq of bio-Myc and Max in E14 and respective controls; Bio-Myc ChIP-seq was performed in a stable clone of E14 mouse embryonic stem cell expressing Biotin-tagged Myc; Mock, Max and IgG were performed in parental wt E14 mESC.

Myc是一种主转录因子，已被证实为胚胎干细胞（embryonic stem cell, ESC）的多能性、自我更新及分化抑制过程所必需。尽管近期已有研究在胚胎干细胞中鉴定出若干Myc靶基因，但受限于当前可用抗体的灵敏度与特异性不足，Myc结合位点的完整列表仍存在大量未被鉴定的空白。为系统鉴定小鼠胚胎干细胞中的Myc结合位点，本研究采用基于链霉亲和素的严格全基因组染色质免疫沉淀测序（chromatin immunoprecipitation sequencing, ChIP-Seq）技术，对生物素标记的Myc（Bio-Myc）进行检测，同时还针对Myc的互作搭档Max开展了ChIP-Seq分析。本次分析共鉴定出4273个Myc结合位点，其中超过85%的位点同时被Max结合，且与转录调控因子、染色质修饰因子以及参与干细胞自我更新的基因所对应的组蛋白H3赖氨酸4三甲基化（histone H3 lysine 4 trimethylation, H3K4me3）阳性启动子和活性增强子区域存在重叠。本次新鉴定的结合位点均经实验验证。本研究为小鼠胚胎干细胞中的Myc与Max结合位点提供了全新的参考数据集。总体实验设计：对E14细胞及其对照样本分别开展Bio-Myc与Max的ChIP-seq检测；Bio-Myc ChIP-seq实验在表达生物素标记Myc的稳定转染E14小鼠胚胎干细胞克隆中完成；Mock对照、Max抗体及IgG对照实验则在野生型亲本E14小鼠胚胎干细胞（mouse embryonic stem cell, mESC）中进行。