Targets of the transcription factor Brachyury in differentiating mouse ES cells

Brachyury (or T) is expressed in the primitive streak, tailbud and notochord of the early mouse embryo (Herrmann et al., 1990; Wilkinson et al., 1990). It plays a key role in early development: mouse embryos lacking functional Brachyury protein fail to gastrulate properly, do not form a differentiated notochord, and lack structures posterior to somite seven (Chesley, 1935; Dobrovolskaïa-Zavadskaïa, 1927; Naiche et al., 2005; Wilson et al., 1995; Wilson et al., 1993; Yanagisawa et al., 1981) We apply a ChIP-on-chip approach to identify targets of Brachyury during mouse ES cell differentiation. ES cells provide an abundant source of differentiating cells and the identification of Brachyury targets in such cells will shed light on the mechanisms of ES cell differentiation and, by analyzing the targets in the developing embryo, will reveal to what extent they provide a bone fide model of early mouse development. The brachyury IP expts used 3 independent biological replicates hybridised to design 1 and 2 chips per replicate Design 1 raw file AE_251471610189 + Design 2 raw file AE_251471710182 = replicate 1 Design 1 raw file AE_251471610716 + Design 2 raw file AE_251471710724 = replicate 2 Design 1 raw file AE_251471610717 + Design 2 raw file AE_251471710725 = replicate 3 in all cases IP DNA label was Cy5 and whole chromatin Cy3 Design 1 files GSM417692 Design 2 files GSM417704 Isotype control experiment performed 1 x ie design 1 raw file AE_251471610714 design 2 raw file AE_251471710722 in GSM417714 and GSM417756 IP DNA label was Cy5 and whole chromatin Cy3 as for brachyury data

Brachyury（亦称为T）在早期小鼠胚胎的原条、尾芽与脊索中表达（Herrmann等，1990；Wilkinson等，1990）。该蛋白在早期发育过程中发挥关键作用：缺乏功能性Brachyury蛋白的小鼠胚胎无法正常完成原肠胚形成，无法分化形成脊索，且缺失第七体节后方的组织结构（Chesley, 1935; Dobrovolskaïa-Zavadskaïa, 1927; Naiche et al., 2005; Wilson et al., 1995; Wilson et al., 1993; Yanagisawa et al., 1981）。本研究采用染色质免疫沉淀芯片（ChIP-on-chip）技术，在小鼠胚胎干细胞（Embryonic Stem Cell, ES细胞）分化过程中鉴定Brachyury的靶标基因。ES细胞是丰富的分化细胞来源，在此类细胞中鉴定Brachyury的靶标将有助于阐明ES细胞分化的分子机制；同时通过分析发育胚胎中的靶标基因，可揭示其在多大程度上可作为早期小鼠发育的真实可信模型。本次Brachyury免疫沉淀（Immunoprecipitation, IP）实验设置3次独立生物学重复，每个重复分别与设计1和设计2的芯片进行杂交。各重复对应的原始文件如下：重复1：设计1原始文件AE_251471610189 + 设计2原始文件AE_251471710182；重复2：设计1原始文件AE_251471610716 + 设计2原始文件AE_251471710724；重复3：设计1原始文件AE_251471610717 + 设计2原始文件AE_251471710725。所有实验中，免疫沉淀所得DNA的荧光标记为Cy5，全染色质的荧光标记为Cy3。设计1的文件对应GSM417692，设计2的文件对应GSM417704。本研究同时设置了1次同型对照实验：对应设计1原始文件AE_251471610714与设计2原始文件AE_251471710722，对应GSM417714与GSM417756，其免疫沉淀DNA与全染色质的荧光标记规则与Brachyury实验一致，即IP DNA标记Cy5、全染色质标记Cy3。