The transcriptional program controlled by Runx1 during early hematopoietic development (expression data)

Transcription factors have long been recognised as powerful regulators of mammalian development, yet it is largely unknown how individual key regulators operate within wider regulatory networks. Here we have used a combination of global gene expression and chromatin-immunoprecipitation approaches across four ES-cell-derived populations of increasing haematopoietic potential to define the transcriptional programme controlled by Runx1, an essential regulator of blood cell specification. Integrated analysis of these complementary genome-wide datasets allowed us to construct a global regulatory network model, which suggested that core regulatory circuits are activated sequentially during blood specification, but will ultimately collaborate to control many haematopoietically expressed genes. Using the CD41/integrin alpha 2b gene as a model, cellular and in vivo studies showed that CD41 is controlled by both early and late circuits in fully specified blood cells, but initiation of CD41 expression critically depends on a later subcircuit driven by Runx1. Taken together, this study represents the first global analysis of the transcriptional programme controlled by any key haematopoietic regulator during the process of early blood cell specification. Moreover, the concept of interplay between sequentially deployed core regulatory circuits is likely to represent a design principle widely applicable to the transcriptional control of mammalian development. 4 samples (Runx1-/VE-cadherin+/Flk1+, Runx1+/VE-cadherin+/CD41+, Runx1+/VE-cadherin+/CD41- and Runx1+/VE-cadherin-/CD41+). 1 x 10^5 undifferentiated ES cells were cultured on confluent OP9 cell layers in 10-cm dishes to induce differentiation. After 6 days of ES cell differentiation, cultured cells were harvested for FACS analysis and sorting.

转录因子（Transcription factors）长期以来被认为是哺乳动物发育的强效调控因子，但目前学界仍未明确单个关键调控因子如何在更广泛的调控网络中发挥功能。
本研究结合全基因组基因表达与染色质免疫沉淀（chromatin-immunoprecipitation）技术，针对四个造血潜能逐步升高的胚胎干细胞（ES cell）衍生群体开展分析，以此明确调控造血细胞特化的关键因子Runx1所控制的转录程序。
通过对这些互补性全基因组数据集进行整合分析，我们构建了一套全局调控网络模型，该模型揭示造血特化过程中核心调控回路会依次激活，最终协同调控众多造血特异性表达基因。
我们以CD41/整合素α2b（integrin alpha 2b）基因为研究模型，细胞与体内实验证实：完全特化的造血细胞中，CD41的表达同时受早期与晚期调控回路共同调控，但其表达起始关键依赖于Runx1介导的晚期亚回路。
综上，本研究首次针对早期造血特化过程中任一关键造血调控因子所控制的转录程序开展了全局分析。此外，依次激活的核心调控回路间存在协同互作，这一概念或许代表了一种普适性的设计原则，可广泛应用于哺乳动物发育的转录调控研究中。
本数据集共包含4组样本：Runx1⁻/VE-钙粘蛋白（VE-cadherin）⁺/Flk1⁺、Runx1⁺/VE-钙粘蛋白⁺/CD41⁺、Runx1⁺/VE-钙粘蛋白⁺/CD41⁻以及Runx1⁺/VE-钙粘蛋白⁻/CD41⁺。
实验操作如下：将1×10⁵个未分化胚胎干细胞接种于铺满汇合态OP9细胞层的10cm培养皿中以诱导分化。待胚胎干细胞分化6天后，收集培养细胞用于荧光激活细胞分选术（Fluorescence-Activated Cell Sorting, FACS）分析与分选。