Hematopoietic Stem Cell Commitment-Molecular Regulation

在个体发育过程中，指导多能造血干细胞建立红系的细胞内信号的研究已经成功地集中在产生红系程序的转录因子上。然而，这些重要因素本身是如何诱导和调节的，以及这与已知的刺激红细胞产生的细胞外分子之间的关系仍然是一个谜。我们将重点放在EKLF(红系Kruppel-like factor)基因的调控上，作为填补这些知识空白的一种手段。EKLF是一种红系细胞特异性转录因子，其生物学特性和基因表达谱使其非常适合于此类分析。这一更新建立在我们之前的研究基础上，这些研究已经确定了EKLF转录单位的染色体结构、序列保守性、体内最小增强子元件以及体外蛋白质-DNA相互作用。我们还实现了对其中一种DNA结合活性的显著纯化，并建立了无血清条件，从而可以监测胚胎干细胞分化过程中EKLF表达对可溶性生长因子的依赖。因此，我们建议通过以下方式来阐明红系细胞特异的细胞内调控分子的产生以及与细胞外信号的关系：(1)鉴定与EKLF增强子元件相互作用的蛋白质(S)；(2)在转基因小鼠中和在分化胚胎干细胞方面测试顺式作用元件的功能重要性，以前只有在转基因小鼠中才有这类元件的特征；(3)通过一种新的方法来鉴定诱导EKLF表达的细胞外分子，该方法利用了在没有血清的情况下将胚胎干细胞分化为类胚体的能力。建立涉及EKLF表达的对照可能会将注意力集中在参与初级红系因子的自动或交叉调节的分子子集及其在信号转导中的作用。因此，这些研究对于描述在哺乳动物早期发育过程中调节造血干细胞承诺的细胞内和细胞外机制是相关的，此外，对于依赖红系祖细胞扩增用于移植的基于干细胞的治疗也具有临床意义。

Investigations of the intracellular signals that direct a multipotent hematopoietic stem cell to establish the erythroid lineage during ontogeny have successfully focused on the transcription factors that generate the erythroid program. Nevertheless, how these important factors are themselves induced and regulated, and how this relates to the extracellular molecules known to stimulate red cell production, remain a mystery. We have focused on the regulation of the EKLF (erythroid Kruppel- like factor) gene as a means to fill in these gaps in knowledge. EKLF is an erythroid cell-specific transcription factor whose biological properties and genetic expression profile make it eminently suitable for such analyses. This renewal builds upon our previous studies, which have determined the chromosomal structure, sequence conservation, minimal in vivo enhancer element, and in vitro protein-DNA interactions of the EKLF transcription unit. We have also achieved significant purification of one of the DNA binding activities, and have established serum-free conditions whereby the dependence on soluble growth factors of EKLF expression during embryonic stem cell differentiation can be monitored. We thus propose to illuminate how production of an erythroid cell-specific intracellular regulatory molecule is accomplished and is related to extracellular signals by: (1) Identifying the protein(s) that interact with the EKLF enhancer element; (2) Testing the functional importance of cis-acting elements, previously characterized only in transfection assays, in transgenic mice and in differentiating embryoid stem cells; (3) Identifying the extracellular molecules that induce EKLF expression by means of a novel assay that takes advantage of the ability to differentiate embryonic stem cells into embryoid bodies in the absence of serum. Establishing the controls involved in EKLF expression will likely focus attention on a subset of molecules involved in auto-or cross-regulation of primary erythroid factors and their role in signal transduction. These studies are therefore relevant to delineating the infra- and extra-cellular mechanisms that regulate commitment of hematopoietic stem cells during early mammalian development, and in addition have clinical relevance for stem cell-based therapies that depend on expansion of erythroid progenitor cells for their use in transplantation.