MYC TRANSCRIPTION FACTOR NETWORK IN STEM CELL BIOLOGY

这项建议的重点是一个新发现的转录组 可能在分化中发挥重要作用的因素， 造血干细胞的增殖。myc家族 原癌基因已被证明与肿瘤形成，细胞凋亡， 分化和增殖。这种基因编码的蛋白质 家族（c-、N-和L-Myc）是基本螺旋环螺旋的成员， 拉链（bHLHZ）类转录因子。证据表明 它们都通过与bHLHZ蛋白Max相互作用而发挥功能。Myc：Max 异二聚体以序列特异性方式结合DNA并激活 在它们的DNA结合位点通过转录 Myc的激活结构域。重要的是，其他蛋白质也与 最大其中一种蛋白质Mad也是bHLHZ组的成员， 似乎通过与Max竞争结合来“对抗”Myc的功能 并作为转录抑制因子。疯狂的表达是诱导后， 在Myc水平升高期间造血细胞的分化 下降这些变化导致从Myc：Max异二聚体转换为 Mad：最大异二聚体作为诱导后的早期事件 分化，并被认为反映了转录开关， 生长调节基因的表达。 最近，我们已经确定了其他四个新的bHLHZ蛋白（MIP 1 -4） 专门与麦克斯互动其中两个与 疯了在本申请中，我们建议研究Mad的潜在作用， MIP 1 -4在造血干细胞生物学中的特异性。我们首先 计划检查分化过程中Mad和MIP 1-4的表达 造血细胞系和正常骨髓细胞中。一旦我们 建立了这些蛋白质的表达模式，我们计划 使用几种诱导性的方法在造血细胞中异位表达它们 向量系统这将使我们能够评估他们的影响。 在增殖细胞周期和分化上的表达。最后 我们计划制备一系列不同的蛋白质表达文库， 造血细胞类型和阶段，以便通过相互作用 克隆策略，另外的Max结合蛋白，其表达可以 细胞类型受限。这些实验可能会导致更好的 理解转录因子和分子机制， 调节正常的造血功能。

This proposal is focused on a newly discovered group of transcription factors that are likely to play important roles in the differentiation and proliferation of hematopoietic stem cells. The myc family of protooncogenes has been shown to be involved in neoplasia, apoptosis, differentiation and proliferation. The proteins encoded by this gene family (c-, N-, and L-Myc) are members of the basic-helix-loop-helix- zipper (bHLHZ) class of transcription factors. Evidence indicates that they all function through interaction with the bHLHZ protein Max. Myc:Max heterodimers bind to DNA in a sequence-specific manner and activate transcription at their DNA binding site through the transcriptional activation domain of Myc. Importantly other proteins also interact with Max. One of these proteins, Mad, is also a member of the bHLHZ group, and appears to "oppose" the function of Myc by competing for binding with Max and acting as a transcriptional repressor. Mad expression is induced upon differentiation of hematopoietic cells during a period when Myc levels decline. These changes lead to a switch from Myc:Max heterodimers to Mad:Max heterodimers as an early event following the induction of differentiation and is thought to reflect a transcriptional switch in expression of growth regulatory genes. Very recently we have identified four other novel bHLHZ proteins (MIP1-4) that specifically interact with Max. Two of these are highly related to Mad. In this application we propose to study potential roles for Mad and MIP1-4 specifically in the biology of hematopoietic stem cells. We first plan to examine the expression of Mad and MIP 1-4 during differentiation of hematopoietic cell lines and in normal bone marrow cells. Once we have established the pattern of expression of these proteins we plan to ectopically express them in hematopoietic cells using several inducible vector systems. This will permit us to assess the effects of their expression on the proliferating cell cycle and on differentiation. Lastly we plan to prepare a series of protein expression libraries from different hematopoietic cell types and stages in order to identify, by interaction cloning strategies, additional Max binding proteins whose expression may be cell-type restricted. These experiments are likely to lead to a better understanding of the transcription factors and molecular mechanisms that regulate normal hematopoiesis.