Mechanism of Hox specificity within the gut endoderm

描述(由申请人提供)HOX转录因子沿前后轴以独特的模式表达，以调节不同的形态发生过程。这个蛋白家族的成员结合DNA，通过与HOX辅因子、牙外环(EXD)和同胸(HTH)形成蛋白质复合体来调节基因的表达。这一发现导致了一种假设，即HOX蛋白通过控制目标基因的独特组合的表达来选择不同的发育途径。然而，有趣的是，许多不同的Hox蛋白复合体与相同或重叠的DNA序列相互作用。因此，HOX转录因子如何在体外结合高度相关的序列，并在体内指导不同的发育途径仍不清楚。最近，我们与M.Affolter的实验室(瑞士巴塞尔)合作，在唇部(LAB)同源框基因中鉴定了一个48个碱基对的增强子元件(1ab48/95)，它包含Lab、EXD和HTH的结合位点。这三种转录因子蛋白在体外与1ab48/95形成一个复合体，这些结合位点中的每一个都需要在体内提供肠道特异的基因表达。然而，这些因素不足以驱动1ab48/95的表达，因为在头部外胚层区域没有检测到活性，在那里所有三种蛋白都同时表达。这些结果表明，1ab48/95增强子的正确空间表达模式需要额外的辅助因子。在这个建议中，我们的目标是：1)鉴定1ab48/95内增强子活性所需的关键核苷酸序列，2)鉴定与Lab/EXD/HTH和1ab48/95 DNA元件相互作用的额外蛋白质，并确定它们在1ab48/95激活中的作用，以及3)建立体内系统来测试额外的HOX转录因子调节内胚层内序列元件的能力。综上所述，我们乐观地认为，HOX基因在内胚层激活的特征将成为发育调节的DNA元件如何在体内实现特异性的模型。

DESCRIPTION (provided by applicant) Hox transcription factors are expressed in unique patterns along the anterior-posterior axis to regulate diverse morphogenetic processes. Members of this protein family bind DNA and regulate gene expression by forming protein complexes with the Hox cofactors, Extradenticle (Exd) and Homothorax (Hth). This finding has led to the hypothesis that Hox proteins select for distinct developmental pathways by controlling the expression of unique combinations of target genes. Interestingly, however, many of the different Hox protein complexes interact with identical or overlapping DNA sequences. Thus, how Hox transcription factors can bind highly related sequences in vitro, and yet direct different developmental pathways in vivo remains unclear. Recently, we, in collaboration with M. Affolter's laboratory (Basel, Switzerland), have characterized a 48 base pair enhancer element (1ab48/95) within the labial (lab) homeobox gene that contains binding sites for Lab, Exd, and Hth. These three transcription factor proteins form a complex with 1ab48/95 in vitro, and each of these binding sites is required to confer gut-specific gene expression in vivo. However, these factors are not sufficient to drive expression of 1ab48/95, as no activity is detected within regions of the head ectoderm, where all three proteins are also co-expressed. These results suggest that additional cofactors are required for the proper spatial expression pattern of the 1ab48/95 enhancer. In this proposal our goals are to: 1) identify the critical nucleotide sequences within 1ab48/95 required for enhancer activity, 2) identify additional proteins that interact with Lab/Exd/Hth and the 1ab48/95 DNA element, and determine their role in the activation of 1ab48/95, and 3) establish an in vivo system to test the ability of additional Hox transcription factors to regulate sequence elements within the endoderm. Taken together, we are optimistic that the characterization of Hox gene activation within the endoderm will serve as a model for how a developmentally-regulated DNA element achieves specificity in vivo.