Mechanism of Hox specificity within the gut endoderm

描述（由申请人提供）Hox转录因子以独特的模式沿着前后轴表达以调节不同的形态发生过程。该蛋白质家族的成员通过与Hox辅因子外齿（Exd）和同胸（Hth）形成蛋白质复合物来结合DNA并调节基因表达。这一发现导致了Hox蛋白通过控制靶基因的独特组合的表达来选择不同发育途径的假设。然而，有趣的是，许多不同的Hox蛋白复合物与相同或重叠的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，但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（巴塞尔，瑞士）已经表征了唇（lab）同源框基因内的48个碱基对增强子元件（1ab 48/95），其含有Lab、Exd和Hth的结合位点。这三种转录因子蛋白在体外与1ab 48/95形成复合物，并且这些结合位点中的每一个都是体内赋予肠道特异性基因表达所需的。然而，这些因子不足以驱动lab 48/95的表达，因为在头外胚层的区域内没有检测到活性，其中所有三种蛋白质也共表达。这些结果表明，额外的辅因子是必要的适当的空间表达模式的1ab 48/95增强子。在本提案中，我们的目标是：1）鉴定1ab 48/95内增强子活性所需的关键核苷酸序列，2）鉴定与Lab/Exd/Hth和1ab 48/95 DNA元件相互作用的其他蛋白质，并确定它们在1ab 48/95激活中的作用，和3）建立体内系统以测试另外的Hox转录因子调节内胚层内的序列元件的能力。综上所述，我们乐观地认为，内胚层内Hox基因激活的表征将作为一个模型，用于研究发育调控的DNA元件如何在体内实现特异性。

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.