Mechanisms of Pbx-directed Genetic &Transcriptional Control of Limb Development

描述(申请人提供)：在脊椎动物中，HOX基因在大多数重要器官的形成中起着重要作用。已经提出，精致的DNA结合特异性允许不同的HOX蛋白调节特定的靶基因，从而指示不同的身体结构的一致性，依赖于与其他同源蛋白的相互作用，这些同源蛋白充当HOX辅助因子。在过去的15年里，基于分子和生化分析，流行的观点是，由PBX基因家族编码的产物TALL同源结构域蛋白是HOX的辅助辅助因子。Pbx1是果蝇外牙周膜蛋白(EXD)的同源基因，在果蝇身体的花纹形成过程中起着关键作用。虽然EXD是果蝇中唯一的PBX编码基因，但老鼠有四个这样的基因(Pbx1-4)。尽管它们在器官发生和身体和肢体轴线的模式形成中起着至关重要的作用，但HOX调节的分子机制仍然难以捉摸。我们的目标是使用小鼠的肢体作为最易处理和已建立的系统来描述是否调节HOX的“共线”表达，这是一种基本而神秘的生物现象，是否受PBX的控制。我们已经确定，不同的PBX基因，类似于HOX基因，在肢体模式和生长中共享重叠的角色。因此，Pbx1/Pbx2双纯合子(Pbx1-/-；Pbx2-/-)胚胎完全没有肢体，而Pbx1-/-；Pbx2突变体的肢体截断与HoxA/D突变体相似。此外，我们还发现Pbx1/Pbx2控制肢体间充质中5‘HoxA/D表达的起始和空间分布。这些发现证实，PBX蛋白分级控制肢体中5‘HoxA/D基因的表达。鉴于这些新的发现，我们的假设提出了一种新的HOX基因调控机制，即5‘HOX基因的表达在转录水平上直接由PBX在花蕾间充质中调控，以促进肢体形态发生和手指形成。我们将使用胚胎学、遗传学和分子生物学方法在小鼠身上验证我们的假设。首先，通过分子方法，我们将确定Pbx1/2是否通过直接控制HoxD GCR来调节5‘HoxD的转录，HoxD GCR是一个控制HoxD在自体中共线表达的基因组区域。然后，我们将通过细胞培养中的瞬时转基因和小鼠的瞬时转基因实验来测试PBX与HoxD GCR的结合是否与转录有功能关系。此外，通过组织特异性和可诱导的遗传消融，使用我们新的Pbx1条件等位基因(在Pbx2缺失的背景上)和可用的Cre系(其中一种在间充质中可诱导)，我们将剖析Pbx1/Pbx2在肢体领域和芽间充质中的空间和时间要求。通过这种方法，我们将确定肢体芽中PBX丢失第一次影响HOX表达的时间。这些研究的完成将确定管理HOX基因转录的新的调控网络，并将直接有助于理解人类先天性肢体畸形。广泛地说，考虑到人类HOX基因与白血病和实体瘤的关系，我们的研究将使我们对HOX的调控也能在人类肿瘤中得到普遍的理解。 公共卫生相关性：HOX蛋白在哺乳动物许多关键器官的形成中起着重要作用，包括肢体；然而，HOX基因调控的分子机制仍然不清楚。这项拟议的研究将使用小鼠模型，为肢体中PBX蛋白对HOX调控的控制提供新的见解。因此，这项工作的广泛影响将是产生关于人类先天性畸形发病机制的新知识，包括那些影响肢体骨骼发育和功能的畸形，大约发生在500名活产婴儿中的1名。

DESCRIPTION (provided by applicant): In vertebrates, Hox genes play major roles in the formation of most vital organs. It has been proposed that the exquisite DNA-binding specificities that allow different Hox proteins to regulate specific target genes, thus instructing the identity of distinct body structures, depend on interactions with other homeoproteins, which act as Hox cofactors. For the last fifteen years, based on molecular and biochemical analyses, the prevailing view has been that TALE homeodomain proteins, which comprise the products encoded by the Pbx gene family, act as ancillary cofactors for Hox. Pbx1 is a homolog of Drosophila extradenticle (exd), which has critical roles in patterning of the fly body. While exd is the sole Pbx-encoding gene in the fly, the mouse has four such genes (Pbx1-4). Despite their paramount roles in organogenesis and patterning of the body and limb axes, the molecular mechanisms of Hox regulation remain elusive. Our objectives are to use the mouse limb as the most tractable and established system to delineate whether regulation of Hox "collinear" expression, a basic and mysterious biological phenomenon, is governed by Pbx. We have established that different Pbx genes, similarly to Hox genes, share overlapping roles in limb patterning and outgrowth. Accordingly, Pbx1/Pbx2 double homozygous (Pbx1-/-;Pbx2-/-) embryos lack limbs altogether, while Pbx1-/-;Pbx2 mutants exhibit limb truncations similar to those of HoxA/D mutants. Additionally, we have found that Pbx1/Pbx2 control the onset and spatial distribution of 5' HoxA/D expression in limb mesenchyme. These findings establish that Pbx proteins hierarchically govern 5' HoxA/D gene expression in the limb. In view of these new findings, our hypothesis proposes a novel mechanism for Hox gene regulation, whereby 5' Hox expression is directly controlled at the transcriptional level by Pbx in the bud mesenchyme for limb morphogenesis and digit formation. We will test our hypothesis using embryologic, genetic and molecular approaches in the mouse. First, by molecular methods, we will determine whether Pbx1/2 regulate 5' HoxD transcription by direct control of the HoxD GCR, a genomic region that governs HoxD collinear expression in the autopod. We will then test whether Pbx binding to the HoxD GCR has functional bearings on transcription by both transient transfections in cell culture and transient transgenesis experiments in the mouse. Moreover, by tissue-specific and inducible genetic ablation, using our new Pbx1 conditional allele (on a Pbx2-deficient background), and available Cre lines (one of which inducible in the mesenchyme), we will dissect Pbx1/Pbx2 spatial and temporal requirements in the limb field and bud mesenchyme. By this approach, we will determine when Hox expression is first affected by Pbx loss in the limb bud. Completion of these studies will define novel regulatory networks that govern transcription of Hox genes and will directly contribute to the understanding of human congenital limb malformations. Broadly, given the involvement of human HOX genes in leukemias and solid tumors, our studies will inform general comprehension of HOX regulation also in human neoplasia. PUBLIC HEALTH RELEVANCE: Hox proteins play essential roles in the formation of many critical organs in mammals, including the limb; however, the molecular mechanisms underlying Hox gene regulation remain elusive. The proposed studies will use mouse models to provide novel insights into the control of Hox regulation by Pbx proteins in the limb. Accordingly, a broad impact of this work will be the generation of new knowledge on the pathogenesis of human congenital malformations, including those that affect limb skeletal development and function, occurring in approximately 1 of 500 human live births.