Targeted Mutagenesis of the Hoxc8 Early Enhancer

这项拨款申请描述了一种新的实验方法来分析脊椎动物的轴形成。前后轴的形成是由哺乳动物的HOX基因控制的，这些基因被组织成四个簇。每个簇包含在大约100kb的跨度内组织的13个原始同源基因的子集。据估计，这些基因簇至少存在了5亿年，这表明这些基因簇之间存在着密切的生物适应性相互作用。Ruddle实验室从分子、遗传、发育和进化的角度深入研究了Hoxc8基因在发育中的小鼠胚胎的臂部和胸部区域轴形成中的作用。PI已经确定了调节Hoxc8转录活性的增强子，并在时间上调节其表达，控制其在A/P轴上的空间分布，并针对其在特定器官中的表达。其中一种增强剂，早期增强剂，已经被相当详细地分析。该基因在羊膜组织中高度保守，全长200个碱基，含有至少10个转录因子结合基序。PI已经收集了与该调控单位相互作用的转录因子的身份的证据，并表明这些因子以类似于增强体的方式协同作用。这项工作的大部分是通过转基因引入早期胚胎的报告结构中使用早期增强子的正常和突变形式完成的。虽然信息量很大，但记者实验并不提供完整基因簇背景下的增强子行为的直接知识。PI现在寻求通过使用胚胎干细胞方法学的同源重组来特异性地修改内源性增强子元件。通过这种方式，他希望获得关于单个增强子基序对发育模式的影响、早期增强子对整个HOXC簇基因顺式调控的影响、以及早期增强子与C簇HOXC8区其他增强子元件之间的相互作用的明确信息。PI相信，这一新的分析路线将极大地加深对HOX基因调节模式形成的方式的理解。

This grant application describes a new experimental approach to the analysis of axis formation in vertebrates. Anterior-posterior axis formation is governed by the Hox genes that are organized into four clusters in mammals. Each cluster contains subsets of thirteen primordial cognate genes organized within a span of approximately 100 kb. The clusters are estimated to have existed over a period of at least 500 million years, suggesting an intimate and biological adaptive interplay among the clustered genes. The Ruddle laboratory has studied the role of the Hoxc8 gene intensively with respect to its contributions to axis formation in the brachial and thoracic regions of the developing mouse embryo from molecular, genetic, developmental, and evolutionary points of view. The PI has identified enhancers that regulate the transcriptional activity of Hoxc8 and regulate its expression temporally, control its spatial distribution on the A/P axis, and target its expression in spedtic organ rudiments. One of these enhancers, the early enhancer, has been analysed in considerable detail. It is highly conserved in amniotes, extends over a 200 bp domain, and contains at least ten transcription factor binding motifs. The PI has assembled evidence for the identities of the transcription factors interacting with this regulatory unit and have shown that the factors interact cooperatively in a manner similar to that of an enhanceosome. Much of this work has been accomplished using normal and mutated forms of the early enhancer in reporter constructs that are introduced into the early embryo by transgenesis. While highly informative, reporter experiments do not provide direct knowledge of enhancer behavior in the context of the intact gene cluster. The PI now seeks to specifically modify the endogenous enhancer element by homologous recombination using embryonal stem cell methodologies. In this way, he expects to obtain unambiguous information on the influence of individual enhancer motifs on developmental patterning, the effect of the early enhancer on cis regulation of genes throughout the Hoxc cluster, and the interplay between the early enhancer and other enhancer elements in the Hoxc8 region of the C cluster. The PI believes that this new line of analysis will greatly deepen understanding of the means by which the Hox genes regulate pattern formation.