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Hox基因控制的，Hox基因在哺乳动物中被组织成四个簇。每个集群包含13个原始同源基因的子集，组织在大约100 kb的范围内。据估计，这些集群至少存在了5亿年，这表明集群基因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臂膀和胸部区域轴形成中的作用。PI已经确定了调控Hoxc8转录活性的增强子，并对其表达进行时间调控，控制其在A/P轴上的空间分布，并以其在生殖器官基本器官中的表达为靶点。其中一种增强子，即早期增强子，已经进行了相当详细的分析。它在羊膜中高度保守，延伸超过200 bp结构域，并包含至少10个转录因子结合基序。PI已经收集了与该调控单元相互作用的转录因子身份的证据，并表明这些因子以类似于增强体的方式相互作用。大部分的工作已经完成，使用正常和突变形式的早期增强子报告结构，通过转基因引入早期胚胎。虽然信息丰富，但报告者实验并不能提供完整基因簇背景下增强子行为的直接知识。PI现在寻求利用胚胎干细胞方法通过同源重组特异性修饰内源性增强子元件。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集群基因顺式调控的影响，以及早期增强子与C集群Hoxc8区域其他增强子元件之间相互作用的明确信息。PI认为，这一新的分析将大大加深对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.