Targeted Mutagenesis of the Hoxc8 Early Enhancer

这项资助申请描述了一种新的实验方法来分析脊椎动物中轴的形成。 前后轴的形成由Hox基因控制，在哺乳动物中被组织成四个簇。 每个簇包含在大约100 kb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Hoxc 8基因在发育中的小鼠胚胎臂区和胸区中轴形成中的作用。 PI已经鉴定了调节Hoxc 8的转录活性并在时间上调节其表达、控制其在A/P轴上的空间分布并靶向其在spedtic器官雏形中的表达的增强子。这些增强子之一，早期增强子，已被相当详细地分析。 它是高度保守的，延伸超过200 bp的结构域，并包含至少10个转录因子结合基序。 PI已经收集了与该调节单元相互作用的转录因子的身份的证据，并且已经表明这些因子以类似于增强体的方式协同相互作用。这项工作的大部分已经完成，使用正常和突变形式的早期增强子的报告构建体，引入到早期胚胎的转基因。虽然信息量很大，但报告基因实验并不能直接了解完整基因簇中增强子的行为。 PI现在寻求通过使用胚胎干细胞方法的同源重组来特异性地修饰内源性增强子元件。 通过这种方式，他希望获得明确的信息，个别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Hoxc 8区域中其他增强子元件之间的相互作用。 PI认为，这种新的分析方法将大大加深对Hox基因调控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.