Genome-wide identification of target promoters of developmental regulators

我们的长期目标是揭示将基因调控和表达的早期模式与结构的最终实现联系起来的步骤，作为信号网络如何协调复杂组织形成的范例。为了实现这一目标，我们正在开发几种结合遗传学和基因组/蛋白质组学的方法来研究肢体发育过程中运作的转录因子和调控级联，最终目的是阐明早期诱导前后（AP）模式和不同指骨（指骨数量、长度和形状不同）形态发生之间的调控层次。了解转录因子如何在正常发育过程中协调生长和形态发生，将促进我们对如何治疗当这些调节成分突变或异常表达时出现的遗传疾病和癌症的理解。肢体发育：Tbx5如何调控肢体发育？Tbx5对心脏和前肢的发育至关重要，人类突变（Holt-Oram综合征）会导致这两个器官系统的严重缺陷。确定tbx5靶标库将阐明肢体生长是如何受到控制的，并为同一因子的环境依赖活性如何调节非常不同的发育程序提供新的见解。我们正在开发胚胎芯片试验，以在全基因组范围内直接鉴定肢体发育转录调控的目标启动子，并揭示在模式形成和形态发生过程中运作的调控网络。Tbx5是一个很好的原型：1)它在前肢早期芽（也包括心脏）中高度表达；Tbx5突变导致人类心脏和肢体异常（Holt-Oram病），并导致小鼠出现类似缺陷。2)确定了Tbx5的直接靶点Fgf10，这将有助于验证和故障排除；然而，大多数目标是未知的。我们已经生成并验证了针对Tbx5的高亲和力多克隆抗体，通过ChIP高效特异性地富集Fgf10启动子，优化了方法，并分析了试点全基因组启动子微阵列，鉴定了大约200个靶点，其中一些是新的靶点。我们现在已经将这种分析扩展到全基因组平铺阵列，并确定了几个新的，意想不到的目标。这些靶点目前正在体外和体内进行验证，使用有条件的Floxed Tbx5基因突变等位基因与他莫昔芬诱导的Cre-deleter系杂交，从不同阶段的肢体芽中去除Tbx5功能。趾形态发生：Hoxd基因如何指导趾身份特征（如关节数量、形状、大小）以及Hoxd与Gli3靶点之间的关系？我们计划将ChIP分析扩展到其他几个在数字规格和模式中重要的发育调节因子。我们制备了Hoxd12和Gli3的多克隆抗体，以确定Hoxd和Gli3蛋白的直接结合靶点，并阐明Hoxd-Gli3相互作用在基因调控中的作用。最近报道了一些5Hoxd的体内靶点（Shh增强子，Hand2启动子），尽管没有很好地表征。定义5Hoxd函数的时间窗（III）对于选择ChIP分析的肢体阶段非常重要。我们已经为ChIP开发了几种抗体，我们也正在与Steve Vokes（美国德克萨斯州，奥斯汀）合作，为ChIP设计一种带有抗标签的表位标记的Hoxd13条件转基因等位基因。5'Hoxd-flox等位基因将促进靶标的生物学验证。结果将与Vokes博士实验室的预期结果相关联，Vokes博士也在分析Gli3靶点。确定5'Hoxd和Gli3靶点将有助于深入了解共调控基因和Gli3- hoxd的作用，并阐明5Hoxd基因在肢体形态发生中的后期效应。Gli和Hox基因也在某些癌症中异常表达，并可能导致其发病机制，这些研究将阐明它们在这些情况下可能发挥的作用。

Our long term goal is to unravel the steps linking early patterns of gene regulation and expression with the ultimate realization of structure to serve as a paradigm for how signaling networks orchestrate the formation of a complex tissue. To accomplish this, we are developing several combined genetic and genomic/proteomic approaches to study transcription factors and regulatory cascades operating during limb development with the ultimate aim of elucidating the regulatory hierarchy between early induction of antero-posterior (AP) pattern and the morphogenesis of distinct digits (with different numbers, lengths and shapes of phalanges). Learning how transcription factors orchestrate growth and morphogenesis during normal development will advance our understanding of how to treat genetic diseases and cancers that arise when such regulatory components are either mutated or expressed abnormally. Limb Initiation: How does Tbx5 regulate limb outgrowth?: Tbx5 is critical for both heart and forelimb development and human mutations (Holt-Oram syndrome) lead to serious defects in both organ systems. Identifying the Tbx5-target repertoire will illuminate how limb outgrowth is controlled and provide new insights on how context-dependent activity of the same factor regulates very different developmental programs. We are developing ChIP assays in embryos for genome-wide direct identification of target promoters in vivo for transcriptional regulators of limb development and unravel the regulatory networks operating during pattern formation and morphogenesis. Tbx5 is an excellent prototype for this analysis: 1) It is highly expressed in early forelimb bud (also heart); Tbx5 mutations cause cardiac and limb abnormalities in humans (Holt-Oram disease) and similar defects in mice. 2) A direct Tbx5 target, Fgf10, has been identified, which will aid in validation and troubleshooting; nevertheless most targets are unknown. We have generated and validated high affinity polyclonal antibodies against Tbx5 that efficiently and specifically enrich the Fgf10 promoter by ChIP, optimized the approach, and have analyzed pilot genome-wide promoter microarrays to identify about 200 targets, some of which are novel. We have now extended this analysis to genome-wide tiling arrays and identified several novel, unexpected targets. These targets are currently being validated both in vitro, and in vivo, using a conditional Floxed Tbx5 gene mutant allele crossed with a tamoxifen inducible Cre-deleter line to remove Tbx5 function from limb buds at different stages. Digit morphogenesis: How do Hoxd genes instruct features of digit identity (such as numbers of joints, shape, size) and what is the relation between Hoxd and Gli3 targets?: We plan to extend ChIP analysis to several other developmental regulators important in digit specification and patterning. We have generated polyclonal antibodies for Hoxd12 and Gli3 to identify direct binding targets for Hoxd and Gli3 proteins and elucidate the role of Hoxd-Gli3 interaction in gene regulation. A few 5Hoxd in vivo targets have been reported recently (Shh enhancer, Hand2 promoter), albeit not well-characterized. Defining time windows for 5Hoxd functions (III) will be important in choice of limb stages for ChIP analysis. We have developed several antibodies for ChIP, and we are also engineering an epitope-tagged Hoxd13 conditional transgene allele for ChIP with anti-tag in collaboration with Steve Vokes (U. Texas, Austin. The 5'Hoxd-flox allele will facilitate biological validation of targets. Results will be correlated with anticipated results from Dr. Vokes lab, who is also analyzing Gli3 targets. Identifying 5'Hoxd and Gli3 targets will provide insight into co-regulated genes and Gli3-Hoxd roles as well as illuminating late effectors of 5Hoxd genes in limb morphogenesis. Gli and Hox genes are also aberrantly expressed in some cancers and may contribute to their pathogenesis, and these studies will shed light on their possible roles in these contexts.