Genome-wide identification of target promoters of developmental regulators

我们的长期目标是解开基因调控和表达的早期模式与结构的最终实现之间的联系，以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，我们正在开发几个相结合的遗传和基因组/蛋白质组学的方法来研究转录因子和调节级联在肢体发育过程中的最终目的是阐明的调控层次之间的早期诱导前后（AP）模式和不同的数字（不同的数量，长度和形状的趾骨）的形态发生。 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遗传疾病和癌症的理解，这些疾病和癌症是在这些调控成分突变或异常表达时出现的。肢体起始：Tbx 5如何调节肢体生长？Tbx 5对心脏和前肢发育至关重要，人类突变（Holt-Oram综合征）导致两个器官系统的严重缺陷。 识别Tbx 5靶谱将阐明肢体生长是如何控制的，并提供关于相同因子的上下文依赖性活动如何调节非常不同的发育程序的新见解。 我们正在开发胚胎中的ChIP检测，用于全基因组直接识别肢体发育转录调节因子的体内靶启动子，并解开模式形成和形态发生过程中的调控网络。Tbx 5是这种分析的一个很好的原型：1）它在早期前肢芽（也是心脏）中高度表达; Tbx 5突变导致人类心脏和肢体异常（Holt-Oram病）以及小鼠的类似缺陷。2)已经确定了一个直接的Tbx 5靶标Fgf 10，这将有助于验证和故障排除;然而，大多数靶标是未知的。我们已经生成并验证了针对Tbx 5的高亲和力多克隆抗体，该抗体通过ChIP有效且特异性地富集Fgf 10启动子，优化了该方法，并分析了试验性全基因组启动子微阵列以识别约200个靶标，其中一些是新的。 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。 这些目标目前正在验证在体外和体内，使用条件Floxed Tbx 5基因突变体等位基因与他莫昔芬诱导的Cre-deleter线交叉，以消除Tbx 5功能从肢芽在不同stages.Digit形态：如何做Hoxd基因指示特征的数字身份（如关节的数量，形状，大小）和什么是Hoxd和Gli 3目标之间的关系？：最近已经报道了一些5 Hoxd体内靶标（Shh增强子、Hand 2启动子），尽管没有很好地表征。 定义5 Hoxd函数的时间窗（III）在选择ChIP分析的肢体分期时很重要。我们已经开发了几种针对ChIP的抗体，并且我们还与Steve Vokes（U.德克萨斯，奥斯汀。5 'Hoxd-flox等位基因将促进靶标的生物学验证。 结果将与沃克斯博士实验室的预期结果相关联，沃克斯博士也在分析Gli 3靶点。鉴定5 'Hoxd和Gli 3靶标将提供对共调节基因和Gli 3-Hoxd作用的深入了解，以及阐明5 Hoxd基因在肢体形态发生中的晚期效应子。 Gli和Hox基因也在一些癌症中异常表达，可能有助于其发病机制，这些研究将揭示它们在这些背景下可能的作用。

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?: 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.