Epigenetic control mechanisms and cell-type-specific responsiveness of Wnt/ß-catenin target genes

在胚胎发育过程中，相同的信号转导机制被反复用于控制各种分化过程。根据细胞环境的不同，任何给定的信号转导通路只作用于其潜在靶标的一个子集。这种选择性是如何实现的？我们正在使用Wnt/β-catenin信号传导及其核效应物- β-catenin和t细胞因子（tcf）作为研究这一基本问题的范例。先前的研究表明，Wnt/β-catenin调控基因的响应和非响应状态与tcf的DNA甲基化、组蛋白修饰和启动子占用的特征模式一致。因此，表观遗传机制可以通过调节tcf的启动子可及性来分离功能不同的基因群。该项目的目标是检验这一假设，并测试表观遗传特征对wnt调节基因的细胞类型和阶段特异性控制的重要性。为了深入了解因果关系，我们将在细胞分化模型中建立染色质修饰、TCF占用和Wnt诱导性变化的时间谱，这些变化伴随着Wnt/β-catenin靶点的功能转变。在Wnt/β-catenin靶基因上，将以位点特异性和全结构域的方式对细胞类型特异性表观遗传景观进行扩展表征，并通过分析表观遗传修饰因子或tcf耗用时结构和功能特性的变化，进一步研究不同染色质结构状态的产生与tcf启动子占用之间的相互依赖性。当tcf与稳定转染的报告基因的启动子区域的结合被结合位点突变阻止时。

During embryonic development the same signal transduction mechanisms are repeatedly used to control a wide variety of differentiation processes. Depending upon cellular context any given signal transduction pathway acts upon only a subset of its potential targets. How is this selectivity achieved? We are using Wnt/β-catenin signaling and its nuclear effectors - β-catenin and the T-cell factors (TCFs) - as a paradigm to investigate this fundamental problem. Previous studies have shown that responsive and non-responsive states of Wnt/β-catenin-regulated genes coincide with characteristic patterns of DNA methylation, histone modifications, and promoter occupancy by TCFs. Therefore, epigenetic mechanisms may be used to separate functionally distinct groups of genes by regulating promoter accessibility for TCFs. The goal of the proposed project is to examine this hypothesis and to test the importance of epigenetic features for cell-type and stage-specific control of Wnt-regulated genes. To gain insight into causal relationships, we will establish temporal profiles of changes in chromatin modifications, TCF occupancy, and Wnt-inducibility which accompany functional transitions of Wnt/β-catenin targets in a cellular differentiation model. An extended characterization of cell-type-specific epigenetic landscapes will be undertaken in a locus-specific and domain-wide manner at Wnt/β-catenin target genes, and interdependencies between the generation of distinct chromatin structural states and promoter occupancy of TCFs will be further examined by analyzing alterations of structural and functional properties in response to depletion of either epigenetic modifiers or TCFs, and when association of TCFs with the promoter regions of stably transfected reporter genes is prevented by binding site mutation.