Mapping Transcriptional Networks in Cardiac Development

描述(由申请人提供)： 心脏发育是多种调控过程的结果。HCCB研究中心的重点是定义其中一个过程--转录调控网络。RNA转录调控被认为在发育过程中起着关键作用，但在心脏胚胎发育过程中整个转录调控因子及其相互作用的定义在很大程度上仍不清楚。虽然已经发现了大量参与心脏发育的转录因子，但激活这些转录因子的过程以及这些转录因子的下游靶点尚不清楚。我们建议使用新的基因组和遗传学方法来定义转录因子网络，该网络调节心脏发育的多个步骤，并将该网络覆盖在这些发育组织中发现的RNA表达谱上。最终，这将为理解心脏发育提供一个RNA蓝图。心脏发育所需网络的定义将加强对最近发现的可能改变发育过程的转录后调控机制(例如microRNAs)的作用的理解。我们将利用新技术来定义RNA表达网络，包括形成初级和次级心腔的基因表达的时间和空间模式。将使用三种不同的新技术来确定每个组织在发育的关键阶段(DSAGE和RNAseq)的RNA图谱，并确定特定转录因子(生物芯片)可能的下游靶标。DSAGE和RNAseq使用高通量DNA测序技术来定义RNA表达的结构和水平，而生物芯片定义了基因组中由特定转录因子结合的启动子位置。这些信息将共同定义转录因子水平及其下游目标基因。被确定为发育步骤中心的分子将通过扰动实验进行验证，包括分子和表型分析。结合人类研究的数据，我们将构建心脏发育蓝图，并询问该蓝图的扰动如何导致先天性心脏病。1)利用DSAGE和RNAseq确定初级心区、次级心区和心腔发育过程中的转录组。2)利用DCHIP技术确定GATA4、F0G2、TBX5、NKX2.5等心脏发育关键转录因子的结合靶点。3)根据模型生物体和人体数据构建心脏发育阶段的发展蓝图。4)通过干扰RNA水平并定义分子和表型结果，验证定义节点在蓝图中的关键作用。相关性(见说明)：这项拟议研究的目标是定义导致成熟心脏形成的转录网络。定义这一网络将为各种先天性心脏病(CHD)的发病机制提供新的见解。最终，这样的研究可能会为治疗冠心病提供新的治疗方法。

DESCRIPTION (provided by applicant): Cardiac development results from a variety of regulatory processes. The HCCB Research Ctr is focused on defining one of these processes-the transcriptional regulatory network. Regulation of RNA transcription is recognized to play critical roles in developmental processes, definition of the entire repertoire of transcriptional regulators and their interactions during cardiac embryogenesis remains largely unknown. While large numbers of transcription factors have been identified that are involved in cardiac development the processes that activate these transcription factors and the downstream targets of these transcription factors are not known. We propose to employ novel genomic and genetic methods to define the network of transcription factors that regulate multiple steps in cardiac development and overlay that network on the RNA expression profile found in these developing tissues. In the end this should provide an RNA blueprint for understanding cardiac development. Definition of the network required for cardiac development would enhance understandings of the roles of recently discovered post-transcriptional regulatory mechanisms (e.g. microRNAs) that may modify developmental processes. We will take advantage of novel technologies to define the RNA expression networks, including both temporal and spatial patterns of gene expression that formation of primary and secondary heart fields to adult chamber formation. Three different novel technologies will be employed to define RNA profiles in each tissue at critical stages of development (DSAGE and RNAseq) and to identify likely downstream targets of particular transcription factors (bioCHIP). DSAGE and RNAseq employ high throughput DNA sequencing technologies to define the structure and level of RNA expression, while bioCHIP defines the promoter sites within the genome that are bound by particular transcription factors. Together this information will define transcription factor levels and their downstream target genes. Molecules that are identified as central for developmental steps will be validated by perturbation experiments, including molecular and phenotypic analyses. Together with data from human studies, we will construct a cardiac developmental blueprint and interrogate how perturbations of this blueprint result in congenital heart disease. 1) Define the transcriptome in the primary heart field, the secondary heart field and in developing cardiac chambers using DSAGE and RNAseq. 2) Identify binding targets of GATA4, F0G2, TBX5, NKX2.5 and other transcription factors critical for cardiac development using DCHIP. 3) Construct a developmental blueprint for developmental stages of cardiac specification and morphogenesis from .model organism and human datasets. 4) Validate crifical role of defines nodes in the blueprint by perturbing RNA levels and defining molecular and phenotypic consequences. RELEVANCE (See instructions): The goal of the proposed research is to define transcriptional networks that lead to formation of the mature heart. Defining this network will provide novel insights into the pathogenesis of a variety of congenital heart diseases (CHD). Eventually such studies may allow new therapeutic approaches to CHD.