Mapping Transcriptional Networks in Cardiac Development

绘制心脏发育中的转录网络

• 批准号: 8663738
• 负责人: JONATHAN G SEIDMAN
• 金额: $ 1.58万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2015-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8663738
• 关键词: Adult; Animal Model; Binding; Biotin; Cardiac; Cataloging; Catalogs; Cells; Chickens; DNA Sequence; Data; Data Set; Development; Developmental Process; Embryonic Development; Gene Expression; Gene Expression Profile; Gene Targeting; Genes; Genetic; Genetic Transcription; Genome; Genomics; Goals; Heart; Heart Diseases; Human; Instruction; Lead; Location; Maps; Methods; MicroRNAs; Molecular; Molecular Profiling; Morphogenesis; Mus; Pathogenesis; Pathway interactions; Pattern; Phosphorylation; Play; Process; Proteins; RNA; RNA Sequences; Regulation; Regulatory Element; Research; Role; Sequence Analysis; Site; Staging; Structure; Technology; Tissues; Transcript; Tube; activating transcription factor; congenital heart disorder; deep sequencing; genome-wide; human data; insight; malformation; new technology; novel; novel therapeutic intervention; promoter; research study; transcription factor; transcriptome sequencing

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.

描述（由申请人提供）： 心脏发育是多种调节过程的结果。 HCCB 研究中心致力于定义这些过程之一——转录调控网络。 RNA 转录的调节被认为在发育过程中发挥着关键作用，但转录调节因子的整个库的定义及其在心脏胚胎发生过程中的相互作用仍然很大程度上未知。虽然已鉴定出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标尚不清楚。我们建议采用新的基因组和遗传学方法来定义调节心脏发育多个步骤的转录因子网络，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 RNA 蓝图。心脏发育所需网络的定义将增强对最近发现的可能改变发育过程的转录后调节机制（例如 microRNA）的作用的理解。我们将利用新技术来定义 RNA 表达网络，包括从初级和次级心脏区域的形成到成人心室形成的基因表达的时间和空间模式。将采用三种不同的新技术来定义每个组织在发育关键阶段的 RNA 谱（DSAGE 和 RNAseq），并确定特定转录因子可能的下游靶标（bioCHIP）。 DSAGE 和 RNAseq 采用高通量 DNA 测序技术来定义 RNA 表达的结构和水平，而 bioCHIP 则定义基因组内与特定转录因子结合的启动子位点。这些信息将共同定义转录因子水平及其下游靶基因。被确定为发育步骤核心的分子将通过微扰实验进行验证，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，并探讨该蓝图的扰动如何导致先天性心脏病。 1) 使用 DSAGE 和 RNAseq 定义初级心域、次级心域和发育心室中的转录组。 2) 使用 DCHIP 识别 GATA4、F0G2、TBX5、NKX2.5 和其他对心脏发育至关重要的转录因子的结合靶点。 3）根据.model有机体和人类数据集构建心脏规格和形态发生的发育阶段的发育蓝图。 4) 通过扰动 RNA 水平并定义分子和表型后果来验证蓝图中定义节点的关键作用。相关性（参见说明）：拟议研究的目标是定义导致成熟心脏形成的转录网络。定义这个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，此类研究可能会为冠心病提供新的治疗方法。