Transcriptional Regulatory Control of Neuronal Cell Type Specification

描述（由申请人提供）：拟议研究的目标是了解通过基本螺旋-环-螺旋（bHLH）转录因子Atoh1控制神经元细胞类型规范的调节机制。d !由于缺乏已知的Atoh1靶点，目前尚不清楚背神经管神经元和小脑颗粒细胞中是否有Atoh1编码。本研究将专注于鉴定作为Atoh1靶点的转录因子，并揭示指定神经元细胞类型的Atoh1顺式调控代码。长期目标是了解神经元bHLH转录因子如何调节神经元细胞类型规范的分子机制，可能导致识别神经退行性疾病，脑和脊髓损伤以及神经元细胞衍生癌症的治疗靶点。具体目标是：1。鉴定Atoh1靶点并确定这些靶点的重要功能调控区域。微阵列实验已经鉴定出在atoh1谱系细胞中富集的转录本。Atoh1的下游靶点将从候选转录因子列表中确定，并在鸡和转基因小鼠实验中检测报告基因的正确表达。Atoh的直接靶点将通过染色质免疫沉淀（ChIP）方法进行鉴定。确定Atoh1指定神经元的顺式调控代码。直接Atoh1靶点的调控区域将探测Atoh1共识结合位点，Atoh1靶点的调控区域将使用生物信息学分析检查常见转录因子结合位点或DNA序列基序。候选的调控代码将通过鸡突变或转基因小鼠报告试验进行功能测试。通过目标1和目标2的迭代循环，将使用监管代码进一步确定Atoh1的靶标。

DESCRIPTION (provided by applicant): The goal of the proposed research is to understand the regulatory mechanisms that govern neuronal cell type specification through the basic helix-loop-helix (bHLH) transcription factor, Atoh1. How d!1 neurons in the dorsal neural tube and granule cells in the cerebellum are encoded by Atoh1 remains undefined due to the lack of known Atoh1 targets. This study will focus on identifying transcription factors that are Atoh1 targets and uncover the Atoh1 cis-regulatory code that specifies neuronal cell types. The long term goal is to understand the molecular mechanisms of how neuronal bHLH transcription factors regulate neuronal cell type specification possibly leading to the identification of therapeutic targets for neurodegenerative diseases, brain and spinal cord injuries, and neuronal cell-derived cancers. The specific aims are:1. Identify Atoh1 targets and determine functionally important regulatory regions of these targets. Microarray experiments have identified transcripts enriched in Atoh1-lineage cells. Downstream targets of Atoh1 will be identified from a candidate list of transcription factors and their regulatory regions tested for proper expression of a reporter gene in chick and transgenic mouse assays. Direct targets of Atoh will be identified by chromatin immunoprecipitation (ChIP) approaches.2. Determine a cis-regulatory code by which Atoh1 specifies neurons. Regulatory regions of direct Atoh1 targets will be probed for an Atoh1 consensus binding site and regulatory regions of Atoh1 targets will be examined for common transcription factor binding sites or DNA sequence motifs using bioinformatics analyses. Candidate regulatory codes will be functionally tested by mutation in the chick or transgenic mouse reporter assays. Regulatory codes will be used to further identify targets of Atoh1 through iterative cycles of Aims 1 and 2. I am trying to understand how neurons form at their most basic level. My research may lead to discoveries in understanding how we can therapeutically restore working nerves in people with brain and spinal cord injuries, neurodegenerative diseases such as Alzheimer's or Parkinson's disease, or pediatric brain tumors.