Characterizing Muscle Regulatory Elements with Mass Spectrometry-Based Proteomics

描述(申请人提供)：下一代测序和染色质免疫沉淀(CHIP)实验正在生成描述细胞状态的表观遗传修饰的全基因组数据集。最近的ENCODE项目已经使用全基因组方法生成了数百个数据集，以绘制蛋白质-DNA相互作用的图谱。这些动态的染色质状态图揭示了基因组中数千个假定的顺式调控模块(CRM)，远远超过了基因的数量。这些顺式调节模块被认为通过招募特定的反式作用因子组合来调节基因表达，例如转录因子(TF)和非编码RNA。同时，全基因组关联研究已经定位了数千个非编码区的单核苷酸多态(SNPs)，这表明多态可能通过影响调节反式因子与CRM的结合来改变基因的表达。尽管有这些先进的技术来定位CRM在基因组中的位置，但我们目前缺乏强大的高通量方法来发现与这些CRM相互作用的蛋白质并表征它们的功能角色。为了实现这一目标，我们建议：(1)通过实验验证肌肉转录因子在新CRM上的动态招募，并利用蛋白质组学分析研究调控基因中蛋白质与CRM的相互作用；(3)开发活体诱饵方法来观察活细胞中蛋白质与DNA的相互作用。通过关注肌肉分化过程中在转录本和蛋白质水平显著上调的基因，我们将比较与新的调控基因位点结合的蛋白质和邻近的控制序列，以确定与候选CRM结合的新的转录因子。除了建立一个强大的工具箱，对与特定基因组座位相互作用的蛋白质进行无偏见的蛋白质组学表征，我们还将应用我们的技术来研究候选CRM和已知的肌肉调控基因座，这些基因围绕着具有良好特征的C2C12肌肉分化模型中的高调控基因。这项工作将为基因组生物学家提供新的方法来识别新的转录因子，并更清楚地理解CRM在基因调控中的功能意义。

DESCRIPTION (provided by applicant): Next-generation sequencing and chromatin immunoprecipitation (ChIP) experiments are generating genome-wide datasets of epigenetic modifications that describe cellular states. The recent ENCODE project has generated hundreds of datasets using genome-wide approaches to map protein-DNA interactions. These dynamic chromatin state maps reveal many thousands of putative cis regulatory modules (CRMs) in the genome, far outnumbering the numbers of genes. These cis regulatory modules are thought to modulate gene expression through the recruitment of specific combinations of trans acting factors, such as transcription factors (TF) and non-coding RNAs. In parallel, genome-wide association studies (GWAS) have mapped thousands of single nucleotide polymorphisms (SNPs) in non-coding regions, suggesting that polymorphisms may be altering gene expression by affecting binding of regulatory trans factors to CRMs. Despite these advanced techniques to localize CRMs in the genome, we currently lack robust high throughput approaches to discover the proteins that interact with these CRMs and characterize their functional roles. To achieve this goal, we propose to: (1) experimentally validate the dynamic recruitment of muscle TFs to novel CRMs and study protein-CRM interactions in regulated genes with proteomics analyses; (3) Develop in- vivo bait approaches to observe protein-DNA interactions in live cells. By focusing on genes significantly up- regulated at the transcript and protein level during muscle differentiation, we will compare proteins bound at novel regulatory loci with neighboring control sequences to identify novel TFs bound at candidate CRMs. In addition to building a powerful toolbox for unbiased proteomic characterization of proteins interacting with specific genomic loci, we will apply our technologies to study candidate CRMs and known muscle regulatory loci surround highly regulated genes in the well characterized C2C12 muscle differentiation model. This work will provide genome biologists with new approaches to identify novel transcription factors and a clearer understanding of the functional significance of CRMs in gene regulation.