Characterizing muscle regulatory elements with mass spectrometry-based proteomics

描述（由申请人提供）：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来绘制蛋白质-DNA相互作用。这些动态染色质状态图揭示了基因组中数千个假定的顺式调节模块（CRM），远远超过了基因的数量。这些顺式调控模块被认为通过募集反式作用因子（如转录因子（TF）和非编码RNA）的特定组合来调节基因表达。与此同时，全基因组关联研究（GWAS）已经绘制了非编码区数千个单核苷酸多态性（SNP）的图谱，表明多态性可能通过影响调节反式因子与CRM的结合来改变基因表达。尽管有这些先进的技术在基因组中定位CRM，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其功能作用。为了实现这一目标，我们提出：（1）实验验证肌肉TF向新型CRM的动态募集，并利用蛋白质组学分析研究受调控基因中的蛋白质-CRM相互作用;（3）开发体内诱饵方法以观察活细胞中的蛋白质-DNA相互作用。通过关注在肌肉分化期间在转录物和蛋白质水平上显著上调的基因，我们将比较结合在新调控基因座处的蛋白质与邻近控制序列，以鉴定结合在候选CRM处的新TF。除了建立一个功能强大的工具箱，用于与特定基因组位点相互作用的蛋白质的无偏见蛋白质组学表征外，我们还将应用我们的技术研究候选CRM和已知的肌肉调控位点，这些位点围绕在特征良好的C2 C12肌肉分化模型中高度调控的基因。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，并更清楚地了解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.