Identification of interaction partners of modified DNA bases and their role in the epigenetic control of gene regulation

表观基因组变化是通过对DNA碱基的化学修饰来实现的，在基因调控和细胞识别中发挥着重要作用。除了作为DNA去甲基化的中间体外，5-甲酰胞嘧啶(5fC)本身也被认为具有重要的生物学作用。最近的研究表明，5fC通过招募特定的转录调控因子在组织特异性和发育基因表达调控中发挥重要作用。然而，与5fC相互作用的蛋白质的身份和功能仍然是该领域的主要悬而未决的问题，这些知识对于全面理解表观遗传调控至关重要。到目前为止，还缺乏一种全面和系统的方法来探索5fC相关蛋白(FAP)，以及其他修饰的碱基，在天然染色质环境中。因此，我建议开发一种新的方法，通过化学标记染色质中与5fC结合或接近5fC的蛋白质，以便能够通过质谱学捕获和鉴定它们。这种方法基于融合到抗坏血酸过氧化物酶(APEX)的工程蛋白，与基因组基因座蛋白质组学(GloPro)相结合，将被用来识别哺乳动物细胞中5fC和其他潜在的氧化胞嘧啶的内源蛋白相互作用。与基因组5fC结合的蛋白质将通过染色质免疫沉淀测序(ChIP-SEQ)进行验证，而生物物理和生化技术将揭示FAP的结合模式、募集及其对5fC动力学的影响。为了确定参与转录调控的候选FAP，我将通过抗体捕获实验以及对基因组数据库的详细生物信息学询问，确定5fC与特征染色质相关蛋白(如启动子相关的组蛋白和转录因子)的共存。这个高度跨学科的项目将利用巴拉苏布拉马尼亚集团S在英国癌症研究所、剑桥研究所和化学系的实验室的独特地位，提供广泛的培训机会。总体而言，这些研究将产生一个全面的、无偏见的内源性5fC基因座的相互作用网络，最终将导致对非规范DNA修饰及其相关蛋白质如何调节重要的细胞过程和功能的新的机械性见解。

Epigenomic changes are mediated by chemical modifications to DNA bases and play an important role in gene regulation and cellular identity. In addition to being a DNA demethylation intermediate, 5-formylcytosine (5fC) has also been attributed to important biological roles in its own right. Recent studies suggest a fundamental role for 5fC in tissue-specific and developmental gene expression control through the recruitment of specific transcriptional regulators. However, the identity and function of proteins that interact with 5fC remain major unanswered questions in the field and such knowledge is vital for a comprehensive understanding of epigenetic regulation. To date, a comprehensive and systematic approach to explore 5fC-associated proteins (FAPs), and also other modified bases, within a native chromatin environment is lacking. I therefore propose to develop a novel method in which proteins bound to or proximal to 5fC in chromatin are chemically tagged to enable their capture and identification by mass spectrometry. This method is based on a engineered protein fused to ascorbate peroxidase (APEX), that combined with genomic locus proteomics (GloPro), will be used to identify endogenous protein interactors of 5fC and potentially other oxidised cytosines in mammalian cells. Protein binding to genomic 5fC sites will be validated by chromatin immunoprecipitation sequencing (ChIP-seq) while biophysical and biochemical techniques will reveal FAP binding modes, recruitment and the effects on 5fC dynamics. To identify candidate FAPs involved in transcriptional regulation, I will determine the co-occurrence of 5fC with characteristic chromatin-associated proteins, such as promoter associated histones and transcription factors using antibody capture experiments as well as detailed bioinformatics interrogation of genomic databases. This highly interdisciplinary project will provide wide ranging training opportunities exploiting the Balasubramanian group´s unique position with laboratories at the Cancer Research UK Cambridge Institute and the Department of Chemistry. Overall, these studies will generate a comprehensive, unbiased interaction network of endogenous 5fC loci that will ultimately lead to novel mechanistic insights into how non-canonical DNA modifications and their associated proteins regulate important cellular processes and functions.