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

点击翻译按钮获取中文摘要

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.