An all-to-all approach to the identification of sequence-specific readers for epigenetic DNA modifications on cytosine

Guang Song,Ximei Luo,Ying Cheng,Hongjun Song,Qifeng Song,Guohua Wang,Heng Zhu,Peng Jin,Jiang Qian,Cedric Moore,Jun Wan

doi:10.1038/s41467-021-20950-w

Abstract

Epigenetic modifications of DNA play important roles in many biological processes. Identifying readers of these epigenetic marks is a critical step towards understanding the underlying mechanisms. Here, we present an all-to-all approach, dubbed digital affinity profiling via proximity ligation (DAPPL), to simultaneously profile human TF-DNA interactions using mixtures of random DNA libraries carrying different epigenetic modifications (i.e., 5-methylcytosine, 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxylcytosine) on CpG dinucleotides. Many proteins that recognize consensus sequences carrying these modifications in symmetric and/or hemi-modified forms are identified. We further demonstrate that the modifications in different sequence contexts could either enhance or suppress TF binding activity. Moreover, many modifications can affect TF binding specificity. Furthermore, symmetric modifications show a stronger effect in either enhancing or suppressing TF-DNA interactions than hemi-modifications. Finally, in vivo evidence suggests that USF1 and USF2 might regulate transcription via hydroxymethylcytosine-binding activity in weak enhancers in human embryonic stem cells.

Highlights

Epigenetic modifications of DNA play important roles in many biological processes
To create an all-to-all approach for unbiased profiling of transcription factor (TF)-DNA interactions, we invented the digital affinity profiling via proximity ligation (DAPPL) approach, which was achieved in five major steps (Fig. 1)
Human proteins were purified as GST fusions and kept on glutathione beads, while a set of DNA barcode sequences were designed such that any two single nucleotide mis-incorporations, insertions, and/or deletions would not lead to misassignment of protein identity (Supplementary Fig. 1a-b)

Summary

Introduction

Epigenetic modifications of DNA play important roles in many biological processes. Identifying readers of these epigenetic marks is a critical step towards understanding the underlying mechanisms. In addition to hmC, fC was found as a stable DNA modification in mammalian genomes[8,16,17,18] These studies indicated that the oxidized forms of mC might have their own physiological functions, and identification of their “readers” and “effectors” might help elucidate their roles in various biological processes[19,20,21,22,23]. In the context of the palindromic CpG dinucleotide, hmCpG presumably exists in a fully hydroxymethylated form in cells; they can transiently become hemi-hmC after semi-conservative DNA replication[34] Following this logic, hemi-fC and hemi-caC should exist, at least transiently, in mammalian genomes. The identification of ‘readers’ and ‘effectors’ for mC, hmC, fC, and caC in both symmetric and hemi-forms will serve as a critical stepping stone to translate epigenetic signals into biological actions and to decipher the epigenetic ‘codes’ governing biological processes

Methods

Results

Conclusion