DNA methylation presents distinct binding sites for human transcription factors

Shaohui Hu,Eric Cox,Hongjun Song,Crystal Woodard,Jiang Qian,Ha Nam Nguyen,Shuang Liu,Jaehoon Shin,Heng Zhu,Herschel Wade,Guo-Li Ming,Huibin Lyu,Jun Wan,Hee Sool Rho,Yaxue Zeng,Qifeng Song,Yijing Su,Shuli Xia

doi:10.7554/elife.00726

Abstract

DNA methylation, especially CpG methylation at promoter regions, has been generally considered as a potent epigenetic modification that prohibits transcription factor (TF) recruitment, resulting in transcription suppression. Here, we used a protein microarray-based approach to systematically survey the entire human TF family and found numerous purified TFs with methylated CpG (mCpG)-dependent DNA-binding activities. Interestingly, some TFs exhibit specific binding activity to methylated and unmethylated DNA motifs of distinct sequences. To elucidate the underlying mechanism, we focused on Kruppel-like factor 4 (KLF4), and decoupled its mCpG- and CpG-binding activities via site-directed mutagenesis. Furthermore, KLF4 binds specific methylated or unmethylated motifs in human embryonic stem cells in vivo. Our study suggests that mCpG-dependent TF binding activity is a widespread phenomenon and provides a new framework to understand the role and mechanism of TFs in epigenetic regulation of gene transcription. DOI:http://dx.doi.org/10.7554/eLife.00726.001.

Highlights

DNA methylation is an ancient and major epigenetic modification that plays an important role in key biological processes, including genomic imprinting, X-chromosome inactivation, suppression of transposable elements, and carcinogenesis (Jaenisch and Bird, 2003; Egger et al, 2004; Robertson, 2005; Feinberg, 2007; Reik, 2007)
We selected a total of 154 DNA motifs (Supplementary file 1A) with the following characteristics: (1) predicted to be potential transcription factor (TF)-binding sites in promoter regions of the human genome (Xie et al, 2005; Yu et al, 2006; Elemento et al, 2007); (2) representative of a subset of the 460 DNA motifs that have been examined for protein-binding activity in our previous study (Hu et al, 2009; Xie et al, 2010); and (3) carrying at least one CpG site
Because our goal was to identify DNA methylation-dependent binding activity, we developed a competition assay on a protein microarray to identify TFs that prefer DNA motifs carrying methylated CpG (mCpG) (Figure 1A)

Summary

Introduction

DNA methylation is an ancient and major epigenetic modification that plays an important role in key biological processes, including genomic imprinting, X-chromosome inactivation, suppression of transposable elements, and carcinogenesis (Jaenisch and Bird, 2003; Egger et al, 2004; Robertson, 2005; Feinberg, 2007; Reik, 2007). Methylation of CpG sites, especially at promoter regions, is generally considered as the hallmark of gene silencing (Baylin, 2005). The molecular consequence of CpG methylation is generally believed to disrupt TF–DNA interactions either directly (Nan et al, 1998), or indirectly by recruiting sequence-independent methylated DNA-binding proteins that occupy the methylated promoters and compete for the TF binding sites (Boyes and Bird, 1991).

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