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Author Notes:

To whom correspondence should be addressed. Tel: +1 713 834 6274; Email: xcheng5@mdanderson.org

Xiaodong Cheng ORCID: https://orcid.org/0000-0002-6967-6362

J.Y. performed protein purification, DNA-binding experiments, mutagenesis and crystallization; J.R.H. performed X-ray data collection and structure determination; D.W. made initial expression construct, performed preliminary purification and DNA binding assays; R.R. assisted in DNA-binding assays and crystallization; J.L, D.S. and Y.H. analyzed published C/EBPβ ChIP-seq data; X.Z. and X.C. organized and designed the scope of the study; R.M.B. performed data analysis and assisted in preparing the manuscript.

We thank B. Baker of New England Biolabs for synthesizing the oligonucleotides.

Conflict of interest statement: None declared.


Research Funding:

U.S. National Institutes of Health (NIH) [GM049245-24 to X.C. and HL134780 to Y.H.]; Cancer Prevention and Research Institute of Texas [RR160029 to X.C. and RR140053 to Y.H.]; American Cancer Society [RSG-18-043-01-LIB to Y.H.].

The open access publication charge for this paper has been waived by Oxford University Press – NAR Editorial Board members are entitled to one free paper per year in recognition of their work on behalf of the journal.


  • CCAAT/enhancer binding proteins
  • C/EBP
  • gene expression
  • developmental stages
  • physiological conditions
  • pathological conditions
  • transcription factors
  • dinucleotides
  • DNA
  • binding affinity
  • methyl group

Structural basis for effects of CpA modifications on C/EBPβ binding of DNA


Journal Title:

Nucleic Acids Research


Volume 47, Number 4


, Pages 1774-1785

Type of Work:

Article | Final Publisher PDF


CCAAT/enhancer binding proteins (C/EBPs) regulate gene expression in a variety of cells/tissues/organs, during a range of developmental stages, under both physiological and pathological conditions. C/EBP-related transcription factors have a consensus binding specificity of 5'-TTG-CG-CAA-3', with a central CpG/CpG and two outer CpA/TpG dinucleotides. Methylation of the CpG and CpA sites generates a DNA element with every pyrimidine having a methyl group in the 5-carbon position (thymine or 5-methylcytosine (5mC)). To understand the effects of both CpG and CpA modification on a centrally-important transcription factor, we show that C/EBPβ binds the methylated 8-bp element with modestly-increased (2.4-fold) binding affinity relative to the unmodified cognate sequence, while cytosine hydroxymethylation (particularly at the CpA sites) substantially decreased binding affinity (36-fold). The structure of C/EBPβ DNA binding domain in complex with methylated DNA revealed that the methyl groups of the 5mCpA/TpG make van der Waals contacts with Val285 in C/EBPβ. Arg289 recognizes the central 5mCpG by forming a methyl-Arg-G triad, and its conformation is constrained by Val285 and the 5mCpG methyl group. We substituted Val285 with Ala (V285A) in an Ala-Val dipeptide, to mimic the conserved Ala-Ala in many members of the basic leucine-zipper family of transcription factors, important in gene regulation, cell proliferation and oncogenesis. The V285A variant demonstrated a 90-fold binding preference for methylated DNA (particularly 5mCpA methylation) over the unmodified sequence. The smaller side chain of Ala285 permits Arg289 to adopt two alternative conformations, to interact in a similar fashion with either the central 5mCpG or the TpG of the opposite strand. Significantly, the best-studied cis-regulatory elements in RNA polymerase II promoters and enhancers have variable sequences corresponding to the central CpG or reduced to a single G:C base pair, but retain a conserved outer CpA sequence. Our analyses suggest an important modification-dependent CpA recognition by basic leucine-zipper transcription factors.

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© The Author(s) 2018. Published by Oxford University Press on behalf of Nucleic Acids Research.

This is an Open Access work distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International License (http://creativecommons.org/licenses/by-nc/4.0/).

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