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Impact of cytosine methylation on DNA binding specificities of human transcription factors
Positives and negatives of methylated CpG
When the DNA bases cytosine and guanine are next to each other, a methyl group is generally added to the pyrimidine, generating a mCpG dinucleotide. This modification alters DNA structure but can also affect function by inhibiting transcription factor (TF) binding. Yin et al. systematically analyzed the effect of CpG methylation on the binding of 542 human TFs (see the Perspective by Hughes and Lambert). In addition to inhibiting binding of some TFs, they found that mCpGs can promote binding of others, particularly TFs involved in development, such as homeodomain proteins.
Structured Abstract
INTRODUCTION
Nearly all cells in the human body share the same primary genome sequence consisting of four nucleotide bases. One of the bases, cytosine, is commonly modified by methylation of its 5 position in CpG dinucleotides (mCpG). Most CpG dinucleotides in the human genome are methylated, but the level of CpG methylation varies with genetic location (promoter versus gene body), whether genes are active versus silenced, and cell type. Research has shown that the maintenance of a particular cellular state after cell division is dependent on faithful transmission of methylated CpGs, as well as inheritance of the mother cells’ repertoire of transcription factors by the daughter cells. These two mechanisms of epigenetic inheritance are linked to each other; the binding of transcription factors can be affected by cytosine methylation, and cytosine methylation can, in turn, be added or removed by proteins that associate with transcription factors.
RATIONALE
The genetic and epigenetic language, which imparts when and where genes are expressed, is understood at a conceptual level. However, a more detailed understanding is needed of the genomic regulatory mechanism by which methylated cytosines affect transcription factor binding. Because cytosine methylation changes DNA structure, it has the potential to affect binding of all transcription factors. However, a systematic analysis of binding of a large collection of transcription factors to all possible DNA sequences has not previously been conducted.
RESULTS
To globally characterize the effect of cytosine methylation on transcription factor binding, we systematically analyzed binding specificities of full-length transcription factors and extended DNA binding domains to unmethylated and CpG-methylated DNA by using methylation-sensitive SELEX (systematic evolution of ligands by exponential enrichment). We evaluated binding of 542 transcription factors and identified a large number of previously uncharacterized transcription factor recognition motifs. Binding of most major classes of transcription factors, including bHLH, bZIP, and ETS, was inhibited by mCpG. In contrast, transcription factors such as homeodomain, POU, and NFAT proteins preferred to bind methylated DNA. This class of binding was enriched in factors with central roles in embryonic and organismal development.
The observed binding preferences were validated using several orthogonal methods, including bisulfite-SELEX and protein-binding microarrays. In addition, the preference of the pluripotency factor OCT4 to bind to a mCpG-containing motif was confirmed by chromatin immunoprecipitation analysis in mouse embryonic stem cells with low or high levels of CpG methylation (due to deficiency in all enzymes that methylate cytosines or contribute to their removal, respectively). Crystal structure analysis of the homeodomain proteins HOXB13, CDX1, CDX2, and LHX4 revealed three key residues that contribute to the preference of this developmentally important family of transcription factors for mCpG. The preference for binding to mCpG was due to direct hydrophobic interactions with the 5-methyl group of methylcytosine. In contrast, inhibition of binding of other transcription factors to methylated sequences was found to be caused by steric hindrance.
CONCLUSION
Our work constitutes a global analysis of the effect of cytosine methylation on DNA binding specificities of human transcription factors. CpG methylation can influence binding of most transcription factors to DNA—in some cases negatively and in others positively. Our finding that many developmentally important transcription factors prefer to bind to mCpG sites can inform future analyses of the role of DNA methylation on cell differentiation, chromatin reprogramming, and transcriptional regulation.
Systematic analysis of the impact of CpG methylation on transcription factor binding.
The bottom left panel shows the fraction of transcription factors that prefer methylated (orange) or unmethylated (teal) CpG sites, are affected in multiple ways (yellow), are not affected (green), or do not have a CpG in their motifs (gray), as determined by methylation-sensitive SELEX (top left). The structure and logos on the right highlight how HOXB13 recognizes mCpG (blue shading indicates a CpG affected by methylation).
Abstract
The majority of CpG dinucleotides in the human genome are methylated at cytosine bases. However, active gene regulatory elements are generally hypomethylated relative to their flanking regions, and the binding of some transcription factors (TFs) is diminished by methylation of their target sequences. By analysis of 542 human TFs with methylation-sensitive SELEX (systematic evolution of ligands by exponential enrichment), we found that there are also many TFs that prefer CpG-methylated sequences. Most of these are in the extended homeodomain family. Structural analysis showed that homeodomain specificity for methylcytosine depends on direct hydrophobic interactions with the methylcytosine 5-methyl group. This study provides a systematic examination of the effect of an epigenetic DNA modification on human TF binding specificity and reveals that many developmentally important proteins display preference for mCpG-containing sequences.
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Supplementary Material
Summary
Figs. S1 to S19
Tables S1 to S6
Data S1 to S3
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References and Notes
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Published In
Science
Volume 356 | Issue 6337
5 May 2017
5 May 2017
Copyright
Copyright © 2017, American Association for the Advancement of Science.
Submission history
Received: 15 September 2016
Accepted: 9 March 2017
Published in print: 5 May 2017
Acknowledgments
We thank S. Linnarsson, N. Zielke, B. Schmierer, F. Zhu, and J. Zhang for critical review of the manuscript; S. Augsten and the Karolinska Institutet Protein Science Facility for protein production and the opportunity to run ITC experiments; and L. Hu, A. Zetterlund, and M. Hoh for technical assistance. Illumina sequencing reads were deposited in the European Nucleotide Archive under accession number PRJEB9797. The experimental data and atomic coordinates have been submitted to the Protein Data Bank with accession codes 5HOD (LHX4), 5EF6 (HOXB13_methDNA), 5EGO (HOXB13:MEIS1_methDNA), 5LUX (CDX1_methDNA), and 5LTX (CDX2_methDNA). This work was supported by the Academy of Finland Center of Excellence in Cancer Genetics, the European Research Area SynBio project MirrorBio, the Center for Innovative Medicine at Karolinska Institutet, the Knut and Alice Wallenberg Foundation, the Göran Gustafsson Foundation, and Vetenskapsrådet. Y.Y., A.J., K.R.N., and J.T. designed the experiments. Y.Y. performed the HT-SELEX, methyl-SELEX, and bisulfite-SELEX experiments. Y.Y. and B.S. performed the ChIP-seq experiment, K.D. performed the ChIP-exo and ATAC-seq experiments, and B.S. performed the whole-genome bisulfite sequencing experiments. S.K.-S. performed the protein-binding microarray experiment. P.K.D. purified proteins for crystallography, E.M. and A.P. performed the x-ray crystallography experiments, and E.M. solved the protein structures. P.A.G. and S.D. generated Tet-TKO ES cells. Y.Y., E.K., K.R.N., A.J., F.Z., T.K., and J.T. contributed to computational analysis. Y.Y., E.K., and K.R.N. prepared the illustrations. Y.Y. and J.T. wrote the manuscript. All authors contributed to data analysis and reviewed the manuscript.
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