Conserved role of intragenic DNA methylation in regulating alternative promoters

Abstract

Although it is known that the methylation of DNA in 5′ promoters suppresses gene expression, the role of DNA methylation in gene bodies is unclear1,2,3,4,5. In mammals, tissue- and cell type-specific methylation is present in a small percentage of 5′ CpG island (CGI) promoters, whereas a far greater proportion occurs across gene bodies, coinciding with highly conserved sequences5,6,7,8,9,10. Tissue-specific intragenic methylation might reduce3, or, paradoxically, enhance transcription elongation efficiency1,2,4,5. Capped analysis of gene expression (CAGE) experiments also indicate that transcription commonly initiates within and between genes11,12,13,14,15. To investigate the role of intragenic methylation, we generated a map of DNA methylation from the human brain encompassing 24.7 million of the 28 million CpG sites. From the dense, high-resolution coverage of CpG islands, the majority of methylated CpG islands were shown to be in intragenic and intergenic regions, whereas less than 3% of CpG islands in 5′ promoters were methylated. The CpG islands in all three locations overlapped with RNA markers of transcription initiation, and unmethylated CpG islands also overlapped significantly with trimethylation of H3K4, a histone modification enriched at promoters16. The general and CpG-island-specific patterns of methylation are conserved in mouse tissues. An in-depth investigation of the human SHANK3 locus17,18 and its mouse homologue demonstrated that this tissue-specific DNA methylation regulates intragenic promoter activity in vitro and in vivo. These methylation-regulated, alternative transcripts are expressed in a tissue- and cell type-specific manner, and are expressed differentially within a single cell type from distinct brain regions. These results support a major role for intragenic methylation in regulating cell context-specific alternative promoters in gene bodies.

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Figure 1: Tissue-specific CpG island methylation is prevalent in gene bodies and rare in 5′ promoter regions.
Figure 2: Differentially methylated intragenic CGIs have features of promoters.
Figure 3: Novel transcripts initiate from differentially methylated, evolutionarily conserved intragenic promoters in a cell context-dependent manner.

Accession codes

Data deposits

Sequencing reads are available through the NCBI SRA, accession number SRP002318 (http://www.ncbi.nlm.nih.gov/sra/?term5SRP002318). Browser tracks (hg18 assembly) are available at http://genome.ucsc.edu/. The sequence data for the novel SHANK3 transcripts, 22t and 32t, have been deposited into the dbEST database (accession numbers GD253656 and GD253657, respectively).

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Acknowledgements

We thank S. Vandenberg for technical assistance and The Pleiades Promoter Project and their funders Genome Canada, Genome British Columbia, GlaxoSmithKline R&D Ltd, BC Mental Health and Addiction Services, Child & Family Research Institute, UBC Institute of Mental Health, and the UBC Office of the Vice President Research. This work was supported in part by an NIH NRSA-F31 fellowship to A.K.M. and an NIH NRSA-F32 fellowship to R.P.N., a grant from the National Brain Tumor Society and Goldhirsh Foundation to J.F.C., and by the British Columbia Cancer Foundation. T.W. was a Helen Hay Whitney Fellow and M.A.M. is a Terry Fox Young Investigator and a Michael Smith Senior Research Scholar.

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A.K.M. conceived and performed SHANK3 experiments; R.P.N. designed and performed MeDIP-seq and MRE-seq and qRT-PCR; M.B., C.D., C.N., Y.Z., G.T. and S.J.M.J. performed and analysed brain ChIP-seq; M.A.M., M.H., Y.Z. supervised and analysed IGAII sequencing, and participated in project coordination; S.D.F. performed bisulphite sequencing. C.H. performed bisulphite sequencing and luciferase assay experiments; B.E.J. helped perform MRE-seq and bisulphite sequencing. A.D. wrote the script to parse the SMART and non-SMART containing tags from RNA-seq data. R.V. performed the iterative alignments from RNA-seq and N.T. generated the gene expression measures from the alignments. K.S., V.M.H. and D.H.R. performed mouse brain dissections and isolated astrocytes, neurons and neuronal precursors; T.W., T.J.B., X.X., C.F. and M.S. performed bioinformatics analyses. D.H. participated in project coordination and SHANK3 genomic conservation analysis. A.K.M., R.P.N., T.W. and J.F.C. coordinated the project, wrote the manuscript and incorporated revisions from co-authors.

Corresponding authors

Correspondence to Ting Wang or Joseph F. Costello.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Information

This file contains Supplementary Tables 1-2, Supplementary Figures S1-S21 with legends, Supplementary Methods, which includes a Supplementary Table, Supplementary References and additional information for Supplementary Data 2. (PDF 5369 kb)

Supplementary Data 1

This file contains MeDIP and MRE datasets for 2 biological replicates. (XLS 3425 kb)

Supplementary Data 2

The file contains methylation analysis of transposable elements (see Supplementary Information file, page 60). (XLS 3851 kb)

Supplementary Data 3

This file contains bisulfite sequencing. (XLS 202 kb)

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Maunakea, A., Nagarajan, R., Bilenky, M. et al. Conserved role of intragenic DNA methylation in regulating alternative promoters. Nature 466, 253–257 (2010). https://doi.org/10.1038/nature09165

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