Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2010 Jul 8;466(7303):253-7.
doi: 10.1038/nature09165.

Conserved role of intragenic DNA methylation in regulating alternative promoters

Alika K Maunakea  1 , Raman P Nagarajan, Mikhail Bilenky, Tracy J Ballinger, Cletus D'Souza, Shaun D Fouse, Brett E Johnson, Chibo Hong, Cydney Nielsen, Yongjun Zhao, Gustavo Turecki, Allen Delaney, Richard Varhol, Nina Thiessen, Ksenya Shchors, Vivi M Heine, David H Rowitch, Xiaoyun Xing, Chris Fiore, Maximiliaan Schillebeeckx, Steven J M Jones, David Haussler, Marco A Marra, Martin Hirst, Ting Wang, Joseph F Costello
Affiliations

Conserved role of intragenic DNA methylation in regulating alternative promoters

Alika K Maunakea et al. Nature. .

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 unclear. 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 sequences. Tissue-specific intragenic methylation might reduce, or, paradoxically, enhance transcription elongation efficiency. Capped analysis of gene expression (CAGE) experiments also indicate that transcription commonly initiates within and between genes. 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 promoters. The general and CpG-island-specific patterns of methylation are conserved in mouse tissues. An in-depth investigation of the human SHANK3 locus 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.

PubMed Disclaimer

Conflict of interest statement

The authors declare no competing financial interests.

Figures

Figure 1
Figure 1. Tissue-specific CpG island methylation is prevalent in gene bodies and rare in 5′ promoter regions
a, Inverse correlation between MeDIP-seq and MRE-seq in 5′ promoter, intragenic, 3′ and intergenic CGIs. Unmethylated CpGs are shown as an MRE score (a normalized number of reads interrogating each CGI, see Supplementary Methods) on the Y-axis. Methylated regions are shown as reads/kb from MeDIP-seq on the X-axis. b, Top, MAPK4 with methylated regions (MeDIP-seq, dark brown) and unmethylated CpG sites (MRE-seq, green). Zoomed-in views of each CGI are shown below, and percent methylation for each CpG site assessed by bisulfite sequencing is graphed to the right. c, Percent of CGIs that exhibit methylation in a particular tissue, methylation in one or more tissues (mouse, at least one cell type), or tissue-specific methylation (mouse, differentially).
Figure 2
Figure 2. Differentially methylated intragenic CGIs exhibit features of promoters
a, Methylated CGIs are indicated above the zero line and unmethylated CGIs are below. For human, the methylation data is from frontal cortex, and CAGE tags are derived from multiple tissues,. For mouse, the methylation data includes the same set of tissues described in figure 1c, and CAGE data are derived from multiple mouse tissues,. 91% of human intragenic CGI CAGE tags mapped outside of exons and are probably not derived from posttranscriptional processing. b, H3K4me3 tissue-ChIP-seq normalized internal coverage (NIC) scores compared to MeDIP- and MRE-seq methylation data at CGIs for human frontal cortex. c, Heatmap view of the status of 8092 intragenic CGIs based on five genome-wide datasets. Each island is coloured according to its status and sorted from top to bottom in the order of increasing signal in MeDIP-seq, then within the three MeDIP-defined subgroups by signals in MRE-seq. This process is performed iteratively based on H3K4me3, RNA-seq TSS and CAGE status. For MeDIP-seq, green indicates unmethylated (0–20 reads/kb), maroon indicates partially methylated (20–50 reads/kb), and red indicates methylated (>50 reads/kb); For MRE-seq, green indicates unmethylated (MRE score 0–5), red indicates methylated (MRE score >5); For H3K4me3 ChIP-seq, green indicates active/with signal, red indicates inactive/without signal. For RNA-seq TSS, green indicates evidence for TSS, red indicates lack of evidence for TSS (see Supplemental Methods). For CAGE, green indicates CAGE tags from one or more tissues that overlap the CGI; red indicates lack of overlapping CAGE tags.
Figure 3
Figure 3. Novel transcripts initiate from differentially methylated, evolutionarily conserved intragenic promoters in a cell context-dependent manner
a, Human frontal cortex MRE-seq, MeDIP-seq and H3K4me3 ChIP-seq at SHANK3 (top). Evolutionarily conserved regions (ECRs) overlap with mouse CAGE tag clusters (arrows), mouse ES H3K4me3 and H3K27me3 bivalent domains and human frontal cortex H3K4me3. ECRs with most or all promoter-associated features are shown with light grey bars. b, Diagram of ECR22 (left) and ECR32 (right) mouse genomic regions displaying from top to bottom ECRs, sequences used for promoter assays, 5′ RACE sequences of 22t and 32t with associated ATGs (arrow), known exons, CpG island (dark green) and CpG-rich (light green) regions, and multi-species DNA sequence conservation. c, In vitro methylation of the mouse SHANK3 intragenic promoters abolished their activity in promoter assays. Me, methylated; Mock, mock treated; Un, untreated. d, Bisulfite sequencing of ECR32 in matched tissues/cells from human and mouse. P=0.018; ANOVA regression analysis. e, Increased 32t transcription in cortical, but not hippocampal astrocytes after treatment with 5azadC by transcript-specific RT-PCR (p<0.05, Student’s t-test). Positive controls: untreated primary cultures of cerebellar granule neural progenitor cells (CGNPs), their in vitro differentiated neurons (CG neurons), and whole brain. The 24-bp size difference in CGNPs and CG neurons is due to alternative splicing within the 32t transcript. Hi., hippocampal; Ctx., cortical. f, Increased expression of full-length SHANK3 detected by qRT-PCR in astrocytes treated with TSA alone or in combination with 5azadC (p<0.05, Student’s t-test) but not 5azadC alone.
See this image and copyright information in PMC

Comment in

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Cokus SJ, et al. Shotgun bisulphite sequencing of the Arabidopsis genome reveals DNA methylation patterning. Nature. 2008;452 (7184):215–219. - PMC - PubMed
    1. Flanagan JM, Wild L. An epigenetic role for noncoding RNAs and intragenic DNA methylation. Genome Biol. 2007;8 (6):307. - PMC - PubMed
    1. Lorincz MC, Dickerson DR, Schmitt M, Groudine M. Intragenic DNA methylation alters chromatin structure and elongation efficiency in mammalian cells. Nat Struct Mol Biol. 2004 - PubMed
    1. Ball MP, et al. Targeted and genome-scale strategies reveal gene-body methylation signatures in human cells. Nat Biotechnol. 2009;27 (4):361–368. - PMC - PubMed
    1. Rauch TA, et al. A human B cell methylome at 100-base pair resolution. Proc Natl Acad Sci U S A. 2009;106 (3):671–678. - PMC - PubMed

Publication types

MeSH terms