Developmentally Programmed 3′ CpG Island Methylation Confers Tissue- and Cell-Type-Specific Transcriptional Activation

Abstract

During development, a small but significant number of CpG islands (CGIs) become methylated. The timing of developmentally programmed CGI methylation and associated mechanisms of transcriptional regulation during cellular differentiation, however, remain poorly characterized. Here, we used genome-wide DNA methylation microarrays to identify epigenetic changes during human embryonic stem cell (hESC) differentiation. We discovered a group of CGIs associated with developmental genes that gain methylation after hESCs differentiate. Conversely, erasure of methylation was observed at the identified CGIs during subsequent reprogramming to induced pluripotent stem cells (iPSCs), further supporting a functional role for the CGI methylation. Both global gene expression profiling and quantitative reverse transcription-PCR (RT-PCR) validation indicated opposing effects of CGI methylation in transcriptional regulation during differentiation, with promoter CGI methylation repressing and 3′ CGI methylation activating transcription. By studying diverse human tissues and mouse models, we further confirmed that developmentally programmed 3′ CGI methylation confers tissue- and cell-type-specific gene activation in vivo. Importantly, luciferase reporter assays provided evidence that 3′ CGI methylation regulates transcriptional activation via a CTCF-dependent enhancer-blocking mechanism. These findings expand the classic view of mammalian CGI methylation as a mechanism for transcriptional silencing and indicate a functional role for 3′ CGI methylation in developmental gene regulation.

SUPPLEMENTAL MATERIAL

Supplemental material for this article may be found at http://dx.doi.org/10.1128/MCB.01124-12.

ACKNOWLEDGMENTS

We thank Mitsuyoshi Nakao for providing the H19 DMR reporter plasmids and Yi Guo, Wei Zhu, Jinming Shu, Wei Wei, Angelique Nelson, Savannah Cook, and Robert Milczarek for technical assistance. We also thank Adam Gillum for assistance with the figures.

This work was supported by grants from the Sidney Kimmel Foundation to L.S., the USDA (CRIS 6250-5100-050) to L.S. and R.A.W., the NIGMS (P01GM081619-01) to C.W., and the NIDDK (1R01DK081557) to R.A.W. and L.S. and by private funding from the Institute for Stem Cell and Regenerative Medicine to C.W.