Pioneer factor interactions and unmethylated CpG dinucleotides mark silent tissue-specific enhancers in embryonic stem cells

doi:10.1073/pnas.0704579104

Pioneer factor interactions and unmethylated CpG dinucleotides mark silent tissue-specific enhancers in embryonic stem cells

*Howard Hughes Medical Institute, Molecular Biology Institute, and Department of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles, CA 90095;
^†Institute of Stem Cell Biology and Regenerative Medicine, Departments of Pathology and Developmental Biology, Stanford University School of Medicine, Stanford, CA 94305-5323; and
^‡Cell and Developmental Biology Program, Fox Chase Cancer Center, Philadelphia, PA 19111

Contributed by Irving L. Weissman, May 26, 2007 (received for review March 5, 2007)

Abstract

Recent studies have suggested that, in ES cells, inactive genes encoding early developmental regulators possess bivalent histone modification domains and are therefore poised for activation. However, bivalent domains were not observed at typical tissue-specific genes. Here, we show that windows of unmethylated CpG dinucleotides and putative pioneer factor interactions mark enhancers for at least some tissue-specific genes in ES cells. The unmethylated windows expand in cells that express the gene and contract, disappear, or remain unchanged in nonexpressing tissues. However, in ES cells, they do not always coincide with common histone modifications. Genomic footprinting and chromatin immunoprecipitation demonstrated that transcription factor binding underlies the unmethylated windows at enhancers for the Ptcra and Alb1 genes. After stable integration of premethylated Ptcra enhancer constructs into the ES cell genome, the unmethylated windows readily appeared. In contrast, the premethylated constructs remained fully methylated and silent after introduction into Ptcra-expressing thymocytes. These findings provide initial functional support for a model in which pioneer factor interactions in ES cells promote the assembly of a chromatin structure that is permissive for subsequent activation, and in which differentiated tissues lack the machinery required for gene activation when these ES cell marks are absent. The enhancer marks may therefore represent important features of the pluripotent state.

Footnotes

^§To whom correspondence may be addressed. E-mail: irv{at}stanford.edu or smale{at}mednet.ucla.edu
Author contributions: J.X., J.L.A., P.P., J.A.W., K.S.Z., I.L.W., and S.T.S. designed research; J.X., S.D.P., A.R.J., J.L.A., P.P., and J.A.W. performed research; J.X., S.D.P., A.R.J., J.L.A., P.P., J.A.W., K.S.Z., I.L.W., and S.T.S. analyzed data; and J.X. and S.T.S. wrote the paper.
Conflict of interest statement: I.L.W. was a member of the scientific advisory board of Amgen, cofounded and is a director of Stem Cells, Inc., and cofounded Cellerant, Inc.
This article contains supporting information online at www.pnas.org/cgi/content/full/0704579104/DC1.
Abbreviations:

BMDM,

bone marrow-derived macrophages;

EB,

embryoid bodies;

HSC,

hematopoietic stem cell.