TY - JOUR
T1 - Whole-genome bisulfite sequencing of two distinct interconvertible DNA methylomes of mouse embryonic stem cells
AU - Habibi, Ehsan
AU - Brinkman, Arie B.
AU - Arand, Julia
AU - Kroeze, Leonie I.
AU - Kerstens, Hindrik H.D.
AU - Matarese, Filomena
AU - Lepikhov, Konstantin
AU - Gut, Marta
AU - Brun-Heath, Isabelle
AU - Hubner, Nina C.
AU - Benedetti, Rosaria
AU - Altucci, Lucia
AU - Jansen, Joop H.
AU - Walter, Jörn
AU - Gut, Ivo G.
AU - Marks, Hendrik
AU - Stunnenberg, Hendrik G.
N1 - Funding Information:
We thank Eva Janssen-Megens, Anita Kaan, and Yan Tan for sequencing, Kees-Jan Françoijs and Simon van Heeringen for bioinformatic assistance, Roberta Menafra and Gianluigi Franci for general practical advice, and Pascal Giehr, Christina LoPorto, Jasmin Gries, and Karl Nordström for help with hairpin BS-seq. Felix Krueger helped with preprocessing of the sequencing data, and Andrew Smith, Qiang Song, Fang Fang, and Meng Zhou helped with the WGBS analysis. Yuanxin Xi provided advice for RRBSMAP data analysis. Joost Martens and Colin Logie gave useful comments on the manuscript. Tüzer Kalkan and Austin Smith provided the Rex/GFP-2i cells, and Alice Jouneau provided the EpiSCs. The research leading to these results has received funding from the Dutch KWF (KUN2008-4130), Netherlands National Computing Facilities foundation grant MP-266-13, the Netherlands Institute for Regenerative Medicine (NIRM), the Epigenomics Flagship Project “EPIGEN” (MIUR-CNR), PON_0101227, Deutsche Forschungsgemeinschaft (DFG) priority program SPP1356 “Pluripotency and Cellular Reprogramming” (grant number WA1029), and the European Union grants SYNERGY (NWO/ Zoom 90201134; within ERASYSBIO+, FP7/2008: 235447), BLUEPRINT (FP7/2011: 282510), and PluriSys (FP7/2009: 223485). E.H. and H.M. carried out most of the wet-lab experiments and F.M. helped with western blots and DNA isolation. M.G., I.B.H., and I.G.G. performed bisulfite conversion and sequencing of the samples. E.H. and H.H.D.K. mapped sequencing data and determined methylation levels. E.H. and A.B.B. performed bioinformatic analysis. J.A., K.L., and J.W. performed hairpin-bisulfite amplicon sequencing and cell stainings. N.C.H. performed MS analysis of DNMT1 and L.I.K. and J.H.J. performed MS analysis of DNA (hydroxy)methylation. R.B. and L.A. measured DNMT1 activity. H.M., E.H., A.B.B., and H.G.S. wrote the manuscript. H.M. and H.G.S. designed and supervised the experiments.
PY - 2013/9/5
Y1 - 2013/9/5
N2 - The use of two kinase inhibitors (2i) enables derivation of mouse embryonic stem cells (ESCs) in the pluripotent ground state. Using whole-genome bisulfite sequencing (WGBS), we show that male 2i ESCs are globally hypomethylated compared to conventional ESCs maintained in serum. In serum, female ESCs are hypomethyated similarly to male ESCs in 2i, and DNA methylation is further reduced in 2i. Regions with elevated DNA methylation in 2i strongly correlate with the presence of H3K9me3 on endogenous retroviruses (ERVs) and imprinted loci. The methylome of male ESCs in serum parallels postimplantation blastocyst cells, while 2i stalls ESCs in a hypomethylated, ICM-like state. WGBS analysis during adaptation of 2i ESCs to serum suggests that deposition of DNA methylation is largely random, while loss of DNA methylation during reversion to 2i occurs passively, initiating at TET1 binding sites. Together, our analysis provides insight into DNA methylation dynamics in cultured ESCs paralleling early developmental processes.
AB - The use of two kinase inhibitors (2i) enables derivation of mouse embryonic stem cells (ESCs) in the pluripotent ground state. Using whole-genome bisulfite sequencing (WGBS), we show that male 2i ESCs are globally hypomethylated compared to conventional ESCs maintained in serum. In serum, female ESCs are hypomethyated similarly to male ESCs in 2i, and DNA methylation is further reduced in 2i. Regions with elevated DNA methylation in 2i strongly correlate with the presence of H3K9me3 on endogenous retroviruses (ERVs) and imprinted loci. The methylome of male ESCs in serum parallels postimplantation blastocyst cells, while 2i stalls ESCs in a hypomethylated, ICM-like state. WGBS analysis during adaptation of 2i ESCs to serum suggests that deposition of DNA methylation is largely random, while loss of DNA methylation during reversion to 2i occurs passively, initiating at TET1 binding sites. Together, our analysis provides insight into DNA methylation dynamics in cultured ESCs paralleling early developmental processes.
UR - http://www.scopus.com/inward/record.url?scp=84884133672&partnerID=8YFLogxK
U2 - 10.1016/j.stem.2013.06.002
DO - 10.1016/j.stem.2013.06.002
M3 - Article
C2 - 23850244
AN - SCOPUS:84884133672
SN - 1934-5909
VL - 13
SP - 360
EP - 369
JO - Cell Stem Cell
JF - Cell Stem Cell
IS - 3
ER -