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Molecular and Cellular Biology, June 2005, p. 4881-4891, Vol. 25, No. 12
0270-7306/05/$08.00+0     doi:10.1128/MCB.25.12.4881-4891.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Reduced Genomic Cytosine Methylation and Defective Cellular Differentiation in Embryonic Stem Cells Lacking CpG Binding Protein

Diana L. Carlone,{dagger} Jeong-Heon Lee,{dagger} Suzanne R. L. Young,{dagger} Erika Dobrota,{dagger} Jill Sergesketter Butler,{dagger} Joseph Ruiz, and David G. Skalnik*

Herman B Wells Center for Pediatric Research, Section of Pediatric Hematology/Oncology, Departments of Pediatrics and Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, Indiana 46202

Received 11 February 2005/ Returned for modification 11 March 2005/ Accepted 21 March 2005

Cytosine methylation at CpG dinucleotides is a critical epigenetic modification of mammalian genomes. CpG binding protein (CGBP) exhibits a unique DNA-binding specificity for unmethylated CpG motifs and is essential for early murine development. Embryonic stem cell lines deficient for CGBP were generated to further examine CGBP function. CGBP–/– cells are viable but show an increased rate of apoptosis and are unable to achieve in vitro differentiation following removal of leukemia inhibitory factor from the growth media. Instead, CGBP–/– embryonic stem cells remain undifferentiated as revealed by persistent expression of the pluripotent markers Oct4 and alkaline phosphatase. CGBP–/– cells exhibit a 60 to 80% decrease in global cytosine methylation, including hypo-methylation of repetitive elements, single-copy genes, and imprinted genes. Total DNA methyltransferase activity is reduced by 30 to 60% in CGBP–/– cells, and expression of the maintenance DNA methyltransferase 1 protein is similarly reduced. However, de novo DNA methyltransferase activity is normal. Nearly all aspects of the pleiotropic CGBP–/– phenotype are rescued by introduction of a CGBP expression vector. Hence, CGBP is essential for normal epigenetic modification of the genome by cytosine methylation and for cellular differentiation, consistent with the requirement for CGBP during early mammalian development.

* Corresponding author. Mailing address: Cancer Research Building, Room W327, 1044 West Walnut St., Indianapolis, IN 46202. Phone: (317) 274-8977. Fax: (317) 274-8679. E-mail: dskalnik{at}iupui.edu.

{dagger} These authors contributed equally to this work.

Molecular and Cellular Biology, June 2005, p. 4881-4891, Vol. 25, No. 12
0022-538X/05/$08.00+0     doi:10.1128/MCB.25.12.4881-4891.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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