Reduced genomic cytosine methylation and defective cellular differentiation in embryonic stem cells lacking CpG binding protein.

Herman B Wells Center for Pediatric Research, Section of Pediatric Hematology/Oncology, Department of Pediatrics, Indiana School of Medicine, Indianapolis, IN 46202, USA.
Molecular and Cellular Biology (Impact Factor: 5.04). 07/2005; 25(12):4881-91. DOI: 10.1128/MCB.25.12.4881-4891.2005
Source: PubMed

ABSTRACT 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.

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    ABSTRACT: The mammalian genome is punctuated by CpG islands (CGIs), which differ sharply from the bulk genome by being rich in G+C and the dinucleotide CpG. CGIs often include transcription initiation sites and display 'active' histone marks, notably histone H3 lysine 4 methylation. In embryonic stem cells (ESCs) some CGIs adopt a 'bivalent' chromatin state bearing simultaneous 'active' and 'inactive' chromatin marks. To determine whether CGI chromatin is developmentally programmed at specific genes or is imposed by shared features of CGI DNA, we integrated artificial CGI-like DNA sequences into the ESC genome. We found that bivalency is the default chromatin structure for CpG-rich, G+C-rich DNA. A high CpG density alone is not sufficient for this effect, as A+T-rich sequence settings invariably provoke de novo DNA methylation leading to loss of CGI signature chromatin. We conclude that both CpG-richness and G+C-richness are required for induction of signature chromatin structures at CGIs.
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    ABSTRACT: CXXC finger protein 1 (Cfp1), encoded by the Cxxc1 gene, binds to DNA sequences containing an unmethylated CpG dinucleotide and is an epigenetic regulator of both cytosine and histone methylation. Cxxc1-null mouse embryos fail to gastrulate, and Cxxc1-null embryonic stem cells are viable but cannot differentiate, suggesting that Cfp1 is required for chromatin remodeling associated with stem cell differentiation and embryogenesis. Mice homozygous for a conditional Cxxc1 deletion allele and carrying the inducible Mx1-Cre transgene were generated to assess Cfp1 function in adult animals. Induction of Cre expression in adult animals led to Cfp1 depletion in hematopoietic cells, a failure of hematopoiesis with a nearly complete loss of lineage-committed progenitors and mature cells, elevated levels of apoptosis, and death within two weeks. A similar pathology resulted following transplantation of conditional Cxxc1 bone marrow cells into wild type recipients, demonstrating this phenotype is intrinsic to Cfp1 function within bone marrow cells. Remarkably, the Lin-Sca-1+c-Kit+ population of cells in the bone marrow, which is enriched for hematopoietic stem cells and multi-potential progenitor cells, persists and expands in the absence of Cfp1 during this time frame. Thus, Cfp1 is necessary for hematopoietic stem and multi-potential progenitor cell function and for the developmental potential of differentiating hematopoietic cells.
    PLoS ONE 12/2014; 9(12):e113745. DOI:10.1371/journal.pone.0113745 · 3.53 Impact Factor

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