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

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.The CpG dinucleotide is an important regulatory component of mammalian genomes. The cytosine of this dinucleotide serves as the target for methylation by DNA methyltransferase (Dnmt) enzymes, which functions as a critical epigenetic modification of DNA. Methylated DNA is correlated with heterochromatin and transcriptionally inactive genes, while actively expressed genes are generally hypomethylated (58). Cytosine methylation may also represent a defense mechanism to silence parasitic repetitive DNA elements present in mammalian genomes (72,78). In addition, cytosine methylation is involved in the processes of genomic imprinting, in which paternal and maternal alleles of a gene exhibit distinct patterns of cytosine methylation and expression (65), and X chromosome inactivation, in which one X chromosome in each cell of a female becomes irreversibly inactivated during early development (52). The CpG dinucleotide is underrepresented in mammalian genomes (5 to 10% of the expected frequency), presumably due to the propensity of 5-methylcytosine to undergo spontaneous deamination to form thymine (8). Approximately 50% of human and mouse genes reside near unmethylated CpG islands, which contain the statistically expected frequency of CpG dinucleotides.Global cytosine methylation patterns inherited from gametes are erased during early embryogenesis (morula), followed by a wave of de novo DNA methylation in the blastocyst upon implantation (44). Dnmt3a and Dnmt3b are de novo methyltransferases that preferentially recognize unmethylated CpG motifs (49), while Dnmt1 is a maintenance methyltransferase that recognizes hemimethylated DNA (5), the imme...

Epigenetics and the plasticity of differentiation in normal and cancer stem cells

“…This was shown in experiments with ES cells deficient either in the DNA methyltransferases Dnmt1, both Dnmt3a and Dnmt3b, or the CpG island-binding protein CGBP that binds to non-methylated DNA. These cells showed severe DNA hypomethylation and a complete differentiation block (Jackson et al, 2004;Carlone et al, 2005). The behavior of ES cells in vitro is like the in vivo regulation of gene expression during early embryonic development, which requires epigenetic reprogramming (Reik et al, 2001;Surani, 2001).…”

Efficient differentiation of murine embryonic stem cells requires the binding of CXXC finger protein 1 to DNA or methylated histone H3-Lys4

“…CXXC1 -/-ES cells additionally exhibit epigenomic defects, including a marked reduction in global 5-methylcytosine levels due to impairment of maintenance DNA methylation, and an increase in global levels of histone H3K4me [11,24]. In the absence of Cfp1, the Setd1a complex and its product, histone H3K4me3, are partially mis-localized to DAPIbright heterochromatin as observed by confocal microscopy.…”

“…3B). Previous studies indicated that ES cells heterozygous for the disrupted CXXC allele (CXXC1 +/-) do not show a phenotype despite expressing ~50% of the WT level of Cfp1 protein [24]. Hence, stably transfected ES clones expressing at least 50% of the WT level of Cfp1 were analyzed by confocal microscopy (Fig.…”

“…Analysis of in vitro differentiation was performed as described [24] in the absence of LIFconditioned media. Histochemical staining for alkaline phosphatase activity was performed using leukocyte alkaline phosphatase kit (Sigma Aldrich, St. Louis, MO) as described [24].…”

“…Cfp1 is also critical for murine hematopoiesis, as ablation of a conditional CXXC1 allele in adult bone marrow cells leads to death within two weeks as a consequence of hematopoietic failure [23]. Murine CXXC1 -/-ES cells are viable, but lack the ability to differentiate in vitro following removal of leukemia inhibitory factor (LIF) [24].…”