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

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 immediate product of DNA replication. Appropriate cytosine methylation in mammals is essential for normal development. Individual ablation of the Dnmt1 or Dnmt3b gene leads to a lethal disruption of murine embryonic development (38, 49). Mice lacking Dnmt3a develop to birth but become runted and die within 4 weeks of age (49). Furthermore, mutations that are predicted to partially inhibit Dnmt3b function are associated with the ICF (immunodeficiency, centromere instability, and facial anomalies) syndrome in humans (77). Overexpression of Dnmt1 in mice leads to global hypermethylation, loss of genomic imprinting, and embryonic lethality (7). A number of DNA-binding factors interact with methylated CpG motifs (27). These include MeCP2, methyl binding domain protein 1 (MBD1) and MBD2, which are involved in repression of gene expression, and MBD4, which functions in DNA repair. Each of these factors contains a conserved methyl-CpG binding domain, but otherwise they exhibit little sequence similarly. Mutations in the methyl-CpG binding protein MeCP2 lead to Rett syndrome, a progressive neurodegenerative disorder (2). Recent reports reveal intricate interrelationships linking cytosine methylation and histone modifications, thus providing a unifying framework for the control of chromatin structure and gene regulation (9). For example, MBD2 and MBD3 are components of the histone deacetylase (HDAC) complexes MeCP1 and Mi-2, respectively (46, 81), and Dnmt proteins also associate with HDAC complexes (20, 21, 55). Furthermore, the chromatin remodeling protein DDM1 in Arabidopsis and the related factor LSH in mammals are required for normal cytosine methylation (15, 29, 30). Disruption of the Suv39h histone methyltransferase gene in murine embryonic stem (ES) cells leads to altered localization of Dnmt3b and decreased cytosine methylation at pericentric satellite repeats (35). Hence, DNA methylation and histone modifications appear to be highly integrated and mutually reinforcing mechanisms that serve to maintain heterochromatin structure and repress gene expression. CpG binding protein (CGBP) exhibits a unique DNA-binding specificity for unmethylated CpG motifs and acts as a transcriptional activator (71). Originally identified in humans, homologues of CGBP have been detected in Drosophila, Caenorhabditis elegans, and both Saccharomyces cerevisiae and Schizosaccharomyces pombe (41, 71). CGBP contains a cysteine-rich CXXC DNA-binding domain (34, 71) which is present in several other proteins, including Dnmt1 (6); human trithorax (HRX) (also known as ALL-1 or MLL), a histone methyltransferase encoded by a gene frequently involved in chromosomal translocations in leukemia (17, 25, 39, 53, 66, 80); MBD1 (14, 27); leukemia-associated protein LCX (50); and MLL-2, which is often amplified in solid tumors (19). CGBP additionally contains two PHD domains, which are characteristic of chromatin-associated proteins and/or regulators of gene expression (1, 71) and often mediate protein-protein interactions (22, 24, 48). CGBP is a component of the nuclear matrix and localizes to nuclear speckles associated with euchromatin (33). Targeted disruption of the CGBP gene results in peri-implantation embryonic lethality in mice (11), a developmental stage associated with global remodeling of chromatin structure and cytosine methylation patterns (32, 37, 54). The molecular mechanisms involved in targeting methylation to specific CpG motifs during development, as well as maintaining hypomethylation of CpG islands, are not well understood. The binding specificity of CGBP for unmethylated CpG motifs suggests a possible role in these events. The early death of embryos lacking CGBP establishes the importance of this protein for mammalian development. However, the severity of this phenotype makes further analysis of this mutant difficult. In the study reported here, murine ES cells lacking CGBP were isolated to permit a more detailed analysis of the CGBP−/− phenotype and provide further insight into CGBP function. The results presented implicate CGBP as a critical regulator of DNA methylation and cellular differentiation.