DEVELOPMENT 1183 DEVELOPMENT AND DISEASE RESEARCH ARTICLE INTRODUCTION ICF syndrome is a rare autosomal recessive disorder characterized by immunodeficiency, centromeric instability and facial anomalies (Ehrlich, 2003; Wijmenga et al., 2000). Individuals with this disease show combined immunodeficiency, including absence or severe reduction of at least two classes of immunoglobulin and a reduced number of T cells, making them prone to infections and death before adulthood. Centromeric instability is characterized by the formation of radiated chromosomes (chromosome 1, 9 and 16) due to demethylation at cytosine residues in classical satellites 2 and 3 at juxtacentromeric regions of these chromosomes. Facial anomalies include hypertelorism, flat nasal bridge, low set ears, protrusion of the tongue, epicanthal folds, micrognathia and high forehead. Most individuals also exhibit growth and mental retardation. Several studies have demonstrated that ICF syndrome is caused by homozygous or compound heterozygous mutations of the DNA methyltransferase 3B (DNMT3B) gene (Hansen et al., 1999; Okano et al., 1999; Xu et al., 1999). Dnmt3b is one of the three active DNA cytosine methyltransferases identified in human and mouse (Okano et al., 1998; Xie et al., 1999). Dnmt3a and Dnmt3b have high structural similarity, and both can carry out de novo methylation in embryonic stem cells and during embryonic development (Okano et al., 1999). Although these two enzymes exhibit overlapping functions during early development, each has distinct expression patterns, genomic targets and functions (Chen et al., 2003; Okano et al., 1999). Although Dnmt3a deficient mice develop to term and appear to be normal at birth, Dnmt3b deficient mice are embryonic lethal. Recently, Dnmt3a, but not Dnmt3b, has been shown to be essential for the establishment of methylation imprints during gametogenesis (Kaneda et al., 2004). Genetic analysis of Dnmt3a and Dnmt3b function in embryonic stem cells has revealed that Dnmt3a and Dnmt3b, and their different variants, have shared as well as distinct DNA targets (Chen et al., 2003). However, the specific functions of Dnmt3a or Dnmt3b in vivo have not been fully analyzed yet. To investigate the function of Dnmt3b in mouse development and to determine whether Dnmt3b mutations result in phenotypes similar to those of individuals with ICF syndrome, we generated mice with point mutations in Dnmt3b corresponding to the mutations found in human patients (ICF mice). Our studies of Dnmt3b null and ICF mutant mice show that Dnmt3b is essential for mouse embryonic development, and that the ICF mice exhibit phenotypes that resemble some of the symptoms of the human ICF syndrome. We also demonstrate that Dnmt3b is essential for the survival of T cells in the thymus of newborn mice. MATERIALS AND METHODS Vectors Construction of the GFP-Dnmt3b1, GFP-Dnmt3b2, GFP-Dnmt3b3, myc- Dnmt3a and myc-Dnmt3b1 vectors has been described previously (Chen et al., 2002; Hata et al., 2002). Human DNMT3B cDNA was synthesized by RT-PCR from total RNA of NT-2 cells, by using the oligonucleotides 5'- ATGAAGGGAGACACCAGGC-3' and 5'-GCCTGGCTGGAACTATT- CAC-3' as primers, and subcloned into pCAG-IRESblast vector. The cDNAs of ICF mutants were generated by a PCR-based site-directed mutagenesis experiment, by using mouse Dnmt3b1 or human DNMT3B cDNA as template, and subcloned into the pEGFP-C1 (CLONTECH) and/or the pCAG-IRESblast vector. The Dnmt3b A609T and D823G knock-in vectors were generated by sequentially subcloning Dnmt3b fragments and a floxed IRES-geo cassette, in which IRES-geo is flanked by loxP sites, into pBluescript II SK. The fragments containing A609T and D823G mutations were generated by PCR using a bacterial artificial chromosome clone as a template and the following oligonucleotides as primers: 5'- CTGGAGCTGCTATATGTGCC-3', 5'-GGAAAAGTACATTACCTCC- GA-3', 5'-CACAGACTTCGGAGGTAATG-3' and 5'-TTGGTGATTTT- CCGGACGTC-3' for A609T; and 5'-CAGACAGGGCAAAAACCAGC- 3' and 5'-CCGCGGCCCATGTTGGACACGCC-3' for D823G. The other Dnmt3b fragments were obtained from a bacterial artificial chromosome clone. The identities of all constructs were verified by DNA sequencing. Roles for Dnmt3b in mammalian development: a mouse model for the ICF syndrome Yoshihide Ueda 1 , Masaki Okano 1,3 , Christine Williams 2 , Taiping Chen 1,4 , Katia Georgopoulos 2 and En Li 1,4, * ICF (Immunodeficiency, Centromeric instability and Facial anomalies) syndrome is a rare autosomal recessive disease caused by mutations in the DNA methyltransferase gene DNMT3B. To investigate the function of Dnmt3b in mouse development and to create animal models for ICF syndrome, we have generated three mutant alleles of Dnmt3b in mice: one carrying a deletion of the catalytic domain (null allele) and two carrying ICF-like missense mutations in the catalytic domain. The Dnmt3b null allele results in embryonic lethality from E14.5 to E16.5 with multiple tissue defects, including liver hypotrophy, ventricular septal defect and haemorrhage. By contrast, mice homozygous for the ICF mutations develop to term and some survive to adulthood. These mice show phenotypes that are reminiscent of ICF patients, including hypomethylation of repetitive sequences, low body weight, distinct cranial facial anomalies and T cell death by apoptosis. These results indicate that Dnmt3b plays an essential role at different stages of mouse development, and that ICF missense mutations cause partial loss of function. These mutant mice will be useful for further elucidation of the pathogenic and molecular mechanisms underlying ICF syndrome. KEY WORDS: DNA methylation, Dnmt3b, ICF syndrome, T cell, Apoptosis Development 133, 1183-1192 (2006) doi:10.1242/dev.02293 1 Cardiovascular Research Center, Massachusetts General Hospital, Harvard Medical School, 149 13th Street, Charlestown, MA 02129, USA. 2 Cutaneous Biology Research Center, Massachusetts General Hospital, Harvard Medical School, 149 13th Street, Charlestown, MA 02129, USA. 3 Center for Developmental Biology, RIKEN, Kobe Hyogo 650-0047, Japan. 4 Epigenetics Program, Novartis Institutes for Biomedical Research, 250 Massachusetts Avenue, Cambridge, MA 02139, USA. *Author for correspondence (e-mail: en.li@pharma.novartis.com) Accepted 18 January 2006