DNA methyltransferase (cytosine-5) 1 (Dnmt1) is the principal enzyme responsible for maintenance of CpG methylation and is essential for the regulation of gene expression, silencing of parasitic DNA elements, genomic imprinting and embryogenesis. Dnmt1 is needed in S phase to methylate newly replicated CpGs occurring opposite methylated ones on the mother strand of the DNA, which is essential for the epigenetic inheritance of methylation patterns in the genome. Despite an intrinsic affinity of Dnmt1 for such hemi-methylated DNA, the molecular mechanisms that ensure the correct loading of Dnmt1 onto newly replicated DNA in vivo are not understood. The Np95 (also known as Uhrf1 and ICBP90) protein binds methylated CpG through its SET and RING finger-associated (SRA) domain. Here we show that localization of mouse Np95 to replicating heterochromatin is dependent on the presence of hemi-methylated DNA. Np95 forms complexes with Dnmt1 and mediates the loading of Dnmt1 to replicating heterochromatic regions. By using Np95-deficient embryonic stem cells and embryos, we show that Np95 is essential in vivo to maintain global and local DNA methylation and to repress transcription of retrotransposons and imprinted genes. The link between hemi-methylated DNA, Np95 and Dnmt1 thus establishes key steps of the mechanism for epigenetic inheritance of DNA methylation.

Imprinted genes are expressed from only one of the parental chromosomes and are marked epigenetically by DNA methylation and histone modifications. The imprinting center 2 (IC2) on mouse distal chromosome 7 is flanked by several paternally repressed genes, with the more distant ones imprinted exclusively in the placenta. We found that most of these genes lack parent-specific DNA methylation, and genetic ablation of methylation does not lead to loss of their imprinting in the trophoblast (placenta). The silent paternal alleles of the genes are marked in the trophoblast by repressive histone modifications (dimethylation at Lys9 of histone H3 and trimethylation at Lys27 of histone H3), which are disrupted when IC2 is deleted, leading to reactivation of the paternal alleles. Thus, repressive histone methylation is recruited by IC2 (potentially through a noncoding antisense RNA) to the paternal chromosome in a region of at least 700 kb and maintains imprinting in this cluster in the placenta, independently of DNA methylation. We propose that an evolutionarily older imprinting mechanism limited to extraembryonic tissues was based on histone modifications, and that this mechanism was subsequently made more stable for use in embryonic lineages by the recruitment of DNA methylation.

Genomic imprinting causes parental origin–specific monoallelic gene expression through differential DNA methylation established in the parental germ line. However, the mechanisms underlying how specific sequences are selectively methylated are not fully understood. We have found that the components of the PIWI-interacting RNA (piRNA) pathway are required for de novo methylation of the differentially methylated region (DMR) of the imprinted mouse Rasgrf1 locus, but not other paternally imprinted loci. A retrotransposon sequence within a noncoding RNA spanning the DMR was targeted by piRNAs generated from a different locus. A direct repeat in the DMR, which is required for the methylation and imprinting of Rasgrf1, served as a promoter for this RNA. We propose a model in which piRNAs and a target RNA direct the sequence-specific methylation of Rasgrf1.

Genomic imprinting plays a fundamental role in cancer and some hereditary diseases, including Beckwith-Wiedemann syndrome (BWS), a disorder of prenatal overgrowth and predisposition to embryonal malignancies such as Wilms tumor. We have previously shown that the K V LQT1 gene on chromosomal band 11p15 is imprinted, with expression of the maternal allele, and that the maternal allele is disrupted in rare BWS patients with balanced germ-line chromosomal rearrangements. We now show that an antisense orientation transcript within K V LQT1, termed LIT1 (long QT intronic transcript 1) is expressed normally from the paternal allele, from which K V LQT1 transcription is silent, and that in the majority of patients with BWS, LIT1 is abnormally expressed from both the paternal and maternal alleles. Eight of sixteen informative BWS patients (50%) showed biallelic expression, i.e., loss of imprinting (LOI) of LIT1. Similarly, 21 of 36 (58%) BWS patients showed loss of maternal allelespecific methylation of a CpG island upstream of LIT1. Surprisingly, LOI of LIT1 was not linked to LOI of insulin-like growth factor II (IGF2), which was found in 2 of 10 (20%) BWS patients, even though LOI of IGF2 occurs frequently in Wilms and other tumors, and in some patients with BWS. Thus, LOI of LIT1 is the most common genetic alteration in BWS. We propose that 11p15 harbors two imprinted gene domains-a more centromeric domain including K V LQT1 and p57 KIP2 , alterations in which are more common in BWS, and a more telomeric domain including IGF2, alterations in which are more common in cancer.