Functional study of KvDMR1, an imprinting control region from mouse distal chromosome 7

Imprinted genes are preferentially expressed from either the maternal or paternal allele and deregulation of imprinting has been implicated in several developmental disorders. The imprinting disorder Beckwith-Wiedemann syndrome can arise from a variety of genetic and epigenetic mechanisms affecting imprinted genes in human chromosome 11p15.5 (mouse distal chromosome 7). In more than 50% of non-UPD (uniparental disomy) sporadic BWS patients, the disease is associated with the loss of methylation (LOM) from the maternal allele of KvDMR1, a differentially methylated CpG island within the KCNQ1 gene. It has been proposed that differentially methylated CpG islands might function as imprinting control regions (ICRs). In order to test if KvDMR1 is an ICR, we generated mouse lines carrying a 2.8-kb deletion of this locus. Embryos and adult mice carrying a paternal deletion of KvDMR1 exhibited a 20% reduction in weight compared to wild-type littermates. Using RT-PCR, the effect of this deletion on imprinted gene expression was assessed in tissues of F1 fetuses from crosses between deletion-carrying animals and PWK mice or C57/BL6 mice congenic for distal chromosome 7 from M. spretus (SD7 mice). Following paternal transmission, the KvDMR1 deletion resulted in biallelic expression of Slc22a1l, Tssc3 (Ipl), Cdkn1c, and Kcnq1 (Kv1qt1), Tssc4 and Ascl2 genes usually transcribed only from the maternal chromosome. Normal maternal-specific expression of these genes was observed in mutant mice that inherited the deletion from their mother. Thus, the unmethylated KvDMR1 regulates mono-allelic expression by repressing genes on the paternal chromosome. These data support the notion that LOM at KvDMR1 on the maternal chromosome results in the repression of CDKNIC expression that leads to BWS.; We also show that KvDMR1 can function as an enhancer-blocker/chromatin insulator. This type of KvDMR1 activity, which appears to be sensitive to DNA methylation, was demonstrated in two different cell-culture-based assays. The enhancer blocking activity of KvDMR1 was mapped to a 600-bp sequence that also binds CTCF protein in vitro. In addition to chromatin insulator, a putative enhancer and a transcriptional promoter were identified at the KvDMR1 locus.