Genomic imprinting is an epigenetic modification that results in expression from only one of the two parental copies of a gene. Differences in methylation between the two parental chromosomes are often observed at or near imprinted genes. Beckwith-Wiedemann syndrome (BWS), which predisposes to cancer and excessive growth, results from a disruption of imprinted gene expression in chromosome band 11p15.5. One third of individuals with BWS lose maternal-specific methylation at KvDMR1, a putative imprinting control region within intron 10 of the KCNQ1 gene, and it has been proposed that this epimutation results in aberrant imprinting and, consequently, BWS1, 2. Here we show that paternal inheritance of a deletion of KvDMR1 results in the de-repression in cis of six genes, including Cdkn1c, which encodes cyclin-dependent kinase inhibitor 1C. Furthermore, fetuses and adult mice that inherited the deletion from their fathers were 20-25% smaller than their wildtype littermates. By contrast, maternal inheritance of this deletion had no effect on imprinted gene expression or growth. Thus, the unmethylated paternal KvDMR1 allele regulates imprinted expression by silencing genes on the paternal chromosome. These findings support the hypothesis that loss of methylation in BWS patients activates the repressive function of KvDMR1 on the maternal chromosome, resulting in abnormal silencing of CDKN1C and the development of BWS.

Loss of imprinting at IGF2, generally through an H19-independent mechanism, is associated with a large percentage of patients with the overgrowth and cancer predisposition condition Beckwith-Wiedemann syndrome (BWS). Imprinting control elements are proposed to exist within the KvLQT1 locus, because multiple BWS-associated chromosome rearrangements disrupt this gene. We have identified an evolutionarily conserved, maternally methylated CpG island (KvDMR1) in an intron of the KvLQT1 gene. Among 12 cases of BWS with normal H19 methylation, 5 showed demethylation of KvDMR1 in fibroblast or lymphocyte DNA; whereas, in 4 cases of BWS with H19 hypermethylation, methylation at KvDMRl was normal. Thus, inactivation of H19 and hypomethylation at KvDMR1 (or an associated phenomenon) represent distinct epigenetic anomalies associated with biallelic expression of IGF2. Reverse transcription-PCR analysis of the human and syntenic mouse loci identified the presence of a KvDMR1-associated RNA transcribed exclusively from the paternal allele and in the opposite orientation with respect to the maternally expressed KvLQT1 gene. We propose that KvDMR1 and͞or its associated antisense RNA (KvLQT1-AS) represents an additional imprinting control element or center in the human 11p15.5 and mouse distal 7 imprinted domains.

The maternally expressed/paternally silenced genes Phlda2 (a.k.a. Ipl/Tssc3), Slc22a1l, Cdkn1c, Kcnq1, and Ascl2 are clustered in an imprinted domain on mouse chromosome 7. Paternal deletion of a cis-acting differentially methylated DNA element, Kvdmr1, causes coordinate loss of imprinting and over-expression of all of these genes and the resulting conceptuses show intrauterine growth restriction (IUGR). To test the specific contribution of Phlda2 to IUGR in the Kvdmr1-knockout, we crossed Kvdmr1(+/-) males with Phlda2(+/-) females. Conceptuses with the (Phlda2(+/+); Kvdmr1(+/-)) genotype showed fetal and placental growth retardation. Restoration of Phlda2 dosage to normal, as occurred in the conceptuses with the (Phlda2(-/+); Kvdmr1(+/-)) genotype, had a marginally positive effect on fetal weights and no effect on post-natal weights, but significantly rescued the placental weights. As we previously reported, loss of Phlda2 expression in the wild-type background (Phlda2(-/+); Kvdmr1(+/+) genotype) caused placentomegaly. Thus Phlda2 acts as a true rheostat for placental growth, with overgrowth after gene deletion and growth retardation after loss of imprinting. Consistent with this conclusion, we observed significant placental stunting in BAC-transgenic mice that over-expressed Phlda2 and one flanking gene, Slc22a1l, but did not over-express Cdkn1c.

Imprinting control regions (ICRs) are known to repress genes by utilizing one of two mechanisms, CTCF‐mediated insulation or the transcription of non‐coding RNAs (ncRNAs). The KvDMR1 ICR contains both the promoter for the Kcnq1ot1 ncRNA and two CTCF‐binding sites located within sequences exhibiting repressive activity in enhancer‐blocking assays. Deletion of KvDMR1 results in ubiquitous biallelic expression of eight maternal‐specific genes in distal chromosome 7. Here we report that while truncation of the Kcnq1ot1 transcript results in the loss of imprinted expression of these genes in the placenta, it does not affect imprinted expression of Cdkn1c in a subset of embryonic tissues despite universal loss of paternal‐specific methylation at Cdkn1c. Consistent with tissue‐specific loss of imprinted expression, growth deficiency of these mutant mice was less severe than that observed previously in mice with deletion of KvDMR1. This study demonstrates that the KvDMR1 locus can silence Cdkn1c by a mechanism independent of Kcnq1ot1 transcription, perhaps by CTCF‐associated repression, making it the first example of an ICR capable of silencing the same gene by two distinct mechanisms.

Paternal deletion of the imprinting control region (ICR) KvDMR1 results in loss of expression of theKcnq1ot1 noncoding RNA and derepression of flanking paternally silenced genes. Truncation of Kcnq1ot1 also results in the loss of imprinted expression of these genes in most cases, demonstrating a role for the RNA or its transcription in gene silencing. However, enhancer-blocking studies indicate that KvDMR1 also contains chromatin insulator or silencer activity. In this report we demonstrate by electrophoretic mobility shift assays and chromatin immunoprecipitation the existence of two CTCF binding sites within KvDMR1 that are occupied in vivo only on the unmethylated paternally derived allele. Methylation interference and mutagenesis allowed the precise mapping of protein-DNA contact sites for CTCF within KvDMR1. Using a luciferase reporter assay, we mapped the putative transcriptional promoter for Kcnq1ot1 upstream and to a site functionally separable from enhancer-blocking activity and CTCF binding sites. Luciferase reporter assays also suggest the presence of an additional cis-acting element in KvDMR1 upstream of the putative promoter that can function as an enhancer. These results suggest that the KvDMR1 ICR consists of multiple, independent cis-acting modules. Dissection of KvDMR1 into its functional components should help elucidate the mechanism of its function in vivo.

The mechanisms underlying the phenomenon of genomic imprinting remain poorly understood. In one instance, a differentially methylated imprinting control region (ICR) at the H19 locus has been shown to involve a methylation-sensitive chromatin insulator function that apparently partitions the neighboring Igf2 and H19 genes in different expression domains in a parent of origin-dependent manner. It is not known, however, if this mechanism is unique to the Igf2/H19 locus or if insulator function is a common feature in the regulation of imprinted genes. To address this question, we have studied an ICR in the Kcnq1 locus that regulates long range repression on the paternally derived p57Kip2 and Kcnq1 alleles in an imprinting domain that includes Igf2 and H19. We show that this ICR appears to possess a unidirectional chromatin insulator function in somatic cells of both mesodermal and endodermal origins. Moreover, we document that CpG methylation regulates this insulator function suggesting that a methylation-sensitive chromatin insulator is a common theme in the phenomenon of genomic imprinting.Human chromosome 11p15.5 and the distal part of chromosome 7 in mouse harbor a well characterized cluster of imprinted genes : Ipl, Orctl2, Cdnk1c, Kcnq1, Mash2, Ins2, Igf2, and H19 (1,2). Disruption of imprinting in 11p15.5 results in the overgrowth and cancer predisposition condition Beckwith-Wiedemann syndrome (BWS) 1 (3). Based on chromosomal break points and methylation changes in BWS patients, a differentially methylated CpG island identified in intron 10 of the Kcnq1 gene has been proposed to be involved in the regulation of imprinting (4,5). This region is methylated on the active maternal allele but unmethylated on the inactive paternal allele of Kcnq1 (1) and overlaps with an oppositely oriented and paternally expressed gene known as Kcnq1 A-S or LIT1 (4, 5). Targeted deletion of this region in the human paternal chromosome 11 propagated in the chicken DT40 cell line resulted in the activation of the normally silent paternal alleles of KCNQ1 and CDNK1C (6). The suggestions that this region, henceforth termed Kcnq1 imprinting control region (ICR), has a pivotal role in the maintenance of imprinting of neighboring genes (6, 7) and has recently been confirmed by targeted deletion experiments in the mouse. 2To examine whether chromatin insulator properties is a common feature of imprinting control regions, we analyzed the activity of the Kcnq1 ICR in enhancer-blocking assays. Our results are consistent with the notion that a CpG methylationsensitive chromatin insulator function is not restricted to the H19 ICR but includes the Kcnq1 ICR as well. EXPERIMENTAL PROCEDURESPlasmid Cloning Strategies-The 3.6-kb Kcnq1 ICR segment was inserted into unique ClaI or NotI sites in both orientations in the E-p-neo-scs vector (8) (see Fig. 1b). To facilitate cloning into episomal plasmids, we inserted a fragment with multiple cloning sites from the parent pREP4 plasmid (amplified using forward primer, AAG CTG ATC TAT CAT GTC TGG ATC...

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