Targeted disruption of the human LIT1 locus defines a putative imprinting control element playing an essential role in Beckwith-Wiedemann syndrome

Human chromosome 11p15,5 harbors an intriguing imprinted gene cluster of 1 Mb, This imprinted domain is implicated in a wide variety of malignancies a nd Beckwith-Wiedemann syndrome (BWS), Recently, several lines of evidence h ave suggested that the BWS-associated imprinting cluster consists of separa te chromosomal domains. We have previously identified LIT1, a paternally ex pressed antisense RNA within the KvLQT1 locus through a positional screenin g approach using human monochromosomal hybrids. KVLQTI encompasses the tran slocation breakpoint cluster in BWS and patients exhibit frequent loss of m aternal methylation at the LIT1 CpG island, implying a regulatory role for the LIT1 locus in coordinate control of the imprinting cluster, Here we gen erated modified human chromosomes carrying a targeted deletion of the LIT1 CpG island using recombination-proficient chicken DT40 cells. Consistent wi th the prediction, this mutation abolished LIT1 expression on the paternal chromosome, accompanied by activation of the normally silent paternal allel es of multiple imprinted loci at the centromeric domain including KVLQTI an d p57(KIP2). The deletion had no effect on imprinting of H19 located at the telomeric end of the cluster. Our findings demonstrate that the LIT1 CPG i sland can act as a negative regulator in cis for coordinate imprinting at t he centromeric domain, thereby suggesting a role for the LIT1 locus in a BW S pathway leading to functional inactivation of p57(KIP2). Thus, the target ing and precise modification of human chromosomal alleles using the DT40 ce ll shuttle system can be used to define regulatory elements that confer lon g-range control of gene activity within chromosomal domains.