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

Abstract

Human chromosome 11p15.5 harbors an intriguing imprinted gene cluster of 1 Mb. This imprinted domain is implicated in a wide variety of malignancies and Beckwith-Wiedemann syndrome (BWS). Recently, several lines of evidence have suggested that the BWS-associated imprinting cluster consists of separate chromosomal domains. We have previously identified LIT1, a paternally expressed antisense RNA within the KvLQT1 locus through a positional screening approach using human monochromosomal hybrids. KvLQT1 encompasses the translocation breakpoint cluster in BWS and patients exhibit frequent loss of maternal methylation at the LIT1 CpG island, implying a regulatory role for the LIT1 locus in coordinate control of the imprinting cluster. Here we generated modified human chromosomes carrying a targeted deletion of the LIT1 CpG island using recombination-proficient chicken DT40 cells. Consistent with the prediction, this mutation abolished LIT1 expression on the paternal chromosome, accompanied by activation of the normally silent paternal alleles of multiple imprinted loci at the centromeric domain including KvLQT1 and 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 island can act as a negative regulator in cis for coordinate imprinting at the centromeric domain, thereby suggesting a role for the LIT1 locus in a BWS pathway leading to functional inactivation of p57(KIP2). Thus, the targeting and precise modification of human chromosomal alleles using the DT40 cell shuttle system can be used to define regulatory elements that confer long-range control of gene activity within chromosomal domains.

Epigenetic deregulation of imprinting in congenital diseases of aberrant growth

“…Furthermore, in the mouse, deletion of the H19 ICR does not affect the KCNQ1 domain and, conversely, targeting of the KvDMR1 does not alter imprinting at the IGF2-H19 domain. (17,34,35) An emerging question is whether, as opposed to what happens in BWS, increased expression of CDKN1C could be causally involved in SRS. In the novel SRS studies, this was explored by analysis of the KvDMR1, but no methylation changes were detected in the patients analysed.…”

Imprinting centers, chromatin structure, and disease

“…In mice, targeted deletion of DMR-Lit1 on the paternal chromosome resulted in biallelic expression of genes that are normally silent on the paternal chromosome. Targeted deletion of human DMR-LIT1 using microcell hybrids produced the same result; when the paternal DMR-LIT1 was deleted, genes normally expressed preferentially from the maternal chromosome, such as KIP2, KCNQ1DN, and KvLQT1, were derepressed on the paternal chromosome [Horike et al, 2000]. The evidence indicates that DMR-LIT1 is an IC for the KIP2/ LIT1 domain and that an unmethylated paternal DMR-LIT1 acts in cis to silence maternalspecific genes on the paternal chromosome in both species.…”

A novel in vitro system for analyzing parental allele-specific histone acetylation in genomic imprinting

“…Using A9 hybrids with human chromosome 11, we found that the silent and hypermethylated LIT1 and H19 alleles were hypoacetylated, although LIT1 and H19 belong to different imprinted subdomains of human chromosome 11p15.5 Mitsuya et al 1999;Horike et al 2000). Conversely, histones of the expressed and undermethylated LIT1 and H19 alleles were hyperacetylated.…”

“…It is well known that DMR play an essential role in the regulation of various imprinted genes and contain important cis-regulatory elements. We examined the CpG islands of LIT1, H19, and SNRPN, which have been demonstrated to be indispensable for imprinted expression of each gene (Huq et al 1997;Dao et al 1999;Horike et al 2000), and found that histone modification of these regions was involved in allelic expression of imprinted genes. Thus, we can directly study the role of chromatin modifications around essential cis-regulatory elements responsible for the regulation of imprinting using our system.…”