Beckwith-Wiedemann syndrome and associated tumors - Identification of factors essential for imprinting mechanisms

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Beckwith-Wiedemann syndrome (BWS) is a complex genetic disorder associated with chromosomal region 11p15.5 in which genomic imprinting plays an essential role. To date a number of causative molecular mechanisms have been described, that either are at a low frequency gene mutations, or for the main part epigenetic alterations in two imprinting clusters (IC1 and IC2). The latter includes microdeletions in the maternal H19/IGF2 imprinting control region (ICR1) recently described by others and us in familial BWS cases. These microdeletions are associated with loss of imprinting (LOI) and transcriptional increase of IGF2. Yet we were able to demonstrate that this LOI is not sufficient to cause BWS. Based on our results from the study of family members with microdeletions in ICR1 from the previous research period and in the context with general epigenetic data, we propose a model that explains how ICR1 regulates transcription of H19 and IGF2 relying on DNA-protein- and enhancer-promoter-interactions. With our proposal we want to test this model by defining combinations and interactions of epigenetic elements in IC1, that are able to cause BWS and associated tumors. We plan to use cellular model assays, modified pull-down and chromatin conformation capture techniques. We will also test the hypothesis of a critical IGF2 threshold dosage for BWS/WT genesis by a xenograft approach. Since imprinting probably evolved as a general mechanism, we searched in parallel for imprinting mediating trans-factors that are able to bind the well defined regulatory region ASSRO in Angelman syndrome (AS) patients on chromosome 15, to test them for the ability to mediate imprinting in the AS area as well as in the BWS associated genomic region. Initial experiments revealed two promising candidate proteins. These AS-SRO binding putative transfactors will be further characterized in respect to its imprinting regulatory ability. The efforts should result in further definition of specific BWS causing mechanisms and the description of DNA-elements relevant for general epigenetic regulation as well as protein factors that bind to them.