Beckwith-Wiedemann syndrome and associated tumors - Identification of factors essential for imprinting mechanisms
Final Report Abstract
Beckwith-Wiedemann syndrome (BWS) is a complex genetic disorder associated with chromosomal region 11p15 in which genomic imprinting plays an essential role. The basic defects involve multiple molecular mechanisms extending from single gene mutations to epigenetic phenomena. To date, no general molecular basis for BWS has been defined. The imprinting center ICR1 is crucial for the imprinted expression of H19 and IGF2. The unmethylated maternal ICR1 binds CTCF and functions as an insulator, preventing access of enhancers to the IGF2 promoters. The methylated paternal ICR1 is not bound by CTCF and methylation spreading seems to inactivate the nearby H19 promoter. The named downstream enhancers now are able to interact with the IGF2 promoters. Current models based on murine models favor a CTCF mediated enhancer blocking mechanism that most likely regulates the interaction of enhancers downstream of H19 with promoters of IGF2 and H19 by chromatin looping. We identified a microdeletion in the maternal ICR1, which was associated with IGF2-loss of imprinting and IGF2 upregulation but without alteration of the affected parental methylation status. Based on this observation and in the light of other microdeletion cases that all have an ICR1 hypermethylation in addition, we proposed a loop model to explain human normal and aberrant ICR1 regulation of H19 and IGF2 transcription relying on DNA-protein- and enhancer-promoter-interactions. We tested this model for the affected maternal 11p15.5 allele with different cellular assays using a specific matUPD11p15.5 case (of a Silver-Russell syndrome patient) and compared it to the available functional information, which up to now is based exclusively on data generated in the murine system. With a modified ChIP assay we determined the ability and potency of the CTS in the human ICR1. The pattern of CTCF binding allowed us to refine the functional model and suggested a hypermethylation of remaining CTS after a microdeletion as being a necessary second alteration for pathogenic LOI of IGF2 in most ICR1 microdeletion cases. We determined the allele specific methylation status of conserved DMR regions in the vicinity of the human IGF2 gene. In a 3C-approach we observed significant chromosomal interaction peaks of the proximal CTS 1-6 with MAR3, DMR0, endodermal enhancers and an interaction with H19 and the mesodermal enhancers, which fit in the model of transcriptional maternal H19 activation as expected from our model. In addition we did also detected strong unexpected interactions with two control-fragments (in the vicinity of IGF2) that will be further analyzed. Finally we also confirmed IGF2 transcription as being the major factor in pathogenesis of BWS (and SRS). In addition, we took advantage of the well defined regulatory region AS-SRO in Angelman syndrome patients (in cooperation with Dres. Buiting and Horsthemke) to isolate and initially characterize two factors putatively involved in mediating imprinting in the chromosome 15 area. As already addressed by the reviewers of our grant proposal, establishment of the 3C technique was more time consuming as initially calculated. This was mainly due to the fact that relevant controls had to be newly established for the human genomic context being analyzed. To our big surprise the most observed interaction of both ICR1 parts seems to take place with a fragment K2 proximal of IGF2, that was meant to be a mere control fragment and of another control fragment K5, that is located proximal to the HUC cluster with the distal CTS 4-6. Both control fragments will be analyzed in detail for sequence motifs that can explain this unexpected finding. In the context of the work on our SPP project, DP was invited as an expert for Beckwith-Wiedemann Syndrome, to explain the basics and implications of this disease on a layman level in the ZDF-TV serial “Volle Kanne”, broadcasted on June sixth, 2009. In addition DP was awarded the Boehringer Ingelheim prize for outstanding research (on BWS) in 2006, a prize being well recognized by the national print- and TV-media.
Publications
- Krebsepigenetik. MedGenetik 3.2005: 296-302; 2005
Prawitt D und Zabel B
- Microdeletion and IGF2 loss of imprinting in a cascade causing Beckwith-Wiedemann syndrome with Wilms' tumor. Nat Genet.; 37(8):785-6; 2005
Prawitt D, Enklaar T, Gartner-Rupprecht B, Spangenberg C, Lausch E, Reutzel D, Fees S, Korzon M, Brozek I, Limon J, Housman DE, Pelletier J, Zabel B
- Microdeletion of target sites for insulator protein CTCF in a chromosome 11p15 imprinting center in Beckwith-Wiedemann syndrome and Wilms' tumor. Proc Natl Acad Sci USA.; 102(11):4085-90; 2005
Prawitt D, Enklaar T, Gartner-Rupprecht B, Spangenberg C, Oswald M, Lausch E, Schmidtke P, Reutzel D, Fees S, Lucito R, Korzon M, Brozek I, Limon J, Housman DE, Pelletier J, Zabel B
- Beckwith-Wiedemann syndrome: multiple molecular mechanisms. Expert Reviews in Molecular Medicine; 8(17):1-19; 2006
Enklaar T, Zabel B, Prawitt D
