Mathias Frevel
Lerner Research Institute; Cleveland Clinic Foundation
Differential DNA methylation of the parental alleles has been implicated in the establishment and maintenance of the monoallelic expression of imprinted genes. H19 and IGF2 are oppositely imprinted with only the maternal and the paternal alleles expressed, respectively. In Wilms' tumor, loss of the H19/IGF2 imprinted expression pattern can result in silencing of H19 and biallelic expression of IGF2. This was shown to be associated with biallelic methylation of the H19 promoter in the tumour and the adjacent kidney tissue suggesting that epigenetic H19 silencing is an early event in Wilms' tumorigenesis. An imprinting mark region characterized by paternal allele specific methylation has been suggested to reside in a GC-rich region of 400 bp direct repeats starting at -2 kb relative to the H19 transcription start and extending upstream. However, the methylation status of this upstream region has not yet been investigated in Wilms' tumors, and the upstream boundary of the potential paternal methylation imprint of the H19 gene has yet to be defined. We sought to define this upstream methylation boundary and investigate whether Wilms' tumors with loss of imprinting are biallelically methylated in this upstream region. The analysis of 6.6 kb of newly generated upstream H19 sequence determined in this study identified a series of the direct 400 bp repeats that extends to approximately -5.3 kb relative to the transcription start. As part of this repeat region upstream of H19, we identified an evolutionarily conserved 42 bp sequence present in human, mouse and rat which is a good candidate for an H19 imprint control element. DNA methylation analyses using the bisulphite genomic sequencing method and Southern blotting indicated that the upstream boundary of the potential imprint may coincide with the 5' end of the upstream repeats. Wilms' tumors with loss of imprinting showed varying degrees of biallelic methylation in the H19 upstream repeat region also affecting one of the 42 bp elements which leads us to suggest that pathological methylation in this region may be the epigenetic error that initiates H19 silencing.