Role of DNA methylation in imprint maintenance in differentiated human cells

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SummaryThe phenomenon of genomic imprinting lies at the heart of a significant number of childhood disease syndromes, including disorders such as Beckwith-Weidemann Syndrome (BWS), Prader-Willi Syndrome (PWS) and Transient neonatal diabetes mellitus (TNDM) type I. Each of these syndromes involves a gene which is imprinted, that is to say is normally only active from one of the two chromosomes in the cell, either the one inherited from the father via the sperm, or the one inherited from the mother via the egg, the genes thus being "imprinted" with a memory of which parent they came from. Patients with imprinted disease syndromes have increased risks for further problems such as diabetes (TNDM) or Wilms' tumour (BWS) in later life as well. The mechanism by which one copy of a gene becomes inactivated, while its identical partner in the same cell nucleus remains active, is still under active research. We know that chemical tags added to the DNA, called DNA methylation, are involved in marking the inactive copy of the imprinted gene, but it is not known whether this is sufficient in all cases to turn off the gene, or whether the proteins associated with the DNA, called histones, are more important. While such mechanistic questions can be addressed in experimental animals such as mice, human studies have either used cells from patients, where it is not possible to tell if the changes seen in the DNA methylation or the histones are causative, or have manipulated DNA methylation levels using drugs in cancer cell lines, which are not genetically stable and thus a poor model. We have developed human cell lines which are non-cancerous and genetically stable, with normal imprinting, and then selectively reduced the levels of DNA methylation only. These cells appear to show loss of imprinting of the genes which cause BWS and PWS and most likely all other imprinted regions, but are unaffected at non-imprinted regions. They have altered growth characteristics and appearance, in line with theories that imprinted genes have important roles in regulating growth and nutrition. They thus represent a unique resource for exploring the importance of DNA methylation versus histone status in regulating imprinted genes and by cross-comparing to cells from BWS patients and others, it should be possible to establish which is more likely to be causative. The cells are a potentially valuable tool for determining the side-effects of pharmacological inhibitors of DNA methylation currently being used in the clinic. We can also use the cells to look for previously unrecognised imprinted genes, which may be linked to additional disorders. Additionally we can use the cells to carry out further mechanistic work, including attempting to identify the factors involved in establishing imprints, which would in turn be candidates for causative agents in these diseases.