Abstract
Background: Methylation of DNA sequences at promoters, CpGislands and other elements plays a vital role in regulating gene activity. In human, loss of methylation is known to play a causativ erole in imprinting disorders and in inappropriate germline gene expression in cancers. While in mouse, loss of function mutants have given great insight into the targets of methylation, functional studies in human have been largely limited to cancer cells and more recently stem cells, not normal adult cells. Methods: Stable knockdown sof the maintenance methyltransferase DNMT1 were generated innormosomic hTERT-immortalised adult fibroblasts. Genome-wide methylation levels were assayed using the Illumina 450K bead array. Results were analysed using RnBeads and Galaxy. Locus specific methylation was verified using pyrosequencing and clonal analysis. Validation was achieved using transient siRNA. Results:Loss of function was poorly tolerated and all clonally-expanded cell lines had spontaneously restored DNMT1 levels by silencing of the shRNA. Evidence for a genome-wide methylation erasure event followed by a wave of remethylation could be clearly traced.Gene bodies and the shores of CpG islands showed the clearest loss of methylation overall. While most CpG islands are normally unmethylated and so unaffected, both imprints and germ line genes fall into the rarer category of normally methylated islands: ofthese two, lasting loss of methylation was much more common among imprints than germ line genes. Conclusions: 1: transient loss of methylation is poorly tolerated; 2: a robust mechanism for remethylation exists even in adult cells; 3: aberrant remethylationis frequent on recovery and 4: Imprints are particularly sensitive.
Original language | English |
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Title of host publication | Unknown Host Publication |
Publisher | Ulster Medical Journal |
Pages | 212-212 |
Number of pages | 1 |
Volume | 84 |
Publication status | Accepted/In press - 3 Sept 2015 |
Event | 18th Meeting of the Irish Society of Human Genetics - Dublin City University Duration: 3 Sept 2015 → … |
Conference
Conference | 18th Meeting of the Irish Society of Human Genetics |
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Period | 3/09/15 → … |
Keywords
- Methylation
- DNMT1
- 450k
- Imprints.