DNA methylation is an important epigenetic mechanism of regulating gene expression that is affected in certain human diseases including imprinting disorders and cancer. In mouse, UHRF1 is an essential cofactor of DNMT1, the enzyme responsible for maintaining methylation patterns. To investigate the effects of loss of UHRF1 on methylation patterns in human cells, UHRF1 levels were decreased in immortalized hTERT fibroblast cell lines using short hairpin RNA. Genome-wide effects on methylation were investigated by the Illumina Infinium HumanMethylation450 BeadChip array. Online bioinformatics software tools were used to identify FDR-significant hypomethylated gene classes, which were then verified by pyrosequencing. Transcriptional effects on these gene classes were investigated by the genome-wide Illumina HumanHT-12 v4 Expression BeadChip array, and verified by RT-qPCR. While UHRF1 depletion caused widespread demethylation, the replication-dependent histone gene cluster and the cancer testis antigen genes were identified as most significantly hypomethylated in UHRF1 knockdown cells. Pyrosequencing confirmed hypomethylation in promoter regions of cancer testis antigen genes TSPY2, MAGEC1, MAGEC2 and MAGEA12, and histone gene HIST2H2AA4 in knockdown cell lines. Hypomethylation in these gene classes correlated with an increase in expression in the knockdown cell line. In addition, cells were rescued using UHRF1 cDNA and showed a return to wild type transcription levels in the rescue cell line. We have shown that these genes are regulated by promoter DNA methylation, confirming the sensitivity of cancer-testis genes to demethylation, supporting possible use of methyltransferase inhibitors to boost antigen presentation in cancers, and the crucial role of UHRF1 in cell cycle regulation.
|Publication status||Published - 2018|
|Event||20th Meeting of the Irish Society of Human Genetics - Croke Park, Dublin, Dublin, Ireland|
Duration: 15 Sep 2017 → 15 Sep 2018
|Conference||20th Meeting of the Irish Society of Human Genetics|
|Period||15/09/17 → 15/09/18|
Scullion, C., Irwin, R. E., Thakur, A., & Walsh, CP. (2018). THE CANCER TESTIS ANTIGEN AND REPLICATION-DEPENDENT HISTONE GENE CLASSES ARE HYPOMETHYLATED IN UHRF1 KNOCKDOWN CELLS, RESULTING IN INCREASED TRANSCRIPTIONAL ACTIVITY.. 54-64. Abstract from 20th Meeting of the Irish Society of Human Genetics, Dublin, Ireland.