Reactivation of a silenced H19 gene in human rhabdomyosarcoma by demethylation of DNA but not by histone hyperacetylation.
BACKGROUND
The active copy of the imprinted gene H19 is turned off by inappropriate methylation in several pediatric tumors including Wilms' Tumour and embryonal rhabdomyosarcoma. H19 controls in cis the linked Insulin-like Growth Factor 2 (IGF2) gene, encoding an important growth factor. Recent work has suggested that methylation of a gene may lead to deacetylation of its associated histones and that silenced genes can be reactivated by increasing histone acetylation levels.


RESULTS
Treatment of a rhabdomyosarcoma cell line which has a silent, methylated H19 gene with histone deacetylase (HDAC) inhibitors under conditions which gave maximal hyperacetylation of histone 4, both globally and at the H19 gene itself could not reactivate H19 or affect the active Insulin-like Growth Factor 2 (IGF2) gene, but caused clear up-regulation of the Tissue-type Plasminogen Activator (TPA) gene, a non-imprinted gene known to respond to changes in histone acetylation. In contrast, mild treatment of the cells with the methylation inhibitor 5-AzaC-2'-deoxycytidine (AzaC) on its own was able to reactivate H19. Combining AzaC treatment with HDAC inhibitors gave a reduced rather than enhanced reactivation. These findings were confirmed in mouse primary liver and kidney explants which maintain normal imprinting, where we also found that the silent Igf2 gene could not be reactivated by HDAC inhibitors.


CONCLUSIONS
These results suggest that DNA methylation rather than histone acetylation is the primary determinant of silencing of H19 in rhabdomyosarcoma.
Lynch
Catherine A
CA
Cancer and Ageing Research Group, School of Biomedical Sciences, University of Ulster, Coleraine BT52 1SA, United Kingdom. c.lynch@ulster.ac.uk
Tycko
Benjamin
B
Bestor
Timothy H
TH
Walsh
Colum P
CP
D000107:Acetylation / Q000187:drug effects; D000818:Animals; D001374:Azacitidine / Q000494:pharmacology; D015152:Blotting, Northern; D015153:Blotting, Western; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D019175:DNA Methylation; D005260:Female; D015870:Gene Expression / Q000187:drug effects; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D056572:Histone Deacetylase Inhibitors; D006655:Histone Deacetylases / Q000378:metabolism; D006657:Histones / Q000378:metabolism*; D006801:Humans; D006877:Hydroxamic Acids / Q000494:pharmacology; D007335:Insulin-Like Growth Factor II / Q000235:genetics; D007668:Kidney / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D008099:Liver / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D011247:Pregnancy; D012313:RNA / Q000235:genetics / Q000378:metabolism; D062085:RNA, Long Noncoding; D022661:RNA, Untranslated / Q000235:genetics*; D012208:Rhabdomyosarcoma / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D010959:Tissue Plasminogen Activator / Q000235:genetics
C094335:H19 long non-coding RNA; D056572:Histone Deacetylase Inhibitors; D006657:Histones; D006877:Hydroxamic Acids; D062085:RNA, Long Noncoding; D022661:RNA, Untranslated; C012589:trichostatin A; D012313:RNA; D007335:Insulin-Like Growth Factor II; D010959:Tissue Plasminogen Activator; D006655:Histone Deacetylases; D001374:Azacitidine