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Reactivation of a silenced H19 gene in human rhabdomyosarcoma by demethylation of DNA but not by histone hyperacetylation.

Lynch CA, Tycko B, Bestor TH, Walsh CP - Mol. Cancer (2002)

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Cancer and Ageing Research Group, School of Biomedical Sciences, University of Ulster, Coleraine BT52 1SA, United Kingdom. c.lynch@ulster.ac.uk

ABSTRACT

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.

Conclusion: These results suggest that DNA methylation rather than histone acetylation is the primary determinant of silencing of H19 in rhabdomyosarcoma.

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Related in: MedlinePlus

Simultaneous treatment of cells to increase histone acetylation and decrease DNA methylation. Rhabdomyosarcoma cells were treated with the indicated amounts of AzaC in the absence (lanes 1–5) or presence (+TSA; lanes 6–10) of 500 nM TSA. RNA was extracted and separated on an agarose gel before transferring to a nylon membrane and hybridizing with the indicated probes. (A)H19 transcription is reactivated by AzaC in a dose-dependent fashion with higher doses resulting in greater reactivation (lanes 1–5). Combining the AzaC treatment with 500 nM TSA however results in lower levels of reactivation of the gene (compare lanes 6–10 with lanes 1–5). (B) The same membrane stripped and rehybridized with a probe for IGF2. Levels of IGF2 show a slight decrease as H19 transcription increases in the cells treated with AzaC alone (lanes 1–5). H19 and IGF2 compete for common enhancer elements, which would explain this effect. The effect is diminished in cells treated with both TSA and AzaC (lanes 6–10), which also show less H19 expression. (C) 28S rRNA loading control for the Northern.
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Figure 3: Simultaneous treatment of cells to increase histone acetylation and decrease DNA methylation. Rhabdomyosarcoma cells were treated with the indicated amounts of AzaC in the absence (lanes 1–5) or presence (+TSA; lanes 6–10) of 500 nM TSA. RNA was extracted and separated on an agarose gel before transferring to a nylon membrane and hybridizing with the indicated probes. (A)H19 transcription is reactivated by AzaC in a dose-dependent fashion with higher doses resulting in greater reactivation (lanes 1–5). Combining the AzaC treatment with 500 nM TSA however results in lower levels of reactivation of the gene (compare lanes 6–10 with lanes 1–5). (B) The same membrane stripped and rehybridized with a probe for IGF2. Levels of IGF2 show a slight decrease as H19 transcription increases in the cells treated with AzaC alone (lanes 1–5). H19 and IGF2 compete for common enhancer elements, which would explain this effect. The effect is diminished in cells treated with both TSA and AzaC (lanes 6–10), which also show less H19 expression. (C) 28S rRNA loading control for the Northern.

Mentions: It has been suggested that some epigenetically silenced genes can only be reactivated by treatment with a combination of histone deacetylase inhibitors and methylation. In order to test whether such a combination treatment might enhance the reactivation of H19 in rhabdomyosarcoma seen using methylation inhibitor alone, we grew RD cells for long or short periods in various combinations of AzaC and TSA. Both long and short-term cultures gave similar results and a typical experiment can be seen in Figure 3 (treatment for 1 week). Here, the silenced H19 allele shows a dose-dependent reactivation in response to AzaC treatment, with higher levels of the drug (0.5 mM to 1 mM) resulting in greater activation (Fig 3A, lanes 1–5). Addition of 500 nM TSA to the culture medium containing various amounts of AzaC resulted in a significant decrease in the level of H19 transcript produced (Fig 3A, lanes 6–10). A decrease in IGF2 transcript levels on addition of TSA was also seen in these cells when compared to treatment with AzaC alone (Fig 3B). These results indicate that a combination of drug treatments do not result in an increase in H19 expression, but rather inhibit reactivation of the gene by demethylation.


Reactivation of a silenced H19 gene in human rhabdomyosarcoma by demethylation of DNA but not by histone hyperacetylation.

Lynch CA, Tycko B, Bestor TH, Walsh CP - Mol. Cancer (2002)

Simultaneous treatment of cells to increase histone acetylation and decrease DNA methylation. Rhabdomyosarcoma cells were treated with the indicated amounts of AzaC in the absence (lanes 1–5) or presence (+TSA; lanes 6–10) of 500 nM TSA. RNA was extracted and separated on an agarose gel before transferring to a nylon membrane and hybridizing with the indicated probes. (A)H19 transcription is reactivated by AzaC in a dose-dependent fashion with higher doses resulting in greater reactivation (lanes 1–5). Combining the AzaC treatment with 500 nM TSA however results in lower levels of reactivation of the gene (compare lanes 6–10 with lanes 1–5). (B) The same membrane stripped and rehybridized with a probe for IGF2. Levels of IGF2 show a slight decrease as H19 transcription increases in the cells treated with AzaC alone (lanes 1–5). H19 and IGF2 compete for common enhancer elements, which would explain this effect. The effect is diminished in cells treated with both TSA and AzaC (lanes 6–10), which also show less H19 expression. (C) 28S rRNA loading control for the Northern.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC119368&req=5

Figure 3: Simultaneous treatment of cells to increase histone acetylation and decrease DNA methylation. Rhabdomyosarcoma cells were treated with the indicated amounts of AzaC in the absence (lanes 1–5) or presence (+TSA; lanes 6–10) of 500 nM TSA. RNA was extracted and separated on an agarose gel before transferring to a nylon membrane and hybridizing with the indicated probes. (A)H19 transcription is reactivated by AzaC in a dose-dependent fashion with higher doses resulting in greater reactivation (lanes 1–5). Combining the AzaC treatment with 500 nM TSA however results in lower levels of reactivation of the gene (compare lanes 6–10 with lanes 1–5). (B) The same membrane stripped and rehybridized with a probe for IGF2. Levels of IGF2 show a slight decrease as H19 transcription increases in the cells treated with AzaC alone (lanes 1–5). H19 and IGF2 compete for common enhancer elements, which would explain this effect. The effect is diminished in cells treated with both TSA and AzaC (lanes 6–10), which also show less H19 expression. (C) 28S rRNA loading control for the Northern.
Mentions: It has been suggested that some epigenetically silenced genes can only be reactivated by treatment with a combination of histone deacetylase inhibitors and methylation. In order to test whether such a combination treatment might enhance the reactivation of H19 in rhabdomyosarcoma seen using methylation inhibitor alone, we grew RD cells for long or short periods in various combinations of AzaC and TSA. Both long and short-term cultures gave similar results and a typical experiment can be seen in Figure 3 (treatment for 1 week). Here, the silenced H19 allele shows a dose-dependent reactivation in response to AzaC treatment, with higher levels of the drug (0.5 mM to 1 mM) resulting in greater activation (Fig 3A, lanes 1–5). Addition of 500 nM TSA to the culture medium containing various amounts of AzaC resulted in a significant decrease in the level of H19 transcript produced (Fig 3A, lanes 6–10). A decrease in IGF2 transcript levels on addition of TSA was also seen in these cells when compared to treatment with AzaC alone (Fig 3B). These results indicate that a combination of drug treatments do not result in an increase in H19 expression, but rather inhibit reactivation of the gene by demethylation.

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Cancer and Ageing Research Group, School of Biomedical Sciences, University of Ulster, Coleraine BT52 1SA, United Kingdom. c.lynch@ulster.ac.uk

ABSTRACT

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.

Conclusion: These results suggest that DNA methylation rather than histone acetylation is the primary determinant of silencing of H19 in rhabdomyosarcoma.

Show MeSH
Related in: MedlinePlus