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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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Effects of histone deacetylase inhibitor on Igf2 knockout cells. Primary embryonic liver cells were derived from mice which carried a paternally-inherited deletion of Igf2 (Pat KO; lanes 1 and 2) or their wild-type littermates (WT; lanes 3 and 4). First passage cells were either treated with 500 nM TSA for 6 hr or left untreated as indicated. (A) Western analysis of protein derived from the cells using the anti-acetylated histone 4 (AcH4) antibody shows that treatment with TSA markedly increases the amount of acetylated histone in the primary cells (lanes 2 and 4). (B) Coomasie stained total protein loading control for the Western. (C) Northern hybridization of RNA derived from the same cells to a mouse probe for Tissue-type plasminogen activator (Tpa). Transcription levels can be seen to increase on TSA treatment (lanes 2 and 4) as in RD cells. (D) The membrane used in (C) was stripped and rehybridized with a probe for Igf2. Mice which carry a deletion of the gene on the paternally inherited allele (Pat KO) are missing the allele which is normally active but retain the silenced maternal allele and show no expression of the gene (lane 1). Reactivation of the silent maternal allele was not seen in the cells treated with TSA (lane 2). The two major transcripts ran as a single band here. (E) Rehybridization with an H19 probe shows no significant difference between the TSA treated and untreated samples, allowing for differences in loading. (F) 28S rRNA loading control for the Northern.
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Figure 4: Effects of histone deacetylase inhibitor on Igf2 knockout cells. Primary embryonic liver cells were derived from mice which carried a paternally-inherited deletion of Igf2 (Pat KO; lanes 1 and 2) or their wild-type littermates (WT; lanes 3 and 4). First passage cells were either treated with 500 nM TSA for 6 hr or left untreated as indicated. (A) Western analysis of protein derived from the cells using the anti-acetylated histone 4 (AcH4) antibody shows that treatment with TSA markedly increases the amount of acetylated histone in the primary cells (lanes 2 and 4). (B) Coomasie stained total protein loading control for the Western. (C) Northern hybridization of RNA derived from the same cells to a mouse probe for Tissue-type plasminogen activator (Tpa). Transcription levels can be seen to increase on TSA treatment (lanes 2 and 4) as in RD cells. (D) The membrane used in (C) was stripped and rehybridized with a probe for Igf2. Mice which carry a deletion of the gene on the paternally inherited allele (Pat KO) are missing the allele which is normally active but retain the silenced maternal allele and show no expression of the gene (lane 1). Reactivation of the silent maternal allele was not seen in the cells treated with TSA (lane 2). The two major transcripts ran as a single band here. (E) Rehybridization with an H19 probe shows no significant difference between the TSA treated and untreated samples, allowing for differences in loading. (F) 28S rRNA loading control for the Northern.

Mentions: Treatment of primary liver or kidney cells which had never been passaged with 500 nM TSA or 3 mM Sodium butyrate for 6 hr resulted in a marked increase in H4 acetylation, as seen previously in RD cells: results are shown for liver cell cultures (Fig 4A). This also resulted in an up-regulation of the mouse tissue-type plasminogen activator gene (Tpa) (Fig 4C), indicating that histone acetylation levels were again being altered to a degree significant enough to effect gene transcription. We found that Igf2 imprinting was maintained in these primary cells, as shown by the lack of expression of the gene in the cultures derived from a mouse with a paternally-inherited Igf2 knockout (Fig 4D, lanes 1–2), whereas Igf2 transcripts were detected in liver explants from wild-type littermates (Fig 4D, lanes 3–4). However, no significant reactivation of the silent maternal Igf2 allele from any of the three embryonic promoters was seen in the samples, even on long exposure of autoradiographs (Fig 4D and data not shown). There was no significant effect seen on the active Igf2 or H19 alleles in this system. Results for sodium butryrate were identical to TSA and no effect of inhibitors on transcription was seen on longer-term culturing of cells (2–15 days) or in primary kidney cells (data not shown).


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)

Effects of histone deacetylase inhibitor on Igf2 knockout cells. Primary embryonic liver cells were derived from mice which carried a paternally-inherited deletion of Igf2 (Pat KO; lanes 1 and 2) or their wild-type littermates (WT; lanes 3 and 4). First passage cells were either treated with 500 nM TSA for 6 hr or left untreated as indicated. (A) Western analysis of protein derived from the cells using the anti-acetylated histone 4 (AcH4) antibody shows that treatment with TSA markedly increases the amount of acetylated histone in the primary cells (lanes 2 and 4). (B) Coomasie stained total protein loading control for the Western. (C) Northern hybridization of RNA derived from the same cells to a mouse probe for Tissue-type plasminogen activator (Tpa). Transcription levels can be seen to increase on TSA treatment (lanes 2 and 4) as in RD cells. (D) The membrane used in (C) was stripped and rehybridized with a probe for Igf2. Mice which carry a deletion of the gene on the paternally inherited allele (Pat KO) are missing the allele which is normally active but retain the silenced maternal allele and show no expression of the gene (lane 1). Reactivation of the silent maternal allele was not seen in the cells treated with TSA (lane 2). The two major transcripts ran as a single band here. (E) Rehybridization with an H19 probe shows no significant difference between the TSA treated and untreated samples, allowing for differences in loading. (F) 28S rRNA loading control for the Northern.
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Figure 4: Effects of histone deacetylase inhibitor on Igf2 knockout cells. Primary embryonic liver cells were derived from mice which carried a paternally-inherited deletion of Igf2 (Pat KO; lanes 1 and 2) or their wild-type littermates (WT; lanes 3 and 4). First passage cells were either treated with 500 nM TSA for 6 hr or left untreated as indicated. (A) Western analysis of protein derived from the cells using the anti-acetylated histone 4 (AcH4) antibody shows that treatment with TSA markedly increases the amount of acetylated histone in the primary cells (lanes 2 and 4). (B) Coomasie stained total protein loading control for the Western. (C) Northern hybridization of RNA derived from the same cells to a mouse probe for Tissue-type plasminogen activator (Tpa). Transcription levels can be seen to increase on TSA treatment (lanes 2 and 4) as in RD cells. (D) The membrane used in (C) was stripped and rehybridized with a probe for Igf2. Mice which carry a deletion of the gene on the paternally inherited allele (Pat KO) are missing the allele which is normally active but retain the silenced maternal allele and show no expression of the gene (lane 1). Reactivation of the silent maternal allele was not seen in the cells treated with TSA (lane 2). The two major transcripts ran as a single band here. (E) Rehybridization with an H19 probe shows no significant difference between the TSA treated and untreated samples, allowing for differences in loading. (F) 28S rRNA loading control for the Northern.
Mentions: Treatment of primary liver or kidney cells which had never been passaged with 500 nM TSA or 3 mM Sodium butyrate for 6 hr resulted in a marked increase in H4 acetylation, as seen previously in RD cells: results are shown for liver cell cultures (Fig 4A). This also resulted in an up-regulation of the mouse tissue-type plasminogen activator gene (Tpa) (Fig 4C), indicating that histone acetylation levels were again being altered to a degree significant enough to effect gene transcription. We found that Igf2 imprinting was maintained in these primary cells, as shown by the lack of expression of the gene in the cultures derived from a mouse with a paternally-inherited Igf2 knockout (Fig 4D, lanes 1–2), whereas Igf2 transcripts were detected in liver explants from wild-type littermates (Fig 4D, lanes 3–4). However, no significant reactivation of the silent maternal Igf2 allele from any of the three embryonic promoters was seen in the samples, even on long exposure of autoradiographs (Fig 4D and data not shown). There was no significant effect seen on the active Igf2 or H19 alleles in this system. Results for sodium butryrate were identical to TSA and no effect of inhibitors on transcription was seen on longer-term culturing of cells (2–15 days) or in primary kidney cells (data not shown).

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