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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

Effects of histone deacetylase inhibitor on rhabdomyosarcoma cells. Cells were treated for 6 hr (lanes 1, 2) or 15 hr (lane 3–5) with 500 nM of either Trichostatin A (TSA) or 5'-aza-2'deoxycytidine (AzaC) as indicated at top. (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 cells at 6 hr (lanes 1 and 2). While treatment for 15 hr also increases acetylation levels, the effect is less marked, presumably due to a compensatory mechanism in the cell (lanes 3 and 4). (B) Coomasie stained total protein loading control for the Western. (C) Northern hybridization of RNA derived from the same cells to a probe for Tissue-type Plasminogen Activator (TPA). Transcription levels can be seen to increase in parallel with the marked increase in histone acetylation (lanes 1 and 2), indicating that acetylation plays an important role in determining levels of TPA transcript in the cell. The less marked increase in acetylation seen at 15 hr has only a slight effect on transcript levels (lanes 3 and 4). (D) The membrane used in (C) was stripped and rehybridized with a probe for H19, which is normally silent in these cells. No signal was detected in the normal or TSA-treated samples (lanes 1–4), but some reactivation could be seen after even a brief treatment with a low level of AzaC (lane 5). (E) Rehybridization with an IGF2 probe shows no difference between the TSA treated and untreated samples at 6 hr (lanes 1 and 2) or 15 hr (lanes 3 and 4). IGF2 transcripts accumulate in the cells during the log phase of growth in culture and at 15 hr, basal levels of IGF2 have increased from those seen at 6 hr (compare lanes 3 and 1). The two major IGF2 transcripts of 6 kb and 4.9 kb are visible. (F) 28S rRNA loading control for the Northern.
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Figure 1: Effects of histone deacetylase inhibitor on rhabdomyosarcoma cells. Cells were treated for 6 hr (lanes 1, 2) or 15 hr (lane 3–5) with 500 nM of either Trichostatin A (TSA) or 5'-aza-2'deoxycytidine (AzaC) as indicated at top. (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 cells at 6 hr (lanes 1 and 2). While treatment for 15 hr also increases acetylation levels, the effect is less marked, presumably due to a compensatory mechanism in the cell (lanes 3 and 4). (B) Coomasie stained total protein loading control for the Western. (C) Northern hybridization of RNA derived from the same cells to a probe for Tissue-type Plasminogen Activator (TPA). Transcription levels can be seen to increase in parallel with the marked increase in histone acetylation (lanes 1 and 2), indicating that acetylation plays an important role in determining levels of TPA transcript in the cell. The less marked increase in acetylation seen at 15 hr has only a slight effect on transcript levels (lanes 3 and 4). (D) The membrane used in (C) was stripped and rehybridized with a probe for H19, which is normally silent in these cells. No signal was detected in the normal or TSA-treated samples (lanes 1–4), but some reactivation could be seen after even a brief treatment with a low level of AzaC (lane 5). (E) Rehybridization with an IGF2 probe shows no difference between the TSA treated and untreated samples at 6 hr (lanes 1 and 2) or 15 hr (lanes 3 and 4). IGF2 transcripts accumulate in the cells during the log phase of growth in culture and at 15 hr, basal levels of IGF2 have increased from those seen at 6 hr (compare lanes 3 and 1). The two major IGF2 transcripts of 6 kb and 4.9 kb are visible. (F) 28S rRNA loading control for the Northern.

Mentions: In order to investigate the effects of increasing histone acetylation levels in RD cells on the silenced H19 gene, cells were grown in culture medium supplemented with a range of concentrations of TSA from 25 nM to 1 mM for periods ranging from 3 hrs to 2 weeks and sodium butryate (3 mM) for various times from 3 hrs to 24 hours. Proteins were then extracted and analyzed by Western blotting using an antibody against acetylated histone 4. Histone acetylation levels are maximally effected by inhibitors at very early time points, with the increase in histone acetylation dropping off rapidly in the first day of treatment and protein samples taken on subsequent days of treatment showed no difference between treated and untreated cells. This effect can be seen in Figure 1, where RD cells were treated with 500 nM TSA for 6 or 15 hours, after which proteins were harvested and analysed by Western hybridisation with an antibody to the acetylated form of histone 4. At 6 hr, acetylation levels have strongly increased (Fig 1A, lanes 1 and 2), but this effect has diminished significantly at 15 hr (Fig 1A, lanes 3 and 4): by 48 hrs, no difference in acetylation levels between treated and untreated samples can be detected (data not shown). Increases in histone acetylation levels were equivalent at 3 hr and 6 hr (data not shown). These data agree with recent studies showing cells can homeostatically adjust to maintain a steady-state level of histone acetylation [13]. Treatment with sodium butyrate (3 mM), another inhibitor of histone deacetylases (HDACs) showed similar effects to 500 nM TSA and results are shown for TSA alone.


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 rhabdomyosarcoma cells. Cells were treated for 6 hr (lanes 1, 2) or 15 hr (lane 3–5) with 500 nM of either Trichostatin A (TSA) or 5'-aza-2'deoxycytidine (AzaC) as indicated at top. (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 cells at 6 hr (lanes 1 and 2). While treatment for 15 hr also increases acetylation levels, the effect is less marked, presumably due to a compensatory mechanism in the cell (lanes 3 and 4). (B) Coomasie stained total protein loading control for the Western. (C) Northern hybridization of RNA derived from the same cells to a probe for Tissue-type Plasminogen Activator (TPA). Transcription levels can be seen to increase in parallel with the marked increase in histone acetylation (lanes 1 and 2), indicating that acetylation plays an important role in determining levels of TPA transcript in the cell. The less marked increase in acetylation seen at 15 hr has only a slight effect on transcript levels (lanes 3 and 4). (D) The membrane used in (C) was stripped and rehybridized with a probe for H19, which is normally silent in these cells. No signal was detected in the normal or TSA-treated samples (lanes 1–4), but some reactivation could be seen after even a brief treatment with a low level of AzaC (lane 5). (E) Rehybridization with an IGF2 probe shows no difference between the TSA treated and untreated samples at 6 hr (lanes 1 and 2) or 15 hr (lanes 3 and 4). IGF2 transcripts accumulate in the cells during the log phase of growth in culture and at 15 hr, basal levels of IGF2 have increased from those seen at 6 hr (compare lanes 3 and 1). The two major IGF2 transcripts of 6 kb and 4.9 kb are visible. (F) 28S rRNA loading control for the Northern.
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Related In: Results  -  Collection

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Figure 1: Effects of histone deacetylase inhibitor on rhabdomyosarcoma cells. Cells were treated for 6 hr (lanes 1, 2) or 15 hr (lane 3–5) with 500 nM of either Trichostatin A (TSA) or 5'-aza-2'deoxycytidine (AzaC) as indicated at top. (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 cells at 6 hr (lanes 1 and 2). While treatment for 15 hr also increases acetylation levels, the effect is less marked, presumably due to a compensatory mechanism in the cell (lanes 3 and 4). (B) Coomasie stained total protein loading control for the Western. (C) Northern hybridization of RNA derived from the same cells to a probe for Tissue-type Plasminogen Activator (TPA). Transcription levels can be seen to increase in parallel with the marked increase in histone acetylation (lanes 1 and 2), indicating that acetylation plays an important role in determining levels of TPA transcript in the cell. The less marked increase in acetylation seen at 15 hr has only a slight effect on transcript levels (lanes 3 and 4). (D) The membrane used in (C) was stripped and rehybridized with a probe for H19, which is normally silent in these cells. No signal was detected in the normal or TSA-treated samples (lanes 1–4), but some reactivation could be seen after even a brief treatment with a low level of AzaC (lane 5). (E) Rehybridization with an IGF2 probe shows no difference between the TSA treated and untreated samples at 6 hr (lanes 1 and 2) or 15 hr (lanes 3 and 4). IGF2 transcripts accumulate in the cells during the log phase of growth in culture and at 15 hr, basal levels of IGF2 have increased from those seen at 6 hr (compare lanes 3 and 1). The two major IGF2 transcripts of 6 kb and 4.9 kb are visible. (F) 28S rRNA loading control for the Northern.
Mentions: In order to investigate the effects of increasing histone acetylation levels in RD cells on the silenced H19 gene, cells were grown in culture medium supplemented with a range of concentrations of TSA from 25 nM to 1 mM for periods ranging from 3 hrs to 2 weeks and sodium butryate (3 mM) for various times from 3 hrs to 24 hours. Proteins were then extracted and analyzed by Western blotting using an antibody against acetylated histone 4. Histone acetylation levels are maximally effected by inhibitors at very early time points, with the increase in histone acetylation dropping off rapidly in the first day of treatment and protein samples taken on subsequent days of treatment showed no difference between treated and untreated cells. This effect can be seen in Figure 1, where RD cells were treated with 500 nM TSA for 6 or 15 hours, after which proteins were harvested and analysed by Western hybridisation with an antibody to the acetylated form of histone 4. At 6 hr, acetylation levels have strongly increased (Fig 1A, lanes 1 and 2), but this effect has diminished significantly at 15 hr (Fig 1A, lanes 3 and 4): by 48 hrs, no difference in acetylation levels between treated and untreated samples can be detected (data not shown). Increases in histone acetylation levels were equivalent at 3 hr and 6 hr (data not shown). These data agree with recent studies showing cells can homeostatically adjust to maintain a steady-state level of histone acetylation [13]. Treatment with sodium butyrate (3 mM), another inhibitor of histone deacetylases (HDACs) showed similar effects to 500 nM TSA and results are shown for TSA alone.

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