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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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Chromatin immunoprecipitation assay of histone acetylation at the H19 locus in treated rhabdomyosarcoma cells. Cells were either treated (TSA) or not treated (Con; control) with histone deacetylase inhibitor (500 nM TSA for 6 hrs). DNA and proteins were cross-linked and precipitated with an antibody to acetylated histone 4, prior to reversing cross-links, amplifying DNA with specific primers and separating the products on a gel. (A) Results obtained using primers specific for the transcribed region of the H19 gene (Region 1), which give an expected band size of 355 bp. Equal amounts of input DNA (In) were used in each immunoprecipitation (lanes 1 and 3) and a no antibody control was run for each sample (lanes 5 and 6). Treatment with TSA gives an increase in signal for the 355 bp target, indicating an increase in acetylated histones associated with the transcribed region (lanes 2 and 4). (B) Primers for a region in the insulator (Region 2) upstream of H19 and downstream of IGF2 give a specific band of 161 bp and this also shows a marked increase after TSA treatment of the cells. Controls are as for (A) above. M; 1 kb DNA size marker (Life Tech.). Minor bands present in some lanes represent non-specific artifacts: primers are visible at the bottom of the gel. Negative images of ethidium-stained gels are shown.
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Figure 2: Chromatin immunoprecipitation assay of histone acetylation at the H19 locus in treated rhabdomyosarcoma cells. Cells were either treated (TSA) or not treated (Con; control) with histone deacetylase inhibitor (500 nM TSA for 6 hrs). DNA and proteins were cross-linked and precipitated with an antibody to acetylated histone 4, prior to reversing cross-links, amplifying DNA with specific primers and separating the products on a gel. (A) Results obtained using primers specific for the transcribed region of the H19 gene (Region 1), which give an expected band size of 355 bp. Equal amounts of input DNA (In) were used in each immunoprecipitation (lanes 1 and 3) and a no antibody control was run for each sample (lanes 5 and 6). Treatment with TSA gives an increase in signal for the 355 bp target, indicating an increase in acetylated histones associated with the transcribed region (lanes 2 and 4). (B) Primers for a region in the insulator (Region 2) upstream of H19 and downstream of IGF2 give a specific band of 161 bp and this also shows a marked increase after TSA treatment of the cells. Controls are as for (A) above. M; 1 kb DNA size marker (Life Tech.). Minor bands present in some lanes represent non-specific artifacts: primers are visible at the bottom of the gel. Negative images of ethidium-stained gels are shown.

Mentions: It is possible that histones at the H19 and IGF2 locus are particularly refractory to inhibitors of histone deacetylases. In order to test this possibility, we carried out chromatin immunoprecipitation experiments to assay whether changes in acetylation levels occur at the H19 locus on treatment with TSA. Chromatin was cross-linked to DNA using formaldehyde, sonicated to reduce the fragment size, then immunoprecipitated with the anti-acetylated H4 antibody. PCR using primers located in the transcribed region (Region 1) or in the upstream insulator region (Region 2) of the H19 gene were used to determine the fraction of total templates which are associated with acetylated histone 4. Input DNA (Fig 2, lanes 1 and 3, In) which has not been treated was used as a control between samples, while a sample of DNA which has been mock-immunoprecipitated without antibody was used as a negative control (Fig 2, lanes 5 and 6, No Ab). We found that after treatment with TSA there was a reproducible increase in the amount of acetylated H4 associated with both the transcribed and the upstream regions (Fig 2A and 2B). Each experiment was carried out independently at least three times. These results indicate that H4 acetylation levels are being increased at the H19 locus in these experiments, but with no detectable effect on H19 transcription.


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)

Chromatin immunoprecipitation assay of histone acetylation at the H19 locus in treated rhabdomyosarcoma cells. Cells were either treated (TSA) or not treated (Con; control) with histone deacetylase inhibitor (500 nM TSA for 6 hrs). DNA and proteins were cross-linked and precipitated with an antibody to acetylated histone 4, prior to reversing cross-links, amplifying DNA with specific primers and separating the products on a gel. (A) Results obtained using primers specific for the transcribed region of the H19 gene (Region 1), which give an expected band size of 355 bp. Equal amounts of input DNA (In) were used in each immunoprecipitation (lanes 1 and 3) and a no antibody control was run for each sample (lanes 5 and 6). Treatment with TSA gives an increase in signal for the 355 bp target, indicating an increase in acetylated histones associated with the transcribed region (lanes 2 and 4). (B) Primers for a region in the insulator (Region 2) upstream of H19 and downstream of IGF2 give a specific band of 161 bp and this also shows a marked increase after TSA treatment of the cells. Controls are as for (A) above. M; 1 kb DNA size marker (Life Tech.). Minor bands present in some lanes represent non-specific artifacts: primers are visible at the bottom of the gel. Negative images of ethidium-stained gels are shown.
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Related In: Results  -  Collection

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Figure 2: Chromatin immunoprecipitation assay of histone acetylation at the H19 locus in treated rhabdomyosarcoma cells. Cells were either treated (TSA) or not treated (Con; control) with histone deacetylase inhibitor (500 nM TSA for 6 hrs). DNA and proteins were cross-linked and precipitated with an antibody to acetylated histone 4, prior to reversing cross-links, amplifying DNA with specific primers and separating the products on a gel. (A) Results obtained using primers specific for the transcribed region of the H19 gene (Region 1), which give an expected band size of 355 bp. Equal amounts of input DNA (In) were used in each immunoprecipitation (lanes 1 and 3) and a no antibody control was run for each sample (lanes 5 and 6). Treatment with TSA gives an increase in signal for the 355 bp target, indicating an increase in acetylated histones associated with the transcribed region (lanes 2 and 4). (B) Primers for a region in the insulator (Region 2) upstream of H19 and downstream of IGF2 give a specific band of 161 bp and this also shows a marked increase after TSA treatment of the cells. Controls are as for (A) above. M; 1 kb DNA size marker (Life Tech.). Minor bands present in some lanes represent non-specific artifacts: primers are visible at the bottom of the gel. Negative images of ethidium-stained gels are shown.
Mentions: It is possible that histones at the H19 and IGF2 locus are particularly refractory to inhibitors of histone deacetylases. In order to test this possibility, we carried out chromatin immunoprecipitation experiments to assay whether changes in acetylation levels occur at the H19 locus on treatment with TSA. Chromatin was cross-linked to DNA using formaldehyde, sonicated to reduce the fragment size, then immunoprecipitated with the anti-acetylated H4 antibody. PCR using primers located in the transcribed region (Region 1) or in the upstream insulator region (Region 2) of the H19 gene were used to determine the fraction of total templates which are associated with acetylated histone 4. Input DNA (Fig 2, lanes 1 and 3, In) which has not been treated was used as a control between samples, while a sample of DNA which has been mock-immunoprecipitated without antibody was used as a negative control (Fig 2, lanes 5 and 6, No Ab). We found that after treatment with TSA there was a reproducible increase in the amount of acetylated H4 associated with both the transcribed and the upstream regions (Fig 2A and 2B). Each experiment was carried out independently at least three times. These results indicate that H4 acetylation levels are being increased at the H19 locus in these experiments, but with no detectable effect on H19 transcription.

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