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Synergism between DNA methylation and macroH2A1 occupancy in epigenetic silencing of the tumor suppressor gene p16(CDKN2A).

Barzily-Rokni M, Friedman N, Ron-Bigger S, Isaac S, Michlin D, Eden A - Nucleic Acids Res. (2010)

Bottom Line: In addition, the inactive-X is marked by the histone macroH2A, a variant of H2A with a large non-histone region of unknown function.Knockdown of macroH2A1 was not sufficient for reactivation of silenced genes.However, when combined with DNA demethylation, macroH2A1 deficiency significantly enhanced reactivation of the tumor suppressor genes p16, MLH1 and Timp3 and inhibited cell proliferation.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell & Developmental Biology, Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem, Israel.

ABSTRACT
Promoter hypermethylation and heterochromatinization is a frequent event leading to gene inactivation and tumorigenesis. At the molecular level, inactivation of tumor suppressor genes in cancer has many similarities to the inactive X chromosome in female cells and is defined and maintained by DNA methylation and characteristic histone modifications. In addition, the inactive-X is marked by the histone macroH2A, a variant of H2A with a large non-histone region of unknown function. Studying tumor suppressor genes (TSGs) silenced in cancer cell lines, we find that when active, these promoters are associated with H2A.Z but become enriched for macroH2A1 once silenced. Knockdown of macroH2A1 was not sufficient for reactivation of silenced genes. However, when combined with DNA demethylation, macroH2A1 deficiency significantly enhanced reactivation of the tumor suppressor genes p16, MLH1 and Timp3 and inhibited cell proliferation. Our findings link macroH2A1 to heterochromatin of epigenetically silenced cancer genes and indicate synergism between macroH2A1 and DNA methylation in maintenance of the silenced state.

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Silencing of CDKN2A is accompanied by macroH2A1 enrichment. (A) Expression and methylation status of CDKN2A in early passage (p18) and late passage (p32) WI-38 cell: western blot for p16 and β-actin showing loss of expression in p32 cells (left panel). Methylation of the CDKN2A promoter in p32 cell confirmed by methylation-specific PCR (MSP) on bisulfite-modified DNA (right panel). (B) ChIP with macroH2A1 antibody. The bound/input ratio for each cell line was calculated using quantitative real-time PCR. To compare between cell lines, enrichment of the bound fraction was normalized according to the positive control α-Crystallin, which is inactive in both cultures (Supplementary Figure S6). An 8.5-fold enrichment for macroH2A1 at the CDKN2A promoter is observed in p32 cells versus p18 cells. Similar results were observed in three biological replicates (see also Figure 5). HoxA9 is another positive control and is not expressed. Aprt and MLH1 are expressed in both samples. The numbers under the gene names indicate position of the interrogated region relative to TSS. Error bars represent standard deviation. (C) ChIP analysis using anti-macroH2A1 or anti-acetylated H3 antibody on HCT116 cells. Input and bound fractions were analyzed by SNaPshot assay to discriminate between the silenced (wild-type) allele of CDKN2A (S) and the active mutant allele (A). The ratio between the two alleles (S/A ratio) was determined based on peak area using Genotyper 2.1 software (ABI PRISM).
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Figure 1: Silencing of CDKN2A is accompanied by macroH2A1 enrichment. (A) Expression and methylation status of CDKN2A in early passage (p18) and late passage (p32) WI-38 cell: western blot for p16 and β-actin showing loss of expression in p32 cells (left panel). Methylation of the CDKN2A promoter in p32 cell confirmed by methylation-specific PCR (MSP) on bisulfite-modified DNA (right panel). (B) ChIP with macroH2A1 antibody. The bound/input ratio for each cell line was calculated using quantitative real-time PCR. To compare between cell lines, enrichment of the bound fraction was normalized according to the positive control α-Crystallin, which is inactive in both cultures (Supplementary Figure S6). An 8.5-fold enrichment for macroH2A1 at the CDKN2A promoter is observed in p32 cells versus p18 cells. Similar results were observed in three biological replicates (see also Figure 5). HoxA9 is another positive control and is not expressed. Aprt and MLH1 are expressed in both samples. The numbers under the gene names indicate position of the interrogated region relative to TSS. Error bars represent standard deviation. (C) ChIP analysis using anti-macroH2A1 or anti-acetylated H3 antibody on HCT116 cells. Input and bound fractions were analyzed by SNaPshot assay to discriminate between the silenced (wild-type) allele of CDKN2A (S) and the active mutant allele (A). The ratio between the two alleles (S/A ratio) was determined based on peak area using Genotyper 2.1 software (ABI PRISM).

Mentions: We used WI-38 human embryonic lung fibroblasts, in which p16Ink4a (CDKN2A) was found to undergo epigenetic silencing through DNA methylation of its promoter. Silencing occurs spontaneously during passage of these cells in culture, while treatment of the non-expressing cells with 5-aza-dC restores p16 expression (44). We compared cells from a late passage (p32) in which CDKN2A is methylated and silenced, to cells from an early passage (p18) which are unmethylated and express p16 (Figure 1A). Using ChIP with anti-macroH2A1 we observed enrichment for macroH2A1 at the CDKN2A promoter only in the late passage cells that have silenced the gene (Figure 1B), demonstrating specific association of macroH2A1 with the inactive state. To validate the efficacy of our ChIP assay, we compared enrichment for macroH2A1 at several non-expressed genes to that of housekeeping genes. MacroH2A1 levels were significantly elevated in the non-expressed tissue-specific genes (Supplementary Figure S1A and B). We also performed ChIP on macroH2A1-deficient ES cells, as negative control confirming that the bound fraction is dependent on macroH2A1 presence (Supplementary Figure 1C).Figure 1.


Synergism between DNA methylation and macroH2A1 occupancy in epigenetic silencing of the tumor suppressor gene p16(CDKN2A).

Barzily-Rokni M, Friedman N, Ron-Bigger S, Isaac S, Michlin D, Eden A - Nucleic Acids Res. (2010)

Silencing of CDKN2A is accompanied by macroH2A1 enrichment. (A) Expression and methylation status of CDKN2A in early passage (p18) and late passage (p32) WI-38 cell: western blot for p16 and β-actin showing loss of expression in p32 cells (left panel). Methylation of the CDKN2A promoter in p32 cell confirmed by methylation-specific PCR (MSP) on bisulfite-modified DNA (right panel). (B) ChIP with macroH2A1 antibody. The bound/input ratio for each cell line was calculated using quantitative real-time PCR. To compare between cell lines, enrichment of the bound fraction was normalized according to the positive control α-Crystallin, which is inactive in both cultures (Supplementary Figure S6). An 8.5-fold enrichment for macroH2A1 at the CDKN2A promoter is observed in p32 cells versus p18 cells. Similar results were observed in three biological replicates (see also Figure 5). HoxA9 is another positive control and is not expressed. Aprt and MLH1 are expressed in both samples. The numbers under the gene names indicate position of the interrogated region relative to TSS. Error bars represent standard deviation. (C) ChIP analysis using anti-macroH2A1 or anti-acetylated H3 antibody on HCT116 cells. Input and bound fractions were analyzed by SNaPshot assay to discriminate between the silenced (wild-type) allele of CDKN2A (S) and the active mutant allele (A). The ratio between the two alleles (S/A ratio) was determined based on peak area using Genotyper 2.1 software (ABI PRISM).
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Figure 1: Silencing of CDKN2A is accompanied by macroH2A1 enrichment. (A) Expression and methylation status of CDKN2A in early passage (p18) and late passage (p32) WI-38 cell: western blot for p16 and β-actin showing loss of expression in p32 cells (left panel). Methylation of the CDKN2A promoter in p32 cell confirmed by methylation-specific PCR (MSP) on bisulfite-modified DNA (right panel). (B) ChIP with macroH2A1 antibody. The bound/input ratio for each cell line was calculated using quantitative real-time PCR. To compare between cell lines, enrichment of the bound fraction was normalized according to the positive control α-Crystallin, which is inactive in both cultures (Supplementary Figure S6). An 8.5-fold enrichment for macroH2A1 at the CDKN2A promoter is observed in p32 cells versus p18 cells. Similar results were observed in three biological replicates (see also Figure 5). HoxA9 is another positive control and is not expressed. Aprt and MLH1 are expressed in both samples. The numbers under the gene names indicate position of the interrogated region relative to TSS. Error bars represent standard deviation. (C) ChIP analysis using anti-macroH2A1 or anti-acetylated H3 antibody on HCT116 cells. Input and bound fractions were analyzed by SNaPshot assay to discriminate between the silenced (wild-type) allele of CDKN2A (S) and the active mutant allele (A). The ratio between the two alleles (S/A ratio) was determined based on peak area using Genotyper 2.1 software (ABI PRISM).
Mentions: We used WI-38 human embryonic lung fibroblasts, in which p16Ink4a (CDKN2A) was found to undergo epigenetic silencing through DNA methylation of its promoter. Silencing occurs spontaneously during passage of these cells in culture, while treatment of the non-expressing cells with 5-aza-dC restores p16 expression (44). We compared cells from a late passage (p32) in which CDKN2A is methylated and silenced, to cells from an early passage (p18) which are unmethylated and express p16 (Figure 1A). Using ChIP with anti-macroH2A1 we observed enrichment for macroH2A1 at the CDKN2A promoter only in the late passage cells that have silenced the gene (Figure 1B), demonstrating specific association of macroH2A1 with the inactive state. To validate the efficacy of our ChIP assay, we compared enrichment for macroH2A1 at several non-expressed genes to that of housekeeping genes. MacroH2A1 levels were significantly elevated in the non-expressed tissue-specific genes (Supplementary Figure S1A and B). We also performed ChIP on macroH2A1-deficient ES cells, as negative control confirming that the bound fraction is dependent on macroH2A1 presence (Supplementary Figure 1C).Figure 1.

Bottom Line: In addition, the inactive-X is marked by the histone macroH2A, a variant of H2A with a large non-histone region of unknown function.Knockdown of macroH2A1 was not sufficient for reactivation of silenced genes.However, when combined with DNA demethylation, macroH2A1 deficiency significantly enhanced reactivation of the tumor suppressor genes p16, MLH1 and Timp3 and inhibited cell proliferation.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell & Developmental Biology, Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem, Israel.

ABSTRACT
Promoter hypermethylation and heterochromatinization is a frequent event leading to gene inactivation and tumorigenesis. At the molecular level, inactivation of tumor suppressor genes in cancer has many similarities to the inactive X chromosome in female cells and is defined and maintained by DNA methylation and characteristic histone modifications. In addition, the inactive-X is marked by the histone macroH2A, a variant of H2A with a large non-histone region of unknown function. Studying tumor suppressor genes (TSGs) silenced in cancer cell lines, we find that when active, these promoters are associated with H2A.Z but become enriched for macroH2A1 once silenced. Knockdown of macroH2A1 was not sufficient for reactivation of silenced genes. However, when combined with DNA demethylation, macroH2A1 deficiency significantly enhanced reactivation of the tumor suppressor genes p16, MLH1 and Timp3 and inhibited cell proliferation. Our findings link macroH2A1 to heterochromatin of epigenetically silenced cancer genes and indicate synergism between macroH2A1 and DNA methylation in maintenance of the silenced state.

Show MeSH
Related in: MedlinePlus