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Dissecting epigenetic silencing complexity in the mouse lung cancer suppressor gene Cadm1.

Reamon-Buettner SM, Borlak J - PLoS ONE (2012)

Bottom Line: Yet, the precise mechanisms are still unclear and complex, involving the interplay of several effectors including nucleosome positioning, DNA methylation, histone variants and histone modifications.Chromatin analysis with micrococcal nuclease also indicated variations in nucleosome positioning to have implications in the binding of transcription factors near nucleosome borders.Chromatin immunoprecipitation showed that histone variants (H2A.Z and H3.3), and opposing histone modification marks (H3K4me3 and H3K27me3) all colocalized in the same nucleosome positions that is reminiscent of epigenetic plasticity in embryonic stem cells.

View Article: PubMed Central - PubMed

Affiliation: Toxicology and Environmental Hygiene, Fraunhofer Institute for Toxicology and Experimental Medicine, Hannover, Germany. reamon-buettner@item.fraunhofer.de

ABSTRACT
Disease-oriented functional analysis of epigenetic factors and their regulatory mechanisms in aberrant silencing is a prerequisite for better diagnostics and therapy. Yet, the precise mechanisms are still unclear and complex, involving the interplay of several effectors including nucleosome positioning, DNA methylation, histone variants and histone modifications. We investigated the epigenetic silencing complexity in the tumor suppressor gene Cadm1 in mouse lung cancer progenitor cell lines, exhibiting promoter hypermethylation associated with transcriptional repression, but mostly unresponsive to demethylating drug treatments. After predicting nucleosome positions and transcription factor binding sites along the Cadm1 promoter, we carried out single-molecule mapping with DNA methyltransferase M.SssI, which revealed in silent promoters high nucleosome occupancy and occlusion of transcription factor binding sites. Furthermore, M.SssI maps of promoters varied within and among the different lung cancer cell lines. Chromatin analysis with micrococcal nuclease also indicated variations in nucleosome positioning to have implications in the binding of transcription factors near nucleosome borders. Chromatin immunoprecipitation showed that histone variants (H2A.Z and H3.3), and opposing histone modification marks (H3K4me3 and H3K27me3) all colocalized in the same nucleosome positions that is reminiscent of epigenetic plasticity in embryonic stem cells. Altogether, epigenetic silencing complexity in the promoter region of Cadm1 is not only defined by DNA hypermethylation, but high nucleosome occupancy, altered nucleosome positioning, and 'bivalent' histone modifications, also likely contributed in the transcriptional repression of this gene in the lung cancer cells. Our results will help define therapeutic intervention strategies using epigenetic drugs in lung cancer.

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ChIP with H2A and H2A.Z in lung cancer cell lines, lung tumor, and normal lung.Results in analyzed nucleosomes are expressed as Percent Input using Ct values. The lung cancer line A2B1 still expresses Cadm1, while A2C12 does not. For quantitative PCR, 20 ng of ChIP DNA was used as template in all samples, including DNA obtained in normal rabbit IgG. The primer sets used and corresponding color coding are indicated on the uppermost right hand corner.
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pone-0038531-g005: ChIP with H2A and H2A.Z in lung cancer cell lines, lung tumor, and normal lung.Results in analyzed nucleosomes are expressed as Percent Input using Ct values. The lung cancer line A2B1 still expresses Cadm1, while A2C12 does not. For quantitative PCR, 20 ng of ChIP DNA was used as template in all samples, including DNA obtained in normal rabbit IgG. The primer sets used and corresponding color coding are indicated on the uppermost right hand corner.

Mentions: On the same ChIP experiment, we carried out quantitative PCR using four primer sets to assay four nucleosomes upstream of TSS using 20 ng of ChIP DNA from A2B1 and A2C12, and a dilution line established from an MNase-digested A2C12 chromatin. Overall, H2A values were greater than H2A.Z; and that A2B1 (H2A > H2A.Z) was lesser than A2C12 (H2A > H2A.Z) (Figure S12C), in agreement with nucleosome depletion associated with gene expression. To confirm results, we performed independent N-ChIP with A2B1 vs. A2C12 and using Ct values as well as Percent Input normalization to interpret results (Figure 5). Altogether, in four independent experiments involving H2A and H2A.Z in A2B1 vs. A2C12, we found that H2A and H2A.Z were higher in A2C12 than in A2B1, a result suggestive of high nucleosome occupancy in transcriptional repression.


Dissecting epigenetic silencing complexity in the mouse lung cancer suppressor gene Cadm1.

Reamon-Buettner SM, Borlak J - PLoS ONE (2012)

ChIP with H2A and H2A.Z in lung cancer cell lines, lung tumor, and normal lung.Results in analyzed nucleosomes are expressed as Percent Input using Ct values. The lung cancer line A2B1 still expresses Cadm1, while A2C12 does not. For quantitative PCR, 20 ng of ChIP DNA was used as template in all samples, including DNA obtained in normal rabbit IgG. The primer sets used and corresponding color coding are indicated on the uppermost right hand corner.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3368868&req=5

pone-0038531-g005: ChIP with H2A and H2A.Z in lung cancer cell lines, lung tumor, and normal lung.Results in analyzed nucleosomes are expressed as Percent Input using Ct values. The lung cancer line A2B1 still expresses Cadm1, while A2C12 does not. For quantitative PCR, 20 ng of ChIP DNA was used as template in all samples, including DNA obtained in normal rabbit IgG. The primer sets used and corresponding color coding are indicated on the uppermost right hand corner.
Mentions: On the same ChIP experiment, we carried out quantitative PCR using four primer sets to assay four nucleosomes upstream of TSS using 20 ng of ChIP DNA from A2B1 and A2C12, and a dilution line established from an MNase-digested A2C12 chromatin. Overall, H2A values were greater than H2A.Z; and that A2B1 (H2A > H2A.Z) was lesser than A2C12 (H2A > H2A.Z) (Figure S12C), in agreement with nucleosome depletion associated with gene expression. To confirm results, we performed independent N-ChIP with A2B1 vs. A2C12 and using Ct values as well as Percent Input normalization to interpret results (Figure 5). Altogether, in four independent experiments involving H2A and H2A.Z in A2B1 vs. A2C12, we found that H2A and H2A.Z were higher in A2C12 than in A2B1, a result suggestive of high nucleosome occupancy in transcriptional repression.

Bottom Line: Yet, the precise mechanisms are still unclear and complex, involving the interplay of several effectors including nucleosome positioning, DNA methylation, histone variants and histone modifications.Chromatin analysis with micrococcal nuclease also indicated variations in nucleosome positioning to have implications in the binding of transcription factors near nucleosome borders.Chromatin immunoprecipitation showed that histone variants (H2A.Z and H3.3), and opposing histone modification marks (H3K4me3 and H3K27me3) all colocalized in the same nucleosome positions that is reminiscent of epigenetic plasticity in embryonic stem cells.

View Article: PubMed Central - PubMed

Affiliation: Toxicology and Environmental Hygiene, Fraunhofer Institute for Toxicology and Experimental Medicine, Hannover, Germany. reamon-buettner@item.fraunhofer.de

ABSTRACT
Disease-oriented functional analysis of epigenetic factors and their regulatory mechanisms in aberrant silencing is a prerequisite for better diagnostics and therapy. Yet, the precise mechanisms are still unclear and complex, involving the interplay of several effectors including nucleosome positioning, DNA methylation, histone variants and histone modifications. We investigated the epigenetic silencing complexity in the tumor suppressor gene Cadm1 in mouse lung cancer progenitor cell lines, exhibiting promoter hypermethylation associated with transcriptional repression, but mostly unresponsive to demethylating drug treatments. After predicting nucleosome positions and transcription factor binding sites along the Cadm1 promoter, we carried out single-molecule mapping with DNA methyltransferase M.SssI, which revealed in silent promoters high nucleosome occupancy and occlusion of transcription factor binding sites. Furthermore, M.SssI maps of promoters varied within and among the different lung cancer cell lines. Chromatin analysis with micrococcal nuclease also indicated variations in nucleosome positioning to have implications in the binding of transcription factors near nucleosome borders. Chromatin immunoprecipitation showed that histone variants (H2A.Z and H3.3), and opposing histone modification marks (H3K4me3 and H3K27me3) all colocalized in the same nucleosome positions that is reminiscent of epigenetic plasticity in embryonic stem cells. Altogether, epigenetic silencing complexity in the promoter region of Cadm1 is not only defined by DNA hypermethylation, but high nucleosome occupancy, altered nucleosome positioning, and 'bivalent' histone modifications, also likely contributed in the transcriptional repression of this gene in the lung cancer cells. Our results will help define therapeutic intervention strategies using epigenetic drugs in lung cancer.

Show MeSH
Related in: MedlinePlus