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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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Related in: MedlinePlus

ChIP with histone variant (H3.3) and histone modifications (H3K4me3, H3K27me3) in lung cancer cells.ChIP experiments were undertaken in a lung cancer cell line with (A2B1) and without (A2C12) Cadm1 gene expression. Results on different nucleosomes are expressed as Percent Input using Ct values. For quantitative PCR, template was adjusted to 20 ng for 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-g006: ChIP with histone variant (H3.3) and histone modifications (H3K4me3, H3K27me3) in lung cancer cells.ChIP experiments were undertaken in a lung cancer cell line with (A2B1) and without (A2C12) Cadm1 gene expression. Results on different nucleosomes are expressed as Percent Input using Ct values. For quantitative PCR, template was adjusted to 20 ng for 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: Besides H2A.Z, we conducted ChIP with the histone variant H3.3, as well as the histone modifications H3K4me3 and H3K27me3, again on A2B1 (with Cadm1 expression) and A2C12 (without Cadm1 expression). ChIP results obtained using Ct values and Percent Input in analyzed nucleosomes in A2B1 vs. A2C12 are shown in Figure 6. For these experiments, H2A served as control for histone integrity. As for A2B1, it showed that enrichment of H3K4me3 and H3K27me3 was not in all nucleosomes. Indeed, the values for these histone modifications in nucleosomes 1 and 3 in A2B1 were higher than in A2C12.


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

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

ChIP with histone variant (H3.3) and histone modifications (H3K4me3, H3K27me3) in lung cancer cells.ChIP experiments were undertaken in a lung cancer cell line with (A2B1) and without (A2C12) Cadm1 gene expression. Results on different nucleosomes are expressed as Percent Input using Ct values. For quantitative PCR, template was adjusted to 20 ng for 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-g006: ChIP with histone variant (H3.3) and histone modifications (H3K4me3, H3K27me3) in lung cancer cells.ChIP experiments were undertaken in a lung cancer cell line with (A2B1) and without (A2C12) Cadm1 gene expression. Results on different nucleosomes are expressed as Percent Input using Ct values. For quantitative PCR, template was adjusted to 20 ng for 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: Besides H2A.Z, we conducted ChIP with the histone variant H3.3, as well as the histone modifications H3K4me3 and H3K27me3, again on A2B1 (with Cadm1 expression) and A2C12 (without Cadm1 expression). ChIP results obtained using Ct values and Percent Input in analyzed nucleosomes in A2B1 vs. A2C12 are shown in Figure 6. For these experiments, H2A served as control for histone integrity. As for A2B1, it showed that enrichment of H3K4me3 and H3K27me3 was not in all nucleosomes. Indeed, the values for these histone modifications in nucleosomes 1 and 3 in A2B1 were higher than in A2C12.

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