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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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Chromatin status in repressed promoter of Cadm1 in lung cancer cells.(A) Annotations of analyzed promoter region of Cadm1, showing location of CpGs, transcription factor binding sites, predicted nucleosome positions with different algorithms, and primers used in amplifying different fragments. Enclosed are positions of fragments analyzed by qPCR. (B) Comparison of total ChIPed DNA from different experiments with a canonical histone (H2A), histone variants (H3.3, H2A.Z) and histone modifications (H3K4me3, H3K27me3), in a lung cancer cell line with (A2B1) and without (A2C12) Cadm1 gene expression. ChIP results are expressed as Percent Input using Ct values (left panel) and as further normalized relative to H2A (right panel). Normalization relative to H2A was undertaken at the level of nucleosomes in each experiment. (C) Amplified fragments in three different lung cancer cell lines after FAIRE method, i.e. formaldehyde crosslinking of chromatin and recovery of DNA fragments not bound by protein in the aqueous phase (boxed). The recovered DNA in the corresponding organic phase is also shown. In the aqueous phase (=open chromatin), banding intensity of amplified fragments in the cell line with Cadm1 gene expression (A2B1) was higher than in the two cell lines without gene expression (GA7, A2C12). In the organic phase (=bound chromatin), fragments were only amplified in A2C12. Both positive control (A2C12 genomic DNA) and negative control (No DNA) for PCR are included during the analysis.
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pone-0038531-g007: Chromatin status in repressed promoter of Cadm1 in lung cancer cells.(A) Annotations of analyzed promoter region of Cadm1, showing location of CpGs, transcription factor binding sites, predicted nucleosome positions with different algorithms, and primers used in amplifying different fragments. Enclosed are positions of fragments analyzed by qPCR. (B) Comparison of total ChIPed DNA from different experiments with a canonical histone (H2A), histone variants (H3.3, H2A.Z) and histone modifications (H3K4me3, H3K27me3), in a lung cancer cell line with (A2B1) and without (A2C12) Cadm1 gene expression. ChIP results are expressed as Percent Input using Ct values (left panel) and as further normalized relative to H2A (right panel). Normalization relative to H2A was undertaken at the level of nucleosomes in each experiment. (C) Amplified fragments in three different lung cancer cell lines after FAIRE method, i.e. formaldehyde crosslinking of chromatin and recovery of DNA fragments not bound by protein in the aqueous phase (boxed). The recovered DNA in the corresponding organic phase is also shown. In the aqueous phase (=open chromatin), banding intensity of amplified fragments in the cell line with Cadm1 gene expression (A2B1) was higher than in the two cell lines without gene expression (GA7, A2C12). In the organic phase (=bound chromatin), fragments were only amplified in A2C12. Both positive control (A2C12 genomic DNA) and negative control (No DNA) for PCR are included during the analysis.

Mentions: A summary of results from different ChIP experiments showed overall enrichments of histone variants and histone modifications of nucleosomes along the Cadm1 promoter region in lung cancer cells in (Figure 7B, left panel). The corresponding results on different nucleosomes are shown in Figure S15. To determine enrichment relative to nucleosome density, we further normalized results relative to the canonical H2A, which was included in each ChIP experiment with the histone variants and modifications. This normalization method is assumed to correct for differences in ChIP signals that are caused by differences in the density of nucleosomes, rather than by changes in histone modification levels [36]. Furthermore, the rationale behind normalization relative to nucleosome density is that histone modifications can only be detected at a specific DNA sequence region if this region is also wrapped into nucleosomes. We found that A2C12 exhibited higher enrichments with respect to histone variants (H2A.Z, H3.3) and histone modifications (H3K4me3 and H3K27me3) than A2B1 (Figure 7B, right panel).


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

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

Chromatin status in repressed promoter of Cadm1 in lung cancer cells.(A) Annotations of analyzed promoter region of Cadm1, showing location of CpGs, transcription factor binding sites, predicted nucleosome positions with different algorithms, and primers used in amplifying different fragments. Enclosed are positions of fragments analyzed by qPCR. (B) Comparison of total ChIPed DNA from different experiments with a canonical histone (H2A), histone variants (H3.3, H2A.Z) and histone modifications (H3K4me3, H3K27me3), in a lung cancer cell line with (A2B1) and without (A2C12) Cadm1 gene expression. ChIP results are expressed as Percent Input using Ct values (left panel) and as further normalized relative to H2A (right panel). Normalization relative to H2A was undertaken at the level of nucleosomes in each experiment. (C) Amplified fragments in three different lung cancer cell lines after FAIRE method, i.e. formaldehyde crosslinking of chromatin and recovery of DNA fragments not bound by protein in the aqueous phase (boxed). The recovered DNA in the corresponding organic phase is also shown. In the aqueous phase (=open chromatin), banding intensity of amplified fragments in the cell line with Cadm1 gene expression (A2B1) was higher than in the two cell lines without gene expression (GA7, A2C12). In the organic phase (=bound chromatin), fragments were only amplified in A2C12. Both positive control (A2C12 genomic DNA) and negative control (No DNA) for PCR are included during the analysis.
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Related In: Results  -  Collection

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pone-0038531-g007: Chromatin status in repressed promoter of Cadm1 in lung cancer cells.(A) Annotations of analyzed promoter region of Cadm1, showing location of CpGs, transcription factor binding sites, predicted nucleosome positions with different algorithms, and primers used in amplifying different fragments. Enclosed are positions of fragments analyzed by qPCR. (B) Comparison of total ChIPed DNA from different experiments with a canonical histone (H2A), histone variants (H3.3, H2A.Z) and histone modifications (H3K4me3, H3K27me3), in a lung cancer cell line with (A2B1) and without (A2C12) Cadm1 gene expression. ChIP results are expressed as Percent Input using Ct values (left panel) and as further normalized relative to H2A (right panel). Normalization relative to H2A was undertaken at the level of nucleosomes in each experiment. (C) Amplified fragments in three different lung cancer cell lines after FAIRE method, i.e. formaldehyde crosslinking of chromatin and recovery of DNA fragments not bound by protein in the aqueous phase (boxed). The recovered DNA in the corresponding organic phase is also shown. In the aqueous phase (=open chromatin), banding intensity of amplified fragments in the cell line with Cadm1 gene expression (A2B1) was higher than in the two cell lines without gene expression (GA7, A2C12). In the organic phase (=bound chromatin), fragments were only amplified in A2C12. Both positive control (A2C12 genomic DNA) and negative control (No DNA) for PCR are included during the analysis.
Mentions: A summary of results from different ChIP experiments showed overall enrichments of histone variants and histone modifications of nucleosomes along the Cadm1 promoter region in lung cancer cells in (Figure 7B, left panel). The corresponding results on different nucleosomes are shown in Figure S15. To determine enrichment relative to nucleosome density, we further normalized results relative to the canonical H2A, which was included in each ChIP experiment with the histone variants and modifications. This normalization method is assumed to correct for differences in ChIP signals that are caused by differences in the density of nucleosomes, rather than by changes in histone modification levels [36]. Furthermore, the rationale behind normalization relative to nucleosome density is that histone modifications can only be detected at a specific DNA sequence region if this region is also wrapped into nucleosomes. We found that A2C12 exhibited higher enrichments with respect to histone variants (H2A.Z, H3.3) and histone modifications (H3K4me3 and H3K27me3) than A2B1 (Figure 7B, right panel).

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