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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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DNA methyltransferase-based single-molecule chromatin (MAP-IT) assay of Cadm1 promoter region.(A) Methylation patterns in clones after treatment with CpG-specific DNA methyltransferase (M.SssI) and scoring of 32 CpGs (−271 to +24 CpGs, TSFR1 fragment) in ‘naked’ mouse-tail genomic DNA, and chromatin from nine pooled normal lungs, three pooled solid lung tumors, and seven different lung cancer cell lines with little or no Cadm1 gene expression. The patterns were obtained with BISMA where blue boxes representing unmethylated CpGs ( = protected) while red boxes, methylated CpGs. In lung tumors and lung cancer cell lines, CpG methylation could be endogenous and/or from the M.SssI treatment. (B) Annotation of analyzed Cadm1 promoter region (CpGs, putative binding sites of lung-specific transcription factors, predicted nucleosomes), and the corresponding sequence-context DNA methylation patterns shown in (A). A stretch of protected CpGs especially within the predicted nucleosome 4 was frequent in many of the 84 clones obtained in lung cancer cell lines.
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pone-0038531-g002: DNA methyltransferase-based single-molecule chromatin (MAP-IT) assay of Cadm1 promoter region.(A) Methylation patterns in clones after treatment with CpG-specific DNA methyltransferase (M.SssI) and scoring of 32 CpGs (−271 to +24 CpGs, TSFR1 fragment) in ‘naked’ mouse-tail genomic DNA, and chromatin from nine pooled normal lungs, three pooled solid lung tumors, and seven different lung cancer cell lines with little or no Cadm1 gene expression. The patterns were obtained with BISMA where blue boxes representing unmethylated CpGs ( = protected) while red boxes, methylated CpGs. In lung tumors and lung cancer cell lines, CpG methylation could be endogenous and/or from the M.SssI treatment. (B) Annotation of analyzed Cadm1 promoter region (CpGs, putative binding sites of lung-specific transcription factors, predicted nucleosomes), and the corresponding sequence-context DNA methylation patterns shown in (A). A stretch of protected CpGs especially within the predicted nucleosome 4 was frequent in many of the 84 clones obtained in lung cancer cell lines.

Mentions: Fragment TSFR1 (345 bp, 37 CpGs, −302 +41) is amplified by primers that contain three CpGs on the forward primer, and two CpGs in the reverse primer (Figure 2). Our previous results showed that the methylation index obtained from this fragment correlated highly with transcriptional repression as compared with the other fragments analyzed in the Cadm1 promoter region. It contains binding sites for Sp1, Zf5, and other predicted transcription factors. Included also are the RefSeq transcription start site (TSS), the translation start site, ATG as well as at least two predicted nucleosomes (nuc 4 and nuc 5). There was no long stretch of unmethylated CpGs, except for 3 of 15 clones in which most of the protected CpGs fall within a predicted nucleosome (nuc 4). In those clones without apparent occupancy of nuc 4, a predicted nucleosome (nuc 5) where the RefSeq TSS as well as the ATG sites are located, appeared to be not present as well, consistent of an open chromatin that is associated with transcription. Furthermore, the −224 CpG in the binding site of Sp1 and the −192 CpG in Zf5, were frequently unmethylated, to support their binding in the promoter region of Cadm1.


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

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

DNA methyltransferase-based single-molecule chromatin (MAP-IT) assay of Cadm1 promoter region.(A) Methylation patterns in clones after treatment with CpG-specific DNA methyltransferase (M.SssI) and scoring of 32 CpGs (−271 to +24 CpGs, TSFR1 fragment) in ‘naked’ mouse-tail genomic DNA, and chromatin from nine pooled normal lungs, three pooled solid lung tumors, and seven different lung cancer cell lines with little or no Cadm1 gene expression. The patterns were obtained with BISMA where blue boxes representing unmethylated CpGs ( = protected) while red boxes, methylated CpGs. In lung tumors and lung cancer cell lines, CpG methylation could be endogenous and/or from the M.SssI treatment. (B) Annotation of analyzed Cadm1 promoter region (CpGs, putative binding sites of lung-specific transcription factors, predicted nucleosomes), and the corresponding sequence-context DNA methylation patterns shown in (A). A stretch of protected CpGs especially within the predicted nucleosome 4 was frequent in many of the 84 clones obtained in lung cancer cell lines.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0038531-g002: DNA methyltransferase-based single-molecule chromatin (MAP-IT) assay of Cadm1 promoter region.(A) Methylation patterns in clones after treatment with CpG-specific DNA methyltransferase (M.SssI) and scoring of 32 CpGs (−271 to +24 CpGs, TSFR1 fragment) in ‘naked’ mouse-tail genomic DNA, and chromatin from nine pooled normal lungs, three pooled solid lung tumors, and seven different lung cancer cell lines with little or no Cadm1 gene expression. The patterns were obtained with BISMA where blue boxes representing unmethylated CpGs ( = protected) while red boxes, methylated CpGs. In lung tumors and lung cancer cell lines, CpG methylation could be endogenous and/or from the M.SssI treatment. (B) Annotation of analyzed Cadm1 promoter region (CpGs, putative binding sites of lung-specific transcription factors, predicted nucleosomes), and the corresponding sequence-context DNA methylation patterns shown in (A). A stretch of protected CpGs especially within the predicted nucleosome 4 was frequent in many of the 84 clones obtained in lung cancer cell lines.
Mentions: Fragment TSFR1 (345 bp, 37 CpGs, −302 +41) is amplified by primers that contain three CpGs on the forward primer, and two CpGs in the reverse primer (Figure 2). Our previous results showed that the methylation index obtained from this fragment correlated highly with transcriptional repression as compared with the other fragments analyzed in the Cadm1 promoter region. It contains binding sites for Sp1, Zf5, and other predicted transcription factors. Included also are the RefSeq transcription start site (TSS), the translation start site, ATG as well as at least two predicted nucleosomes (nuc 4 and nuc 5). There was no long stretch of unmethylated CpGs, except for 3 of 15 clones in which most of the protected CpGs fall within a predicted nucleosome (nuc 4). In those clones without apparent occupancy of nuc 4, a predicted nucleosome (nuc 5) where the RefSeq TSS as well as the ATG sites are located, appeared to be not present as well, consistent of an open chromatin that is associated with transcription. Furthermore, the −224 CpG in the binding site of Sp1 and the −192 CpG in Zf5, were frequently unmethylated, to support their binding in the promoter region of Cadm1.

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