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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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Genomic DNA sequence-based bioinformatic predictions of nucleosome positions and transcription factor binding sites along the promoter region of Cadm1.(A) Position of five analyzed nucleosomes (blue rectangles) and binding sites of transcription factors. Nucleosomes are arbitrarily numbered starting from the farthest (e.g. nuc 1) towards the RefSeq transcription start site (TSS) and the translation start site ATG, which are both located at nucleosome 5 (nuc 5). Nucleotide numbering with +1 corresponds to A of the ATG. (B) Predicted nucleosomes and location of CpGs (vertical stripes) and the CpG island along the Cadm1 promoter. (C) Five fragments covering analyzed CpGs in bisulfite-treated genomic DNA and predicted nucleosomes. (D) Possible nucleosome positions from three different algorithms.
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pone-0038531-g001: Genomic DNA sequence-based bioinformatic predictions of nucleosome positions and transcription factor binding sites along the promoter region of Cadm1.(A) Position of five analyzed nucleosomes (blue rectangles) and binding sites of transcription factors. Nucleosomes are arbitrarily numbered starting from the farthest (e.g. nuc 1) towards the RefSeq transcription start site (TSS) and the translation start site ATG, which are both located at nucleosome 5 (nuc 5). Nucleotide numbering with +1 corresponds to A of the ATG. (B) Predicted nucleosomes and location of CpGs (vertical stripes) and the CpG island along the Cadm1 promoter. (C) Five fragments covering analyzed CpGs in bisulfite-treated genomic DNA and predicted nucleosomes. (D) Possible nucleosome positions from three different algorithms.

Mentions: To determine whether CpG methylation could influence nucleosome occupancy leading to epigenetic silencing, as previously shown [30], we used bioinformatic tools to predict nucleosome positions, and to annotate the Cadm1 promoter region. Using a nucleosome positioning prediction based on genomic DNA sequence (Segal, see Materials and Methods), we located at least five possible nucleosome positions approximately 1000 bp towards the transcription start site (TSS) and the translation start site (ATG). The RefSeq TSS (NM_001025600.1) is located at –21 of ATG. The predicted nucleosomes are designated arbitrarily relative to the ATG, as nuc 1 (−1011 to −865), nuc 2 (−697 to −551), nuc 3 (−417 to −271), nuc 4 (−230 to −84) and nuc 5 (−41 to +106). The binding sites of predicted sequence-specific transcription factors lie at the borders or within these nucleosomes, and highly concentrated at the nucleosomes most adjacent to the TSS (Figure 1A). Many CpGs are found inside nucleosomes, especially those within the CpG island in the promoter region of Cadm1 (Figure 1B). The 1000-bp region is covered by five fragments of sizes 124–349 bp we used to analyze CpG methylation in bisulfite-treated genomic DNA, particularly in conjunction with DNA methyltransferase-based single-molecule chromatin (MAP-IT) assay (Figure 1C, Table S1). During the study, other nucleosome positioning algorithms became available (e.g. NuPOP, ICM, see Materials and Methods) and comparison showed overlap in predictions among the three methods used (Figure 1D). Nonetheless, the position of three nucleosomes (designated nuc 1, 3, 4) appeared to be more or less consistent.


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

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

Genomic DNA sequence-based bioinformatic predictions of nucleosome positions and transcription factor binding sites along the promoter region of Cadm1.(A) Position of five analyzed nucleosomes (blue rectangles) and binding sites of transcription factors. Nucleosomes are arbitrarily numbered starting from the farthest (e.g. nuc 1) towards the RefSeq transcription start site (TSS) and the translation start site ATG, which are both located at nucleosome 5 (nuc 5). Nucleotide numbering with +1 corresponds to A of the ATG. (B) Predicted nucleosomes and location of CpGs (vertical stripes) and the CpG island along the Cadm1 promoter. (C) Five fragments covering analyzed CpGs in bisulfite-treated genomic DNA and predicted nucleosomes. (D) Possible nucleosome positions from three different algorithms.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3368868&req=5

pone-0038531-g001: Genomic DNA sequence-based bioinformatic predictions of nucleosome positions and transcription factor binding sites along the promoter region of Cadm1.(A) Position of five analyzed nucleosomes (blue rectangles) and binding sites of transcription factors. Nucleosomes are arbitrarily numbered starting from the farthest (e.g. nuc 1) towards the RefSeq transcription start site (TSS) and the translation start site ATG, which are both located at nucleosome 5 (nuc 5). Nucleotide numbering with +1 corresponds to A of the ATG. (B) Predicted nucleosomes and location of CpGs (vertical stripes) and the CpG island along the Cadm1 promoter. (C) Five fragments covering analyzed CpGs in bisulfite-treated genomic DNA and predicted nucleosomes. (D) Possible nucleosome positions from three different algorithms.
Mentions: To determine whether CpG methylation could influence nucleosome occupancy leading to epigenetic silencing, as previously shown [30], we used bioinformatic tools to predict nucleosome positions, and to annotate the Cadm1 promoter region. Using a nucleosome positioning prediction based on genomic DNA sequence (Segal, see Materials and Methods), we located at least five possible nucleosome positions approximately 1000 bp towards the transcription start site (TSS) and the translation start site (ATG). The RefSeq TSS (NM_001025600.1) is located at –21 of ATG. The predicted nucleosomes are designated arbitrarily relative to the ATG, as nuc 1 (−1011 to −865), nuc 2 (−697 to −551), nuc 3 (−417 to −271), nuc 4 (−230 to −84) and nuc 5 (−41 to +106). The binding sites of predicted sequence-specific transcription factors lie at the borders or within these nucleosomes, and highly concentrated at the nucleosomes most adjacent to the TSS (Figure 1A). Many CpGs are found inside nucleosomes, especially those within the CpG island in the promoter region of Cadm1 (Figure 1B). The 1000-bp region is covered by five fragments of sizes 124–349 bp we used to analyze CpG methylation in bisulfite-treated genomic DNA, particularly in conjunction with DNA methyltransferase-based single-molecule chromatin (MAP-IT) assay (Figure 1C, Table S1). During the study, other nucleosome positioning algorithms became available (e.g. NuPOP, ICM, see Materials and Methods) and comparison showed overlap in predictions among the three methods used (Figure 1D). Nonetheless, the position of three nucleosomes (designated nuc 1, 3, 4) appeared to be more or less consistent.

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