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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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M.SssI maps in normal lung, and a lung cancer cell line (A2C12) with no Cadm1 gene expression, before and after treatment with 5-aza-dC.(A) Location of the five fragments analyzed in the Cadm1 promoter region that cover 69 CpGs −944 to +41, relative to the translation start site, ATG. CpGs are represented by stripes. (B-D) Methylation maps of normal lung and A2C12; blue boxes represents unmethylated CpGs ( = protected) while red boxes, methylated CpGs. The fragments are presented with respect to their location i.e. from BFR to TSFR1. The CpGs in the core sequence of Sp1 and Zf5 binding sites are indicated by arrows. (D) After 5-aza-dC treatment and slight gene re-expression, some clones resemble patterns found in normal lung (e.g. in TFSR1, enclosed), to suggest nucleosome remodeling (eviction) in gene expression.
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pone-0038531-g003: M.SssI maps in normal lung, and a lung cancer cell line (A2C12) with no Cadm1 gene expression, before and after treatment with 5-aza-dC.(A) Location of the five fragments analyzed in the Cadm1 promoter region that cover 69 CpGs −944 to +41, relative to the translation start site, ATG. CpGs are represented by stripes. (B-D) Methylation maps of normal lung and A2C12; blue boxes represents unmethylated CpGs ( = protected) while red boxes, methylated CpGs. The fragments are presented with respect to their location i.e. from BFR to TSFR1. The CpGs in the core sequence of Sp1 and Zf5 binding sites are indicated by arrows. (D) After 5-aza-dC treatment and slight gene re-expression, some clones resemble patterns found in normal lung (e.g. in TFSR1, enclosed), to suggest nucleosome remodeling (eviction) in gene expression.

Mentions: Further to mapping with M.SssI, we conducted chromatin analysis with micrococcal nuclease (MNase), an enzyme that preferentially cuts within nucleosomal linker regions and therefore useful in determining nucleosome positions [34]. MNase digestion of native chromatin in different samples resulted mainly in fragments of about 150–200 bp (mononucleosomes), but not in control ‘naked’ genomic DNA. Mononucleosomes were gel-isolated and interrogated by normal- and quantitative-PCR using a panel of PCR primers (see Table S2) amplifying within or at the left or right boundaries of predicted nucleosomes (Figure 4, Figures S9, S10). The PCR products obtained with ‘middle’ (65–113 bp), ‘left’ (132–168 bp), and ‘right’ (93–222 bp) primers were verified by sequencing. The ‘naked’ genomic DNA was completely digested and no expected PCR products were obtained (FigureS9).


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

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

M.SssI maps in normal lung, and a lung cancer cell line (A2C12) with no Cadm1 gene expression, before and after treatment with 5-aza-dC.(A) Location of the five fragments analyzed in the Cadm1 promoter region that cover 69 CpGs −944 to +41, relative to the translation start site, ATG. CpGs are represented by stripes. (B-D) Methylation maps of normal lung and A2C12; blue boxes represents unmethylated CpGs ( = protected) while red boxes, methylated CpGs. The fragments are presented with respect to their location i.e. from BFR to TSFR1. The CpGs in the core sequence of Sp1 and Zf5 binding sites are indicated by arrows. (D) After 5-aza-dC treatment and slight gene re-expression, some clones resemble patterns found in normal lung (e.g. in TFSR1, enclosed), to suggest nucleosome remodeling (eviction) in gene expression.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0038531-g003: M.SssI maps in normal lung, and a lung cancer cell line (A2C12) with no Cadm1 gene expression, before and after treatment with 5-aza-dC.(A) Location of the five fragments analyzed in the Cadm1 promoter region that cover 69 CpGs −944 to +41, relative to the translation start site, ATG. CpGs are represented by stripes. (B-D) Methylation maps of normal lung and A2C12; blue boxes represents unmethylated CpGs ( = protected) while red boxes, methylated CpGs. The fragments are presented with respect to their location i.e. from BFR to TSFR1. The CpGs in the core sequence of Sp1 and Zf5 binding sites are indicated by arrows. (D) After 5-aza-dC treatment and slight gene re-expression, some clones resemble patterns found in normal lung (e.g. in TFSR1, enclosed), to suggest nucleosome remodeling (eviction) in gene expression.
Mentions: Further to mapping with M.SssI, we conducted chromatin analysis with micrococcal nuclease (MNase), an enzyme that preferentially cuts within nucleosomal linker regions and therefore useful in determining nucleosome positions [34]. MNase digestion of native chromatin in different samples resulted mainly in fragments of about 150–200 bp (mononucleosomes), but not in control ‘naked’ genomic DNA. Mononucleosomes were gel-isolated and interrogated by normal- and quantitative-PCR using a panel of PCR primers (see Table S2) amplifying within or at the left or right boundaries of predicted nucleosomes (Figure 4, Figures S9, S10). The PCR products obtained with ‘middle’ (65–113 bp), ‘left’ (132–168 bp), and ‘right’ (93–222 bp) primers were verified by sequencing. The ‘naked’ genomic DNA was completely digested and no expected PCR products were obtained (FigureS9).

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