Dissecting Epigenetic Silencing Complexity in the Mouse Lung Cancer Suppressor Gene Cadm1

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.

Mechanisms underlying epigenetic and transcriptional heterogeneity in Chinese hamster ovary (CHO) cell lines

“…To determine epigenetic and transcriptional heterogeneity in the PT1-CHO cell lines, we used approaches and protocols, as previously described [ 52 , 53 ]. We predicted nucleosome positions along the promoter sequence (1261-bp) of EEF1A1 using bioinformatic tools such as: (1) nucleosome prediction by genomic sequence ( http://genie.weizmann.ac.il/software/nucleoprediction.html ) [ 54 – 56 ]; (2) NuPoP:Nucleosome Positioning Prediction Engine ( http://nucleosome.stats.northwestern.edu/) [ 57 ]; and (3) The ICM Web ( http://dna.ccs.tulane.edu/icm/ ) [ 58 ].…”

“…We analyzed recombinant mRNA essentially as described previously [ 52 ]. We isolated total RNA from frozen cell pellets with the RNeasy Mini kit using the recommended protocol (Qiagen, Hilden, Germany).…”

“…We performed DNA methylation analysis as previously described [ 52 ]. Briefly, genomic DNA was isolated from frozen cell pellets of PT1-CHO cell lines using Nucleo Spin Tissue (Macherey-Nagel, Düren, Germany).…”

“…The protocols used for the various experiments on chromatin, i.e. chromatin isolation, M.Sss I treatment, micrococcal nuclease (MNase) digestion, CHIP, and FAIRE have been described earlier [ 52 ]. ChIP experiments on histones were done with N-ChIP, which uses native chromatin fragmented by MNase digestion, thus yielding a nucleosome-based resolution.…”

The Function of Histone Modifications in Chronic Obstructive Pulmonary Disease

“…De novo methyltransferases 3A and 3B catalyse the creation of new methyl groups in DNA. In general, gene silencing occurs when CpG islands in promoters become hypermethylated (148,149), whereas active transcription occurs when CpG islands become hypomethylated (13,150). The inactivation of transcription that occurs as a result of changes to DNA methylation has been proposed to be explained by two different mechanisms.…”