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.