Despite the fact that 45% of all human gene promoters do not contain CpG 
islands, the role of DNA methylation in control of non-CpG island promoters is 
controversial and its relevance in normal and pathological processes is poorly 
understood. Among the few studies which investigate the correlation between DNA 
methylation and expression of genes with non-CpG island promoters, the majority 
do not support the view that DNA methylation directly leads to transcription 
silencing of these genes. Our reporter assays and gene reactivation by 
5-aza-2'-deoxycytidine, a DNA demethylating agent, show that DNA methylation 
occurring at CpG poor LAMB3 promoter and RUNX3 promoter 1(RUNX3 P1) can directly 
lead to transcriptional silencing in cells competent to express these genes in 
vitro. Using Nucleosome Occupancy Methylome- Sequencing, NOMe-Seq, a 
single-molecule, high-resolution nucleosome positioning assay, we demonstrate 
that active, but not inactive, non-CpG island promoters display a 
nucleosome-depleted region (NDR) immediately upstream of the transcription start 
site (TSS). Furthermore, using NOMe-Seq and clonal analysis, we show that in 
RUNX3 expressing 623 melanoma cells, RUNX3 P1 has two distinct chromatin 
configurations: one is unmethylated with an NDR upstream of the TSS; another is 
methylated and nucleosome occupied, indicating that RUNX3 P1 is monoallelically 
methylated. Together, these results demonstrate that the epigenetic signatures 
comprising DNA methylation, histone marks and nucleosome occupancy of non-CpG 
island promoters are almost identical to CpG island promoters, suggesting that 
aberrant methylation patterns of non-CpG island promoters may also contribute to 
tumorigenesis and should therefore be included in analyses of cancer 
epigenetics.