Differentially expressed genes are marked by histone 3 lysine 9 trimethylation in human cancer cells

Histone H3 lysine 9 trimethylation (H3K9Me3) has been associated with transcriptional repression, but recent findings implicate this chromatin modification in transcriptional activation and mRNA elongation by RNA polymerase II. Here, we applied immunoprecipitation (IP) with a custom DNA tiling microarray containing many transcription factors important in development and cancer (for example homeotic genes; N=683 total genes) to explore the relationship between H3K9Me3 and other histone modifications with the differential expression of genes. Cancer cell lines derived from different tissues (2 leukemia, 2 medulloblastoma) were characterized with IP antibodies to H3K9Me3, H3K4 dimethylation (H3K4Me2) and H3K9 acetylation (H3K9Ac). MV4-11 is known to overexpress the HOXA9 and MEIS1 genes, whereas D283 overexpresses the OTX2 homeobox gene. Gene expression was assessed by Affymetrix U133 array. Mapping the number and size of histone markings demonstrated significant colocalization of H3K9Ac and H3K4Me2 with H3K9Me3, indicating a pattern of putative 'activating' and 'repressive' markings. The median site size was 600-821 bp and 72-95% or 53-80% of chromatin signal sites were located within 1 kb or 500 bp of transcription start sites (TSS), respectively. A relatively small number of genes displayed additional H3K9Me3 sites in the 5'-region distant from the TSS. Comparing genes with modification sites to those without sites in their promoters confirmed the positive associations of H3K9Ac and H3K4Me2 with gene expression and revealed that H3K9Me3 is associated with active genes rather than being a repressive marking as previously thought. The positive regulatory effect of all three types of modifications were quantitatively correlated with site size, and applied to absolute gene expression within a single cell line as well as relative expression among pairs of cell lines. Extended patterns of H3K9Me3 upstream of some genes (for example HOXA9 and OTX2) may result from the action of multiple promoter elements. We found an inverse relationship between promoter DNA hypermethylation and H3K9Me3 in three studied genes (HOXA9, TMS1, RASSF1A). The localization of H3K9Me3 downstream of the TSSs of expressed genes and not within promoter regions of hypermethylated and silenced genes is consistent with the proposed coupling of H3K9Me3 with RNA polymerase II. Our results indicate a need for revising aspects of the histone code involving H3 lysine methylation. Awareness of H3K9Me3 as a mark of gene activity, not repression, is especially important for the classification of human cancer using chromatin and histone profiles.