Previous research seemed to point to the idea that CpG islands–short DNA stretches containing lots of two-nucleotide cytosine-guanosine sequences–were necessary for controlling nearby transcriptional promoters. Methylating a CpG island turns a gene off–shutting down RNA transcription from that site–while demethylating the island turns a gene on. And methylating CpGs in a CpG-poor promoter seemed to have little or no effect.
About half of promoter sequences don’t have nearby CpG islands, which seems a little strange, since many of these promoters control genes that’re important only for specific tissues. After all, methylation is one of the major epigenetic tricks for controlling tissue-specific expression–and some of these special genes have few CpG sites that are indeed methylated, even though the genes have promoters without proper CpG islands nearby.
Having seen throttled gene expression in methylated non-CpG island genes, Han Han and colleagues at the University of Southern California took a closer look at a couple of these gene, LAMB3 and RUNX3. Using bisulfite sequencing, they found that both genes show methylation that appears to be specific for particular tissues–LAMB3 was methylated in sperm, testis, lung, and white blood cells, but not placenta lining, while the RUNX3 P1 promoter region was methylated in most tissues they looked at, except for T-cells. (RUNX3 also has a P2 promoter that is near a CpG island, but it apparently produces a distinct transcript.)
To cells that don’t usually express these genes–HaCaT and 623melanoma cells–the USC researchers added 5-Aza-CdR to inhibit methylation. But while this increased RUNX3 P1 expression in 623melanoma cells, it didn’t work for LAMB3 in HaCaT cells. So they next got clever:
Methylated and unmethylated pCpGL-LAMB3 and pCpGL-RUNX3P1 were transfected into HaCaT cells and 623melanoma cells. In agreement with their endogenous expression status, high luciferase activities were observed when unmethylated promoters, including mock-treated control plasmids, were transfected into cells which are competent to express each gene. In contrast, low luciferase activities were observed when the promoters were methylated, indicating methylation can directly suppress promoter activity.
There’s more, of course, including a corroborating look at nucleosome occupancy upstream of the promoters of each gene, and consistent histone modifications. The results probably aren’t quite enough to be considered conclusive, but I’d most like to hear whether anyone else considers this no-CpG-islands-required finding surprising. Or is it something everyone suspected all along?
(Flickr user Jo@net‘s photo of “James Bond Island” in Phuket, Thailand, used under a Creative Commons license.)
Han H, Cortez CC, Yang X, Nichols PW, Jones PA, & Liang G (2011). DNA methylation directly silences genes with non-CpG island promoters and establishes a nucleosome occupied promoter. Human molecular genetics PMID: 21835883