static melanomas had a mutation affecting BRAF led to a new targeted therapy (vemurafenib) for this aggressive disease. But genes actually account for only about 2% of the genome. With the increased power of whole genome sequencing technology, the importance of some of the non-coding ‘dark matter’ is coming to light. David Altshuler, MD, PhD (MGH, Broad), helped develop technologies to probe the whole genome for single DNA changes, called single nucleotide polymorphisms or SNPs, that influence hereditary human traits, ranging from height and weight to the risk of developing heart disease, diabetes, or cancer. Surprisingly, most of these risk variants were outside of genes, and many appeared to be in regulatory elements known as promoters and enhancers. Promoters essentially prepare genes for expression, while enhancers are dimmer dials for the level of expression.
How Enhancers Interact With Their Target Genes
Matthew Freedman studies enhancers, which are regulatory elements that often occur a long distance from the gene they regulate. Enhancers and genes are thought to interact when they come into contact because of how the DNA strands loop inside the nucleus. Chromosome Conformation Capture (3C) is a technique that determines which regions of the genome physically interact with each other, and then re-configures the DNA sequence into a format amenable to copying by polymerase chain reaction (PCR) for further study. Credit: Matthew Freedman.
As a postdoctoral researcher, Matthew Freedman, MD (DFCI), worked with Altshuler on whole genome sequencing, and now in his own lab he studies SNPs affecting enhancers. Freedman wants to connect cancer-risk enhancers to the genes they regulate. But whereas promoters can be found adjacent to the gene they regulate, enhancers can occur a long distance from the gene they influence, even on a different chromosome, which complicates the task.
However, enhancers interact with genes by coming into physical contact as DNA strands loop and twist in the nucleus, and a method called Chromosome Conformation Capture (3C) can visualize where a regulatory element touches the DNA loop, providing a clue of where to look in more detail. Freedman also compares the number of RNA transcripts of genes in cells with and without a SNP, looking for correlations that suggest a candidate gene for further study. Applying these methods to prostate and breast cancer, Freedman identified novel genes not previously associated with cancer risk. He hopes such work can provide new genes to target therapeutically, and insights into prevention for those with inherited cancer risks.
A Promoter Linked to Melanoma
Several years ago, Levi Garraway’s team had generated enough whole genome sequences of melanoma that they decided to look for cancer risk mutations in the non-coding regions of the genome. A very simple first pass identified two novel single-DNA changes. One or the other mutation occurred in 17 of the 19 samples they could examine in detail, and they each occurred in the same promoter. “We didn’t believe it at first because the mutations were so prevalent,” recalls Garraway. “We had imagined that we might someday find a non-coding mutation that was as important as a coding mutation, but together these were more prevalent than the BRAF mutation in melanoma.”
But it began to make sense. They were somatic mutations, not hereditary, and they involved a C to T change in DNA base pairs – typical of the ultraviolet light-induced DNA damage that is important to melanoma development. Also, the mutations occurred in a promoter that regulates the TERT gene. TERT produces part of the telomerase enzyme that maintains the ends of chromosomes, called telomeres. Telomeres normally shorten with each cell cycle, eventually causing the cell to undergo apoptosis. By activating telomerase and maintaining these tips, cancer cells become “immortalized” – able to divide indefinitely.
The two mutations were also common in a larger sample set Garraway analyzed later (and in some bladder and liver cancers). But is telomerase dysregulation