UM Researchers Revolutionize the Process of Identifying Individual Gene Boundaries & Their Switches
Mathias G. Lichtenheld, M.D., Director of the Interdisciplinary Biomedical Studies Program at the University of Miami Leonard M. Miller School of Medicine, and his former student, Matthew E. Pipkin, Ph.D., were working on perforin, a molecule that plays a critical role in the defeat of viral infections and tumors.
Researchers have long known that genes are activated by promoters near the structural part of the gene. Promoters govern the flow of genetic information, encoded by DNA, into the actual building blocks of cells that determine their function. However, promoters and the structural aspects of genes only account for about two percent of all DNA contained on our chromosomes. Think of the promoter and gene as the house in the center of a large field. Currently most “house” areas of genes are well understood, but the large area surrounding each house is poorly understood, although it contains information that is critical for the proper function of genes. “Ninety-eight percent of the chromosome is unstructured, but it’s the key to getting electricity to the house,” Lichtenheld said.
Lichtenheld originally found with small experimental portions of the perforin gene and promoter that, even when the promoter should have been switched on, it remained off. At that time in 2001, when Pipkin began his Ph.D. studies, there wasn’t an efficient way to examine large regions of our chromosomes to see where additional switches other than the promoter may lie. Now there is. “In this one piece of work we were able to deduce everything,” said Lichtenheld, who is also an associate professor in the Department of Microbiology and Immunology and at the University of Miami Sylvester Comprehensive Cancer Center.
“I said, listen, the way that these experiments have been done is just crazy,” recalled Lichtenheld. “Typically you are able to look at a maximum of 10,000 base pairs of the gene, and we’ve just got to improve this to be able to look at 100,000 base pairs.” They improved on existing techniques, combining them all in a creative new way to develop the Mega-DNaseI hypersensitivity analysis, or MDHA, which they first reported in 2006.
Pipkin and Lichtenheld began the work with one major goal in mind. They wanted to be able to see all the switches controlling the gene they were studying, human perforin – not just a tiny portion of it but exactly where the overall territory of one gene ended and where its neighbor began in a chromosome. “After about three months Matthew had the key ingredient for how to do it,” Lichtenheld said. “Then it took much, much longer to really apply it and do all the fine tuning of the method.” The next step was to test the work.
The perforin gene is active in killer lymphocytes in the blood and plays a role in rejecting tumors and fighting viral infections, but it also exists in all other types of cells, where it is normally turned off. For example, fibroblasts are connective-tissue cells found under the skin and do not express perforin – the perforin gene is turned off in these cells. Yet Pipkin found that the genes on either side of perforin were active in fibroblasts. “So you would imagine that there had to be a fence between the three genes because if the two on the ends were on why wouldn’t the one in the middle be on,” said Pipkin.
Using their new method, and the idea of the two fences as guides, Pipkin and Lichtenheld sought to identify the entire territory encompassing the perforin gene that is responsible for perforin being turned on in killer lymphocytes and turned off in fibroblasts. To visualize this, they lifted out the human chromosome that includes the perforin gene from fibroblasts, and then they stuck it into killer lymphocytes. The perforin gene turned on in the killer lymphocytes. “Not only did it become active but it was now active at biological levels,” said Pipkin. By moving the chromosome into the killer lymphocytes, they switched on a formerly “silent” gene, and then mapped the territory that was “reprogrammed” between the fences. Then they did it six more times with the same successful result.
To make sure they really understood what was happening, they also snipped out one of the fences from the territory and implanted the remaining portion into the neighborhood of other genes, on different chromosomes. The section without the fence that they transplanted instantly behaved like the surrounding genes rather than the way the perforin gene should behave. “But every time before, when we had the fences on there, we could put that piece of DNA [the perforin territory] in the desert and it could function as perforin, even in the desert,” Lichtenheld said. “It functioned perfectly.”
The work immediately garnered attention from colleagues. “They saw this as something that would be powerful for finding long-range regulatory regions and realized they would find them by applying our strategy,” said Pipkin. “It gives anyone who works on any gene in any lab anywhere the confidence to say, ‘I want to find everything for that gene,’ and they can put their effort toward that, knowing that in the end they’re going to figure it out. This gives them those tools.”
It also throws open the door to new therapeutic targets. As an example, human perforin plays an important role against viruses and cancer, and an adverse role in transplantation, contributing to organ rejection. Being able to study the entire gene instead of just two percent of it offers researchers many more opportunities to find or develop drugs to switch killer lymphocytes on or off, as needed.
For this work Pipkin earned his Ph.D. at the UM Miller School of Medicine and is now a postdoctoral fellow at the CBR Institute for Biomedical Research at Harvard University. He remains in touch with his former mentor and their groundbreaking work continues in two labs. “There can be dozens of switches that can be spread over hundreds of thousands of base pairs of DNA,” said Lichtenheld. “It’s very, very difficult to get a grasp on them, but they are the ones that really control whether you’re male or female or whether your eyes are blue or brown. It’s not just one switch, it’s a combination of switches. It’s like a mathematical program.” But now there is a formula to solve it. After January 11, the study will be available online at http://www.immunity.com/content/article/abstract?uid=PIIS1074761306005644.
UM/Sylvester opened in 1992 to provide comprehensive cancer services and today serves as the hub for cancer-related research, diagnosis, and treatment at the University of Miami Leonard M. Miller School of Medicine. UM/Sylvester handles 1,400 inpatient admissions annually, performs 3,000 surgical procedures, and treats 3,000 new cancer patients. All UM/Sylvester physicians are on the faculty of the Miller School of Medicine, South Florida’s only academic medical center. In addition, UM/Sylvester physicians and scientists are engaged in 200 clinical trials and receive more than $31 million annually in research grants. UM/Sylvester at Deerfield Beach opened in 2003 to better meet the needs of residents of Broward and Palm Beach Counties. This 10,000 square-foot facility at I-95 and S.W. 10th Street offers appointments with physicians from six cancer specialties, complementary therapies from the Courtelis Center, and education and outreach events.