August 31, 2007

Remodeling proteins drive immune system gene shuffle

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Robin Cobb, Ph.D., and Oleg Osipovich, Ph.D., are on the team studying how regions of DNA are opened or closed. (photo by Neil Brake)

Remodeling proteins drive immune system gene shuffle

DNA is something of a celebrity molecule – access to it is tightly controlled. Cells keep the genetic code under wraps by winding it around histone proteins and compacting the whole mix, called chromatin, into the nucleus.

This densely packed chromatin presents a challenge to the cell: it must make the DNA available to factors that turn genes on or off, or that recombine gene segments.

Eugene Oltz, Ph.D., professor of Microbiology & Immunology, and colleagues are studying chromatin accessibility – how regions of DNA are opened or closed – in a particular gene locus in immune system cells. They report in the August issue of Nature Immunology that a bit of DNA called a promoter attracts a chromatin-remodeling complex, and that this combo is required for appropriate gene recombination.

Gene recombination, or rearrangement, in the immune system is critical for producing the “antigen receptor” molecules that protect us against a universe of pathogens. Within each developing B and T cell are “enormous collections of gene pieces,” Oltz said. Each B and T cell randomly selects three pieces from these collections and fuses them in the genome. Think of picking one card from each of three large decks to produce a unique combination of cards, Oltz explained.

“Each cell, because of its random selection will have a binding specificity for foreign molecules that's different from the next cell,” Oltz said, “and that way we can create billions of different antibodies and T cell receptors so that we can be ready for almost any of the pathogens Mother Nature throws at us.”

If this “V(D)J recombination” process doesn't work at all, it is not possible to generate mature B and T cells, resulting in severe immunodeficiencies – the boy in the plastic bubble syndrome. And if the recombination process doesn't work correctly, it can introduce genetic mistakes called chromosomal translocations that are at the heart of almost all leukemias and lymphomas, Oltz noted.

“We need to properly target these recombination events, and our current findings suggest that chromatin remodelers are an integral part of the targeting mechanism,” he said.

Chromatin remodelers are sets of proteins that use cellular energy to slide histone proteins along the DNA, in order to open genetic regions and make them accessible to other factors.

Oleg Osipovich, Ph.D., research instructor, and Robin Cobb, Ph.D., postdoctoral fellow, demonstrated that in the T cell receptor-beta gene locus – one of the antigen receptors – a certain promoter region of the DNA attracts a chromatin-remodeling complex to the site.

To test the role of the chromatin-remodeling complex, they tethered it to the gene locus in an engineered system missing the promoter and found that recombination still occurred. In developing T cells they showed that the remodeling proteins bind next to the gene segments awaiting recombination and that removal of the promoter sequence causes loss of remodeling protein binding.

In the “clinching experiment,” the investigators showed that knocking down expression of a critical component of the remodeling complex in developing T cells blocked recombination in the gene locus.

The study is the first to demonstrate that chromatin-remodeling complexes participate in driving rearrangement of a real locus in a real cell and that promoter regions are critical for the process, Oltz said.

Because promoter regions are also involved in regulating gene expression, the findings suggest that the two processes – gene expression and gene recombination – may use similar mechanisms. In future studies, the investigators plan to probe how the promoter attracts chromatin remodelers and what other proteins are involved in docking the complex.

The current findings add a chapter to a growing story.

“We're excited by the forward progression of our studies,” Oltz said. “We're getting down to the nuts and bolts of how this recombination process works.”

“It's like Harry Potter…we're all waiting for the next book,” Cobb added with a laugh.

The research was supported by the National Institutes of Health and the Vanderbilt-Ingram Cancer Center. Cobb is a trainee of the Cellular and Molecular Microbiology Training Program.