The human body continually faces a variety of threats, both foreign—pathogens like pollens, bacteria, parasites, and viruses—and “domestic,” such as dysfunctional cells that form tumors. Among the first responders are B-cell antibodies and T-cell receptors, which identify and bind to invaders, marking them for destruction by other components of the immune system.
For these front-line defenders to identify the widest possible variety of targets, three distinct types of segments in their genes—variable (V), diversity (D), and joining (J)—must be shuffled into the largest number of combinations possible. A genetic process called V(D)J recombination is responsible for cutting and rejoining those gene segments, producing an arsenal with maximum diversity.
At the same time, the immune response must also be highly specific, each antibody or receptor matching only one type of target.
However, researchers have long been puzzled by a genetic “distance” issue. The V, D and J segments that code for antibody variation are spread over huge distances on the chromosome. Until now researchers could only speculate as to how V(D)J recombination is guided, or regulated, over long chromosomal distances.
Through an elegant series of experiments in mice, an international research team led by Frederick Alt, the Charles A. Janeway Professor of Pediatrics at Children’s Hospital Boston and Harvard Medical School and president of the Immune Disease Institute, has discovered a regulatory region of the chromosome that fits the bill, creating diversity while maintaining specificity; they have named this region InterGenic Control Region 1 (IGCR1).
These findings are published online September 11 in Nature.
The researchers found that the stretch of the chromosome called IGCR1 guides proper assembly of antibodies; one way is by bending the chromosome into large loops that bring together distant parts of the antibody gene to produce a huge variety of antibodies. In addition, IGCR1 also prevents excessive recombination of a small number of gene segments located near each other, a sort of local “inbreeding” that would greatly limit the diversity of the immune response.
Since the 1980s, the Alt lab has been at the forefront of exploring the mechanisms that regulate V(D)J recombination. They showed that rearrangement follows a specific developmental order: Ds join with Js, then Vs join with DJ complexes. The lab also demonstrated that when one version of an antibody is correctly rearranged within a cell and made into a protein, the other potential version is held back from developing further.
The Alt lab also showed that only one enzyme was responsible for rearranging one set of segments and then the other. This led them to believe that something in the chromosome itself must be in charge. In addition, though specificity is created when the V is joined to the DJ segment, the Vs are located at an enormous chromosomal distance from the DJ segments. For this new study, the Alt group hypothesized that some master regulator must be located between the V and the D segments.
Many labs, including Alt’s, have been interested in the activities of a common protein called CTCF, already known to be active in various aspects of genetic regulation, and the points at which it binds to the chromosome, called CBEs (CTCF binding elements). Many such binding elements exist on the chromosome; the IGCR1 contains two.
Chunguang Guo, a postdoctoral researcher in Alt’s lab who shares first authorship on the new study, deleted the entire IGCR1 region, including the two CBEs it contains, from the V-to-D region, producing mice with clear defects in B cell development, as hypothesized.
However, to make sure that it was deletion of the CBEs and not something else in the region that was responsible for the defects, a more specific experiment was necessary. In another set of mice, Guo carefully mutated only the CBE sites, scrambling their genetic sequence so they no longer bound the CTCF protein. The same dramatic defects were produced in the immune cells of these mice: their developmental antibody gene assembly stages occurred in the wrong order; antibody genes, which are normally assembled only in B cells, were now assembled in T lymphocytes; and cells lost ability to produce just one kind of antibody. Moreover, mice with this mutation produced antibodies of very limited diversity, because primarily a single V region was undergoing rearrangement.
Thus, the Alt team showed that IGCR1, through its binding of the protein CTCF, is required for control of all of the major known regulatory aspects of antibody gene V(D)J recombination.
In the group’s newly proposed model, IGCR1 is represented as a brick wall, blocking premature V-to-D recombination and preventing recombination of neighboring Vs. The model also proposes that loops are created between IGCR1 and other distant CBEs, allowing orderly transcription and recombination of Ds and Js while delaying any recombination among Vs. When the “wall” created by IGCR1 was removed, a few proximal genes dominated recombination, greatly reducing diversity; in addition, cells produced more than one type of antibody, blunting the specificity of the immune response.
Next steps for the Alt lab include deeper explorations of the actions of IGCR1, CTCF, and other binding elements using the mutation techniques they describe in the study. They believe characterization of IGCR1 may become a prototype for studies of long-range genetic regulation across the genome.
This research was funded by the National Institutes of Health.