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January 8, 1999 GENETICS Building Proper Brain May Depend on DNA Cutting and PastingImmunologist's Discovery Could Lead to New Understanding of Nervous System Development Researchers at the Howard Hughes Medical Institute at Children's Hospital, the Center for Blood Research (CBR), HMS, and other institutions have made a discovery that could help solve one of the central riddles of biology--how the brain, with its dazzling display of cell types, develops from a relatively undistinguished pool of progenitor cells. For years, immunologists have known that the immune system, with its multitude of T cell receptors and antibodies, is produced not by a finite set of pre-existing genes but through a cutting and pasting of DNA fragments. This process, V(D)J recombination, is carried out by a set of proteins that essentially snip genes into bits and then join the broken DNA ends, forming a nearly infinite supply of new genes. It now appears that two of these "paste" proteins are also required during a specific period in brain development--when progenitor cells are developing into different kinds of neurons. The discovery by Yijie Gao and Fred Alt, of HMS, the CBR, and the Howard Hughes Medical Institute at Children's, and their colleagues appears in the December 23 Cell.
"The exciting possibility is that there could be some program--gene rearrangement or something equivalent to that--that's occurring at a very specific time in brain development," says Alt, who is a professor of genetics and the Charles A. Janeway profesor of pediatrics. "It may not be as complicated as putting whole sets of genes together, as in the immune system. But one could imagine simpler things like molecular switches--DNA segments that are flipped or deleted to turn genes on and off in an irreversible fashion--which in turn could result in differentiation." Brain Cells Die To arrive at their findings, Gao, who is a research fellow in genetics, generated mice lacking the gene for the paste protein XRCC4. A mouse lacking another paste protein, DNA ligase IV, was made by Karen Frank and JoAnn Sekiguchi, clinical fellow in pathology and research fellow in genetics, respectively, at Children's Hospital. In addition to severe immunological deficiencies and increased radiation sensitivity, both strains of mice displayed holes in regions of their brains, signifying areas of cell death. The combined defects were so serious that the mice died before birth. It is the first time that mice lacking a V(D)J protein have been observed to die prenatally.
What was especially striking about the observations, which were made in collaboration with Children's Hospital colleagues Michael Greenberg, professor of neurology, and Stuart Orkin, Howard Hughes investigator and the Leland Fikes professor of pediatric medicine, is that the damage to the mutant mouse brains occurred during a very specific stage in neural development. "It's a very narrow window," Alt says. Another surprise was that the brain cells died not during division, when chromosomes stretch out and become vulnerable, but just afterwards. "The onset of cell death is very tightly linked to the period when dividing progenitors are differentiating into neurons. That makes it potentially very exciting," Alt says. Although he is not certain what is killing the brain cells, he believes it is "very likely" that they are dying because they have DNA breaks that they are not mending. "The question then is why do they have the DNA breaks during this specific period?" Alt says. "The possibility arises that there's some specific process at that time. That's very exciting to us because we know that process is either happening just before the neural progenitors begin differentiating or it's happening just after. So that means we know where to look for this event." The Function of DNA Breaks Alt and his colleagues are developing assays to see if genes expressed during differentiation require breaks for their expression. The first step will be to see if genes expressed during or just after the critical period in normal brains are lacking in the brains of mice without XRCC4 or ligase IV. "I suspect we'll find things. The next step would be to see if these genes in normal mice are associated with DNA breaks," Alt says. He points out that the DNA breaks occurring in V(D)J recombination in the immune system do not occur randomly but, instead, at specific sites on the chromosome. Presumably, cut and repair proteins in the brain could work to create flips and deletions with the same precision. In the immune system, cut and pasted genes code for T cell receptors and antibodies. What might break-dependent genes in the brain be expressing? "I don't know," Alt says. "It could be a receptor or some other protein that ultimately leads to expression of a surface receptor." So far, total deletions of XRCC4 and ligase IV have not shown up in humans. Presumably, such mutations would be embryonically lethal, Alt says, "but it is possible that more subtle defects in the genes could show up." Defects in other V(D)J genes have been found in people with severe immunodeficiency disease. "Maybe now we will evaluate these people for neurological manifestations." Alt believes it might be possible to correct these defects with gene therapy. "But that's way down the road," he says. The research was supported in part by the National Institutes of Health.
--Misia Landau |
Alt
and his colleagues subsequently developed two mouse strains--one
lacking Ku70 (the partner protein of Ku80) and the other the
DNA-PKcs gene. Both displayed immunodeficiencies and increased
radiation sensitivity, but they did not die. The finding that
mice lacking either DNA ligase IV or XRCC4 die before birth
came as a surprise. Alt believes one possible explanation
for the difference is that XRCC4 and DNA ligase IV may play
a more needed role in joining reactions, such as those in
V(D)J recombination, than the roles played by other paste
proteins (see figure). He speculates that Ku80 and its partner
Ku70 may work early, spotting broken DNA ends created by the
cutting of DNA, and possibly attracting DNA-PKcs to the scene.
DNA-PKcs may then help prepare the broken ends. XRCC4 and
DNA ligase IV may play the critical task of gluing back together
the cut DNA fragments.