Dr. Crabtree is also a professor of pathology and of developmental biology at Stanford University School of Medicine.
ATP-Dependent Chromatin Remodeling in Development and Disease
Growing up on a small West Virginian farm in Potrock Hollow, population 13, Gerald Crabtree was in contact with the everyday realities of biology, chemistry, and physics. When he wasn't in his basement chemistry lab making explosives for fun, he was feeding chickens, plowing fields, or trying to understand how the farm equipment worked. By high school, Crabtree had read his older brother's college chemistry texts, and his love of rockets and explosives piqued his interest in the underlying chemical and physical principles. But to understand the laws of nature, he had to learn some mathematics. He became primed for that task by helping his father solve Martin Gardner's Puzzlers in Scientific American. His ninth-grade math teacher was also a puzzle fan, and she recognized Crabtree's talent. "Although my interest in chemistry and math were well developed by this time, I hesitated before enrolling in a nearby college," Crabtree recalls. "I thought it would be more fun to join my father, who designed and built houses."
Crabtree entered West Liberty State College in West Virginia to study chemistry and mathematics. Finishing his degree before the end of the draft for the Vietnam War, he had to decide whether to become a conscientious objector, flee to Canada, or apply to medical school. At the last minute, he entered Temple University Medical School, which he enjoyed far more than he expected. While there, he developed an appreciation for the complexities of medicine and the impression that few human diseases can be effectively treated. So as soon as he finished, he rushed back to the laboratory, where he has remained ever since.
His research career began at Dartmouth College, where he worked with Allan Munck on the biochemistry of steroid hormone action. During a subsequent two-year stint as a senior investigator at the National Institutes of Health, Crabtree began to study gene regulation, using primitive bioinformatics to discover a DNA-binding protein, hepatic nuclear factor 1, and to attempt large-scale genomics. He also became interested in how signals are transmitted to the nucleus to bring about changes in the phenotypes of cells. Therefore, when he set up his lab at Stanford University in 1985, he decided to focus on interactions between cells and their environments. His personal environment is eight acres in California's Santa Cruz Mountains, where new ideas about signaling pathways bubble to the surface during his daily walks.
Choosing the early activation genes in T lymphocytes as a target for signals from the cell membrane, Crabtree discovered a transcription complex, nuclear factor of activated T cells (NFAT), that bound to the regulatory regions of those genes and coordinated their activation. In the late 1980s, he found that cyclosporine A, a drug used to prevent transplant rejection, blocked the assembly of this complex. A few years earlier, cyclosporine had revolutionized transplant therapy, but no one knew how it worked. Crabtree's observation that it immediately inhibited NFAT assembly provided a perfect starting place for understanding the mechanism.
In 1991, Crabtree received a phone call from Stuart Schreiber (now an HHMI investigator) at Harvard. Schreiber had discovered that cyclosporine and a related immunosuppressant, FK506, bound the intracellular proteins cyclophilin and FK506-binding protein, respectively. After talking, the two men became friends and collaborators. Within a couple of years, they had uncovered the broad outlines of one of the first known pathways that transmit information from the cell membrane to the nucleus. This calcium-calcineurin-NFAT pathway, originally discovered in white blood cells, is now known to be important for the development of many, if not most, vertebrate organs and tissues. Crabtree's group has been especially interested in its role in the nervous, immune, and cardiovascular systems, though they recently showed that its operation is also essential for bone development, insulin production, myelination, and neural crest diversification. Hence, the pathway has become a focus for scientists interested in osteoporosis and diabetes.
The group discovered that the calcium-calcineurin-NFAT pathway helps nerve cells make connections with each other. By using mice lacking either active calcineurin or NFAT, they showed that the pathway normally enables developing nerve cells to respond to certain growth factors by putting out the long "telephone wires" called axons. Using microarrays of DNA developed by another HHMI investigator, Pat Brown, Crabtree's group identified the genes that must be activated by the pathway if axonal outgrowth is to occur.
The mutant mice also led to the discovery that small structures in the developing heart called cardiac cushions, which are remodeled into heart valve leaflets, fail to develop if the calcium-calcineurin-NFAT pathway is not operating. Surprisingly, valve primordia (cells that line the cardiac cushions) from the mutant mice prevented valve primordia from normal mice from becoming leaflets. The group then determined that the middle, muscular layer of the heart wall, which tells the inner layer to form cardiac cushions, made three times as much vascular endothelial growth factor (VEGF) in the mutant mice as in normal mice. Removing VEGF protein allowed the heart valves to develop normally. Thus, the calcium-calcineurin-NFAT pathway is essential to heart valve development because it lowers the concentration of VEGF in specific regions of the heart where the valves will form. One big puzzle was how the pathway can have such tissue-specific functions when it is found in nearly all cells. Crabtree's group found that earlier developmental events prime the chromatin of certain genes so those genes can respond when the pathway is later activated. This led the group to isolate a 12-protein complex, similar to yeast SWI/SNF, that uses the energy of ATP to remodel chromatin. Remarkably, they discovered that these BAF complexes are made by mixing and matching the products of several gene families to produce hundreds of varieties. Further studies showed that neurons contain a family of specialized BAF complexes. The group's current work is revealing that different complexes interact with chromatin at different locations and therefore allow different sets of genes to be transcribed. Thus, some genes are accessible to signaling pathways in some cells, whereas different genes are accessible in others. The researchers have also found that BAF complexes are involved in the development of T lymphocytes.
Some of the group's findings suggest medical applications. For example, the researchers discovered that slightly increasing NFAT activity dramatically promotes bone growth. Thus, NFAT or proteins that regulate it might be suitable targets for drugs designed to treat thinning bones. They also determined why inhibiting calcineurin with, for example, immune-suppressing drugs, can lead to diabetes, and they found that 10 diabetes-related genes are regulated by the calcium-calcineurin-NFAT pathway. These insights have led to new ways of thinking about diabetes treatment.
In studies that hark back to his teenage interest in chemistry, Crabtree has worked with Stuart Schreiber's group to develop small molecules that regulate proteins by bringing them closer together. These molecules are presently being investigated for gene therapy to control levels of proteins such as insulin and growth hormone. In a variation on this work, Crabtree's group recently synthesized molecules that allow precise timing of the degradation of an individual gene in a developing mouse. This allowed them to dissect the function of a protein kinase called GSK3β, which exports the NFATc proteins from the nucleus. They were able to define the time that GSK3β executes its different biologic functions and unexpectedly found that it was used for about 24 hours in the formation of the palate, and hence might have an important role in cleft palate in humans.
In the next few years, Crabtree hopes to elucidate the general rules of how these bifunctional molecules can be used to modulate a wide variety of protein-protein interactions for therapeutic and experimental purposes. "We also hope to further understand how chromatin is prepared in a tissue-specific way to receive signals from the cell membrane and how these tissue-specific patterns of chromatin evolve from the pluripotent state found in stem cells," he says.
The best thing about being a scientist, he adds, is the astounding freedom to pick an area of interest and pursue it in one's own way. "When I give a talk," he says, "I feel I am the writer, producer, director, choreographer, and actor."