Bar Harbor, Maine -- Jackson Laboratory Professor Luanne Peters, Ph.D., and colleagues discovered that the mouse mutant Nan, a model for severe inherited anemia, carries a single-gene mutation, but one that selectively interferes with a cascade of events critical to normal red blood cell formation.
The discovery could point the way to future treatments for beta thalassemia, sickle cell disease or other blood disorders, and appears to represent a previously unknown mechanism of inherited disease.
Peters explains that the anemic Nan is one of dozens of mouse models of blood disease that she and her laboratory have studied over the past two decades. Mary Lyon of MHC Harwell in England had published a description of the mutation in the old Mouse Newsletter in 1983, and Peters asked Lyon to send her the mouse for analysis in 1994.
Years ago the Peters lab mapped the Nan mutation and found a single amino-acid change in one of the so-called zinc fingers of EKLF, erythroid Kru¨ppel-like factor, which orchestrates genes that control development of blood cells. Zinc fingers are protein components that turn genes on and off. Like hands reading Braille, they search out specific DNA sequences, then bind to the DNA and insert the correct “on-off” control proteins into the target site.
“It was a pretty conservative change,” Peters says. “Glutamic acid to aspartic acid, E to D, not something by itself that you’d get wildly excited about.” Even her collaborator Dr. James Bieker of New York’s Mount Sinai School of Medicine, one of the world’s leading experts in EKLF, was “underwhelmed” with this information, she notes. “So frankly, for a while we didn’t believe it was EKLF and we kept looking for a different gene.” Adding to the mystery: Knockout mouse models lacking EKLF have a different phenotype than the Nan mouse.
The breakthrough came when Peters and her Mount Sinai collaborators conducted a simple gel-shift experiment, which showed that the Nan version of EKLF failed to bind DNA normally. Notably, this failure was selective, depending on the DNA binding site.
EKLF has three zinc fingers, each of which binds to a specific base triplet in its DNA binding site. The Nan mutation is in zinc finger 2. If the DNA binding site contains a C base, the mutant Nan EKLF binds the DNA and transactivates the gene normally. If on the other hand that triplet contains a T, “it won’t work, and all is not well,” Peters says. “A subset of erythroid genes is not expressed properly.” It was the first demonstration of a sequence-selective transcription factor defect like this, and it distinguished the Nan allele of EKLF from the null allele in the knockout models.
“It was one of those voila moments,” Peters says.
In the Nan mice, embryonic globins, which are normally down-regulated, or suppressed, after birth, are highly expressed. EKLF is a major regulator of the switch to adult globin expression. “This suggests the possibility of a drug intervention that tinkers with EKLF in order to reactivate embryonic globins in patients with beta thalassemia or sickle cell disease.”
The research findings are published in the Proceedings of the National Academy of Sciences.
The Jackson Laboratory is an independent, nonprofit biomedical research institution based in Bar Harbor, Maine, with a facility in Sacramento, Calif. Its mission is to discover the genetic basis for preventing, treating and curing human diseases, and to enable research and education for the global biomedical community.
Siatecka et al.: Severe anemia in the Nan mutant mouse caused by sequence-selective disruption of erythroid Kru¨ppel-like factor. Proceedings of the National Academy of Sciences, www.pnas.org/cgi/doi/10.1073/pnas.1004996107
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