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Genetics, Vol. 167, 1697-1709, August 2004, Copyright © 2004
doi:10.1534/genetics.104.029017
Identification of Residues of the Caenorhabditis elegans LIN-1 ETS Domain That Are Necessary for DNA Binding and Regulation of Vulval Cell Fates
Ginger R. Miley*,
Douglas Fantz*,1,
Danielle Glossip*,
Xiaowei Lu
,2,
R. Mako Saito
,
Robert E. Palmer
,3,
Takao Inoue,
Sander van den Heuvel,
Paul W. Sternberg and
Kerry Kornfeld*,4
* Department of Molecular Biology and Pharmacology, Washington University School of Medicine, St. Louis, Missouri 63110
Howard Hughes Medical Institute, Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139
Massachusetts General Hospital Cancer Center, Charlestown, Massachusetts 02129
Howard Hughes Medical Institute, Division of Biology, California Institute of Technology, Pasadena, California 91125
4 Corresponding author: Department of Molecular Biology and Pharmacology, Washington University School of Medicine, Campus Box 8103, 660 S. Euclid Ave., St. Louis, MO 63110.
E-mail: kornfeld{at}molecool.wustl.edu
LIN-1 is an ETS domain protein. A receptor tyrosine kinase/Ras/mitogen-activated protein kinase signaling pathway regulates LIN-1 in the P6.p cell to induce the primary vulval cell fate during Caenorhabditis elegans development. We identified 23 lin-1 loss-of-function mutations by conducting several genetic screens. We characterized the molecular lesions in these lin-1 alleles and in several previously identified lin-1 alleles. Nine missense mutations and 10 nonsense mutations were identified. All of these lin-1 missense mutations affect highly conserved residues in the ETS domain. These missense mutations can be arranged in an allelic series; the strongest mutations eliminate most or all lin-1 functions, and the weakest mutation partially reduces lin-1 function. An electrophoretic mobility shift assay was used to demonstrate that purified LIN-1 protein has sequence-specific DNA-binding activity that required the core sequence GGAA. LIN-1 mutant proteins containing the missense substitutions had dramatically reduced DNA binding. These experiments identify eight highly conserved residues of the ETS domain that are necessary for DNA binding. The identification of multiple mutations that reduce the function of lin-1 as an inhibitor of the primary vulval cell fate and also reduce DNA binding suggest that DNA binding is essential for LIN-1 function in an animal.
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