Identification of residues of the Caenorhabditis elegans LIN-1 ETS domain that are necessary for DNA binding and regulation of vulval cell fates.
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.
Miley
Ginger R
GR
Department of Molecular Biology and Pharmacology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Fantz
Douglas
D
Glossip
Danielle
D
Lu
Xiaowei
X
Saito
R Mako
RM
Palmer
Robert E
RE
Inoue
Takao
T
Van Den Heuvel
Sander
S
Sternberg
Paul W
PW
Kornfeld
Kerry
K
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D017173:Caenorhabditis elegans / Q000235:genetics*; D029742:Caenorhabditis elegans Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D018509:DNA, Helminth / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics; D005260:Female; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D010641:Phenotype; D017354:Point Mutation; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D014844:Vulva
D029742:Caenorhabditis elegans Proteins; D018509:DNA, Helminth; D004268:DNA-Binding Proteins; C097533:Lin-1 protein, C elegans; D014157:Transcription Factors