Identification of interaction partners for the basic-helix-loop-helix protein E47.
Helix-loop-helix proteins constitute a family of transcription factors with the potential to form homo- and hetero-dimers mediated by the helix-loop-helix domain. Oncogenic mutations in such genes can disrupt the equilibrium of protein-protein interactions in the affected cell. In order to assess the biological consequences of such mutations, the full complement of interacting proteins must be known. To identify proteins interacting with the basic-helix-loop-helix domain of the ubiquitously expressed E47 protein, a 'sandwich'-screening procedure was developed which distinguishes between homo- and hetero-oligomers, and specifically excludes the detection of complexes which cannot bind DNA. Nine distinct cDNAs were identified which encode proteins with apparent basic-helix-loop-helix domains, including a novel clone termed eip1 which is distantly related in the basic-helix-loop-helix domain to the Drosophila enhancer-of-split m7 protein. Using epitope-tagging, interaction of E47 basic-helix-loop-helix protein with the eip1 protein encoded by this novel cDNA was confirmed by immunoprecipitation experiments in COS7 cells. Interaction was also observed in the yeast two-hybrid system. Three cDNAs encoding proteins without basic-helix-loop-helix domains were also found to interact in the sandwich-expression screen. Interactions with human PARP and mouse replication factor 1a were confirmed using glutathione transferase-tagged cDNAs. A cDNA encoding part of the nucleolin protein sequence interacted with the E47 basic-helix-loop-helix only when fused to a beta-galactosidase tag.
Dear
T N
TN
Deutsches Krebsforschungszentrum, Heidelberg, Germany.
Hainzl
T
T
Follo
M
M
Nehls
M
M
Wilmore
H
H
Matena
K
K
Boehm
T
T
D000595:Amino Acid Sequence; D000818:Animals; D051792:Basic Helix-Loop-Helix Proteins; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular / Q000379:methods; D018076:DNA, Complementary / Q000235:genetics; D004268:DNA-Binding Proteins / Q000378:metabolism*; D018257:Helix-Loop-Helix Motifs; D018398:Homeodomain Proteins; D006801:Humans; D046911:Macromolecular Substances; D051379:Mice; D008969:Molecular Sequence Data; D011233:Precipitin Tests; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism; D051820:TCF Transcription Factors; D058125:Transcription Factor 7-Like 1 Protein; D014157:Transcription Factors / Q000378:metabolism*; D051720:Upstream Stimulatory Factors
D051792:Basic Helix-Loop-Helix Proteins; D002352:Carrier Proteins; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D051820:TCF Transcription Factors; D058125:Transcription Factor 7-Like 1 Protein; D014157:Transcription Factors; D051720:Upstream Stimulatory Factors; C107989:BHLHE40 protein, human; C491539:Bhlhe40 protein, mouse; C544912:TCF7L1 protein, human; C544913:Tcf7l1 protein, mouse