The use of inducible engrailed fusion proteins to study the cellular functions of eukaryotic transcription factors.
Transcription factors determine cell lineages, control cell fate, and regulate cellular responses to stimuli. Many methods are currently used to study the function of transcription factors in a cellular context and several of these involve overexpressing a constitutively active form of the protein and studying its effects. Here we outline an alternative approach involving the inducible expression of dominant-negative transcription factors in human cell lines. Dominant-negative transcription factors can be used to investigate the effect of signaling pathways on complex cellular processes that are regulated by a particular transcription factor. Potent dominant-negative transcription factors can be created by using fusions to the engrailed repressor domain. These fusion proteins can be coupled to inducible expression systems such as the ecdysone-inducible system. The ability to control protein expression has several benefits including the ability to create stable cell lines that express potentially cytotoxic proteins. Therefore when used in tandem, these two methods constitute a new and improved approach for dissecting the cellular role and transcriptional targets of many transcription factors. Here we illustrate this integrated approach by using a conditional dominant-negative Elk-1 protein to identify candidate Elk-1-regulated target genes.
Vickers
Elaine R
ER
School of Biological Sciences, University of Manchester, 2.205 Stopford Building, Oxford Road, Manchester, M13 9PT, UK.
Sharrocks
Andrew D
AD
D000818:Animals; D017209:Apoptosis; D002453:Cell Cycle; D002454:Cell Differentiation; D002460:Cell Line; D019070:Cell Lineage; D004268:DNA-Binding Proteins; D029721:Drosophila Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004331:Drosophila melanogaster; D004440:Ecdysone / Q000494:pharmacology; D005057:Eukaryotic Cells / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000187:drug effects; D005799:Genes, Dominant / Q000235:genetics; D017930:Genes, Reporter; D018398:Homeodomain Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D008967:Molecular Biology / Q000379:methods*; D020411:Oligonucleotide Array Sequence Analysis; D017434:Protein Structure, Tertiary; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D015854:Up-Regulation / Q000187:drug effects; D050807:ets-Domain Protein Elk-1
D004268:DNA-Binding Proteins; D029721:Drosophila Proteins; C494112:ELK1 protein, human; C063459:En protein, Drosophila; D018398:Homeodomain Proteins; D011518:Proto-Oncogene Proteins; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D050807:ets-Domain Protein Elk-1; D004440:Ecdysone