Target gene identification: target specific transcriptional activation by three murine homeodomain/VP16 hybrid proteins in Saccharomyces cerevisiae.
The mammalian homeodomain proteins encoded by Hox genes play an important role in embryonic development by providing positional queues which define developmental identities along the anteroposterior axis of developing organisms. These proteins bind DNA specifically through their homeodomain to sequences containing ATTA cores, and thereby are thought to exert their effect regulating downstream genes. Little is known about the specificity of binding of homeodomain proteins to their sequences and the identity of their target genes. We have developed a transcriptional activation assay in yeast which employs a homeobox/VP16 fusion gene as a transcriptional activator and a target construct in which test fragments of DNA are inserted upstream to a reporter gene. Using this assay, we compared transcriptional activation by three chimeric proteins containing the homeodomains of the mouse homeobox genes, Hoxa-5, Hoxb-6, and Hoxc-8. When tested on previously defined target sequences, strong differential specificities of activation were observed. In an effort to identify enhancers that normally respond to homeodomain transcriptional activators, random fragments of mouse genomic DNA were cloned upstream of the reporter gene. Genomic DNA fragments with distinct activation profiles were obtained and were found to share matches beyond the ATTA core with previously described enhancers. These results demonstrate that the transcriptional activation system in yeast can be used as a convenient system to detect DNA motifs which bind homeodomain proteins, and subsequently, to identify authentic target genes responsive to Hox gene proteins.
Friedman-Einat
M
M
Department of Biology, Yale University, New Haven, Connecticut 06511, USA.
Einat
P
P
Snyder
M
M
Ruddle
F
F
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D017405:Herpes Simplex Virus Protein Vmw65 / Q000096:biosynthesis / Q000235:genetics*; D018398:Homeodomain Proteins / Q000096:biosynthesis / Q000235:genetics*; D051379:Mice; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D015533:Transcriptional Activation
D017405:Herpes Simplex Virus Protein Vmw65; D018398:Homeodomain Proteins; D011994:Recombinant Proteins