I want to identify if my transcription factor has enhancer or repressor activity by inhibiting the promoter (whether by introducing mutations on it, or by using inhibitors). To track this activity I will use reporter gene: luciferase. The promoter will be a pathway-specific promoter. What is the read out that I should expect when my the TF works as enhancer or repressor?
You do not need to use inhibitors or mutations. If you have a promoter of interest, clone it upstream of luciferase. Then transiently transfect the luciferase reporter with increasing amounts of your protein in a cell of interest. The promoter-luc construct will have some base line activity. The question then is does your protein increase or decrease this activity? This should get you started.
You need a plasmid with your promoter cloned in a luciferase plasmid. Then you transfect this plasmid with an expression vector of the transcription factor (and a group with only the promoter-lic construction). If the luciferase activity is higher when the transcription factor is cotransfected it is a activator and the opposite if the transcription factor decrease the luciferase activity.
good luck!
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