I never worked on promoter regions and we are currently trying to characterize one. We started from ENCODE data on chromatine state to identify the area of interest and will probably go for some ChIP and qPCR for TF that should bind there (again according to ENCODE data). Also, we plan some luciferase to understand how modulation of upstream affects promoter activity.
What are some tips and tricks that you could suggest for a "promoter newbie"?
There are many promoter and motif analysis tools (see www.startbioinfo.com) and I could begin with some of the most commonly used ones. But in general, I would use the following strategy: a) literature search to find out what is known about the regulation of expression of the gene of interest in the species of interest and those which are closely related; b) perform multiple sequence alignment of promoters of the gene of interest from closely related species, especially if the gene expression is conserved across them - determine potential regions of functional significance (which are likely to be more similar); c) analyze the transcription factor binding sites using MEME, JASPER and/or Transfac; d) Analyse CpG islands, if any; e) look at existing ChIP data for the tissue and condition of interest - and then design the experiments.
I agree with all of the steps suggested above. Using sequence alignment is an excellent way to determine what sequences might be important, and it can also help you narrow down the "proximal promoter" region to use for your luciferase assays. Use the UCSC genome browser's conservation track to visualize the approximate length of conserved regions upstream from the 5' end and it may help you decide what the size of your initial promoter construct should be. If you have identified this gene based on your interest in the protein, do not forget to check for alternative promoters or alternative splice variants in both the refseq genes and in EST tracks in UCSC as well.
To really map promoter activity well using luciferase assays, you can do a series of deletion mutants. It is possible to design the first set of deletion mutants based on the location of restriction enzyme sites already present in your sequence and the luciferase vector to get a rough idea of what parts are functionally important (in the cell line you are using).
You should be able to find free programs online that you can use in conjunction with the databases mentioned above for searching for promoter motifs.
HTH - Good Luck!
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