Direct or indirect protein-DNA interaction

[nukeman]

Greetings,

How would one go about deducing whether a certain DNA binding transcription factor has a direct interaction with a gene of interest (we know that the TF has impact on expression because a mutation in the gene coding for it caused a 10-fold decrease in expression of my gene of interest), or if it's a middle step in a series of regulations eventually leading to the gene of interest?

All I have to work with is the regulatory region of the gene of interest.

I'm trying to think of ways to assay this, but I can't come up with anything useful.

Perhaps some kind of reporter gene assay might work? But reporter genes can't tell me what (if it's the TF I suspect, or another one) drives the expression, only that something does or not.

[Cat]

How about gel shift assay?

See http://www.piercenet.com/browse.cfm?fld ... 7A8280C68F