I think you mean regulatory factors. There're some database or websites (like TESEARCH) you can use to analyse your promoter and predict potential binding sites. But still you must do further experiment to validate it.

Hi,

You might be planning to 'in silico' translate the potential regulatory genes, model their structure if possible using template then see if you can identify their potenital binding sites....but such a study is too theoretical and may need experimental validations

bye

Hi Rasbin,

I thought, your mean were Transcription factors and wanted to predict their targets. PlantPAN V.2 (http://plantpan2.itps.ncku.edu.tw/) is the best database which you can help. The part of TFBS is the best section for your mean which I thought.

Hi Rasbin,

In silico analyses might give you a first idea about possible targets, but still you need to confirm those experimentally. One option is to accomplish Chromatin Immunoprecipitation (ChIP).  The idea behind the protocol is that you immunoprecipitate your protein (regulatory factor) together with the chromatin that is bound to it at a certain time.  Then you probe this chromatin against primers for the promoter regions of suspected targets. It is crucial that you have an antibody that specifically recognizes your protein or if this is not the case, a transgenic line tagged to something that has an Ab, for instance GFP works well. you may check Plant Mol. Biol. 2012; 80:405-418 for more detailed protocol.

Hope it helps, Best

Plantpan is great, many thanks Reyhaneh. 

In Silico analysis seems interesting, I havent done such experiment before, so its not getting into my head.

All I want is, I have got some regulatory genes in rice with AP2 domain, I want to find which structural genes or promoters they act(bind) on. In arabidopsis, I found that they bind on BCCP2 promoters. How to find out ??

you can perform CHIP-seq, and find which site you regulators binding.

Hi Rasbin i m agree with Ana Elisa you can do a chromatin immunoprecipitation(Chip)

to see in wich region of promoter gene there will be your regulatory protein.

in these days i design different set of primer in particular 4 that recognize promoter region of the gene that i use in PCR and after I see the different product of pcr generate from fragments of chromatin that i have before immunoprecipitated.

this primers seems work very well.For the design of the primer is very important that the product of pcr is very short around 150 bp and that for your interest region design more set of primers that cover all promoter region .

After you see you product of PCR on agarose gel

I hope that I m clear

Good Luck

Daniela Cangiano,PhD

Hi Rasbin,

 You can find some info about AP2 domains in SMART (http://smart.embl-heidelberg.de/smart/do_annotation.pl?ACC=SM00380)

Still, I think you will need you confirm experimentally your putative targets.

Best, AnaElisa

Hi Rasbin,

If I understood you correctly you want to find if your regulatory gene having AP2 domain interacts with other 'gene factors' (proteins) or can bind to promoters. As suggested by others ChIP is one option if you have a good antibody for your favourite protein in question. You can do EMSA using purified protein. You can actually do screens to find our which DNA sequence they can bind (HT-SELEX). you can do phage /yeast hybrid screens. It totally depend on how high throughput you want your search to be, or if you have some nice indications that it might act with few candidate DNA regions, you cans design your experiment accordingly. To find out interacting factors you might consider co-immunoprecipitations, protein-protein interactions pathways (in-silico) to validate them later with experimentation, functional interactional assays. The methods available till date can go on and on, but all it depends what kind of search you want to do. 

If you are looking to identify cognate gene promoter for your regulatory gene (element) then you can think of using chromosome conformation capture based high resolution 4C technology. HR e4C seq would allow you to characterise any un assigned regulatory element to its cognate gene promoter/s.

or 

If you are looking for regulatory gene (which perhaps codes of a transcription factor (TF) protein binding to specific gene promoters then you require to perform chromatin immune precipitation provided you have a specific antibodies for that TF. You can think of performing ChIP seq method to characterise all those chromatin regions (including gene promoters) in unbiased way.