How to find transcription factors?

Question

I need to find master transcription factor(s) regulating secreted proteases in a fungus to be disrupted toward upregulation and overexpresion of extracellular recombinant proteins. How can I detect transcription factors and preferably master TFs (master regulators) in an organism? Does NGS applicable to find TFs and preferably the novel ones? I know ChIP-seq is used to identify TF binding sites but how about detection of the TFs that bind to these sites?

Answer 1

How can I detect transcription factors and preferably master TFs (master regulators) in an organism?

The easiest approach to find transcription factors for any given organism would be based on sequence homology with other transcription factors. In order for this to work your organism of interest does have to be sequences, you can check this (e.g.) on the NCBI genomes page. By comparing the genome or the genes of your organism with other known (fungi) transcription factors, you can find most of the transcription factors of that organism.

Does NGS applicable to find TFs and preferably the novel ones?

Both NGS and ChIP-Seq only allow you to determine DNA (or RNA if you reverse transcribe first) sequences. Therefore these techniques alone will not help you in identifying which TFs act on your genes.

I know ChIP-seq is used to identify TF binding sites but how about detection of the TFs that bind to these sites?

I can't currently think of any 'classical' or standard experiments to determine which TFs bind to a given gene or promoter. One possibility would be to use CoIP (co-immuno precipitation) on the RNA polymerase complex and use Mass-spectronomy to identify the TFs in that complex. The problem with this is going to be, that you need a way to only get the RNA polymerase complex for your genes of interest and not all the other ones in the cell. Maybe you can use columns with DNA probes that can hybridise with your genes instead of standard antibody based CoIP.

Another way to determine which TFs bind the promoters of your genes is to analyse the respective promoter sequences. However, without knowledge of most (or at least some) of the binding sequences of the TFs in your organism, this will most likely not work very well. Edit: As @AlanBoyd commented there is a database for fungal transcription factors. However, it seems like they mainly annotate which fungi genes are transcription factors, but do not cover binding motifs of these factors.

Answer 2

The best way is to determine the TFs in a specific organism with specific function first, then you need to check if they are transcription factors and find their domains on http://smart.embl-heidelberg.de/.