I want to explore the transcription factor candidate for a human promoter gene. I want to mean that, let I do not know about any possible factor. So, now how can I find a transcriptional factor candidate for the promoter gene? What are the methods- using software only? Is there any manual method through which I can explore my query?
Please share as I am new in this cellular biology field.
The best way would be to use in silico i.e. software analysis of your target promoter to predict putative transcription factors binding sites (TFBS).
As you are interested in TFBS on human promoter sequence, there is lot of data already available in public domain.
eg. https://genome.ucsc.edu/
On the above site, you can zoom in to your target sequence and examine TFBS. The database is updated with many whole genome chromatin immunoprecipitation data.
If you do not find TFBS for your desired sequence, then go for oligonucleotide pull down assay to validating TF. Briefly the method is described below:
Design oligonucleotides for the putative region of transcription factor binding site and add biotin label at 5 prime end of the oligos. Then mix this nuclear extract, and fish out the mixture using streptavidin-agarose solution. strepatavidin binds to biotin.
Then you can boil the mixture with Laemmli buffer and do Western blot analysis with antibody for your specific transcription factor.
You may also use oligos with different polymorphism to verify direct binding of TF to the DNA sequence. Remember, the putative TF binding site should be in the middle of oligo sequence.
You could also design several oligos, mix them with nuclear extract separately and do as described above and then instead of boiling the mixture with Laemmli buffer, you could run the samples directly on SDS-PAGE, Do silver staining to make the bands visible, cut them out and analyze via mass spectrometry.
You may start looking for putative binding sites in the promoter sequence using a library of positional weight matrices that describe the TFBSs. This has been described in our publication "MatInspector and beyond: promoter analysis based on transcription factor binding sites", Bioinformatics, Cartharius et al., 2005. I co-authored that paper.
However, in silico sites are never enough evidence for a regulatory function. You may look in silico for conserved combinations of TFBS in orthologous promoters from several species, which has a better evidence for a regulatory function than a single TFBS match.
The proof that a certain transcription factor is involved in the regulation of the promoter in question must be by experimental evidence. In silico analysis can give you an idea which TFs or TF - families are worth a closer look.
Apart from predictions there are many sites that have been experimentally defined by ENCODE and other projects for a range of tissues. ENCODE data is easily accessible. It may also provide additional information that is valuable i.e. the epigenetic status largely defines the permission for TF binding. The absence of a permissive chromatin state in particular will prevent TF binding.
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