I am a first year Phd student and I am dealing with cellular immunology. I realized my project needs some bioinformatic DATA to design experiments well.
I have one target gene (RUNX3) and two transcription factors (KLF4; PU.1) of interest. I can find the gene (RUNX3) on chromosome 1 and also retrieve of it`s and the promoter`s sequence. I would like to investigate how many potential binding sites are available on the the two promoters (P1 and P2) of the gene (RUNX3). A paper published data about two promoters exist for this gene. But one of the databases I checked for promoters (prediction on a given loci) gave me 3 promoters. What should I follow? My aim is to check the transcription factor binding sites on the promoter regions of the gene. I found some core binding sequence motifs within the CDS – are these artifacts or can be active binding sites (like duons or something) – how can I check it in silico? If I am right enhancer elements can also contain potential binding sites – how can I predict or determine the length and the position of the enhancer region of this gene (RNUX3) before the promoter region and right after the coding sequence? How should I determine the extra amount of DNA what I should add as extra before and after the gene to check as enhancer region? I have some data about one of the promoters (CHIP-seq) and primers. Is there any software which is able to give me the DNA sequence which is situated within the primers or I have to do it manually?
To summarize my questions:
- How can I validate in silico the predicted promoters of a gene?
- I don`t have access to Transfac – which software would you recommend to use for prediction and identification of transcription factor binding sites at a given region of DNA?
- How can I validate in-silico transcription factor binding sites within the CDS region?
- How can I predict the enhancer region of a gene (upstream and downstream direction) or just simply how many extra basepairs should I add in u. and d. direction to the gene when I want to get the DNA (to be sure I cover the enhancer regions) to identify potential binding sites?
Thank you very much!!!