Can a gene's regulatory region overlap with another gene? I'm looking for promoter region of a gene. If I take 1kb upstream of TSS, the sequence will overlap with an upstream gene. Should I take the region between the upstream gene and TSS, or take the 1kb upstream of TSS for test?
Sometimes intron of one gene can be a regulatory region for a gene really far from it
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2386664/.
For enhancer analysis, its also better to start with a bigger sequence and then chop it into overlapping smaller segments to look for the enhancer. Also if you can find out some ChIP studies for your regulator/gene of interest than you can have some idea where the regulatory sequence might be. Similarly doing conserved sequence analysis increases the probability of finding an evolutionary important enhancer region.
@Abel @Piyush Thanks for the answer!
For the enhancer analysis, usually how many kb should I start with?
Also, after I get this sequence, should I do a transcription factor binding site prediction on this sequence to see any TF possibly bind to it? And see if the predicted binding site is conserved?
@Wjkp
While there is no optimal size, however, you would think that a closer site would be important. So you can start with 1kb and than keep on increasing until you find any. Now if you find a site that is 20 kb away, that will be doubtful as that site could be regulating the gene closer to it, which is not the case all the time as shown in the reference I sent you, but wouldn't be worth trying unless you have lot of funds.
Another important segment to include in your search are the introns in the gene that you think is being regulated, as regulatory sites could be present in the first or second intron of the gene itself. So start 1kb upstream of your TSS and include the first two intronic region.
For TF binding analysis, you can go on this site http://cisbp.ccbr.utoronto.ca/TFTools.php. It is also important to check if the region is conserved among related organisms. For example if it is vertebrate, then check for conservation between mouse, human, frog, chicken, zebrafish etc.
But it will be better to look for ChIP seq studies in embryonic stem cells or the particular cell type you are interested. Additionally, colocalization of histone modification (ChIP seq for them are also available sometimes), together with binding of regulator could suggest that the site is indeed active.
@Piyush Very clear and helpful! I will take 1kb and 2 introns for TF analysis and conservation check. Thanks a lot!
Any recommendations for websites of ChIP seq studies?
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3614620/
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC525685/
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