Hi,

I would try get the FASTA sequence of your enhancer region (you can also export it from IGV browser, for example) and submit it to AME:

http://meme-suite.org/tools/ame

This analysis will result in a list of transcription factors, whose binding motifs were enriched in your FASTA sequence (compared to a random nucleotide sequence).

Best,

Michail

Hi Michail Yekelchyk , thank you for your answer.

I have all the FASTA sequence, actually I have a FASTA file with the enhancer sequence in 42 species which I submited to MEME and it gave me back some discovered motifs, which I submited to GOMo and it gave me predictions of the biological roles of the motifs. It gave me back things as " immune response". Does it means that it is a motif for binding TF for immune response? Or that the motif acts in a immune response, in which case I should be talking about a protein and not an enhancer? Sorry if my doubts are so basic, I'm very confused with all the tools in MEME-suite.

I'm gonna try AME, from your answer I think that this is the tool that will give me what I'm looking for and not the one I was using before, right? Please, do you know if is it enough to use as control the "Shuffled input sequences"? And I'm not sure which I choose in "sequence scoring method" and " motif enrichment test". Do I use the default?

Thank you very much for your help and time

Best,

Gabrielle

I was not using the GOMo after the AME... Though I assume that when you feed your motifs (basically, the predicted transcription factors binding events) to it, you end up with a set of genes, where such motifs were found in promoters/enhancers. Subsequently you check those genes for gene ontology terms enriched, which in your case says "immune response". Which says, that the set of motifs you found, was enriched in the group of genes, related to "immune response". Of course, it is also connected to the transcription factors themselves (=proteins), but you should be aware that some motifs can share the sequence among a family of transcription factors and vice versa.

I was using the default. Shuffled input sequences means that it compare your input FASTA (where you have some motifs) with the random ATGC sequence (where you have no motifs enriched). The final data for you will be the matrix, containing: Motifs, predicted transcription factors, species and p-value (adjusted p-value).

Good luck!

Michail

Hi Michail Yekelchyk , actually i used GOMo afeter MEME, not after AME, that's where I think I went wrong, I suppose.

I used AME but I can't understand the results, would you please help me? This is the output http://meme-suite.org/opal-jobs/appAME_5.0.41548871604396488735666/ame.html

Is these "logo" the predict motifs and the "ID" the predict TF that will bind to these motifs? The problem is, theses "motifs" are not in the original sequences I put as the input. I put a fasta file with 42 sequences (different species) of the same enhancer. Should I have used just one sequence (of one specie) for each analyses? How do I interpretad these different nucleotides in the same site?

Thank you so much for your help!

The Logo is depicting the motif (height of the letter is representing its probability on particular position) , that's correct. ID and Alt ID show the name of TF. For example, Nkx2-5. I would rather use repetitively this tool for different species and see if the enriched list will change, or which TF motifs are conserved. Anyway, you should discuss the biological relevance of your findings with your supervisor.

Good luck!

Michail

Michail Yekelchyk , I tried separating the sequences and using the tool for each species. In a first moment I tried just 4 because this is the maximum jobs the software allows you to do at the same moment. Well for 3 of the species it didn't had any result, it said No motifs passed the E-value threshold (10). I increased the threshold and I am trying again. But, one of the sequence, the Homo sapiens one, it turn back with a positiv result, but I still don't understand the motif it gave me. It gave me this http://floresta.eead.csic.es/footprintdb/index.php?tf=d5adaa1da6d0a96c0de482ad2fc21da6 and the way I understan the motif is this consensus sequence right? The rATCAATGkyATTGAtr one? But there isn't such sequence in the whole sequence that was the input!

Thank you again for your help, I'm sorry I'm not understanding this =(

The "rATCAATGkyATTGAtr" is a sequence, to which the transcription factor can bind. It is also called a "motif". r,k,y,t,r, are the positions, where could be different bases (see attached table from wikipedia). It means, that "r" on the first position (for example) may be either A or G. So to find this motif in your FASTA you need to look for either "AATCAATG..." or "GATCAATG...". Similarly for other non-fixed bases.

Best,

Michail

You can use MEME also, which is an unbiased way of TF binding. Take atleast 100 bps upstream and downstream from summit and you can use the tool. This will give you a list of prediction sites for the TF binding. I hope this helps.

Hi Michail Yekelchyk , actually, I did understand that R means A or G (but thank you very much for this table, it's really helpful), but I can't find neither of this sequences in my original input. I even tried checking if was the reverse complementary strand that gave the motif, but couldn't find it either. The small motifs it gave back I can find when using this table, but most of them are actually the same region, as examples: YYAATTAR, NYAATTAA, YYYAATTANV, BYAATTAR. It's the same AATTA, just the ones around that chance. But any other motif in the list I can't find. As a example, the one that is ARGGTCAAAAGGTCA, the R is just in the beginning, and I can't find GGTAAAAAGGTCA in my sequences.

I'm almost giving up on AME =(

Hi Prit Benny Malgulwar

Could you explain a bit more why should I take the 100 bps upstream and downstram of my enhancer when using MEME? My enhancer it's located in a intron, does it help?

If you take 100bps then your chances of getting TFBs at particular enhancer summit is increased. You can also tweak a bit with respect to size of region where you want to see.

Enhancers can be anywhere. Its possible that your enhancer of interest can modulate the expression via intronic region.

Hope this helps.