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11.1 years ago
Manvendra Singh
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2.2k
I have analyzed the ChIP-seq data from mouse. I just wanna see what are genomic regions in Human where my txn factor would probably bind. Please help
Have you considered doing motif discovery with MEME-Chip and then just searching the human genome for the resulting motif(s)?
Please accept my sincere thanks. You always help dpryan. Yes, I have done the same and Now I do have motif. I have to perform some ChIP-PCR/qPCR in Human lines. So basically I was searching for positive control where we should be assured that this transcription factor would bind. For this, I was just finding peaks in regulatory regions of Mouse genes and was wondering that if we get similar sequence in Human genome then it could be used to design primers.
There's no a priori guarantee that the binding motif will be completely identical in humans. If these are immortalized cell lines, you might just amplify a nice peak region from the mouse genome and then use it to transfect a dish of the human cell line. Performing the qPCR after that should let you know if everything is working properly.
Oh, This is great suggestion. I would do that. I think this should work for sure. Thanks Dear
Now your question is, how do I find occurrences of sequence motif (ambiguous/unambiguous)? This is totally different from your initial wording of the question.
I am sorry Michael if it had reflected this impression but I this was my hypothesized step somewhere in the middle of experimental plan. My Main motto was to get very high probable candidate which could be used as positive control in Human ChIP-qPCR experiment. I am glad that you mentioned the way peak sequences motifs are occurring, I was actually doing that but could not find the UCR/UCE list or other data-set to intersect my peaks and to have look up.
You seem to make some erroneous assumptions about TFs and therefore your question is ill posed: "100% conservation" (you seem to mean identity) is neither a sufficient nor necessary criterion. There could be large stretches of 100% identical sequences which are never bound by any TF, on the other hand even slight differences in binding motifs might still facilitate TF-binding. Therefore, the best approach is possibly outlined in dpryan's comment.
Dear Michael, Many Thanks for your response. Actually I have to perform some ChIP-PCR in Human lines, whereas I do have so many candidates from the analysis of ChIP-seq data from Mouse. So basically I was searching for positive control where we should be assured that this transcription factor would bind in Human genome. For this, I was just finding peaks in regulatory regions of Mouse genes and was wondering that if we get similar sequence for Human gene regulatory regions then it could be used to design primers........ Thanks again.
Again, in the same direction as dpryan, finding the motif is not a 100% guarantee of TF binding to the identical motif in another species, because - to be picky - there is only "this transcription factor" in mouse, the human TF is not the same bot a homologous one, but is it identical? Also, motif alone might give you a too large number of false positives to effectively screen. I would in addition check for presence of motif in regulatory regions of highly conserved orthologues, for improving chances of a success.
Yes Michael, Thanks again,,, Exactly, So After this ultra useful discussion I have decided to find the motifs in orthologue regulatory regions ( do not know how to get their conservation score between Human and mouse) in hg19. and then would transfect this in hiPS cells ( I have shRNA and expression constructs ready for this txn factor) and then would do qPCR for those genes, top expressing genes should be candidates.
I'm glad we could help, often the most important step is to get the question right. I'll leave it to dpryan or you to write a formal answer. The answer to getting the conservation score is probably Is any database provide human genes' conservation scores ?.
Please, could you read these ten rules ? http://www.ploscollections.org/article/info%3Adoi%2F10.1371%2Fjournal.pcbi.1002202;jsessionid=A2C2B677241104800E044DA36AFB577B
Thanks for this link, I have just gone through it but this is not what I am asking for. This is my first post so it could be forgiven, from next post onwards, I would try and follow all possible rules