11/2/2014
Customer has produced ChIP-seq data using the wild-type and mutant forms of a transcription factor (each with multiple replicates), and she wishes to find out whether the mutant form binds DNA with the same selectivity as the wild-type transcription factor.
Mon, 11/3/2014 at 8:36 AM
AccuraScience LB: Could you tell me a little more about the nature of this "selectivity" difference, in particular, would it likely to be difference in "specificity", or it is more likely to be difference in "affinity"? The former would be reflected in different binding motif sequences, and the latter would be reflected in different binding strength on the same motif sequences. These two types of changes would leave different signatures in the ChIP-seq data.
Mon, 11/3/2014 at 10:52 AM
Customer: Since the mutation is not in the DNA-binding domain, it may be that the DNA-binding sites are the same as for the wild-type transcription factor. We don't know the answer and that's why we performed the genome-wide ChIP study. An alternative to the "selectivity hypothesis" under which the DNA binding site is different, is that the mutant transcription factor interacts with different co-regulators.
Mon, 11/3/2014 at 2:35 PM
AccuraScience LB:Indeed coregulators could lead to change in binding specificity of transcription factors, and there may be multiple mechanisms how they may do so - these two papers describe some of the possibilities: http://www.ncbi.nlm.nih.gov/pubmed/22146299 and http://www.ncbi.nlm.nih.gov/pubmed/22153072.
With the current understanding of your project, I would propose that the following be tried: (1) Use the same stringency (in terms of FDR cutoff) in peak determination between for the wild-type and mutant, then examine whether the number of peaks obtained are similar between the wild-type and the mutant. Multiple FDR levels are to be used. (2) Use an alternative way of defining stringency, that is, requiring that the same peak to be called simultaneously in replicate experiments, and repeat (1). If the peak number differ substantially between the wild-type and the mutant, then difference in binding affinity (rather than specificity) is the likely explanation. (3) Compare the motifs enriched in peaks between the wild-type and mutant, to see if there is any quantitative difference. (4) Try to obtain enriched motifs in those peaks identified in the wild-type samples, but not in the mutant samples, and vice versa. If any meaningful motifs differing from the "common" motif are identified, this would support a different specificity hypothesis.
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