Answer the questions for the following article: https://www.ncbi.nlm.nih.gov/pmc
ID: 85764 • Letter: A
Question
Answer the questions for the following article: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4907330/
1Describe two methods for determining transcription factor binding sites in vitro.
2What are the disadvantages of these in vitro methods for determining binding sites that are relevant in vivo?
3What is Halo used for in the DAP-seq method?
4Is genomic DNA the same thing as chromatin? Please provide your rationale.
5Describe how a peak identified from DAPseq differs from a transcription factor motif.
6How did the authors determine the validity of the DAPseq approach in terms of identifying transcription factor motifs? Which figure(s) and panel(s) show this?
7Where are TF motifs found relative to gene features?
8How were functions for transcription factors determined from the DAPseq data? What other data sources did they use?
9Describe an example of a new function for one of the transcription factors described in the paper.
10Do similar transcription factors (in terms of amino acid identity) have similar function? Please use Figure 4 as a reference when giving your answer.
11Provide at least two reasons why a ChIP-seq peak may not overlap with a DAP-seq peak.
12What were the repeat-specific binding patterns for ARF transcription factors?. How can these binding patterns be explained by transcription factor dimerization models?
13. Two assays – one computational and one experimental, were used to identify methylated transcription factor binding sites. Please describe these two methods and how they differ.
14 What are the three general rules with respect to methylation context and transcription factor binding?
15 In Figure 7C what is the difference between methylC and methylCG?.. From this figure, give an example of which transcription factor’s binding is the MOST affected by cytosine methylation and one which is the LEAST affected by cytosine methylation.
16How would you test functionally if a change in cytosine methylation results in a change of gene expression?
Explanation / Answer
Answer 1:- i] The one of the method is DNA affinity purification (DAP)sequencing method. This is a high-throughput assay. This assay uses in-vitro-expressed transcription factors to interrogate naked genomicDNA fragments to establish binding locations (peaks) and sequence motifs. This DAPchip method can be used for any purified regulator in any organism with a sequenced genome. The protocol involves a separate reaction, where an affinity-purified TF is prepared by in vitro expression, bound to ligand- coupled beads, and washed to remove nonspecific cellular components. The gDNA library is added to the affinity-bound TF and the unbound DNA is washed away . The bound fraction is eluted, amplified with PCR primers and the DNA is sequenced. By mapping the reads to a reference genome, enriched loci (peaks) can be used to identify TFBS and motifs
Ii] The another in vitro approach is systematic evolution of ligands by exponential enrichment (SELEX). This method is used to enrich small populations of bound DNAs from a random sequence pool by PCR amplification. It provides a powerful way to determine the in vitro binding specificities of DNA-binding proteins such as transcription factors.
Answer 2:- Disadvantage of invitro method such as SELEX and electrophoretic mobility shift assays (EMSA) is that, these assays employ synthetic DNA that lacks genomic DNA properties known to impact TF binding, including primary sequence context and chemical modifications, such as the widespread and tissue-specific 5-methylcytosine found in plants and animals.
Answer 3:- Halo is used in the DAP-seq protocol for affinity tagging the transcription factor.
Answer 4:- Genomic DNA is organized three-dimensionally in the nucleus. It forms compact chromatin domains. Genomic DNA exists in Chromatin form in the nucleus. Thus genomic DNA is same as that of chromatin.
Answer 5:- Many DNA functional motifs tend to accumulate or cluster at specific gene locations. These locations can be detected, as a group of gene sequences, as high frequency ‘peaks’ with respect to a reference position, such as the transcription start site. Thus peaks in DAP seq refer to such cluster of motifs.
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