Exercise 2 - Task 2

Transcriptional regulation / Transcription factor binding sites

• Use the UCSC genome browser to retrieve promoter sequences (-700 ... +300) for the human genes in the list below and use the PFM for the upregualting response elements from the previous task to search for potential TFBS (transcription factor binding sites).
  
  
  • Gene list
  • Use FIMO from the meme suite
  
  
  FIMO from the meme siute, can use the PFM as produced by the sequence logo program.
  
  


• How many p53 target genes could you identify (generate a list of these genes)?


• For the most significant target, find a publication that confirms the regulation by p53.


• Hints

• Map the p53 peak regions from a ChIPseq result file (BED file format, genome assembly hg19/GRCh37) to the human genome using the UCSC genome browser.
  
  ChIPseq peak-file: p53_macs_peaks.bed
  
  
  • Make sure to select the correct genome assembly version. (*)
  • Add the data as custom track (rename the track to “p53”)
  • display the region around the CDKN1A gene
  • Show the mapped regions together with other known regulatory features
    • CpG islands
    • ENCODE Regulation
    • TFBS conserved
  
  
  
  • Where are the p53 peaks located in respect to the TSS (near/far/upstream/downstream)?
  • Find all peaks that overlap with a CpG site.
  • Find all peaks that overlap with a DNase cluster.
    
    
  • Extract the peak sequence from the peak that overlaps with CDKN1A
  
  
  
  • Find the exact TFBS in this peak with your p53 PFM (upregulated)
  • Map the exact binding site back to the ChIPseq peak regions in UCSC genome browser and display the result.
    
    
    
    
  • Hints

• Predict a TFBS motif using peak sequences from ChIPseq data
  
  ChIPseq peak-file: p53_macs_peaks.bed
  
  
  • Find the top 300 ChIPseq peaks, according to their -log10(p-value) in the 5th column:
    Import p53_macs_peaks.bed in Excel and sort descending, and save the first 300 lines as new BED file.
  • Extract the peak sequences from the top 300 ChIPseq peaks using the UCSC table browser.
  • Discover a TFBS motif with MEME-ChIP using the sequences from the top 300 peaks
  • How many sites contained the discovered motif?
  • Compare the motif (logo) obtained from the ChIPseq data with the motif(s) you generated in task 2.1 and the motifs from the Jaspar database.
  • Discuss the differences (if any). Which are the most similar motifs that can be found in the databases?