Exploiting transcription factor binding site clustering to identify cis-regulatory modules involved in pattern formation in the Drosophila genome

1
Exploiting transcription factor binding site
clustering to identify cis-regulatory modules
involved in pattern formation in the Drosophila
genome

• ECS289A Presentation
• By Hua Chen
• 2003-3-3

2
Background Knowledge

• A significant character of cis-regulatory sites
  the multiple binding sites for different
  transcriptional factors tend to cluster together
  in one region around the gene, forming the
  Cis-Regulatory Modules (CRM).
• The searching of cis-regulatory sites gives out
  too many candidate positions, which make it
  difficult to tell the true ones
• The character of CRM provides a feasible method
  to identify the cis-regulatory sites in the
  genome.

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One example of CRM in Drosophilaeve gene
4
Targets
The System Investigated

• Adopt the clustering of cis-regulatory modules as
  a method to identify the functional motifs
• Test the method with some known real CRM regions
• Search the genome to discover CRMs and confirm
  the results by experiments.

• The early Drosophila embryo.
• Five transcriptional factors Bcd, Cad, Hb, Kr
  and Kni are investigated.

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Methods

• Collecting Transcription Factor Binding Sequences
  in preceding lab works and doing Alignment
• Construction of Position Weight Matrices (PWM)
  for the conserved motifs.
• Test the method with the known CRMs
• Genome-wide Searching for unknown regulatory
  regions
• mRNA Hybridization and Microarray hybridization
  to test whether the predicted regions are near to
  genes under regulation of the Transcription
  Factors
• One special case giant gene, further
  investigated by Transgenics and Mutant Embryo.

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Step1 Collection and Alignment of TF Binding
Sites

• Bcd, Cad, Hb, Kr, Kni binding sequences are
  determined by in vitro DNAse protection assays
• The sequences are aligned with MEME.

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(No Transcript)
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Step 2 Construction of PWMs and Searching

• Patser is used to construct the Position Weight
  Matrix
• Cis-Analyst is used to identify the potential
  binding sites matching to the PWM in the
  Drosophila genome.
• A user-defined cutoff parameter (site_p) to
  eliminate predicted low-affinity sites
• Search the sequence with a specified window
  length
• Retain the windows that contain at least
  min_sites binding sites
• Merge all overlapping windows into a cluster.

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Binding Site Sequence for Cad
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Binding Sites
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(No Transcript)
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Step 3 Collection of Known CRMs
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Successful Result 14/19with the searching
criteria window-size700 bp, number of predicted
sitesgt13
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Step 4 Genome-wide Searching

• 28 clusters identified
• 23 out of 28 fall in regions between genes
• 5 in the intron regions
• 49 genes in the nearby regions.

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Step 5 Examine the expression pattern of the 49
genes by RNA in situ hybridization and microarray
hybridization

• The 49 genes are examined by hybridizations to
  see whether they show the pattern of under
  regulation of the TFs
• 10 out of the 28 clusters are near to at least
  one gene show the anterior-posterior expression
  pattern (Under regulation of the five TFs).

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Step 6 The special case giant gene

• The posterior expression is regulated by
  Cad,Hb,Kr
• The cis-regulatory sites are still unknown
• The predicted CRM nearest to the giant gene is
  cloned to the upstream of lacZ reporter gene.
• The lacZ gene show a similar expression pattern
  as the giant mRNA.

• / Kr/Kr

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Conclusions

• Binding site clustering is an effective method to
  identify cis-regulatory modules
• A major block is the paucity of the binding data
  for most transcription factors, which need a
  systematical work
• The real CRM structures is more complex, it needs
  to incorporate more complex rules in the method.

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Reference

• Berman, B.P., Nibu, Y. et al. 2001. Exploiting
  transcription factor binding site clustering to
  identify cis-regulatory modules involved in
  pattern formation in the Drosophila genome.
  P. N. A. S. 99757-762