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

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Title: 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.

3
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.

5
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.

6
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.

7
(No Transcript)
8
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.

9
Binding Site Sequence for Cad
10
Binding Sites
11
(No Transcript)
12
Step 3 Collection of Known CRMs
13
Successful Result 14/19with the searching
criteria window-size700 bp, number of predicted
sitesgt13
14
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.

15
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).

16
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

17
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.

18
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
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