Identification of a Novel cis-Regulatory Element Involved in the Heat Shock Response in Caenorhabditis elegans Using Microarray Gene Expression and Computational Methods

1
Identification of a Novel cis-Regulatory Element
Involved in the Heat Shock Response in
Caenorhabditis elegans Using Microarray Gene
Expression and Computational Methods

• Debraj Guha Thakurta, Lisanne Palomar, Bary D.
  Stormo, Pat Tedesco, Thomas E. Johnson, Davis W.
  Walker, Gordon Lithgow, Stuart Kim, and
  Christopher D. Link
• Presented by
• Abel G. Gezahegne
• ECS 289A
• February 24, 2003

2
Overview

• Monitor 12,000 genes from C. elegans to
  determine genes up-regulated on heat shock (HS).
• Analyze the upstream regions of these genes using
  computational DNA pattern recognition methods to
  identify any cis-regulatory motifs.
• Determine the significance of these motifs using
  statistical methods.
• Perform comparative sequence analysis to
  determine if any cross-species conservations
  exist.

3
Microarray Experiment

• Determine Gene expression patterns before and
  after HS using DNA Microarray for 11,917 known
  and predicted C. elegans genes.
• Animals were harvested as young adults and then
  split in two halves HS population and control
  population.
• 5 independent HS experiments at 35OC In two
  experiment animals were harvested after 1 hr of
  HS. In three experiments animals were heat
  shocked for 2 hrs and allowed to recover at 20OC
  for 2 hrs then harvested.

4
Software Tools

• Consensus a greedy algorithm that searches for
  a matrix with a low probability of
  occurring by chance.
• ANN-Spec an algorithm based on Artificial
  Neural Network and Gibbs sampling method
  to discover un-gapped patterns in
  DNA sequences
• GLASS Graphical Language for Assembly of
  Secondary Structures a sequence
  alignment algorithm.
• Patser given weight matrix identifies high
  scoring subsequences and
  calculates p values.

5
Gene Identification

• Identified 28 genes induced in at least four of
  the five experiments and over-expressed by a
  factor of two or more.
• Because of noise in DNA Microarray considered
  only genes up-regulated by an average factor of
  four or more.

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Gene Identification (cont.)

• Used 500 bp upstream from transcription start
  site to select candidates for promoter elements.
• Two DNA motifs identified by Consensus and
  ANN-Spec.
• HSE - TTCTAGAA, a well known DNA binding site
  for HS Transcription Factors (HSF).
• HSAS - GGGTGTC, un unknown motif that does not
  correspond to any known TF binding site.

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Mathematical Model

• Probability of a protein binding to a site with a
  score s
• P(bounds) ? es
•  When multiple binding sites exist, probability
  of binding
• Pmseq ?sites es
•  Geometric Mean of the pp-values
•  lt Pmseq gt ?Sseq ?sites es 1/N
• Difference of the log geometric means of the
  pp-values
• DLGM log lt Pmseq gtHS - lt Pmseq gtRand
•  

8
Statistical Significance

• Use the DLGM to determine the cutoff scores using
  the 13 up-regulated genes and 3000 random genes
  from the C. elegans genome.
• DLGM log lt Pmseq gtHS - log lt Pmseq gtRand
• At a low cutoff value there are substantial
  amount of low scoring sequences thus DLGM is low.
• At a high cutoff even the high scoring sequences
  are not being used thus DLGM drops.
• The cutoff score that maximizes DLGM is chosen as
  the appropriate cutoff value.

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Cross-Species Conservation

• To study conservation of regulatory sites across
  related species two orthologous gene pairs were
  examined between C. elegans and C. briggsae.
• The pattern of HSE and HSAS sites on the
  promoters indicate conservation across closely
  related species.
• Output from VISTA (VISualization Tools for
  Alignment.

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Cross-Species C. (cont)

• The gene structure and distances between the
  genes are similar in both organisms.
• The two genes share 450 nt in the upstream DNA
  sequence.
• Output from GLASS alignment algorithm.

11
Mutant Promoter Construct

• A single mutation of HSE or HSAS still results in
  a significant expression level of GFP (green
  fluorescence protein).
• Mutation of all three or two sites of HSEs or
  one HSEs and the HSAS results in dramatic
  reduction is expression level.

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Remarks and Conclusion

• Since Microarray data was conducted for 2/3 of
  the C. elegans genes, there may exist other HS
  induced genes.
• Through experiments and statistical methods the
  novel cis-regulatory element discovered has been
  shown to play a significant role in heat shock
  response.
• This has also shown computational methods can be
  a valuable tool in discovery of novel regulatory
  elements.