1. Nora Pierstorff Dept. of Genetics University of Cologne 30.8.2005
Combined ab initio and comparative analysis of putative regulatory regions
Nora Pierstorff
Dept. of Genetics
University of Cologne

2. Outline Introduction Ab Initio Approach Datasets
Comparative Analysis of Enhancers and Results
Combination of Both Approaches and Results
Discussion

3. Eukaryotic regulation model

4. 3 Approaches
Search for binding sites of known transcription factors using Position Weight Matrices.
Search for conserved motifs in upstream-regions of homolog or coregulated genes.
Search statistical overrepresented motifs

5. Outline Introduction Ab Initio Approach Datasets
Comparative Analysis of Enhancers and Results
Combination of Both Approaches and Results
Discussion

6. Ab Initio Approach (overrepresented patterns)
overrepresented patterns are frequent in the DNA => many false positive predictions
amount of available data is not large enough to find additional reliable universally valid rules

7. Outline Introduction Ab Initio Approach Datasets
Comparative Analysis of Enhancers and Results
Combination of Both Approaches and Results
Discussion

8. Dataset (collected by Nazina et al. 2003)
target-species: Drosophila melanogaster
reference species:
D. yakuba
D. ananassae
D. pseudoobscura
D. virilis
# sequences: 39 # bp:
# regulatory regions: 87 # bp in enh:
enhancer/sequence: 2.462
amount of bp in enhancers:
Dorsal motif     dorsal matches

9. Outline Introduction Ab Initio Approach Datasets
Comparative Analysis of Enhancers and Results
Combination of Both Approaches and Results
Discussion

10. Are enhancers alignable?
Emberly et al. (2003)
the overlap of binding sites and conserved sequence blocks is not much greater than by chance, but still statistically significant
compared organisms: D. melanogaster and D. pseudoobscura
alignment methods: LAGAN, SMASH (construct chains of local alignments)

11. Assumptions about enhancer conservation
binding sites contain core sequences essential to bind transcription factor
core sequences are conserved between binding sites of one species and between species
binding sites are indicated by short, exactly conserved, overrepresented patterns

12. Alignment of short exact matches
input: chain of high scoring fragments from blastn alignment of each sequence pair
output: regions containing a high amount of short conserved stretches

13. Outline Introduction Ab Initio Approach Datasets
Comparative Analysis of Enhancers and Results
Combination of Both Approaches and Results
Discussion

14. Result using only comparative approach with 5 species
m8 region
score = number of short conserved stretches in a 200bp window

16. Outline Introduction Ab Initio Approach Datasets
Comparative Analysis of Enhancers and Results
Combination of Both Approaches and Results
Discussion

17. searching overrepresented motifs in conserved region
input: all short conserved words
1. step: counting the occurrence of all 5bp-substrings of the word in the 1000 surrounding basepairs
2. calculating one observed/expected ratio for every species
output: conserved stretches containing at least one 5mer which is overrepresented in each species

18. Outline Introduction Ab Initio Approach Datasets
Comparative Analysis of Enhancers and Results
Combination of Both Approaches and Results
Discussion

19. Improvement by combination
m8 region
score = number of short conserved stretches in a 200bp window
m8 region
score = number of short conservedoverrepresented stretches in a 200bp window

20. improvement by combination

21. Outline Introduction Ab Initio Approach Datasets
Comparative Analysis of Enhancers and Results
Combination of Both Approaches and Results
Discussion

22. Discussion use of a combination of methods improves predictions
in nearest future regulatory regions can be found without knowing the binding transcription factors, if enough related species are known.
more features to differ between conserved regulatory regions and other functional conserved regions need to be found

23. References
E. Emberly, N. Rajewsky, E. Siggia (2003) Conservation of regulatory elements between two species of Drosophila BMC Bioinformatics 2003, 4:57
A. Nazina, D. Papatsenko (2003) Statistical extraction of Drosophila cis-regulatory modules using exhaustive assessment of local word frequency. BMC Bioinformatics Dec 22;4:65.