Genome scan for cis -regulatory DNA motifs associated with social behavior in honey bees

1. Genome scan for cis-regulatory DNA motifs associated with social behavior in honey bees Saurabh Sinha, Xu Ling, Charles W. Whitfield, Chengxiang Zhai, Gene E. Robinson PNAS 2006

2. Motivation • Background • Social regulation: nursing vs. foraging • Switching by age • Switching by need • Differential gene expression in the brain

3. Microarray analysis Individual brain gene expression profiles predict behavioral phenotype Genes are socially regulated C. W. Whitfield et al., Science 302, 296 -299 (2003)

4. Motivation • Question to answer cis-regulatory code social regulation of gene expression

5. Data Resources • Newly sequenced honey bee genome • Microarray analyses of genes in the brain in the context of socially regulated division of labor (3129 genes, ~25% of genes in bee genome)

6. Gene sets associated with socially regulated division of labor

7. Data Resources • Experimentally validated Drosophila motifs, mostly involved in the regulation of embryonic development in Drosophila.

8. Drosophila melanogastercis-regulatory motifs used in this study

9. Hmm… We have • Bee sequences • Bee gene sets associated with social regulation • Fly motifs Using all these data resources, what can we do? What questions can we answer?

10. Overview of methods • Hypothesis • Transcriptional networks involved in the regulation of embryonic development in Drosophila are also used to regulate adult behavioral development in honey bees. • General Scheme • Genome scan for cis-regulatory DNA motifs associated with social behavior

11. Hmm… How to answer this question using all these data?

13. Results #1 (test 1A) • Behaviorally related genes in honey bees have high G/C-content promoters • Most of the detected 41 motif–gene set associations involved G/C-rich motifs. • The overall Pearson’s correlation coefficient between G/C content of motif and the number of gene sets enriched for it was 0.7

14. Results #1 • The promoters for these gene sets were in general high in G/C content ? • Three gene sets were significantly enriched for G/C-rich promoters. (test 1B) • Four gene sets with G/C content correlated significantly with brain expression levels. (“test 2”) • Using a simple threshold-based classifier, promoter G/C content can classify a significant fraction of genes in some gene sets as being up- or down-regulated (eg., 74.6%manganese-responsive genes) (test 3)

15. Results #1 • G/C enrichment reflects regulatory phenomenon common to Drosophila too? No! • Hypergeometric tests: no association between Drosophila orthologs of the bee gene sets and high G/C promoter content • Gene Ontology (GO) analysis: The bee genes with the highest G/C-content promoters are significantly over-represented in transcriptional regulation and ectoderm, midgut, heart, and nervoussystem development. But not for Drosophila genes.

16. Hmm… • Why high G/C? • Any thoughts? • Take a guess of any reasons?

17. Question #1 The reasons for these bee/fly differences: • Involve enhanced transcription factor binding to known G/C-rich motifs in bees? • The presence of additional, as yet unidentified G/C-rich motifs in bees? • Species differences in methylation? • Species differences in gene expression? • Genes with brain-specific patterns of expression are known to have high G/C content promoters in humans

18. Results #2 • Behaviorally related genes in honey bees are enriched for cis-regulatory motifs of Hairy, CF1, Adf1, Dri and Snail • Factor out the effects of G/C content • Use local background in Stubb searching, and require more enriched for the motif than for G/C content • Use partial correlation coefficients

19. motif-gene set associationsvia enrichment or correlation analysis

20. Results #2 • Other evidences: • “Randomized” promoter sequence as negative control: same enrichment analysis revealed no statistically significant associations • For these 5 motifs, the genome has more promoters with extremely high Stubb scores than expected by chance • GO analysis: genes • for Adf1 and Dri are significantly associated with nervous system development • for Hairy are significantly associated with sensory organ development

21. Results #2 • Known functions of the corresponding transcription factors • Orthologs in bee genome show high degree of sequence identity for the DNA binding domains • However, the motif-gene set associations we found for honey bee were not detected in fly => specific to honey be social behavior?

22. Result #3 • Patterns of occurrence of Hairy, Snail and GAGA-binding factor classify expression patterns of honey bee behaviorally related genes • Cases where motif-based classification accuracy is statistically significant and G/C content-based classification accuracy is less so • SVM classified up- and down- regulated using all motifs got 71% accuracy with p=0.00018. (p>0.1 for using G/C content alone)

23. Classification of brain gene expression (up- or down-regulated) on basis of pattern of (single) motif occurrences

24. Result #4 • Cis-regulatory motifs show combinatorial regulation of honey bee behaviorally related genes • Pairs of cis-regulatory binding sites co-occur in the promoter regions of our genes • A cohesive set of 7 transcription factors (Adf1, Hairy, CF1 and GAGA again, plus AbdB, Zeste, Eve) have significant pairwise interaction

25. Combinatorial interactions of transcription factors

26. Hmm… • How to identify the pairs of cis-regulatory binding sites co-occur in the promoter regions ?

27. Conclusion • Differences in motif-gene set associations and promoter G/C content between honey bee and Drosophila might reflect unique aspects of gene regulation associated with social regulation • Demonstrate a robust association for social behavior, brain gene expression, and distributions of transcription factor binding sites throughout the genome • Transcriptional networks involved in the regulation of Drosophila embryonic development are re-used by nature for adult behavioral functions

28. Thanks!

29. Outline • Motivation • Data resources • Overview of methods • Results & discussion • Conclusion