1. http://cs273a.stanford.edu [Bejerano Fall10/11] 1

2. 2 Lecture 15: HW2 Feedback Ultraconservation

3. http://cs273a.stanford.edu [Bejerano Fall10/11] 33 GGTGCCAGGGAAAGGGCAGGAGGTGAGTGCTGGGAGGCAGCTGAGGTCAACTTCTTTTGAACTTCCACGTGGTATTTACTCAGAGCAATTGGTGCCAGAG GCTCAGGGCCCTGGAGTATAAAGCAGAATGTCTGCTCTCTGTGCCCAGACGTGAGCAGGTGAGCAGCTGGGGCGAAAGACCTGTTGGAGGCTATGAATGC AATCAAGGTGACAGACAACTGGTGCAATGATGGTAGTGGAAATGGAGGAGAGGGGATTGATTCAAGATGCATTTAGGACCAAGAATCGGGAGCTTGTGAA CGTGTGTATGAGTACTGTAGACGGAGTGGGTGTGTCATCAGAGAAGATCTGAGCATTTGGGCTTGCTCTCCTCAGAGGCCCTGCGAGTGGAGTTCAGCTT TTCCTCATGGGGCAAATCTCACTTTCGCTCCAGTTCCTGGGGCTCAGAGTCCCTGGCCCAGATGCCTCTTGCCATCTCATCTTCACCCTGCCTGGCTTCC CTTGCTTGTTCCAGGATTGTTTCATAAAGAGGGATGTGGTTGGTCTTTAACCCTATGAATGCTGGCTGAGGATGCCTGCGGAACCTGTAGTGAAGCTTTC AGGGGCTGCTCGGGTTCTGGCTGGTAGGTGAACACTGTCCATCTTGCCGGCTGGGACACAGTGACTCTGGGTAGTTGTGTAAGAGAGGGGCCCTTGGCAG ACAAACAGGTTCTTCTCTGTTGGTGGGCCAGCCAGCAGGTCAGTGGGAAGGTTAAAGGTCATGGGGTTTGGGAGAACTGGGTGAGGAGTTCAGCCCCATC CCCCGTAAAGCTCCTGGGAAGCACTTCTCTACTGGGGCAGCCCCTGATACCAGGGCACTCATTAACCCTCTGGGTGCCAGGGAAAGGGCAGGAGGTGAGT GCTGGGAGGCAGCTGAGGTCAACTTCTTTTGAACTTCCACGTGGTATTTACTCAGAGCAATTGGTGCCAGAGGCTCAGGGCCCTGGAGTATAAAGCAGAA TGTCTGCTCTCTGTGCCCAGACGTGAGCAGGTGAGCAGCTGGGGCTGTCTGCTCTCTGTGCCCAGACGTGAGCAGGTGAGCAGCTGGGGCTGTCTGCTCT CTGTGCCCAGACGTGAGCAGGTGAGCAGCTGGGGCTGTCTGCTCTCTGTGCCCAGACGTGAGCAGGTGAGCAGCTGGGGCTGTCTGCTCTCTGTGCCCAG GAAAGACCTGTTGGAGGCTATGAATGCAATCAAGGTGACAGACAACTGGTGCAATGATGGTAGTGGAAATGGAGGAGAGGGGATTGATTCAAGATGCATT TAGGACCAAGAATCGGGAGCTTGTGAACGTGTGTATGAGTACTGTAGACGGAGTGGGTGTGTCATCAGAGAAGATCTGAGCATTTGGGCTTGCTCTCCTC AGAGGCCCTGCGAGTGGAGTTCAGCTTTTCCTCATGGGGCAAATCTCACTTTCGCTCCAGTTCCTGGGGCTCAGAGTCCCTGGCCCAGATGCCTCTTGCC ATCTCATCTTCACCCTGCCTGGCTTCCCTTGCTTGTTCCAGGATTGTTTCATAAAGAGGGATGTGGTTGGTCTTTAACCCTATGAATGCTGGCTGAGGAT GCCTGCGGAACCTGTAGTGAAGCTTTCAGGGGCTGCTCGGGTTCTGGCTGGTAGGTGAACACTGTCCATCTTGCCGGCTGGGACACAGTGACTCTGGGTA GTTGTGTAAGAGAGGGGCCCTTGGCAGACAAACAGGTTCTTCTCTGTTGGTGGGCCAGCCAGCAGGTCAGTGGGAAGGTTAAAGGTCATGGGGTTTGGGA GAAACTGGGTGAGGAGTTCAGCCCCATCCCCCGTAAAGCTCCTGGGAAGCACTTCTCTACTGGGGCAGCCCCTGATACCAGGGCACTCATTAACCCTCTG GGTGCCAGGGAAAGGGCAGGAGGTGAGTGCTGGGAGGCAGCTGAGGTCAACTTCTTTTGAACTTCCACGTGGTATTTACTCAGAGCAATTGGTGCCAGAG GCTCAGGGCCCTGGAGTATAAAGCAGAATGTCTGCTCTCTGTGCCCAGACGTGAGCAGGTGAGCAGCTGGGGCGAAAGACCTGTTGGAGGCTATGAATGC AATCAAGGTGACAGACAACTGGTGCAATGATGGTAGTGGAAATGGAGGAGAGGGGATTGATTCAAGATGCATTTAGGACCAAGAATCGGGAGCTTGTGAA CGTGTGTATGAGTACTGTAGACGGAGTGGGTGTGTCATCAGAGAAGATCTGAGCATTTGGGCTTGCTCTCCTCAGAGGCCCTGCGAGTGGAGTTCAGCTT TTCCTCATGGGGCAAATCTCACTTTCGCTCCAGTTCCTGGGGCTCAGAGTCCCTGGCCCAGATGCCTCTTGCCATCTCATCTTCACCCTGCCTGGCTTCC CTTGCTTGTTCCAGGATTGTTTCATAAAGAGGGATGTGGTTGGTCTTTAACCCTATGAATGCTGGCTGAGGATGCCTGCGGAACCTGTAGTGAAGCTTTC AGGGGCTGCTCGGGTTCTGGCTGGTAGGTGAACACTGTCCATCTTGCCGGCTGGGACACAGTGACTCTGGGTAGTTGTGTAAGAGAGGGGCCCTTGGCAG ACAAACAGGTTCTTCTCTGTTGGTGGGCCAGCCAGCAGGTCAGTGGGAAGGTTAAAGGTCATGGGGTTTGGGAGAACTGGGTGAGGAGTTCAGCCCCATC CCCCGTAAAGCTCCTGGGAAGCACTTCTCTACTGGGGCAGCCCCTGATACCAGGGCACTCATTAACCCTCTGGGTGCCAGGGAAAGGGCAGGAGGTGAGT GCTGGGAGGCAGCTGAGGTCAACTTCTTTTGAACTTCCACGTGGTATTTACTCAGAGCAATTGGTGCCAGAGGCTCAGGGCCCTGGAGTATAAAGCAGAA TGTCTGCTCTCTGTGCCCAGACGTGAGCAGGTGAGCAGCTGGGGCTGTCTGCTCTCTGTGCCCAGACGTGAGCAGGTGAGCAGCTGGGGCTGTCTGCTCT CTGTGCCCAGACGTGAGCAGGTGAGCAGCTGGGGCTGTCTGCTCTCTGTGCCCAGACGTGAGCAGGTGAGCAGCTGGGGCTGTCTGCTCTCTGTGCCCAG Ultraconserved Elements in the Human Genome: The Hip & The Hype Dept. of Developmental Biology Dept. of Computer Science Stanford University Gill Bejerano

4. http://cs273a.stanford.edu [Bejerano Fall10/11] 4 Sequence Conservation implies Function which (but which function/s?...) human mouse mammalian ancestor...CTTTGCGA-TGAGTAGCATCTACTATTT......ACGTGGGACTGACTA-CATCGACTACGA... functional region! Comparative Genomics of related species highlights:

5. http://cs273a.stanford.edu [Bejerano Fall10/11] 5 Human Genome: 3*10 9 letters Human Genome full of Conserved Non-Coding Elements 1.5% known function >50% junk 3x more functional DNA than known! compare to other species >5% human genome functional ~10 6 genomic loci do not code for protein What do they do then?

6. http://cs273a.stanford.edu [Bejerano Fall10/11] 6 Conserved elements in the Human Genome all human-mouse alignments human-mouse ancestral repeats alignment Difference: 5% of Human Genome [Mouse consortium, Nature 2002] election human-mouse ancestral repeats alignment 85%id on average

7. http://cs273a.stanford.edu [Bejerano Fall10/11] 7 Conserved elements in the Human Genome all human-mouse alignments human-mouse ancestral repeats alignment Difference: 5% of Human Genome election human-mouse ancestral repeats alignment 85%id on average Ultraconservation

8. http://cs273a.stanford.edu [Bejerano Fall10/11] 8 Typical DNA Conservation levels Conserved elements between human and mouse are on average 85% identical. [mouse consortium, 2002] (dot = base identical to human)

9. http://cs273a.stanford.edu [Bejerano Fall10/11] 9 Ultraconserved Elements [Bejerano et al., Science 2004] fish 481 elements perfectly conserved (100%id) over 200bp or more between human, mouse and rat. using 2 vs. 3 species

10. http://cs273a.stanford.edu [Bejerano Fall10/11] 10 Contamination

11. http://cs273a.stanford.edu [Bejerano Fall10/11] 11 What exactly is an Ultraconserved Element? Aha!! using 3 vs. 43 species

12. http://cs273a.stanford.edu [Bejerano Fall10/11] 12 Ultraconservation as a Phenomenon Few species More and more species Hmmm….

13. http://cs273a.stanford.edu [Bejerano Fall10/11] 13 Ultraconserved Elements: Why? Hundreds of long genomic regions identical between amniotes  they must have rejected many different changes. But... all functions we understand in our genome are encoded using redundant codes. * * * * * CDSncRNATFBS seq.

14. http://cs273a.stanford.edu [Bejerano Fall10/11] 14 Conserved elements in the Human Genome all human-mouse alignments human-mouse ancestral repeats alignment Difference: 5% of Human Genome election human-mouse ancestral repeats alignment 85%id on average Ultraconservation Why did I look at the tail?

15. http://cs273a.stanford.edu [Bejerano Fall10/11] 15...ACGTACGACTGACTAGCATCGACTACGA........TCTGACTAGCATCGACTACGA... DNA Replication is Imperfect It’s imperfect on all scales: small, medium and large. In particular it begets novel functional entities:...ACGTACGACTGACTAGCATCGACTACGA......ACGTACGACTGACTAGCATCGACTACGA........TCTGACTAGCATCGACTACGA... functional junk functional functional’’ functional’ regional duplication functional divergence Protein & RNA gene families come to life this way. What else does?

16. http://cs273a.stanford.edu [Bejerano Fall10/11] 16 Computational Approach I Group them into paralog families of human functional regions of common origins: Annotated members induce function on all. Examine core, substitutions in family. Test for “guilt by association”. [Bejerano et al., ISMB 2004].....ACGTGCATGACTGACTAGCATCAGACGACTAC..GATAATACGCTACGACTAGCTAC..... human DNA...TGACTAGCATCGACTAC..GATAATACGAC......CATCGACTAC..GATAATACGACGGTTGGT... AC T ~400bp

17. http://cs273a.stanford.edu [Bejerano Fall10/11] 17 Functional Annotation by Families [Bejerano et al., ISMB 2004] Puzzling News: 96% of the 700,000 appear unique(!) Good News: We still find 12,027 families novel putative ncRNAs, cis-regulatory elements, etc. After removing from top 5% Human all annotated regions, and more: 700,000 elements, covering 3.5% Human Genome

18. http://cs273a.stanford.edu [Bejerano Fall10/11] 18 human mouse rat related genes related elements (75%id over 200bp) same element 96%id over 200bp same element 95%id over 200bp Computational Approach II Classical Biological approach: experiment to understand these regions Computational approach: how many regions like this or “better” are there?

19. http://cs273a.stanford.edu [Bejerano Fall10/11] 19 Out popped the Ultraconserved Elements Puzzling News: 96% of the 700,000 conserved non-coding elements appear unique(!) Same with Ultras

20. http://cs273a.stanford.edu [Bejerano Fall10/11] 20 What could ultras be doing? exonic non possibly

21. Associating distal peaks in a gene-based context is statistically inappropriate 21 Gene transcription start site Ultraconserved Element Ontology term (e.g. ‘development’) http://cs273a.stanford.edu [Bejerano Fall10/11] N = 8 genes in genome K = 3 genes annotated with n = 3 genes selected by proximal peaks k = 2 selected gene annotated with P = Pr(k ≥1 | n=2, K =3, N=8) 1.Set gene regulatory domain. 2.Associate Ultras with genes. 3.Per ontology term, count annotated genes selected. 4.Rank terms by enrichment hypergeometric p-value. Evolved into http://great.stanford.edu/

22. Enrichment Association of Ultraconserved Elements 22 Exonic Ultras Non-exonic Ultras http://cs273a.stanford.edu [Bejerano Fall10/11]

23. 23 Ultras are Functional Back in 2004 we hypothesized: 481 ultraconserved elements exonic subset – post transcriptional regulation [Ni et al., Genes Dev.; Lareau et al., Nature, 2007] “nonexonic” subset – transcriptional regulators [Pennacchio et al., Nature, 2006]

24. http://cs273a.stanford.edu [Bejerano Fall10/11] 24 Ultraconserved Non-coding RNA [Calin et al, Cancer Cell, 2007] miRNA complementarity About 1/3 of all ultras are expressed. Some are predicted to provide microRNA targets. A few are anti-correlated with miRNA expression levels. A few even act as oncogenes.

25. http://cs273a.stanford.edu [Bejerano Fall10/11] 25 Ultras are Under Strong Human Selection Ultra DAF NonSyn DAF [Katzman et al, Science,2007] Mutational cold spots? NO. Rare (new) mutations are introduced to the population. Fierce purifying selection? YES. Very few of these get anywhere near fixation. chimp A humans G A A A

26. http://cs273a.stanford.edu [Bejerano Fall10/11] 26 Touch an Ultra And You - DIY [Ahituv et al., PLoS Biology, 2007]

27. http://cs273a.stanford.edu [Bejerano Fall10/11] 27 What can’t we measure in the lab? N e is population size, s selective dis/advantage. Both of which are VERY wrong in the lab.

28. http://cs273a.stanford.edu [Bejerano Fall10/11] 28 So it can happen – but does it FIX? t DNA element mouse

29. http://cs273a.stanford.edu [Bejerano Fall10/11] 29 Count Fraction Lost, Binned by %id t human macaque dog mouse rat 100bp sliding window count_all count_hole bin by %id human dog rat mouse macaque

30. http://cs273a.stanford.edu [Bejerano Fall10/11] 30 Quite Some Time Later [McLean & Bejerano, Genome Res., 2008]

31. http://cs273a.stanford.edu [Bejerano Fall10/11] 31 Ultras are Fiercely Retained through Evolution Ultras are >300 fold more persistent than neutral DNA (25% deleted) genomic deletion the genomic deletion is 100%id primates-dog: 1,691,090bp rodents deleted: 1,447bp (0.086%)

32. http://cs273a.stanford.edu [Bejerano Fall10/11] 32 How special are the Ultras? election Ultraconservation

33. http://cs273a.stanford.edu [Bejerano Fall10/11] 33 Ultraconservation as a Phenomenon Few species More and more species Hmmm…. We do not see a bump in the curve

34. Ultraconserved Elements: What do we know? Excessive sequence conservation exists. Set is heterogeneous from a functional perspective. Four can be KO-ed with no clear phenotype. Yet, the set is under extreme selection in natural populations, both for mutations and deletions. Most ultras have deep orthology, and no paralogy. One ultra comes from a mobile element co-option events. Others may have come from similar events. Ultras appear the tip of a continuum, not a unique peak. http://cs273a.stanford.edu [Bejerano Fall10/11] 34

35. Ultraconserved Elements: What we don’t What maintains so much conservation? http://cs273a.stanford.edu [Bejerano Fall10/11] 35 * * * * *