Lecture 15: HW2 Feedback Ultraconservation

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

2. Lecture 15: HW2 Feedback Ultraconservation http://cs273a.stanford.edu [Bejerano Fall10/11]

3. Ultraconserved Elements in theHuman Genome: The Hip & The Hype 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 Gill Bejerano Dept. of Developmental Biology Dept. of Computer Science Stanford University http://cs273a.stanford.edu [Bejerano Fall10/11] 3

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

5. Human Genome full of Conserved Non-Coding Elements Human Genome: 3*109 letters 1.5% known function compare to other species >50% junk >5% human genome functional 3x more functional DNA than known! ~106 genomic loci do not code for protein What do they do then? http://cs273a.stanford.edu [Bejerano Fall10/11]

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

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

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

9. Ultraconserved Elements fish using2 vs. 3species 481 elements perfectly conserved (100%id) over 200bp or more between human, mouse and rat. http://cs273a.stanford.edu [Bejerano Fall10/11] [Bejerano et al., Science 2004]

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

11. What exactly is an Ultraconserved Element? using3 vs. 43species Aha!! http://cs273a.stanford.edu [Bejerano Fall10/11]

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

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

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

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

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

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

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

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

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

23. Ultras are Functional Back in 2004 we hypothesized: 481 ultraconserved elements “nonexonic” subset – transcriptional regulators exonic subset – post transcriptional regulation [Pennacchio et al., Nature, 2006] [Ni et al., Genes Dev.; Lareau et al., Nature, 2007] http://cs273a.stanford.edu [Bejerano Fall10/11]

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

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

26. No Apparent Phenotype Touch an Ultra And You - DIY [Ahituv et al., PLoS Biology, 2007] http://cs273a.stanford.edu [Bejerano Fall10/11]

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

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

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

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

31. Ultras are Fiercely Retained through Evolution No Apparent Phenotype 100%id primates-dog: 1,691,090bp rodents deleted: 1,447bp (0.086%) Ultras are >300 fold more persistent than neutral DNA But Doomed ... the genomic deletion is (25% deleted) http://cs273a.stanford.edu [Bejerano Fall10/11]

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

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

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]

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