MW 12:50-2:05pm in Beckman B302 Profs: Serafim Batzoglou & Gill Bejerano

1. CS273A Lecture 15: Evolutionary Genomics MW  12:50-2:05pm in Beckman B302 Profs: Serafim Batzoglou & Gill Bejerano TAs: HarendraGuturu & PanosAchlioptas http://cs273a.stanford.edu [BejeranoFall13/14]

2. Announcements • Projects are rolling.. http://cs273a.stanford.edu [BejeranoFall13/14]

3. Sweeps Negative Selection Positive Selection http://cs273a.stanford.edu [BejeranoFall13/14]

4. The Species Tree S S Sampled Genomes S Speciation Time

5. EvoDevo Evolutionary developmental biology (evolution of development or informally, evo-devo) is a field of biology that compares the developmental processes of different organisms to determine the ancestral relationship between them, and to discover how developmental processes evolved. … it now appears that just as evolution tends to create new genes from parts of old genes (molecular economy), evo-devo demonstrates that evolution alters developmental processes to create new and novel structures from the old gene networks or will conserve a similar program in a host of organisms. http://cs273a.stanford.edu [BejeranoFall13/14]

6. What makes us molecularly human? Searching Near … http://cs273a.stanford.edu [BejeranoFall13/14]

7. Why compare to Chimp? http://cs273a.stanford.edu [BejeranoFall13/14]

8. Humans and Chimpanzees Possess Many Vastly Different Phenotypes A: Chimp B: Human A B A B 8 [Varki, A. and Altheide, T., Genome Res., 2005]

9. Disease Susceptibility Differences http://cs273a.stanford.edu [BejeranoFall13/14]

10. What human-chimp changes do we find? Small Large Medium http://cs273a.stanford.edu [BejeranoFall13/14]

11. Large differences Fusion (HSA 2) 18 pericentromeric inversions http://cs273a.stanford.edu [BejeranoFall13/14]

12. Medium Sized Differences Gene families expandand contract Mobile element insertion and mediated deletion http://cs273a.stanford.edu [BejeranoFall13/14]

13. Small Differences 1% difference at the base level http://cs273a.stanford.edu [BejeranoFall13/14]

14. Genetic basis of human phenotypes? Phenotype Genotype Number of rearrangements Most mutationsare near/neutral. How do we know? 4D sites, ARs. http://cs273a.stanford.edu [BejeranoFall13/14]

15. What functional instructions can change? • Human/chimp genome: ~3*109bp • Rough composition: • Genes 2% • Non-coding RNAs 1% • Regulatory DNA 10-20% • (Repeats 40%) • (Other 30-40%) http://cs273a.stanford.edu [BejeranoFall13/14]

16. Is it our genes? • 70-80% of all human-chimp orthologous proteins differ. • On average they differ by 1-2 amino acids. • Which amino acid changes matter? • One can also compare non-synonymous amino acid substitutions with synonymous changes, and look for proteins unusually enriched from the former.Those may be evolving under positive selection. http://cs273a.stanford.edu [BejeranoFall13/14]

17. Positive and negative gene selection in the human genome http://cs273a.stanford.edu [BejeranoFall13/14]

18. Candidate genes for human specific evolution ... http://cs273a.stanford.edu [BejeranoFall13/14]

19. The KE family This is a unique family, 3 generations, in which 15 out of 24 members suffer from severe speech and language difficulties. Remaining relatives are unaffected.

20. FOXP2 – evolutionary perspective • Mutations in a single gene explain family KE. • There are 3 aa differences between human and mouse but two of them occurred after the divergence of chimpanzee.

21. What if we did an unbiased search? rapid change Human Chimp Chimp Human conserved • HAR1: • Novel ncRNA • 18 unique human substitutions [Pollard, K. et al., Nature, 2006] [Beniaminov, A. et al., RNA, 2008] Human-specific substitutions in conserved sequences 21

22. Human Accelerated Regions rapid change Human Chimp • HAR2/HACNS1: • Between CENTG2 / GBX2. • Mouse element is not an enhancer (where assayed). conserved Human-specific substitutions in conserved sequences 22 [Prabhakar, S. et al., Science, 2008]

23. Biased Gene Conversion http://cs273a.stanford.edu [BejeranoFall13/14]

24. Different Unbiased Search: Loss vs Gain Human Accelerated Regions rapid change Human • 4-18 unique human substitutions • Pollard, K. et al., Nature, 2006 • Prabhakar, S. et al., Science, 2008 Chimp conserved Human Conserved Sequence Deletions (hCONDELs) deleted! Human • Complete human loss of sequence • Likely to confer human-specific phenotypes Chimp [McLean, Reno, Pollen et al., Nature, 2011] conserved http://cs273a.stanford.edu [BejeranoFall13/14]

25. Identifying hCONDELs deleted! Human Chimp conserved http://cs273a.stanford.edu [BejeranoFall13/14]

26. hCONDEL genomic distribution • Median size: 2.8kb • Not enriched in highly variable genomic regions • Most do not disrupt proteins: only 1 validated exonic deletion http://cs273a.stanford.edu [BejeranoFall13/14]

27. Deletions of functional non-coding DNA ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) Gene Gene Gene Gene Gene Gene Gene Gene Gene Gene Gene with function e.g. “neuronal gene” Gene without function Conserved element hCONDEL Gene Gene http://great.stanford.edu [McLean et al., Nat. Biotechnol., 2010] http://cs273a.stanford.edu [BejeranoFall13/14]

28. Functional enrichments of hCONDELs Ontology Term p-value Gene Ontology Steroid hormone receptor activity 3.73 x 10-4 InterPro Fibronectin, type III 1.01 x 10-4 Zinc finger, nuclear hormone receptor type 1.80 x 10-4 CD80-like, immunoglobulin C2 set 1.37 x 10-3 Entrez Gene Neuronal genes 1.11 x 10-4 Monoallelically-Expressed Genes Monoallelic expression 8.62 x 10-3 These enrichmentsare unique to hCONDELs http://great.stanford.edu http://cs273a.stanford.edu [BejeranoFall13/14]

29. hCONDEL near Androgen Receptor The deletion appears fixed in humansand appears deleted in Neandertal. http://cs273a.stanford.edu [BejeranoFall13/14]

30. Androgen Receptor chimpanzee enhancer assay Androgen Receptor Human Chimp Genomic fragment Hsp68 promoter LacZ reporter gene [Phil Reno, David Kingsley] http://cs273a.stanford.edu [BejeranoFall13/14]

31. The human deletion near AR acts as an enhancer within known AR expression domains Sensory whiskers Genital tubercle Chimp enhancer E16.5 E16.5 E16.5 Penile spines Mouse enhancer 8 weeks E16.5 E16.5 [Phil Reno, David Kingsley] http://cs273a.stanford.edu [BejeranoFall13/14]

32. Androgen Receptor Testosterone Nucleus AR+T dimer AndrogenReceptor Cell Androgen Receptor Human Chimp http://cs273a.stanford.edu [BejeranoFall13/14]

33. Androgen responsiveness in domains of expression Penile spines Sensory whiskers Sensory whisker length (mm) Sensory Penile whiskers spines Mice with Ar coding region mutations lack penile spines Galago [Murakami, 1987] [Ibrahim & Wright 1983] [Dixson, 1976] http://cs273a.stanford.edu [BejeranoFall13/14]

34. Could sequence loss lead to tissue gain? ( ) • hCONDELs enriched for suppressors of cell proliferation or cell migration expressed in cortex (P=1.3 x 10-3) Non-human mammals Humans Do not suppress proliferation Suppress proliferation http://cs273a.stanford.edu [BejeranoFall13/14]

35. hCONDEL 75 kb downstream of GADD45g • GADD45g expressed in the developing forebrain subventricular zone • GADD45g represses cell cycle and activates apoptosis • GADD45g expression loss linked to human pituitary adenoma growth The deletion appears fixed in humans and appears deleted in Neandertal. http://cs273a.stanford.edu [BejeranoFall13/14]

36. Deletion near GADD45g is an enhancer for a subset of GADD45g expression domains • SVZ is a progenitor amplification domain for neurons both in ventral regions and neocortex VT VT VZ SVZ Se POA Se POA E14.5 E14.5 E14.5 Ventral forebrain expression domains: Septum (Se), Preoptic area (POA), Ventral thalamus (VT) Kriegstein et al., 2006; Taglialatela et al., 2004; Gelman et al., 2009 [Alex Pollen, David Kingsley] http://cs273a.stanford.edu [BejeranoFall13/14]

37. What makes us molecularly human? … Searching Far http://cs273a.stanford.edu [BejeranoFall13/14]

38. The Vertebrate-Invertebrate Divide Nematode Tunicate Fly Human NOT just ultras, ALL conserved non-coding sequence DISAPPEARS at the vertebrate/invertebrate boundary. http://cs273a.stanford.edu [BejeranoFall13/14]

39. The Bilaterian Tree of Life Human Zebrafish Tunicates Amphioxus Acorn Worm Sea Urchin Insects Tick Nematode Sea Hare Owl Limpet Ancestor to all bilaterians ~650 MYA

40. Conserved embryonic morphologies Bilateral symmetry Human Zebrafish Tunicates Amphioxus Acorn Worm Sea Urchin Insects Tick Nematode Sea Hare Owl Limpet Ancestor to all bilaterians ~650 MYA

41. Conserved signaling pathways Human Zebrafish Tunicates Amphioxus Acorn Worm Sea Urchin Insects Tick Nematode Sea Hare Owl Limpet Ancestor to all bilaterians ~650 MYA

42. Conserved developmental transcription factors Hox genes Human Zebrafish Tunicates Amphioxus Acorn Worm Sea Urchin Insects Tick Nematode Sea Hare Owl Limpet Ancestor to all bilaterians ~650 MYA

43. Ancient Regulatory Circuits Signaling pathway Transcription factor Enhancer Target gene ?

44. Hypotheses • The enhancer components of ancient regulatory circuits are entirely newly evolved. • Ancient regulatory circuits do utilize ancient enhancers, but these enhancers have gone undetected by standard approaches.

45. If At First You Don't Succeed, Try, Try Again Thousands of putative vertebrate enhancers All publicly available non-vertebrate metazoan sequence data Very sensitive sequence alignment Filters for alignment quality Filter for gene synteny [Clarke et al., PLoS Genetics, 2012]

46. Needles in a haystack: 2 hits in 255Gb

47. Bilaterian Conserved Regulatory Elements (Bicores) Bicore2 Bicore1 Human Zebrafish Tunicates Amphioxus Acorn Worm Sea Urchin Insects Tick Nematode Sea Hare Owl Limpet Ancestor to all bilaterians ~650 MYA

48. The “model” invertebrates are the most diverged bilaterians Adapted from Srivastava et al. Nature 2010 and Putnam et al. Science 2007

49. Bicores have maintained synteny Human Bicore2 Bicore2 target gene In vertebrates, the Bicore2 target gene patterns the hindbrain 1 kb

50. Bicores have maintained synteny Human Sea urchin Tick 1 kb