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

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

3. 3 Sweeps Positive Selection Negative Selection

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

5. Evo Devo 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]5

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

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

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

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

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

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

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

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

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

15. http://cs273a.stanford.edu [BejeranoFall13/14] 15 What functional instructions can change? Human/chimp genome: ~3*10 9 bp Rough composition: Genes 2% Non-coding RNAs 1% Regulatory DNA 10-20% (Repeats 40%) (Other 30-40%)

16. 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.

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

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

19. 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. 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. 21 What if we did an unbiased search? Human-specific substitutions in conserved sequences 21 [ Pollard, K. et al., Nature, 2006] [Beniaminov, A. et al., RNA, 2008] Human Chimp Human rapid change HAR1: Novel ncRNA 18 unique human substitutions conserved Chimp

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

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

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

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

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 26 http://cs273a.stanford.edu [BejeranoFall13/14]

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

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 enrichments are unique to hCONDELs http://great.stanford.edu http://cs273a.stanford.edu [BejeranoFall13/14] 28

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

30. Androgen Receptor chimpanzee enhancer assay [Phil Reno, David Kingsley] Androgen Receptor Human Chimp Genomic fragmentHsp68 promoterLacZ reporter gene http://cs273a.stanford.edu [BejeranoFall13/14] 30

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

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

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

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 ( ) Suppress proliferation Do not suppress proliferation 34 http://cs273a.stanford.edu [BejeranoFall13/14]

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

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

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

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

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

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

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

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

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. Data source Number of species Total sequence (gigabases) Genbank species with at least 10kb of sequence 5,27821.5 Trace archive134218.5 Genome assembly4715.6 1.Very sensitive sequence alignment 2.Filters for alignment quality 3.Filter for gene synteny If At First You Don't Succeed, Try, Try Again All publicly available non-vertebrate metazoan sequence data Thousands of putative vertebrate enhancers [Clarke et al., PLoS Genetics, 2012]

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

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

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 1 kb Human Bicore2 Bicore2 target gene In vertebrates, the Bicore2 target gene patterns the hindbrain

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

51. Transgenic enhancer assay Human Bicore2Minimal promoterReporter gene DNA construct Construct injected into embryo

52. Bicore2 is a hindbrain enhancer Human Bicore2 Julie VanderMeer Expression pattern of bicore2 target gene

53. Bicore2 encodes conserved instructions Sea urchin Bicore2 Human Bicore2 Tick Bicore2 71 % identity 58 % identity 65 % identity

54. Human Bicore2 Urchin Bicore2 Tick Bicore2 Julie VanderMeer Bicore2 encodes conserved instructions

55. Summary of Bicores ElementLengthTarget gene Vertebrate expression pattern Urchin functionSignaling Bicore298bp Transcriptional repressor HindbrainEctodermWnt Bicore1100bp Transcriptional repressor Fore, mid & hindbrain, dorsal neural tube Dorsal ectoderm Bmp/Tgf- beta

56. Search faster (search more), use more elaborate comparison functions http://cs273a.stanford.edu [BejeranoFall13/14] 56