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CS273A
Lecture 15: Evolutionary Genomics
MW 12:50-2:05pm in Beckman B302
Profs: Serafim Batzoglou & Gill Bejerano
TAs: Harendra Guturu & Panos Achlioptas
http://cs273a.stanford.edu [BejeranoFall13/14]
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Announcements
• Projects are rolling..
http://cs273a.stanford.edu [BejeranoFall13/14]
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Sweeps
Positive Selection
Negative Selection
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The Species Tree
S
S
Sampled Genomes
S
Speciation
Time4
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), evodevo 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]
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What makes us molecularly human?
Searching
Near …
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Why compare to Chimp?
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Humans and Chimpanzees Possess
Many Vastly Different Phenotypes
A: Chimp B: Human
A
A
B
B
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[Varki, A. and Altheide, T., Genome Res., 2005]
Disease Susceptibility Differences
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What human-chimp changes do we find?
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Large differences
Fusion (HSA 2)
18 pericentromeric inversions
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Medium Sized Differences
Gene families expand
and contract
Mobile element insertion
and mediated deletion
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Small Differences
1% difference at the base level
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Genetic basis of human phenotypes?
Phenotype
Number of rearrangements
Genotype
Most mutations
are near/neutral.
How do we know?
4D sites, ARs.
http://cs273a.stanford.edu [BejeranoFall13/14]
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What functional instructions can change?
Human/chimp genome: ~3*109 bp
Rough composition:
• Genes 2%
• Non-coding RNAs 1%
• Regulatory DNA 10-20%
• (Repeats 40%)
• (Other 30-40%)
http://cs273a.stanford.edu [BejeranoFall13/14]
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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]
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Positive and negative gene selection in the human genome
http://cs273a.stanford.edu [BejeranoFall13/14]
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Candidate genes for human specific evolution
...
http://cs273a.stanford.edu [BejeranoFall13/14]
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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.
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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.
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What if we did an unbiased search?
Human-specific substitutions in conserved sequences
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]
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Human Accelerated Regions
Human-specific substitutions in conserved sequences
rapid
change
Human
Chimp
conserved
HAR2/HACNS1:
• Between CENTG2 / GBX2.
• Mouse element is not
an enhancer
(where assayed).
[Prabhakar, S. et al., Science, 2008]
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Biased Gene Conversion
http://cs273a.stanford.edu [BejeranoFall13/14]
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Different Unbiased Search: Loss vs Gain
rapid
change
Human Accelerated Regions
Human
Chimp
• 4-18 unique human substitutions
• Pollard, K. et al., Nature, 2006
• Prabhakar, S. et al., Science, 2008
conserved
Human
Human Conserved Sequence Deletions
(hCONDELs)
Chimp
• Complete human loss of sequence
• Likely to confer human-specific phenotypes
deleted!
conserved
[McLean, Reno, Pollen et al., Nature, 2011]
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Identifying hCONDELs
deleted!
Human
Chimp
conserved
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hCONDEL genomic distribution
• Median size: 2.8kb
• Not enriched in highly variable genomic regions
• Most do not disrupt proteins: only 1 validated exonic deletion
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Deletions of functional non-coding DNA
Gene
( )
Gene
( )
Gene
Gene
( )
Gene
( )
Gene
Gene with function
e.g. “neuronal gene”
( )
Gene
( )
Gene
( )
Gene
( ) ( )
Gene
( )
Gene without function
Gene
Gene
hCONDEL
Conserved element
( )
http://great.stanford.edu
http://cs273a.stanford.edu [BejeranoFall13/14]
[McLean et al., Nat. Biotechnol., 2010]
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Functional enrichments of hCONDELs
Ontology Term
p-value
Gene Ontology
Steroid hormone receptor activity
InterPro
Fibronectin, type III
Zinc finger, nuclear hormone receptor type
CD80-like, immunoglobulin C2 set
Entrez Gene
Neuronal genes
Monoallelically-Expressed Genes
Monoallelic expression
3.73 x 10-4
1.01 x 10-4
1.80 x 10-4
1.37 x 10-3
1.11 x 10-4
8.62 x 10-3
These enrichments
are unique to hCONDELs
http://cs273a.stanford.edu [BejeranoFall13/14]
http://great.stanford.edu
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hCONDEL near Androgen Receptor
The deletion appears fixed in humans
and appears deleted in Neandertal.
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Androgen Receptor chimpanzee enhancer assay
Androgen Receptor
Human
Chimp
Genomic fragment Hsp68 promoter LacZ reporter gene
http://cs273a.stanford.edu [BejeranoFall13/14]
[Phil Reno, David Kingsley]
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Mouse enhancer Chimp enhancer
The human deletion near AR acts as an
enhancer within known AR expression domains
Genital tubercle
E16.5
Sensory whiskers
E16.5
E16.5
Penile spines
E16.5
E16.5
http://cs273a.stanford.edu [BejeranoFall13/14]
[Phil Reno, David Kingsley]
8 weeks
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Androgen Receptor
Testosterone
Androgen
Receptor
AR+T
dimer
Androgen Receptor
Nucleus
Cell
Human
Chimp
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Androgen responsiveness in domains of expression
Penile spines
Sensory whisker length (mm)
Sensory whiskers
Sensory
whiskers
Penile
spines
Mice with Ar coding
region mutations lack
penile spines
Galago
http://cs273a.stanford.edu [BejeranoFall13/14]
[Ibrahim & Wright 1983] [Dixson, 1976] [Murakami, 1987]
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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
( )
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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]
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Deletion near GADD45g is an enhancer for
a subset of GADD45g expression domains
VT
VT
SVZ VZ
Se
POA
Se
E14.5
POA
E14.5
E14.5
Ventral forebrain expression domains:
Septum (Se), Preoptic area (POA), Ventral thalamus (VT)
• SVZ is a progenitor amplification domain for
neurons both in ventral regions and neocortex
Kriegstein et al., 2006; Taglialatela et al., 2004; Gelman et al., 2009
http://cs273a.stanford.edu [BejeranoFall13/14] [Alex Pollen, David Kingsley]
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What makes us molecularly human?
… Searching
Far
http://cs273a.stanford.edu [BejeranoFall13/14]
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The Vertebrate-Invertebrate Divide
Tunicate
Fly
Nematode
Human
NOT just ultras, ALL conserved non-coding sequence
DISAPPEARS at the vertebrate/invertebrate boundary.
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The Bilaterian Tree of Life
Human
Zebrafish
Tunicates
Amphioxus
Acorn Worm
Ancestor to all
bilaterians
~650 MYA
Sea Urchin
Insects
Tick
Nematode
Sea Hare
Owl Limpet
Conserved embryonic
morphologies
Bilateral symmetry
Human
Zebrafish
Tunicates
Amphioxus
Acorn Worm
Ancestor to all
bilaterians
~650 MYA
Sea Urchin
Insects
Tick
Nematode
Sea Hare
Owl Limpet
Conserved signaling pathways
Human
Zebrafish
Tunicates
Amphioxus
Acorn Worm
Ancestor to all
bilaterians
~650 MYA
Sea Urchin
Insects
Tick
Nematode
Sea Hare
Owl Limpet
Conserved developmental
transcription factors
Hox genes
Human
Zebrafish
Tunicates
Amphioxus
Acorn Worm
Ancestor to all
bilaterians
~650 MYA
Sea Urchin
Insects
Tick
Nematode
Sea Hare
Owl Limpet
Ancient Regulatory Circuits
Signaling pathway
Transcription factor
Enhancer
?
Target gene
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.
If At First You Don't Succeed, Try, Try Again
Thousands of putative
vertebrate enhancers
All publicly available non-vertebrate
metazoan sequence data
Data source
Genbank species with at
least 10kb of sequence
Trace archive
Genome assembly
Number of
species
Total sequence
(gigabases)
5,278
21.5
134
47
218.5
15.6
1. Very sensitive
sequence alignment
2. Filters for alignment
quality
3. Filter for gene synteny
[Clarke et al., PLoS Genetics, 2012]
Needles in a haystack: 2 hits in 255Gb
Bilaterian Conserved Regulatory Elements
(Bicores)
Bicore1
Human
Zebrafish
Tunicates
Amphioxus
Acorn Worm
Ancestor to all
bilaterians
~650 MYA
Sea Urchin
Insects
Tick
Nematode
Sea Hare
Owl Limpet
Bicore2
The “model” invertebrates are the
most diverged bilaterians
Adapted from Srivastava et al. Nature 2010 and Putnam et al. Science 2007
Bicores have maintained synteny
Human
1 kb
Bicore2
Bicore2 target gene
In vertebrates, the Bicore2 target gene patterns the hindbrain
Bicores have maintained synteny
Human
1 kb
Sea urchin
Tick
Transgenic enhancer assay
Human Bicore2
Minimal promoter
Reporter gene
DNA construct
Construct injected into embryo
Bicore2 is a hindbrain enhancer
Human Bicore2
Expression pattern of bicore2 target gene
Julie VanderMeer
Bicore2 encodes conserved instructions
Human
Bicore2
71 %
identity
Sea urchin
Bicore2
Tick
Bicore2
65 %
identity
58 %
identity
Bicore2 encodes conserved instructions
Human Bicore2
Urchin Bicore2
Tick Bicore2
Julie VanderMeer
Summary of Bicores
Vertebrate
expression pattern
Urchin
function
Element
Length
Target gene
Signaling
Bicore2
98bp
Transcriptional
Hindbrain
repressor
Bicore1
100bp
Transcriptional Fore, mid & hindbrain, Dorsal
Bmp/Tgfrepressor
dorsal neural tube
ectoderm beta
Ectoderm Wnt
Search faster (search more), use more
elaborate comparison functions
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