Comparative Genomics and Evolution Pollard, K.S., et al., Forces Shaping the Fastest Evolving...
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![Page 1: Comparative Genomics and Evolution Pollard, K.S., et al., Forces Shaping the Fastest Evolving Regions in the Human Genome. PLoS Genetics 2(10), 2006. McLean,](https://reader036.fdocuments.in/reader036/viewer/2022062516/56649d605503460f94a41c8b/html5/thumbnails/1.jpg)
Comparative Genomics and Evolution
Pollard, K.S., et al., Forces Shaping the Fastest Evolving Regions in the Human Genome. PLoS Genetics 2(10), 2006.
McLean, C., and Bejerano, G., Dispensability of Mammalian DNA. Genome Research 18, 1743-1751 (2008).
Image source: http://mbbnet.umn.edu
Image from: McLean, C., and Bejerano, G., Dispensability of Mammalian DNA. Genome Research 18, 1743-1751 (2008).
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“Forces shaping the fastest evolving regions in the human genome”
by Katherine S. Pollard et al.
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What’s the difference?
Image sources: http://pro.corbis.com, http://www.science.psu.edu
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What’s the difference?
• Humans have higher “brainpower”
• Examples: creativity, problem solving, language
• What part of the genome is the cause?
Image source: http://www.spaceflight.esa.int
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What’s the difference?
• Human and chimpanzee DNA is 98% similar
• The 2% difference is 29 million bases (mostly in non-coding DNA)
Image source: http://en.wikipedia.org
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Comparative Genomics
• Human and rodent genomes are often compared to identify conserved (presumably functional) elements.
• Humans and chimpanzees are compared to understand what is uniquely human about our genome.
Image source: http://genome.ucsc.edu
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Comparative Genomics
• Look at HARs in human genome
• HAR - human accelerated region. High rate of nucleotide substitution in humans, low in other vertebrates.
• Fastest is HAR1 – novel RNA gene expressed in development of neocortex (language, conscious thought).
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HARs
~ 100 bp, mostly non-coding
• Function is likely to be gene regulation.
• Seem to have been under strong negative selection up to common ancestor of chimp and human.
• Rapid positive selection then started in humans only.
Image source: http://www.shutterstock.com
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Finding HARs
• Evolutionary tree based on the comparison of conserved regions in whole-genome alignments between species.
Branch lengths given in substitutions per base, or in millions of years
Evolution of vertebrates
Image from: Pollard, K.S., et al., Forces Shaping the Fastest Evolving Regions of the Human Genome.
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Finding HARs
• Find HARs by using LRT, the likelihood ratio test.
• In statistical hypothesis testing, the likelihood ratio (Λ) is the ratio of the maximum probability of a result under a null hypothesis and alternative hypothesis.
• The LRT decides between the two hypothesis based on the value of the likelihood ratio.
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• Two models were used for genomic LRT.
Model 1: human substitution rate is held proportional to the other substitution rates in the evolutionary tree.
Model 2: human substitution rate can be accelerated relative to the rates in the rest of the tree.
Finding HARs
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Finding HARs
. . .
. . .
Human
Another vertebrate . . .
. . . . . .
All the conserved alignments
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Finding HARs
. . .
. . .
Human
Another vertebrate . . .
. . . . . .
Determine 1st set of rates
Determine 2nd set of rates
Determine 3rd set of rates
Scale all by the same amount
Model 1
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Finding HARs
Human
Another vertebrate . . .
. . . . . .
Scale all by the same amount
Model 2
. . .
. . .
Scale the human rates separately
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Identify regions conserved between human and other vertebrates (34,498 of them)
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Identify regions conserved between human and other vertebrates (34,498 of them)
For all regions, fit model 1 and determine the proportional rates that maximize the likelihood of the tree
Obtain P1(max probability 1)
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Identify regions conserved between human and other vertebrates (34,498 of them)
For all regions, fit model 1 and determine the proportional rates that maximize the likelihood of the tree
Loop over all conserved regions. For each region, do:
Obtain P1(max probability 1)
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Identify regions conserved between human and other vertebrates (34,498 of them)
For all regions, fit model 1 and determine the proportional rates that maximize the likelihood of the tree
Loop over all conserved regions. For each region, do:
Fit model 2 to the region in human, find acceleration for that region that maximizes the likelihood of the tree
Obtain P1(max probability 1)
Obtain P2(max probability 2)
Calculate LRT for the region as Λ = log(P2 / P1)
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Finding HARs
• Big LRT value indicates an HAR. How big is big?
• Do 1 million simulations of the 34,498 conserved alignments.
• To create each simulation, use the model 1 proportional rates.
• Repeat the LRT calculation for each simulation.
• Then for each region, find proportion of simulated LRTs that are bigger than its original LRT.
• That proportion is a p-value that tells if the region is an HAR.
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Finding HARs
• Note on methods: vertebrates that were used in selecting the conserved regions (chimp, macaque, mouse, rat, rabbit) were omitted from any LRT analysis.
• This ensured that the LRT test is independent of the method used to select the conserved regions.
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Finding HARs
• Result: 202 HARs were found in the human genome.
Image source: http://www.3dscience.com
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Results for Conserved Elements
• 80.4% of the 34,498 conserved regions are non-coding.
• 45.4% of non-coding regions are intronic, 31% are intergenic,
• Non-coding regions are enriched for transcription factors, DNA-binding proteins, regulators of nucleic acid metabolism
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Results for HARs
• 202 HARs have p < 0.1, 49 of them have p < 0.05
• HAR1 through HAR5 have p < 4.5e-4, very accelerated
• Most HARs are non-coding
• 66.3% are intergenic, 31.7% are intronic, only 1.5% are coding
• Results support the hypothesis (King and Wilson) that most chimp-human differences are regulatory.
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Results: Confirming Accelerated Selection in HARs
• Are the HARs just due to relaxation of negative selection?
• No. Compare to neutral rate for 4D sites to see.
Negative selection
Positive selection
Image source: http://cs273a.stanford.edu [Bejerano Aut 08/09]
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The chimp rates in all five elements fall well below the human rates, which exceed the background rates by as much as an order of magnitude. H, human; C, chimp.
Genome-wide neutral rate for 4D sites in human and chimp
Genome-wide neutral rate for 4D sites in human and chimp in chromosome end bands
Image from: K.S. Pollard et al., Forces Shaping the Fastest Evolving Regions of the Human Genome.
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Results: W S Bias in HARs
• Dramatic AT GC bias was observed in HARs.
AT GC substitution bias in HARs
HAR1 – HAR5
HAR6 – HAR49
HAR50 – HAR202GC AT
AT GC
Rest of ~ 34000 conserved elements
Image from: Pollard, K.S., et al., Forces Shaping the Fastest Evolving Regions of the Human Genome.
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Results: W S Bias in HARs
• Top 49 HARs are 2.7 times as likely to be located near final chromosomal bands as the other conserved elements
• Interestingly, HAR1 and HAR5 are also in end regions in other mammals, but are not accelerated.
Image source: http://www.intelihealth.com
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• HARs tend to be located in regions of high recombination in humans.
• All of this evidence points to biased gene conversion (BGC) as the driving force behind HARs.
Results: W S Bias in HARs
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Genetic Recombination
• Paired chromosomes can exchange homologous pieces
• Typically occurs during meiosis
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maternal chromosome Apaternal chromosome A
diploid germ cellMeiosis
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maternal chromosome Apaternal chromosome A
centromeresister chromatids
DNA replication
diploid germ cellMeiosis
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maternal chromosome Apaternal chromosome A
centromeresister chromatids
DNA replication
Recombination
diploid germ cellMeiosis
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maternal chromosome Apaternal chromosome A
centromeresister chromatids
DNA replication
Recombination
Segregation
diploid germ cellMeiosis
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maternal chromosome Apaternal chromosome A
centromeresister chromatids
DNA replication
Recombination
Segregation
haploid gametes
diploid germ cellMeiosis
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Recombination
Recombination hotspot
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Genetic Recombinationduplex 1
duplex 2
Formation of Holliday Junction intermediate
Vertical resolution with crossover
Horizontal resolution with gene conversion
Mismatch repair
or
Image source: http://www.sanger.ac.uk
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Genetic Recombination: Chromosomal Crossover
• Chromosomal crossover results in exchange of DNA pieces
Homologous chromosomes
Recombinant chromatids
Image source: http://www.emc.maricopa.edu
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Genetic Recombination: Gene Conversion
• Gene conversion results in nonreciprocal transfer of DNA
Mismatch repair causes DNA to revert back to its original formRecombinant
chromatids
Image source: http://www.emc.maricopa.edu
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Genetic Recombination: Gene Conversion
• The result is a nonstandard ratio of alleles, such as 3:1
• This causes homogenization of a species’ gene pool
haploid gametes
Image source: http://www.emc.maricopa.edu
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Biased Gene Conversion
• DNA repair machinery likes to replace weak pairings with strong pairings during gene conversion.
A - T is a weak pairing
G - C is a strong pairing
Image source: http://commons.wikimedia.org
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• Biased gene conversion results in G – C enrichment of a species’ gene pool (in addition to causing homogenization)
Recombinant chromatids
Biased Gene Conversion
A – T replaced by G – C during mismatch repair
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HARs and Recombination Hotspots
• HARs tend to be located near recombination hotspots in humans
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Recombination Hotspots
• Mysterious
• Extremely different between chimps and humans (change rapidly during evolution)
• Not caused by the local DNA sequence (it is the same in human and chimp)
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Some
HARsRecombination
hotspots ?
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Possible Conclusion
• Recombination-caused BGC (often seen negatively) played a big role in the development of our species.
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Alternative Explanation
• Isochore – DNA region (~100 kb) with high gene concentration
• Isochores are stabilized by many strong (GC) pairings
HAR HAR
Isochore
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• Theory (Bernardi et al.) that weakly deleterious changes drive isochore to a critical point of destabilization
• At critical point, GC content cannot decrease – otherwise isochore becomes unstable
• AT GC substitution in the isochore suddenly gains selective advantage and sweeps through the population
Alternative Explanation
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• Isochore selective sweep theory vs. the BGC theory.
• Isochore sweep has a different DNA signature than BGC
Alternative Explanation
~ 100 kb
GC GC GC GCGC GC GC
Isochore selective sweep
~ 100 bases
GC GC GC GCGC GC GC
Biased gene conversion
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• Evidence so far favors the BGC explanation for HARs
• However, the results are not yet conclusive
Alternative Explanation
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“Dispensability of Mammalian DNA”
by Gill Bejerano and Cory McLean
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Are mammalian CNEs dispensable?
• CNE – conserved non-exonic element
• Examples: cis-regulatory DNA, ultraconserved DNA
?
Image source: http://apps.co.marion.or.us
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Cis-regulatory DNA elements
promoter or inhibitor
Image source: http://cnx.org
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Cis-regulatory DNA elements
Image source: http://cnx.org
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Ultraconserved elements
• 200 bp and up, many seem to be regulatory
• “100% identity with no insertions or deletions between orthologous regions of the human, rat, and mouse genomes.”
• “Nearly all of these segments are also conserved in the chicken and dog genomes, with an average of 95 and 99% identity, respectively. Many are also significantly conserved in fish.”
(quotes from “Ultraconserved elements in the human genome” by Bejerano et al.)
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Are mammalian CNEs dispensable?
• About 20% of gene knockout experiments, including cis-regulatory and ultraconserved knockouts, produce no phenotype measurable in lab settings.
Image source: http://www.sciencedaily.com
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Are mammalian CNEs dispensable?
Do CNEs have functional redundancy?
OR
Are CNEs indispensable, but in a way that cannot be observed in the lab?
• Approach: look at CNEs lost in rodents due to evolution
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Finding CNEs lost by rodents
Computational Pipeline
Identify conserved mammalian sequences
Pick out the ones absent in rodents
Remove artifacts
due to assembly, alignment, structural RNA migration
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Image from: McLean, C., and Bejerano, G., Dispensability of Mammalian DNA.
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Image from: McLean, C., and Bejerano, G., Dispensability of Mammalian DNA.
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Use UCSC chains and nets
To avoid assembly artifacts
Ignore multi-level nets
Image from: McLean, C., and Bejerano, G., Dispensability of Mammalian DNA.
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Identify lost
DNAValidate quality of results
Image from: McLean, C., and Bejerano, G., Dispensability of Mammalian DNA.
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Look at the aligned orthologous sequences in primates (human, macaque), dog, and rodents (mouse, rat).
Identifying DNA lost by rodents
primates
A
G
dog
rodents
primates
dog
Different bases between primates and dog
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100 bp window
Compute primate-dog %id (percentage of identical alignment columns)
Identifying DNA lost by rodents
primates
A
G
dog
rodents
primates
dog
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Compute primate-dog %id
Identifying DNA lost by rodents
primates
A
G
dog
rodents
primates
dog
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primates
A
G
dog
rodents
Compute primate-dog %idDeletion in rodents
Identifying DNA lost by rodents
primates
dog
!
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primates
A
G
dog
rodents
Ultraconserved-like element between primates-dog
Identifying DNA lost by rodents
primates
dog
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primates
A
G
dog
rodents
Ultraconserved-like element that was lost in rodents
Identifying DNA lost by rodents
primates
dog
!
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Results for non-exonic ultras
• 1,691,090 bp of ultraconserved-like sequences were found
• 1147 bp of these sequences were lost in rodents
• Thus only 0.086% of ultras is lost in rodents
• In comparison, ¼ of neutrally-evolving DNA (50%id – 65%id) is lost in rodents
• Thus ultraconserved-like sequences are 300 times more indispensable than neutrally-evolving DNA
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Results for neutral DNA
• Expected uniform rate of lost neutrally-evolving DNA
• Observed that less conserved sequences are more retained
Image from: McLean, C., and Bejerano, G., Dispensability of Mammalian DNA.
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Results for neutral DNA
• Phenomenon due to poorly conserved sequences being adjacent to exons, and thus shielded from being lost
• Larger deletions are biased away from gene structures
Image from: McLean, C., and Bejerano, G., Dispensability of Mammalian DNA.
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• Moving away from 100%id, there is a mixing of DNA under purifying selection and neutrally evolving DNA
Separating DNA under selection from neutral DNA
Image from: McLean, C., and Bejerano, G., Dispensability of Mammalian DNA.
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• To distinguish neutral DNA from conserved DNA in the mix, use longer evolutionary tree branch lengths
Separating DNA under selection from neutral DNA
Image from: McLean, C., and Bejerano, G., Dispensability of Mammalian DNA.
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• Example: human-dog-horse alignment has longer cumulative branch length than human-macaque-dog
Separating DNA under selection from neutral DNA
Image from: McLean, C., and Bejerano, G., Dispensability of Mammalian DNA.
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• Example: human-dog-horse alignment has longer cumulative branch length than human-macaque-dog
Separating DNA under selection from neutral DNA
Image from: McLean, C., and Bejerano, G., Dispensability of Mammalian DNA.
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• Thus human-dog-horse alignment has lower %id for neutral DNA than human-macaque-dog
• This shifts the neutral DNA curve shifts to the right
Separating DNA under selection from neutral DNA
Image from: McLean, C., and Bejerano, G., Dispensability of Mammalian DNA.
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Results for DNA under purifying selection
Image from: McLean, C., and Bejerano, G., Dispensability of Mammalian DNA.
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Results for DNA under purifying selection
• 80%id to 100%id identified as DNA under purifying selection
• As is visible from the figure, practically none of this DNA is lost in the primates (only 0.154% of bases are lost)
Image from: McLean, C., and Bejerano, G., Dispensability of Mammalian DNA.
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Results for DNA under purifying selection
• The previous results were for CNEs
• Those results compare to the numbers for lost coding DNA:
Fraction of lost CNEs: 0 at 100%id, 0.00122 at 80%id
Fraction of lost exons: 0 at 100%id, 0.0000861 at 80%id
Image from: McLean, C., and Bejerano, G., Dispensability of Mammalian DNA.
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Results for DNA under purifying selection
• Thus CNEs under purifying selection are indispensable, similarly to coding elements.
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CNE dispensability ranking
In rodents In primates
Deepest in vertebrate tree, so corresponds to the most indispensable CNEs
Region of high conservation (CNEs)
• Left plot explanation (right plot is similar): take the h-m-d alignments, find their conservation %id in each of the shown species. Then for each of those species, plot the fraction of DNA lost in rodents vs the %id.
Image from: McLean, C., and Bejerano, G., Dispensability of Mammalian DNA.
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CNE dispensability ranking
Image from: McLean, C., and Bejerano, G., Dispensability of Mammalian DNA.
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Conclusion
• Many mammalian CNE knockouts produce no observable phenotype in the lab, suggesting great functional redundancy.
• However, evolutionary analysis shows that the CNEs, and particularly ultraconserved regions, are indispensable.
• Seems like the phenotype in knockouts is subtle, but very important.
Image source: http://apps.co.marion.or.us