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Carol Eunmi LeeUniversity of Wisconsin

Evolution 410

Molecular Evolution I

Molecular Evolution:pertains to evolution at the levels of

DNA, RNA, and proteins

Outline(1) Types of Mutations that could affect

functionStructural ChangesRegulatory Changes

Cis vs. Trans-regulatory

(2) Which predominate?(3) Case study with Human Brain Evolution

Previously, we had delved into some aspects of molecular evolution when we discussed Mutations

Mutations: any change in the genetic code, including those that arise from errors in DNA replication or errors in DNA repair

Most Mutations have no Effect(most mutations are neutral)

• 3.12 billion nucleotides in the human genome

• Most of the genome is non-coding sequence and has no function (up to 95%):

– Mutations here are “Neutral”

• Mutations that affect function are what matter(within genes, or within regulatory sequences that affect the expression of genes)

Mutations

• So, where in the genome do the mutations matter???

• What exactly are these mutations doing?

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What are the mutations doing?

• So, let’s go into more detail on what exactly these mutations are doing…

• And how they affect expression and function of genes and proteins

Diagram of eukaryotic gene Each gene is composed of

regulatory and coding region

Eukaryotic gene (DNA)

Futuyma D.J. (2009)

Gene Regulation (expression)

(cis-acting factor: promoter or enhancer)

trans-acting factor (e.g. transcription factor, microRNA, repressor, etc.)

gene is expressed

ACAGTGA

So… different types of mutations could include:

• STRUCTURAL: Affects the Property of the gene product (changes to the allele itself)– A mutation could occur within the coding sequence of a gene,

and change the amino acid composition of a protein (structural)

• REGULATORY: Typically affects the Amount of the gene product– A mutation could occur within a regulatory element, like a

promoter or enhancer near the gene (cis-regulatory)– A mutation could occur within a regulatory element, like a

transcription factor that is encoded far away from the gene (trans-regulatory)

STRUCTURAL• Primary: Amino Acid composition (Amino

Acid substitutions)• Secondary, Tertiary, Quaternary structure

REGULATORY• Protein expression (transcription, RNA

processing, translation, etc)• Protein activity (allosteric control,

conformational changes, receptors)

Hierarchical processes that are affected by Mutations

Once these mutations have occurred, creating genetic variation, selection could then act on genes, gene expression, and on genetic architecture (allelic and gene interactions) – by acting on the phenotype

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STRUCTURAL evolutionary changes

• Mutations in DNA or mRNA that result in changes in the amino acid composition of a protein

• Some amino acid changes alter the activity and/or function of proteins (and enzymes)

“The Central Dogma” of Molecular Biology

Francis Crick (1958)

Amino Acid Codons § In the case of amino

acids

§ Mutations in Position 1, 2 lead to Amino Acid change

§ Mutations in Position 3 often don’t matter

REGULATORY CHANGES

• The protein structure itself does not change

• Changes in Gene Expression– Change in amount of expression (amount

of protein made - transcribed or translated)

– Changes in location, timing of expression

REGULATORY

Protein Expression• Transcription: Mutations at promoters, enhancers

(CIS), transcription factors (TRANS), etc

• RNA Processing: Mutations at splice sites, sites of polyadenylation, sites controlling RNA export

• Translation: Mutations in ribosomes, regulatory regions, etc

Protein activity (allosteric control, conformational changes, receptors)

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Gene Expression• Focusing on Transcription alone:

Cis-regulation (at or near the gene)Examples: Promoters

EnhancersLocal repressor

Trans-regulation (somewhere else in the genome)Examples: Gene regulatory proteins (trans-acting factors,

like transcription factors)

• Reading by Emerson et al. -- Focus reading mainly on Part 1 (Intro) and Part 2

Gene Expression

• Focusing on Trans-Acting Factors:

• Transcription Factors: proteins that bind to specific DNA sequences, thereby controlling transcription and gene expression.

– Usually regulates many genes and therefore often has large pleiotropic effects

Gene Regulation (expression)

cis-acting factor (e.g., promoter or enhancer)

trans-acting factor (e.g. transcription factor, microRNA, repressor)

gene is expressed

ACAGTGA

Which types of mutations predominately contribute to

adaptive evolution?

Structural or Regulatory Changes?

• Structural Evolution: Hopi Hoekstra & Jerry Coyne (2007):

– Adaptation and speciation probably proceed substantially

through selection on structural mutations

– “There is no evidence at present that cis-regulatory changes

play a major role—much less a pre-eminent one—in adaptive

evolution.”

• Regulatory Evolution: David Stern (2000), Sean Carroll (2000)

– Cis-regulatory elements are the most likely target for the

evolution of gene regulation

– Most mutations causing morphological variation are expected

to reside in the cis-regulatory, rather than the coding, regions

of developmental genes

Which types of mutations predominately contribute to adaptive in evolution?

So who is right?

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So what’s the answer?• The “debate” between the importance of

structural vs regulatory evolution arose in part because of lack of communication between population geneticists and developmental biologists

• Developmental Biology and the importance of evolution of gene regulation was in fact left out of the evolutionary synthesis of the 1930s and 1940s.

So what’s the answer?

• This is a not a good question (binary thinking)• Obviously, structural and regulatory changes

both contribute to adaptive evolution…

• But, there does appear to be general trends on which type of mutations predominatedepending on the level of divergence among taxa (refer to optional slides if you are interested)

Patterns of Evolution

• It appears that patterns of structural, cis-, trans-regulatory changes varies depending on levels of divergence among taxa

• Evolution in different kinds of populations and over different evolutionary time scales may result in selection of different kinds of mutations.

Structural or Regulatory?• David Stern 2008 performed the first comprehensive

review:

• According to Stern’s analysis, Cis-regulatory mutations represented ~22% of 331 identified genetic changes; although, the number of cis-regulatory changes published annually is rapidly increasing (there is a bias in the literature as more studies have examined amino acid changes)

• Above the species level, cis-regulatory mutations altering morphology are more common than protein coding changes (supporting the argument of Sean Carroll, at least at greater divergences)

Cis- vs Trans-Regulatory?

Cis-regulatory changes account for a greater proportion of the expression differences observed between rather than within species. Specifically, cis-regulatory changes seem to accumulate preferentially over time (Wittkopp et al. 2008).

Cis- vs Trans-Regulatory?

• The position of a gene in a regulatory network is an important parameter to consider when determining whether cis-regulatory or trans-regulatory are more likely to contribute to phenotypic evolution.

Gene Network and Pleiotropy

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• Conversely, a seemingly unbeneficial trait might get selected for because the gene that codes for it also enhances fitness

• Pleiotropy could sometimes lead to evolutionary tradeoffs (you can have evolutionary tradeoffs that are not pleiotropic– between traits encoded by different genes)

Pleiotropy: when a gene affects many traits or functions

• Selection might not be able to act on trait if the gene that codes the trait is Pleiotropic, and also affects other traits.So, changing the gene could negatively affect the other traits

Gene Network

• Trans-acting factors, such as transcription factors, often affect many other genes… and are highly pleiotropic

Cis- vs Trans-Regulatory?

• Cis-regulation contributes disproportionately to gene expression divergence between species, relative to its contribution within species (Wittkopp et al. 2008)

• This is in agreement with expectations that cis-regulatory alleles may be preferentially fixedbecause of their weaker pleiotropic effects.

Prevalence of cis vs trans-regulatory mutations

• Trans-regulatory mutations arise more often (greater mutational target... More DNA encodes a transcription factor than a promoter sequence)

• But more cis-regulator mutations persist over time (due to less pleiotropic constraint)

• HOWEVER, the trans-regulatory mutations that persist over time tend to have large impacts

Trans-regulatory evolution

• Trans-regulatory mutations tend not to persist over longer evolutionary time (due to pleiotropic constraints)

• BUT, the trans-regulatory mutations that do persist are responsible for major evolution of animal body plans (animal phyla) and differences between chimps and humans (Homo)

• As we know from previous lectures, it is thought that regulatory evolutionary changes that are due to cis-regulatory elements would occur more readily (than trans), because of the increased pleiotropy of trans-acting elements

• However, trans-regulatory changes can have profound (large) impacts because of the many genes they regulate

• Also, due to the pleiotropy, trans-regulatory changes could have high costs (leading to tradeoffs)

Trans-regulatory evolution and the costs

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Figure from Carl Zimmer, The Tangled Bank 2010

Hominin Evolution

• Large Brains (1600cm3, larger than us): same size at birth as us, but more rapid growth during development

• More brain function devoted to vision and movement (http://www.sciencedaily.com/releases/2013/03/130319093639.htm)

Homo neanderthalensis

Reconstruction of Neanderthal child from Gibraltar (Anthropological Institute, University of Zurich)

Rapid Evolution in Homo

• Evolution of gene regulation à Evolution of Development

• Humans display a 3–5 times faster evolutionary rate in divergence of developmental patterns in some tissues, compared to chimpanzees.

• Particularly in brain tissue… affected in many cases by trans-acting (regulatory) elements

Most different in the Brain tissue between Humans and other primates (Enard et al. 2002)

Changes in developmental genes and patterns of gene expression are greater in brain tissue than other tissues in humans relative to other primates

• FOXP2: gene implicated in language (a transcription factor)

• 2 amino acid substitutions in Homo relative to chimps

• Neanderthals share the same derived allele (but regulation of these alleles might differ between them and us)

Enard et al. Nature 2002

§Microcephalin (MCPH1): a gene that regulates brain size during development and has experienced positive selection in Homo

§ Thought a derived allele (Haplogroup D) introgressed into H. sapiensby mating with extinct Homo species http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1635020/)

Alleles unique to Homo

• But, most differences between Homo and Chimps are due to evolutionary changes in gene expression, quantitative changes,

• …rather than the presence of unique alleles

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• The accelerated evolution of human brain expression appears to mainly involve remodeling of developmental patterns (evolution of development)

• Much of the changes are due to trans-regulatory evolution (transcription factors and micro RNAs)

• Some changes due to gene deletions in the Homo lineage (mostly regulatory regions, like the enhancers mentioned)

Evolution of the Homo brain PNAS 2009, 106: 22358-22363http://www.pnas.org/content/106/52/22358.abstract

• Nowick et al (2009) identified 90 TF genes with significantly different expression levels in human and chimpanzee brains,

among which the rapidly evolving KRAB-zinc finger genes are markedly over-represented.

• KRAB-zinc fingers have on average accumulated more amino acid differences between humans and chimpanzees than other genes, indicating that they may have contributed disproportionately to the phenotypic differences between these species

• KRAB-ZNFs affect transcription; they are transcription factors with an N-terminal KRAB domain and C-terminal zinc fingers

• The functions of many of these TFs are unknown

Trans-regulatory evolution in the Humans (focusing mainly on the brain)

Differential gene expression between humans and chimps

Nowick et al. 2009 PNAS

• Most expression differences were seen in testis• 25% of transcription factors examined (18 out of 79) in this

study showed changes in expression in the human brain

Evolution of gene regulatory networks in the Human Brain (Nowick et al. 2009)

• The transcription factors (TFs) form 2 tight gene networks (Fig 3), coordinately-regulated in expression in the brain (co-regulating 2 sets of functional categories of genes, Fig. 5), indicating dramatic shifts within larger biological pathways

• The TFs are involved in regulation of energy metabolism, vesicle transport, and related functions required to build and maintain the larger and more complex human brain

• Human TFs are more interconnected with each other than those of chimpanzees

Network of transcription factor (TF) genes that show evolutionary shifts in the human brain

Module 1:Dominated by TF genes up-regulated in human brain

Module 2:Dominated by downregulated TFs

Both modules are enriched for primate-specific KRAB-ZNF genes

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Network of transcription factor (TF) genes that show evolutionary shifts in the human brain

Module 1:Dominated by TF genes up-regulated in human brain

Module 2:Dominated by downregulated TFs

The coordinated regulation suggests that expression of these transcription factors is regulated by a common regulatory element or interacting elements (perhaps transcription factors)…

So, maybe: Master transcription factors à regulate transcription factors downstream

Increases in links of human transcription factors

• So, what functions are these transcription factors regulating?

• What are the differences between humans and chimps?

Functions of genes associated with transcription factors that underwent evolution in the human brain (2 functional modules)

• Module 1- Upregulated in Humans relative to chimpanzee: TF-associated genes highly enriched for functional categories involved in transcription, ubiquitination, and vesicular transport

• Module 2- Downregulated in Humans relative to chimpanzee: TF-associated genes, over-represented with functional categories corresponding to translation, mitochondrial function and energy metabolism

Functions of genes associated with transcription factors that underwent evolution in the human brain (now shown in comparison to the chimpanzee)

Evolution at the level of trans-regulators

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Figure 3. Trans-effects on developmental pattern divergence in the prefrontal cortex of the brain (Somel et al. 2011 PLoS Biology)

Human vs Chimp divergence

Evolution of trans-regulators,i.e. transcription factors and microRNAs in the brain

Same figure from your assigned reading

Type III: fundamental changes in the trajectories of gene expression patterns between humans and other primates

MORE RECENT UPDATES

• Perdomo-Sabogal et al. 2016. Human Lineage-Specific Transcriptional Regulation through GA-Binding Protein Transcription Factor Alpha (GABPa). Molecular Biology and Evolution. https://academic.oup.com/mbe/article/33/5/1231/2579702

• Perdomo-Sabogal et al. 2014. The role of gene regulatory factors in the evolutionary history of humans. Curr Opin Genet Dev.

Genomic deletions in the Human relative to chimpanzee

McClean et al. 2011 Nature

(not all evolution changes in humans is trans-regulatory)

Specific Deletions in the Human genome(McClean et al. 2011 Nature)

• 510 deletions in humans relative to chimpanzees • The deletions are almost exclusively in non-coding regions

and affect gene regulation• The deletions are enriched near genes involved in steroid

hormone signaling and neural function

• Deletions of tissue-specific enhancers may thus accompany both losses and gains of traits in the human lineage, and provide specific examples of the kinds of regulatory alterations and inactivation events long proposed to have an important role in human evolutionary divergence.

• One deletion in Homo removes a sensory vibrissae and penile spine enhancer from the human androgen receptor (AR) gene, a molecular change correlated with the anatomical losses of androgen-dependent sensory vibrissae (whiskers) and penile spines (penis spines) in Homo (loss at ~700,000 yrs ago)

• Another deletion removes a forebrain subventricular zone enhancer near the tumor suppressor gene: growth arrest and DNA-damage inducible, gamma (GADD45G), a loss correlated with expansion of specific brain regions in humans

Primate penile spines. Philip Reno, Stanford University

Specific Deletions in the Human genome (some are cis-regulatory)

(McClean et al. 2011 Nature)

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Evolutionary Tradeoffs associated with rapid brain evolution

• Particularly since much of the evolution of the brain appears to be due to trans-regulatory evolution,

• …greater pleiotropic constraint of trans-acting factors (leading to consequences for other functions)

• Gene deletions could also lead to costs

• Loss of tumor suppressor gene in Homo àpromoting excessive brain growth

• Humans appear to be less efficient than chimpanzees in carrying out programmed cell death

• Might in part be why humans have a much higher rate of cancer than chimpanzees

Gaurav Arora, Nalini Polavarapu, John F. McDonald. 2009. Did natural selection for increased cognitive ability in humans lead to an elevated risk of cancer? Medical Hypotheses

Evolutionary Tradeoff between Large Brain and Cancer Susceptibility?

Rapid evolution of the brain in Homo

• Rapid evolution à new opportunities, but also new problems: Evolution of many psychiatric disorders

• Humans are unique among animals in being susceptible to certain neuropathologies

Neurodegeneration with Aging

• Alzheimer's disease in the later stages of life

• Even healthy aging in humans is marked by variable degrees of neural deterioration and cognitive impairment, such as shrinking of the brain not found as much in aging chimpanzees

Schizophrenia and other psychiatric disorders

• Khaitovich et al. 2008. Metabolic changes in schizophrenia and human brain evolution. Genome Biology. http://genomebiology.com/2008/9/8/R124

• American Journal of Human Genetics, Song and Lowe et al.: "Characterization of a Human-Specific Tandem Repeat Associated with Bipolar Disorder and Schizophrenia" https://www.cell.com/ajhg/fulltext/S0002-9297(18)30238-6

• Popular Press: https://www.eurekalert.org/pub_releases/2018-08/cp-eci080218.php

Many genes that are expressed in Schizophrenia are genes that experienced rapid positive selection in Homo

Patterns of Molecular Evolution• What are mutations? How would structural vs regulatory

mutations affect function?

• Would you expect structural or regulatory evolutionary differences to predominate?

• What are cis- vs. trans-regulatory mutations?

• When would you expect cis-regulatory evolutionary differences to predominate? Over which evolutionary time scales? And Why?

• What about trans-regulatory differences?

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1.When comparing DNA sequences that encode a protein between two species, the number of substitutions at nonsynonymous was found to be much higher than those at synonymous sites. This result suggests evidence for:

(a) Non-adaptive evolution(b) Adaptive evolution(c) Negative selection(d) Evolutionary constraint(e) Preferential fixation of synonymous sites

2.Which statement is FALSE regarding genetic differences

between humans and other apes?(a) There are structural evolutionary differences in the alleles of FOXP2 that are unique to Homo sapiens and Homo neanderthalensis versus those of chimpanzees (b) The genome sequences of humans and other great

apes are mostly identical, with less than 2% differences in coding sequences(c) Some key differences between human and chimpanzee gene expression appear to be due to differences in expression of transcription factors

(d) The transcription factors that differ in expression between humans and chimpanzees form networks of coordinately-regulated genes (all up or down in expression as a unit)(e) Most key genetic differences between human and chimp

brain development appear to be due to cis-regulatory changes

3. Which type of mutations (among those that contribute to phenotypic evolution) would most likely persist over longer evolutionary time scales?(2 answers)

(a) Mutations in promoter sequence(b) Mutations in microRNA sequence(c) Mutations in transcription factor sequence(d) Mutations in enhancer sequence(e) Mutations in repressor sequence

• 1B• 2E• 3A, D

• Optional slides (Not required)

Cis- vs Trans-Regulatory?

• Both contribute to evolutionary change

• There are also other issues (see optional slides if interested)

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Cis- vs Trans-Regulatory?

• Cis-regulation contributes disproportionately to gene expression divergence between species, relative to its contribution within species (Wittkopp et al. 2008)

• Cis-regulatory variants alter allele-specific expression, whereas trans-regulatory factors influence expression of both alleles in a diploid cell.

Dominance between alleles

(1) Cis- vs Trans-Regulatory?• A promoter or enhancer (cis-) is only going to

regulate the gene that is downstream to it, and not the other allele (allele-specific expression).

• However, a transcription factor (trans-) will regulate both alleles (not allele-specific)

• Thus, a fundamental difference in coefficients of dominance between cis- and trans-regulatory variation might play significant roles

• Lemos et al. 2008. PNAS. http://www.pnas.org/content/105/38/14471.full

Cis- vs Trans-Regulatory?• Cis-regulated alleles tend to have additive effects

(each regulated independently by its own cis-regulatory elements – promoters, etc.)

• Cis variation is additive and therefore accessible to positive selection

• A Trans-regulatory protein could have a deleterious mutation that is masked from selection by the other functional allele (due to dominance)

• When Trans variation is masked in the recessive state it is not purged by negative selection

Selection on cis- vs trans- regulatory elements

• If a cis-regulatory element has a deleterious effect, it will be exposed to selection, they will be expressed independently

Allele 1

Allele 2

Allele 1 Allele 2

• If a trans-acting factor has a deleterious mutation, the trans-factor might be masked from selection, as the functional allele might compensate for reduced function in the other (dominance)

Deleterious trans-acting factor

Deleterious cis-regulatory element expressed

functional trans-acting factor takes over (dominant)

expressed

expressed

reduced or no expression

(2) Cis- vs Trans-Regulatory?

• The mutation variance for trans-variation is substantially larger than the mutation variance for cis-variation.

• More mutational targets in a trans-acting factor (of a protein) than for a promoter or enhancer

Also… Differences in Mutational Targets between cis- and trans-regulatory elements:

Cis- vs Trans-Regulatory?• So, what this means is that deleterious

mutations could accumulate more in trans-alleles à greater mutational target + masking in the recessive state…

• Such deleterious mutations could accumulate over time… such that over greater evolutionary distances the mutational load is higher in the trans- alleles…

• So, over greater evolutionary distances, cis-alleles might be favored over trans-alleles