Mehdi Layeghifard

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Mehdi Layeghifard

• Evolutionary Mechanisms Underlying the
  Functional Divergence of Vertebrates Circadian
  Rhythm Genes

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Gene duplication

• is one of the most important mechanisms in the
  evolution of gene diversity
• is any duplication of a region of DNA that
  contains a gene it may occur as an error in
  homologous recombination, a retrotransposition
  event, or duplication of an entire chromosome

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Gene duplication

• Duplications arise from an event termed unequal
  crossing-over that occurs during meiosis between
  misaligned homologous chromosomes

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Gene duplication
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Evolutionary Mechanisms

• What is the evolutionary fate of duplicates?
• How duplicate genes are retained in a genome?

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Natural Selection

• If reproductive success is impeded by a mutation,
  then selection of organisms with the mutation is
  Negative
• If reproductive success is promoted then the
  selection is Positive
• In the middle is Neutral selection, that may lead
  to either weak positive or weak negative
  selection

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Natural Selection
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Concerted Evolution

• The extra amount of a gene product is sometimes
  beneficial (dosage effect)
• Daughter genes may become fixed through strong
  Purifying Selection
• Duplicate genes will have very similar sequences
  and functions and will be prevented from being
  diverged 

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Concerted Evolution
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Neofunctionalization

• Genetic redundancy after duplication
• Gain of a new function by one of the duplicates
• Includes two scenarios

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Neofunctionalization

• First scenario
• Functional redundancy leads to the fixation of
  random mutations in one duplicate under Relaxed
  Functional Constraint
• Later, when the environment or genetic
  background is altered, the fixed mutations may
  induce a change in gene function (DykhuizenHartl
  effect)

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Neofunctionalization

• Second scenario
• After duplication, a new but weak function maybe
  created by a few neutral or nearly neutral
  mutations
• Positive Darwinian selection then, accelerates
  the fixation of advantageous mutations that
  enhance the activity of novel function

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Subfunctionalization

• Genetic redundancy after duplication
• Both duplicates undergo Relaxed Functional
  Constraint
• Includes two scenarios

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Subfunctionalization

• Division of Expression scenario
• Random fixations of complementary degenerate
  mutations under Relaxed Functional Constraint are
  the main causes of duplicates fixation in the
  genome

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Subfunctionalization

• Functional Specialization scenario
• Ancestral gene already has dual functions and
  the duplication provides the opportunity for each
  duplicate to adopt one ancestral function and
  further substitutions under positive Darwinian
  selection can refine the function

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Evolutionary Mechanisms
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Evolutionary Mechanisms
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Circadian Rhythms

• Important in determining the sleeping and feeding
  patterns of all animals
• Have been described in many eukaryotic and
  prokaryotic species
• Living organisms use this endogenous circadian
  clock, which can be synchronized to daily and
  seasonal changes in light and temperature,
  to anticipate environmental transitions,
  perform activities at biologically
  advantageous times during the day, and
  undergo characteristic seasonal responses

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Circadian Genes

• Circadian rhythm pathway of vertebrates is
  consisted of seven groups of genes
• Per, Clock, Bmal, NR1D, DEC, Cry, and CKI
• The circadian system, like many other multigene
  families, has undergone gene duplication, and so
  circadian genes that are found in single copies
  in insects are duplicated in vertebrates

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Circadian Genes
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Material Methods

1. Data sets and phylogenetic analysis
2. Protein domain analysis
3. Analysis of functional divergence
4. Analysis of positive selection
5. Analysis of recombination

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Material Methods

• Data sets and phylogenetic analysis
• a) Retrieving gene sequences
• 164 sequences from 7 groups
• b) Constructing phylogenies

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Material Methods

• 2) Protein domain analysis
• The Simple Modular Architecture Research Tool
  (SMART) is an online resource used for protein
  domain identification and the analysis of protein
  domain architectures
• We used this tool to identify the potential
  domains of circadian proteins in order to better
  predict the functional properties of these single
  domains and also to depict their role in the
  functional divergence of circadian proteins

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Material Methods

• 3) Analysis of functional divergence
• Gene family evolution reflects a balance between
  homogenization by unequal crossing over and gene
  conversion and diversification by mutation
• Among these mechanisms, only mutation followed
  by positive Darwinian selection or relaxation of
  functional constraints can account for the
  evolution of new functions, although the two
  other factors play an important role in the
  evolutionary fate of duplicated genes

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Material Methods

• 3) Analysis of functional divergence
• DIVERGE
• Type I functional divergence refers to the
  evolutionary process that results in altered
  selective constraints (different evolutionary
  rates) between two duplicate genes, regardless
  of the underlying evolutionary mechanisms,
  developed by Gu (1999)
• SHIFT-FINDER
• Uses the same approach used by DIVERGE but with
  more sensitivity

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Material Methods

• 4) Analysis of positive selection
• Site-based methods
• Using several codon models of molecular
  evolution that allow for heterogeneous dN/dS
  ratios at sites
• SLAC method
• Conservative tests of positive selection based
  on Suzuki and Gojobori (1999) method
• Branch-site method
• Using branch-site test 2 (also called the
  branch-site test of positive selection)
  developed by Zhang et al. (2005)

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Material Methods

• 5) Analysis of recombination
• Recombination can play a dominant role in the
  generation of novel patterns of genetic variation
  through the rearrangement of existing genetic
  variation generated through mutation. These
  patterns of genetic variation can closely
  resemble the effects of positive selection
• GENECONV
• MaxChi
• Bootscan

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Results Discussion

• Phylogeny
• The phylogenetic analysis indicated that
  members of each group of circadian genes were
  generated by a gene duplication event in the
  early stages of vertebrates' evolution
• The duplication events of Cry and Bmal groups
  were seemingly occurred in teleosts and those of
  other groups were occurred before the evolution
  of teleosts

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Results Discussion

• Functional divergence
• Site-specific altered selective constraint
  after the gene duplications was statistically
  significant for all groups
• Functionally important sites were mapped on the
  sequences

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Results Discussion

• Positive selection
• The SLAC method, like codon models of PAML,
  predicted no positively selected site in
  circadian genes with probabilities above 95
• Periods of positive Darwinian selection
  following the duplication events were found in
  Clock, NPAS2, PER1,2,3, and NR1D1 lineages by
  branch-site method

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Results Discussion

• Recombination
• Those events that were detected by at least two
  of the three implemented methods were only
  considered
• Zero to three recombination events for gene
  groups
• Low levels of recombination (fewer than three
  events in a dataset of about ten sequences) have
  no significant effect on positive selection
  analysis

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Results Discussion

• Molecular evolutionary history of duplicates
• The data obtained from all the molecular
  evolutionary analyses were used to provide a
  clearer picture of mechanisms behind the
  functional divergence of circadian genes

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Results Discussion

• Molecular evolutionary history of duplicates
• a) NR1Ds
• 1) Significant functional divergence was
  mainly because of amino acid changes occurred
  in HOLI domain
• 2) HOLI is a highly conserved DNA-binding
  domain
• 3) Functional divergence between these
  proteins may due to recognizing different DNA
  sequences

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Results Discussion

• Molecular evolutionary history of duplicates
• a) NR1Ds
• 4) Two positively selected sites with P lt 0.05
  in NR1D1
• 5) 1 of NR1D2 amino acids have experienced
  positive selection
• 6) Functional Specification is probably the
  most suitable model for describing the
  evolutionary fates of these duplicates

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Results Discussion

• Molecular evolutionary history of duplicates
• b) CKIs
• 1) Only three functionally important sites
  between CKId and CKIe
• 2) None of the detected sites was mapped to
  Pkinase domain
• 3) Majority of negative sites were located in
  Pkinase domain

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Results Discussion

• Molecular evolutionary history of duplicates
• b) CKIs
• 4) Pkinase domain is highly conserved
• 5) Might have only experienced the purifying
  selection
• 6) Occurrence of functional divergence without
  the operation of positive selection
  DykhuizenHartl effect

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Results Discussion

• Molecular evolutionary history of duplicates
• c) DECs
• 1) Detection of functional divergence between
  DEC proteins without signatures of positive
  selection
• 2) DykhuizenHartl effect as the most suitable
  model

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Results Discussion

• Molecular evolutionary history of duplicates
• d) CLOCK and NPAS2
• 1) Positive selection and significant
  functional divergence were found
• 2) Neofunctionalization with positive
  selection or Functional Specification
• 3) Experimental studies have shown that both
  genes play almost the same role in circadian
  rhythm pathway, but in different tissues

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Results Discussion

• Molecular evolutionary history of duplicates
• d) CLOCK and NPAS2
• 4) Ancestral gene might have been active in
  all tissues
• 5) Duplicates underwent Functional
  Specification
• 6) CLOCK is active in central circadian clock
  in suprachiasmatic nuclei, while NPAS2 is
  active in peripheral oscillator within other
  tissues like liver

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Results Discussion

• Molecular evolutionary history of duplicates
• e) BMALs
• 1) Significant functional divergence and
  positive Darwinian selection
• 2) Neofunctionalization with positive
  selection model suggested as the most suitable
  explanation 

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Results Discussion

• Molecular evolutionary history of duplicates
• f) CRYs
• 1) No signatures of positive selection
• 2) Experimental and computational evidence of
  functionally divergence
• 3) DykhuizenHartl effect as the most suitable
  model

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Results Discussion

• Molecular evolutionary history of duplicates
• g) PERs
• 1) PER family members acquired their new
  functions through mutations followed by
  positive selection after gene duplication
• 2) Neofunctionalization with positive
  selection model suggested as the most suitable
  explanation

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Final Words

• We showed that the evolution of circadian genes
  have depended on gene duplication and functional
  divergence and that each group of genes involved
  in circadian rhythm pathway (which are duplicates
  of one-copy ancestral genes) has experienced an
  independent evolutionary fate following
  duplication, i.e., there have been different
  forces behind the functional divergence detected
  between circadian rhythms gene
• This research also showed the importance of
  molecular evolution approaches in finding
  supporting evidence for experimental results as
  well as proposing new hypotheses to be tested by
  experimental research