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Mehdi Layeghifard Evolutionary Mechanisms Underlying the Functional Divergence of Vertebrates’ Circadian Rhythm Genes
Mehdi Layeghifard
Evolutionary Mechanisms Underlying the Functional Divergence of Vertebrates’ Circadian
Rhythm Genes
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
Gene duplication
Duplications arise from an event termed unequal crossing-over that occurs during meiosis between misaligned homologous chromosomes
Gene duplication
Evolutionary Mechanisms
• What is the evolutionary fate of duplicates?
• How duplicate genes are retained in a genome?
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
Natural Selection
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
Concerted Evolution
Neofunctionalization
• Genetic redundancy after duplication
• Gain of a new function by one of the duplicates
• Includes two scenarios
Neofunctionalization
First scenario:Functional redundancy leads to the fixation of random mutations in one duplicate under Relaxed Functional ConstraintLater, when the environment or genetic background is altered, the fixed mutations may induce a change in gene function (Dykhuizen–Hartl effect)
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
Subfunctionalization
• Genetic redundancy after duplication
• Both duplicates undergo Relaxed Functional Constraint
• Includes two scenarios
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
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
Evolutionary Mechanisms
Evolutionary Mechanisms
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
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
Circadian Genes
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
Material & Methods
1) Data sets and phylogenetic analysis
a) Retrieving gene sequences
164 sequences from 7 groups
b) Constructing phylogenies
Material & Methods
2) Protein domain analysisThe 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
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
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
Material & Methods
4) Analysis of positive selectionSite-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) methodBranch-site method
Using branch-site test 2 (also called the branch-site test of positive selection) developed by Zhang et al. (2005)
Material & Methods
5) Analysis of recombinationRecombination 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
GENECONVMaxChiBootscan
Results & Discussion
• PhylogenyThe 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
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
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
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
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
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
Results & Discussion
• Molecular evolutionary history of duplicates
a) NR1Ds
4) Two positively selected sites with P < 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
Results & Discussion
• Molecular evolutionary history of duplicates
b) CKIs
1) Only three functionally important sites between CKIδ and CKIε
2) None of the detected sites was mapped to Pkinase domain
3) Majority of negative sites were located in Pkinase domain
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 theoperation of positive selection Dykhuizen–
Hartl effect
Results & Discussion
• Molecular evolutionary history of duplicates
c) DECs
1) Detection of functional divergence between DEC proteins without signatures of positive
selection
2) Dykhuizen–Hartl effect as the most suitable model
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
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
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
Results & Discussion
• Molecular evolutionary history of duplicates
f) CRYs
1) No signatures of positive selection
2) Experimental and computational evidence of functionally divergence
3) Dykhuizen–Hartl effect as the most suitable model
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
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
