Mehdi Layeghifard

1. Mehdi Layeghifard Evolutionary Mechanisms Underlying the Functional Divergence of Vertebrates’ Circadian Rhythm Genes

2. 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

3. Gene duplication Duplications arise from an event termed unequal crossing-over that occurs during meiosis between misaligned homologous chromosomes

4. Gene duplication

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

6. 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

7. Natural Selection

8. 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 

9. Concerted Evolution

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

11. 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 (Dykhuizen–Hartl effect)

12. 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

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

14. 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

15. 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

16. Evolutionary Mechanisms

17. Evolutionary Mechanisms

18. 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

19. 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

20. Circadian Genes

21. Material & Methods • Data sets and phylogenetic analysis • Protein domain analysis • Analysis of functional divergence • Analysis of positive selection • Analysis of recombination

22. Material & Methods • Data sets and phylogenetic analysis a) Retrieving gene sequences 164 sequences from 7 groups b) Constructing phylogenies

23. 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

24. 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

25. 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

26. 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)

27. 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

28. 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

29. 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

30. 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

31. 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

32. 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

33. 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

34. 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

35. 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

36. 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 Dykhuizen–Hartl effect

37. 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

38. 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

39. 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

40. 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 

41. 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

42. 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

43. 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