1. Origins and Evolution of Novel Genes
Aoife McLysaght
Trinity College Dublin

2. Novelty novel genes increase complexity perform novel functions
role in speciation?
consider difficulty of a single gene acquiring a new function that does not hinder the original function compared to the evolution of a new function in an entirely new locus

3. Promoter
exon
intron
exon

4. Bricolage
Long et al., 2003

5. Polyploidy – whole genome duplication
Aneuploidy – chromosomal duplication
Partial chromosome duplication
Gene duplication
Partial gene duplication
Q: Where do new genes come from?
A: Other genes.
Genes don’t appear by chance from random DNA (or at least, they only do so very rarely)

6. Gene Duplication Create new genes
Generate multigene families / multidomain genes
gene duplication and corresponding deletion
length of dup/del depends on extent of misalignment
**unequal crossing over is facilitated by the presence of repeated sequences ... can get more tandem duplication

7. Exon/domain shuffling
Gene
structure
Protein domains
Domain complexity increases with organismal complexity
Rubin et al., Science, 2000

8. Survivorship/maintenance of gene duplicates may depend on:
Duplicability
Survivorship/maintenance of gene duplicates may depend on:
protein function
higher duplicability of metabolic genes in yeast (Marland et al, 2004)
network centrality
more highly connected proteins have lower duplicability in yeast but higher duplicability in human
evolutionary rate
higher duplication of slowly evolving genes (Davis and Petrov, 2004)
dosage balance
dosage-balanced genes are retained after whole genome duplication (WGD) but unlikely to experience small-scale duplication (SSD)

9. Fate of Duplicated Genes: Examples
Neofunctionalisation
GLUD2 in primates has a new role in neurotransmitter flux
Thrombin (cleaves fibrinogen during clotting) and trypsin (digestive enzyme) are derived from a complete gene duplication
Lactate dehydrogenase can be converted into malate dehydrogenase with a single amino acid replacement (out of total protein length of 317 amino acids)
Subfunctionalisation
SIR3 and ORC1 gene pair in yeast
Have divergent functions, but single ancestral-type protein from another yeast has both functions
Dosage increase
Esterase B in mosquito
increased gene dosage confers greater pesticide resistance
Functional compensation
Many duplicated genes shelter the organism from deleterious mutations in the other copy (shown in yeast and worm)

10. Functional compensation of duplicate genes
Essentiality of duplicated genes
Liao BY and Zhang. Trends in Genetics (2007)
Duplicate genes usually overlap in function.
Nematode
Sequence divergence of duplicated genes correlates with their capacity for back up function.
Conant GC and Wagner A. Proc. R. Soc. Lond. B (2004)

11. Polyploidisation Global increase in genome
Addition of one or more complete chromosome sets
2 copies : diploid
3 : triploid (sterile)
4 : tetraploid
6 : hexaploid
Polyploidisation
global increase in genome
entire genome duplicated
Organism with two copies of every chromosome: diplod
three tetraploid (infertile)

12. Examples of Paleopolyploids
Yeast
Arabidopsis
Wheat
Fish
Ancestral vertebrate (2R)

13. Loss or retention of genes duplicated by WGD (ohnologs)
Most duplicates are subsequently lost
Biased retention of certain classes of genes
Retained duplicates are enriched for:
Developmental genes
Transcription factors
Metabolic genes
Protein complex membership

14. Dosage-balance hypothesis
Dosage-balanced genes are not robust to gene loss and gene duplication.
Gene A
Gene B
Gene C
Gene D
Gene E
Pathway
Pathway
Gene A
Gene B
Gene C
Gene D
Gene E

15. Whole genome duplication and dosage-balanced genes
Gene A
Gene B
Gene C
Gene D
Gene E
Pathway
Gene A
Gene B
Gene C
Gene D
Gene E
Pathway
WGD duplicates all genes simultaneously and therefore does not perturb relative dosages. Whereas SSD of dosage-balanced genes is likely to be deleterious, WGD should be neutral. Furthermore, once duplicated by WGD they are unlikely to be lost

16. De novo origins Conversion of 3’ UTR into coding sequence
Incorporation of transposable elements into coding sequence

17. De novo origin of whole protein-coding genes
Origin of an open reading frame (ORF) from ancestrally non-coding sequence
Single-base substitutions or small indels that remove a stop codon
Acquisition of expression activity
Considered to be very rare events

18. New genes in Drosophila
Levine et al. 2006, PNAS
Five de novo originated genes found in Drosophila melanogaster
Begun et al. 2007, Genetics
11 genes that likely appeared in D. yakuba or the D. yakuba / D. erecta ancestor were identified using testis-derived ESTs
Testis biased expression
Often X-linked
Zhou et al., 2008, Genome Research
9 genes (some overlap with previous papers)
Estimate 12% of new genes arose de novo

19. New genes in Saccharomyces
Cai et al. 2008, Genetics
BSC4 identified as a de novo gene in S. cerevisiae (132 aa)
DNA similarity but no ORF in closely-related yeasts S. paradoxus, S. mikatae and S. bayanus
Transcibed in these other yeast lineages
Origin of protein-coding gene from RNA gene
Deletion of DUN1 or RPN4 is lethal if BSC4 is also deleted
PeptideAtlas evidence supports translation
Purifying selection
Possibly involved in the DNA repair pathway

20. De novo origin of mouse-specific gene
Heinen et al., 2009, Current Biology
Non-coding RNA gene
3 exons, alternatively spliced
Specifically expressed in post-meiotic cells of the testis
Indel mutations in 5’ regulatory region
Possible selective sweep

21. Novel primate genes

22. Human-Chimp Divergence
99% identity of alignable sequence
High colinearity of gene order
What is the genetic distinction?
Regulatory differences?
Differential gene duplication and loss?
40-45Mb of species-specific euchromatic sequence
Unique genes?

23. Differential gene duplication and loss
Demuth et al, 2006
Hahn et al, 2007

24. Genome Quality Issues } EnsEMBL family containing the
Centaurin Gamma 2
gene
Within synteny blocks
Out of synteny blocks
Hs Chr10
Pt Chr10
Genomic location of the human Chr10 genes }
Hs Chr7
Hs Chr10
Genes
Pt Chr2b
Hs Chr2
Pt Chr12
Hs Chr12
Hs Chr7
Pt Chr7
Hahn et al 2007 Genetics

25. De novo origins of monkey genes
Toll-Riera et al., (2009) MBE
Examined “primate orphans”
Protein-coding
Present in human and macaque but absent in older lineages

26. Have new genes arisen de novo recently in the human lineage?
This study:
Have new genes arisen de novo recently in the human lineage?

27. Unique human genes?
All-against-all BLASTP search identified 644 human genes with no match in the chimp genome
Candidate novel genes
examine these in great detail

28. Genome Quality Issues
Several spurious/trivial causes of apparent gene gain
candidate novel gene is spurious (human genome annotation error)
sequence gaps – gene is present but unsequenced
Chimp genome annotation error – gene is sequenced but unannotated

29. ? Strategy Synteny-based approach
Gene order is conserved between close taxa
Regions of conserved gene order are likely to be ancestral
The expected location of a gene can be identified and carefully examined
Human ?
Chimp

30. Synteny Blocks
Blocks with conserved gene order built using unambiguous orthologs:
String of orthologs no more than 10 genes apart in either genome.
Small local gene order differences permitted.

31. Expected location definition

32. 644
450
194 3

33. Novel human protein-coding genes
All short ORFS
No introns within coding sequence

34. ORF origins Examine orthologous DNA from chimp and macaque
Identify “disablers” - sequence differences that obstruct the ORF
Single base differences that cause an early stop
Frame-shift inducing indels that result in an early stop codon
Absence of a start codon

35. CLLU1 Chronic Lymphocytic Leukemia Upregulated gene 1 (CLLU1)
Start
Chronic Lymphocytic Leukemia Upregulated gene 1 (CLLU1)
Identified in a search for differentially expressed genes in chronic lymphocytic leukemia (Buhl et al 2006)
Located in a EST dense region
Overlapping another gene, CLLU1OS, in the opposite strand

36. Human origin or parallel primate inactivation of ancient gene?

37. CLLU1 Chronic Lymphocytic Leukemia Upregulated gene 1 (CLLU1)
Start
Chronic Lymphocytic Leukemia Upregulated gene 1 (CLLU1)
Identified in a search for differentially expressed genes in chronic lymphocytic leukemia (Buhl et al 2006)
Located in a EST dense region
Overlapping another gene, CLLU1OS, in the opposite strand

38. CLLU1

39. C22orf45

40. DNAH10OS

41. Are these ORFs actually genes?
The presence of an ORF does not guarantee that the gene is coding, i.e., that a protein is produced
PRIDE
PRoteomics IDEntifications is a public database for proteomics data
Peptide Atlas
Public database of peptides identified by mass spectrometry

42. Proteomics support

43. CLLU1
DNAH10OS
C22orf45

44. Human population polymorphism
ORF is present intact in all sequenced individuals (public data)
No convincing evidence for a selective sweep from published genome-wide scans of HapMap data.

45. How might these genes arise?
Sequence analysis traced the origin of the ORF, but these must also be expressed.
Expression of a new gene
ENCODE project indicated that much of the genome is transcribed
All three of these genes overlap other genes
CLLU1 is in a permissive expression environment

46. 3 identified cases under strict criteria
De novo genes: Summary
3 identified cases under strict criteria
Estimate about 18 should exist
All have evidence of transcription and translation
ORF formation allowed by human specific mutation in all cases
No “re-use” of coding sequence of previously-existing genes, but perhaps re-use of regulatory sequences.

47. Gene duplication: consequences
Innovation
Robustness
Consequences. NB, NOT causes
Neofunctionlisation
Functional compensation

48. Defining essential genes
A gene is considered “essential” if its removal results in a lethal or sterile phenotype.
Essential genes
(Lethal or sterile)
Non-essential genes
(other phenotypes)
Wild type
eyeless
vestigial
Fly
Kolodziej PA et al. Neuron (1995)
Wild type
Mouse
foxn1
Garacia MU et al. PNAS (2005)
Fly: 2540 essential and 5197 non-essential genes
Mouse: 2109 essential and 2969 non-essential genes

49. Evolutionary impact of gene duplications
PE - proportion of essential genes
Singletons
Duplicates
count lethal
knockouts
count lethal
knockouts
PE singletons
PE duplicates
>> =

50. Evolutionary impact of gene and genome duplications
PE - proportion of essential genes
Singletons
Duplicates
count lethal
knockouts
count lethal
knockouts
PE singletons
PE duplicates
>>
Functional compensation =

51. Functional compensation of duplicate genes
Essentiality of duplicated genes
Liao BY and Zhang. Trends in Genetics (2007)

52. All duplicates are not created equal
Whole Genome Duplication (WGD)
Small-Scale Duplication (SSD)
Differ in extent and frequency
Also differ in evolutionary impact???
WGD occurred in yeast, plant and animal.
SSD is ongoing.
WGD
Fly
Ascidian
Fish
Chicken
Mouse

53. Evolutionary impact of gene and genome duplications
PE - proportion of essential genes
SSD
WGD
count lethal
knockouts
count lethal
knockouts
PE SSD duplicates
PE WGD duplicates
>> =
<<

54. Essentiality of WGD and SSD duplicated genes in yeast
Correlation between sequence divergence and the proportion of essential genes for SSD duplicated genes
WGD duplicated genes are less essential than SSD duplicated genes in yeast.
Guan Y et al. Genetics (2007)

55. Defining duplicates and singletons
Duplicated genes and singletons
All-against-all blastp search for mouse (fly) (Ensembl 50)
E-value threshold: e-20
WGD duplicated genes in mouse
Human WGD duplicated genes
One-to-one orthology (Ensembl 50)
Mouse WGD duplicated genes
SSD duplicated genes in mouse
All duplicated genes excluding WGD duplicated genes

56. PE for WGD and SSD genes in mouseKA
(SSD: R = 0.94, P = 0.017)
SSD duplicated genes (38.1%)
< singletons (42.2%)
(P = 0.027)
SSD duplicated genes (38.1%)
< WGD duplicated genes (45.4%)
(P = 3.1 x 10-6, χ2 test)
No difference in essentiality between WGD duplicated genes (45.4%)
and singletons (42.2%)
(P = 0.10)
SSD duplicated genes carry out the expected backup role, but WGD duplicated genes are equally as essential as singletons in mammalian genomes.

57. PE for developmental genes
Duplicate developmental genes created by WGD were preferentially retained in vertebrate genomes.
Blomme T et al. Genome Biol. (2006)
Developmental genes: genes with GO: (multicellular organismal development) or GO: (cell differentiation)
Developmental genes in mouse:
singletons < duplicated genes
(P = , χ2 test)
Non-developmental genes in mouse and fly:
singletons > duplicated genes
(Mouse: P = )
(Fly: P = 2.8 x 10-8)
Developmental genes in fly:
Singletons ≈ duplicated genes
(P = 0.98)
Non-developmental genes: genes with other GO ids

58. Functional compensation of duplicate genes
Essentiality of duplicated genes
Liao BY and Zhang. Trends in Genetics (2007)
Data bias:
Developmental genes represent 37% of knockout data but only 11% of genome

59. Why is the essentiality of WGD genes high?
Dosage balance hypothesis:
Gene A
Gene B
Gene C
Gene D
Gene E
Pathway
Gene A
Gene B
Gene C
Gene D
Gene E
Pathway
Gene A
Gene B
Gene C
Gene D
Gene E
Pathway
Gene A
Gene B
Gene C
Gene D
Gene E
Pathway
WGD creates a unique opportunity for the duplication of dosage-balanced genes

60. Known categories of dosage-balanced genes
Developmental genes, transcription factors and protein complex members
1. Enrichment of developmental genes
WGD duplicated genes in our dataset are significantly enriched for the functional category ‘transcription regulator activity’.
2. Enrichment of transcription factors
WGD duplicated genes (21.8%; 388/1781)
vs. Total dataset (17.9%; 910/5078)
3. Enrichment of protein complex membership
(P = , χ2 test)
WGD duplicated genes are likely to be dosage-balanced genes.

61. Dosage balance hypothesis
Test: Are ohnologs refractory to changes in dosage?
SSD
Individual gene duplication within the vertebrate lineage
CNV (Copy Number Variation)
Recent (polymorphic) gene duplication in human populations

62. Recent SSD (within the tetrapod lineage)
Reconstructed “tetrapod gene families” based on inferred gene complement just after fish-tetrapod split
Two categories of family
Containing ohnologs
Not containing ohnologs
Count fraction of families that include at least one SSD event

63. Ohnologs are less likely to experience SSD
Along the human lineage
6.7% of ohnolog families have experienced subsequent SSD
10.1% of other genes duplicated in the same time period (P = 4.8 x 10-15)

64. Resistance to SSD predates WGD event
In pre-WGD lineages
Ascidian singletons (no lineage-specific SSD) are more likely to be orthologs of human ohnologs (30.1%; 1804/5998) than ascidian duplicates (20.6%; 649/3147; P < 2.2 x 10-16).
Similarly for fly, worm and sea anemone
Fly
Ascidian
Fish
Chicken
Human

65. Ohnologs also less likely to experience CNV
PCNV = Proportion of genes with copy number variation
Genome average = 29.3%, 6136/20907
Ohnologs = 22.6%, 1648/7294
SSD paralogs = 36.6%, 3306/9027
Ohnologs are unlikely to experience CNV whereas SSD-paralogs are likely to also display CNV

66. Many ohnologs are dosage-balanced
Retained ohnologs are resistant to duplication (SSD or CNV), even in distantly-related lineages that did not experience WGD.
Over 60% of ohnologs (4638/7294) are free of subsequent SSD and CNV
These are dosage-balanced ohnologs (DBOs)

67. DBOs are associated with disease
Data used to search for CNV was from healthy individuals
Studies have reported a link between CNV and disease
Duplication of a DBO is expected to be deleterious and lead to disease
DBOs identified here are enriched for disease genes in OMIM (P < 2.2x10-16)

68. Trisomy 21 – Down’s Syndrome
Extreme example of CNV
CNV of an entire chromosome
1.5-fold increase in dosage of some chr 21 genes results in Down’s Syndrome
Most commonly observed human trisomy
1/1000 individuals
Other trisomy mutations occur, but are lethal. Trisomy 21 has the least severe phenotypic consequences.
DBOs are significantly under-represented on chr 21 (obs. 40 vs. exp. 56.1, P=0.010)

69. Trisomy 21 candidate genes
75% (12/16) of reported DS candidates are also DBOs, which is significantly more than expected (2; P = 5.9 x 10-8)

70. Conclusions: novel genes
We show the first evidence of de novo formation of unique human genes
Newly formed genes show simple ORFs
Regions/tissues with permissive expression environments may favor this process
SSD duplicated genes have a backup role for their duplicated copies in mouse and in fly.
WGD-duplicated genes do not have a backup role and have high essentiality, because of the enrichment of dosage-balanced genes.
The evolutionary profile of gene duplication and retention can suggest a role in human disease.

71. Acknowledgements David Gonzalez Knowles Takashi Makino
Knowles & McLysaght, Genome Research (2009)
Takashi Makino
Makino et al., TiG (2009) and Makino & McLysaght PNAS (2010)

72. Human-chimp sequence divergence
Total (and nonsynonymous) base subsitutions pooled over all three genes
5 (2)
12 substitutions
Use macaque to orient the changes
5 in human, 7 in chimp
In human, 2 non-synonymous
In chimp, 3 non-synonymous-like
7 (3)