2. Human Evolutionary Genomics: Lessons from DUF1220 Protein Domains, Cognitive Disease and Human Brain Evolution James M. Sikela, Ph.D. Department of Biochemistry & Molecular Genetics Human Medical Genetics and Neuroscience Programs University of Colorado School of Medicine Advanced Genome Analysis Course University of Colorado School of Medicine March 5, 2015

3. Primate Evolution New World Monkeys (e.g. squirrel monkey,spider monkey) Old World Monkeys (e.g. baboon, rhesus, etc.) Gibbons Orangutan Gorilla Human Chimp Bonobo B/C = ~ 2 C/H = ~ 5 HC/G = ~ 8 HCG/O = ~ 13 HCG/O/Gib = ~20 Hom/OWM = ~ 25 HomOWM/NW = ~ 40 40 MYA 25 MYA 20 MYA 13 MYA 8 MYA 5 MYA 2 MYA

4. Chimpanzee Gorilla Bonobo Orangutan

5. More Primates! ---- some things have changed!

6. Human Characteristics Body shape and thorax Cranial properties (brain case and face) Small canine teeth Skull balanced upright on vertebral column Reduced hair cover Enhanced sweating Dimensions of the pelvis Elongated thumb and shortened fingers Relative limb length Body shape and thorax Cranial properties (brain case and face) Small canine teeth Skull balanced upright on vertebral column Reduced hair cover Enhanced sweating Dimensions of the pelvis Elongated thumb and shortened fingers Relative limb length Neocortex expansion Enhanced language & cognition Advanced tool making Neocortex expansion Enhanced language & cognition Advanced tool making modified from S. Carroll, Nature, 2005

7. Reports of “human-specific” genes FOXP2 –Mutated in family with language disability ASPM/MCPH –Mutated in individuals with microcephaly HAR1F –Gene sequence highly changed in humans SRGAP2 (neuronal migration?) –Partial human-specific gene duplication DUF1220 protein domains –Highly increased in copy number in humans; expressed in important brain regions FOXP2 –Mutated in family with language disability ASPM/MCPH –Mutated in individuals with microcephaly HAR1F –Gene sequence highly changed in humans SRGAP2 (neuronal migration?) –Partial human-specific gene duplication DUF1220 protein domains –Highly increased in copy number in humans; expressed in important brain regions

8. HAR1F Gene Marques-Bonet, et al Ann Rev Genomics 2009

10. Molecular mechanisms driving genome evolution Single nucleotide substitutions - change gene expression - change gene structure Genome rearrangement Gene/segmental duplication - copy number change - value of redundancy Single nucleotide substitutions - change gene expression - change gene structure Genome rearrangement Gene/segmental duplication - copy number change - value of redundancy

11. Gene Duplication & Evolutionary Change “There is now ample evidence that gene duplication is the most important mechanism for generating new genes and new biochemical processes that have facilitated the evolution of complex organisms from primitive ones.” - W. H. Li in Molecular Evolution, 1997 “Exceptional duplicated regions underlie exceptional biology” - Evan Eichler, Genome Research, 2001

12. Fig 1. Measuring genomic DNA copy number alteration using cDNA microarrays (array CGH). Fluorescence ratios are depicted in a pseudocolor scale, such that red indicates increased, and green decreased, gene copy number in the test (right) compared to reference sample (left). Interhominoid cDNA Array-Based Comparative Genomic Hybridization (arrayCGH)

14. Fortna, et al, PLoS Biol. 2004 Human & Great Ape Genes Showing Lineage-Specific Copy Number Gain/Loss

15. Human GorillaOrang Chimp Bonobo IMAGE:814107 IMAGE:261219 IMAGE:665496 H B C G O BAC-FISH with clone containing SLC35F5 gene

16. PLA2G4B/SPTBN5 gene copy number increases in African great apes

19. Human lineage-specific amplification of AQP7 9p22 9q22 Oranutan Chimpanzee Baboon Marmoset Lemur Human Bonobo Gorilla Gibbon Macaque Test/Reference Ratio: 2.5 r 2 =0.9532 AQP7 Human Chromosome 9

20. SMA Chr5q13 Williams Beuren Chr7q11.2 Prader-Willi Chr15q11.1 DiGeorge Chr22q11

21. *

22. DUF1220 Repeat Unit Popesco, et al, Science 2006

23. Synonymous and Nonsynonymous Differences Between Aligned Sequences Synonymous and Nonsynonymous Differences Between Aligned Sequences Ks = Average number of synonymous changes Ka = Average number of nonsynonymous changes Ks = Average number of synonymous changes Ka = Average number of nonsynonymous changes

24. Nonsynonymous and Synonymous Sites in Codons Nonsynonymous and Synonymous Sites in Codons N N N N N N N N 1/3 S S S 2/3 N What will be the Ka/Ks values for most proteins?

25. Intra-primate comparison mean:0.91 Rodent-primate comparison mean: 0.61 Number of genes per bin Ka/Ks Distribution Ka/Ks Value

26. DUF1220 shows greatest human specific copy number expansion of any protein coding sequence in the human genome Show signs of positive selection Human increase primarily due to domain amplification (rather than gene duplication) GenomePDE4DIP Total DUF1220 NBPF Genes Human227223 Chimp312519 Gorilla39915 Orangutan49211 Gibbon35310 Macaque13510 Marmoset13111 Mouse Lemur121 Bushbaby132 Tarsier110 Rabbit183 Pika110 Mouse110 Rat110 Guinea Pig111 Squirrel111 Tree Shrew143 Cow173 Dolphin141 Pig131 Horse183 Dog131 Panda121 Cat132 Megabat110 Microbat110 Hedgehog110 Shrew110 O’Bleness et al. Evolutionary History and Genome Organization of DUF1220 Protein Domains. G3 (Bethesda). Sept (2012).

28. * Branch points in millions of years. A Chronology of DUF1220 Domain Evolution O’Bleness, et al, G3: Genes, Genomes, Genetics, 2012

29. Consensus Tree of Evolutionary Relationships of 429 Primate DUF1220 Sequences

30. Ancestral DUF1220 found in human PDE4DIP NBPF-type DUF1220 Domains Clades CON1-3 are conserved DU1220 sequences among primates Clades HLS1-3 refers to a three-DUF1220 domain unit that has expanded only in the human lineage CON1 CON2 HLS1 HLS2 HLS3 CON3 DUF1220 triplet NBPF12 HLS1 HLS2 HLS3 CON1CON2 DUF1220 triplet HLS1 HLS2 HLS3 CON3 DUF1220 Duplication and Protein Domain Classifications

31. ChimpanzeeHuman DUF1220/NBPF Genome Organization in Chimp & Human O’Bleness, et al, G3: Genes, Genomes, Genetics, 2012

32. Prestained Marker Frontal Lobe Temporal Lobe Parietal Lobe Occipital Lobe Cerebellum Placenta Western analysis of Normal Adult Human Brain regions with DUF1220 antibody: Total protein lysates (50ug) from normal adult human brain regions (male and female; ages ranging from 22-82yrs) were electrophoresed on 4-20% denaturing SDS-PAGE gels and blotted with: A) DUF1220 affinity purified antibody B) GAPDH. 36kDa 50 37.5 25 GAPDH A B Popesco, et al Science 2006

33. DUF1220 antibody staining in the human cerebellum (77yr old white female). A) DUF1220 affinity purified antibody; B) Double labeling with DUF1220 affinity purified antibody and Neurofilament 160kDa; C) same as B-higher magnification; D) Double labeling with DUF1220 affinity purified antibody and GFAP; E) DUF1220 preimmune and GFAP; F) DUF1220 Adsorption control. Blue labeling represents DAPI for nuclear staining. D E F AB C P den igl ml DUF1220 Protein Expression in Adult Human Brain Popesco et al Science 2006

34. (30yr old female) Hippocampus- CA regions- DUF1220 Affinity purified + GFAP + DAPI GFAPDAPI DUF1220 Affinity Purified Antibody

35. (30yr old female) Cortical regions- Hippocampus- DUF1220 Affinity purified + GFAP + DAPI GFAP DUF1220 Affinity Purified Antibody DAPI

36. Noteworthy DUF1220 Copy Number Totals DUF1220 Copies Total in Human Genome272 Total in Chimp Genome (CLS)125 (23) Total in Last Common Ancestor of Homo/Pan102 Total of Newly Added Copies in Human Lineage167 Total Human-Specific Copies Added via Domain Amplification146 Total Human-Specific Copies Added via Gene Duplication21 Avg. Number Added to Human Lineage Every Million Years28 O’Bleness, et al, G3: Genes, Genomes, Genetics, 2012

37. Sequences Encoding DUF1220 Domains Show the largest human lineage-specific increase in copy number of any protein coding region in the genome (160 HLS; >270 total in haploid genome) Show the largest human lineage-specific increase in copy number of any protein coding region in the genome (160 HLS; >270 total in haploid genome) Show signs of positive selection especially in primates Show signs of positive selection especially in primates In brain, are expressed only in neurons In brain, are expressed only in neurons Are highly amplified in human, reduced in great apes, further reduced in monkeys, single-or-low copy in prosimians and non-primate mammals, and absent in non-mammals Are highly amplified in human, reduced in great apes, further reduced in monkeys, single-or-low copy in prosimians and non-primate mammals, and absent in non-mammals Have increased in human primarily by domain hyper- amplification involving DUF1220 triplet Have increased in human primarily by domain hyper- amplification involving DUF1220 triplet

38. Key Human-Specific Evolutionary Features of 1q21.1 Region O’Bleness, et al, Nat Rev Genet, 2012 ‡*

39. 1q21.1 Deletions linked to Microcephaly* 1q21.1 Duplications linked to Macrocephaly* Recurrent Reciprocal 1q21.1 Deletions and Duplications Associated with Microcephaly or Macrocephaly and Developmental and Behavioral Disorders Brunetti-Pierri, et al, Nature Genetics 2008 Recurrent Rearrangements of Chromosome 1q21.1 and Variable Pediatric Phenotypes Mefford, et al, N. Engl. J. Med. 2008 *Implies the copy number (dosage) of one or more genes in this region is influencing brain size in a dose-dependent manner These CNVs encompass or are immediately flanked by DUF1220 sequences (Dumas & Sikela, Cold Spring Harbor Symposium Quant. Biol., 2009)

40. DUF1220/NBPF Sequences & Recurrent Disease-associated 1q21.1 CNVs

41. Human Evolutionary Genomics: Relevant Reviews Sikela, J.M. (2006). The Jewels of Our Genome: The Search for the Genomic Changes Underlying the Evolutionarily Unique Capacities of the Human Brain. PLoS Genet. 2, e80. O’Bleness, M.S., Searles, V., Varki, A., Gagneux, P., and Sikela, J.M. (2012). Evolution of genetic and genomic features unique to the human lineage. Nat. Rev. Genet., 13, 853-866.