DUF1220 Protein Domains: Lessons from Human Brain Evolution

1. 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

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

3. Chimpanzee Bonobo Orangutan Gorilla

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

5. 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 Human Characteristics modified from S. Carroll, Nature, 2005

6. 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

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

9. 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

10. 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

11. Interhominoid cDNA Array-Based Comparative Genomic Hybridization (arrayCGH) 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).

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

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

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

18. Human Chromosome 9 AQP7 AQP7 Human Bonobo Chimpanzee Test/Reference Ratio: Gorilla Oranutan Gibbon < 0.4 1 > 2.5 Macaque r2=0.9532 Baboon Marmoset Lemur Human lineage-specific amplification of AQP7 9p22 9q22

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

20. *

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

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

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

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

25. 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) O’Bleness et al. Evolutionary History and Genome Organization of DUF1220 Protein Domains. G3 (Bethesda). Sept (2012).

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

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

29. DUF1220 Duplication and Protein Domain Classifications 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 CON1CON2HLS1HLS2HLS3CON3 DUF1220 triplet DUF1220 triplet NBPF12 HLS1HLS2HLS3 HLS1HLS2HLS3 CON1 CON2 CON3

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

31. 50 37.5 25 Prestained Marker Temporal Lobe Occipital Lobe Parietal Lobe Frontal Lobe Placenta Cerebellum A 36kDa B GAPDH 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. Popesco, et al Science 2006

32. C A B ml P den igl F E D DUF1220 Protein Expression in Adult Human Brain 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. Popesco et al Science 2006

33. (30yr old female) Hippocampus- CA regions- DUF1220 Affinity purified + GFAP + DAPI DUF1220 Affinity Purified Antibody GFAP DAPI

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

35. Noteworthy DUF1220 Copy Number Totals O’Bleness, et al, G3: Genes, Genomes, Genetics, 2012

36. 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 signs of positive selection especially in primates • 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 • Have increased in human primarily by domain hyper-amplification involving DUF1220 triplet

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

38. 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)

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

40. 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.