2. DUF1220 Domains & the Search for the Genes that Made Us Human James M. Sikela, Ph.D. Human Medical Genetics, Neuroscience, & Comparative Genomics Programs, Department of Biochemistry & Molecular Genetics, University of Colorado School of Medicine Genomics Course February 28, 2012 James M. Sikela, Ph.D. Human Medical Genetics, Neuroscience, & Comparative Genomics Programs, Department of Biochemistry & Molecular Genetics, University of Colorado School of Medicine Genomics Course February 28, 2012

3. Key Points First gene-based and first genome-wide study of lineage-specific gene duplication and loss in human and primate evolution Dramatic human-specific increase in copy number of DUF1220 protein domains DUF1220 copy number linked to evolution of brain size Selection of evolutionarily adaptive genome sequences may be driving disease, e.g. 1q21.1 First gene-based and first genome-wide study of lineage-specific gene duplication and loss in human and primate evolution Dramatic human-specific increase in copy number of DUF1220 protein domains DUF1220 copy number linked to evolution of brain size Selection of evolutionarily adaptive genome sequences may be driving disease, e.g. 1q21.1

4. 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

5. Chimpanzee Gorilla Bonobo Orangutan

6. More Primates! ---- something has changed!

7. 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

8. 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 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 DUF1220 protein domains –Highly increased in copy number in humans; expressed in important brain regions

9. Molecular Mechanisms Underlying Genome Evolution Single nucleotide substitutions - change gene expression & structure Genome rearrangements Gene duplication - copy number change: gene dosage - redundancy as a facilitator of innovation Single nucleotide substitutions - change gene expression & structure Genome rearrangements Gene duplication - copy number change: gene dosage - redundancy as a facilitator of innovation

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 11:653-656, 2001

11. 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 (aCGH)

12. Experimental Design Carry out pairwise cDNA aCGH comparisons between human and other hominoid species Use a >39,000 cDNA microarray representing >29,000 human genes Hybridize human genomic DNA (reference sequence: cy3/green) and other hominoid genomic DNAs (test sequence: cy5/red) simultaneously to the microarray Visualize aCGH signals “gene-by-gene” along each chromosome across five species: human (n=5), bonobo (n=3), chimpanzee (n=4), gorilla (n=3) and orangutan (n=3) Carry out pairwise cDNA aCGH comparisons between human and other hominoid species Use a >39,000 cDNA microarray representing >29,000 human genes Hybridize human genomic DNA (reference sequence: cy3/green) and other hominoid genomic DNAs (test sequence: cy5/red) simultaneously to the microarray Visualize aCGH signals “gene-by-gene” along each chromosome across five species: human (n=5), bonobo (n=3), chimpanzee (n=4), gorilla (n=3) and orangutan (n=3)

13. Whole Genome Caryoscope Image of Interhominoid aCGH Data

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

16. Summary of Human/Primate ArrayCGH Results First genome-wide and first gene-based aCGH comparison of human and nonhuman primate gene copy number variation (Fortna, et al 2004) 1,004 (4,159) genes identified that showed lineage- specific changes in copy number Time machine of evolutionary copy number change Gene candidates to underlie lineage-specific traits Genes identified represent most of major lineage- specific gene duplications and losses over the last 60 million years of human and primate evolution (Dumas, et al 2007) First genome-wide and first gene-based aCGH comparison of human and nonhuman primate gene copy number variation (Fortna, et al 2004) 1,004 (4,159) genes identified that showed lineage- specific changes in copy number Time machine of evolutionary copy number change Gene candidates to underlie lineage-specific traits Genes identified represent most of major lineage- specific gene duplications and losses over the last 60 million years of human and primate evolution (Dumas, et al 2007)

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

19. “This (Fortna, et al, 2004) is the first time that copy number changes among apes have been assayed for the vast majority of human genes, and we can expect that the biological consequences of the 140 human- specific copy number changes identified in this study will be heavily investigated over the coming years. “ ---M. Hurles, PLoS Biol. 2004 ---M. Hurles, PLoS Biol. 2004

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

21. InterPro-predicted DUF1220-containing proteins (NBPF family*) * Vandepoule, et al, Mol. Biol. & Evol, 2005

22. 70 60 50 40 30 20 10 0 Q-PCR Predicted Copy Number Copy Number of DUF1220 (Q8IX62/17-33) Sequences in Primate Species Human BonoboChimp Gorilla Orangutan GibbonMacaqueBaboon

23. Summary of aCGH, Q-PCR and BLAT results: DUF1220 domains are highly amplified in human, reduced in great apes, further reduced in Old & New World monkeys, single or low copy non-primate mammals and absent in non-mammals

24. DUF1220 copy number in Animal Genomes GenomePDE4DIP DUF1220 Total DUF1220 NBPF genes Human226821 Chimp312515 Gorilla39915 Orangutan49211 Macaque13510 Marmoset13010 Rabbit183 Mouse110 Rat110 Guinea Pig110 GenomePDE4DIP DUF1220 Total DUF1220 NBPF genes Cow162 Pig131 Horse183 Dog131 Panda121 Opposum110Euarchotanglines Laurasiatheria Afrotheria Metatheria Elephant111Prototheria Platypus110 Chicken000 Lizard000 Frog000 Zebrafish000 Other Vertebrates A total of 40 genomes were searched, but only the 22 with 4X coverage or higher are displayed.

25. DUF1220 Copy Number Statistics in hg19 build DUF1220 Copies Total in Human Genome272 Total amplified HLS DUF1220 Triplets129 Total DUF1220 in Last Common Ancestor of Homo/Pan102 Total of Newly Added Copies in Human Lineage167 Total Copies Added via Domain Amplification146 Total Copies Added via Gene Duplication21 Average Number Added to Human Lineage every million years28 This table shows the unprecedented DUF1220 copy number increase in the human lineage. The primary mechanism for this expansion was domain amplification via hyper-amplification of the HLS DUF1220 triplet.

26. Sequences encoding DUF1220 domains Show a major copy number burst in primates Are increasingly amplified generally as a function of a species evolutionary proximity to humans, where the greatest number of copies (270) is found Show signs of positive selection Are highly expressed in brain regions associated with higher cognitive function In brain show neuron-specific expression preferentially in cell bodies and dendrites Show a major copy number burst in primates Are increasingly amplified generally as a function of a species evolutionary proximity to humans, where the greatest number of copies (270) is found Show signs of positive selection Are highly expressed in brain regions associated with higher cognitive function In brain show neuron-specific expression preferentially in cell bodies and dendrites Popesco, et al, Science 2006

28. Recurrent Reciprocal 1q21.1 Deletions and Duplications Associated with Microcephaly or Macrocephaly and Developmental and Behavioral Abnormalities 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 Recurrent Reciprocal 1q21.1 Deletions and Duplications Associated with Microcephaly or Macrocephaly and Developmental and Behavioral Abnormalities 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 1q21.1 Deletions* Linked to Microcephaly 1q21.1 Duplications* Linked to Macrocephaly 1q21.1 Deletions* Linked to Microcephaly 1q21.1 Duplications* Linked to Macrocephaly We note that these CNVs encompass or are immediately flanked by DUF1220 sequences (Dumas & Sikela, Cold Spring Harbor Symposium Quant. Biol., 2009) *Implies human brain size directly related to the dosage of one or more genes in these 1q21.1 CNVs

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

30. Association (p<0.0001) of human head circumference (FOC Z-score) & DUF1220 copy number

31. Copy number of genes in the 1q21.1-q21.2 region versus brain size 46 1q21.1 genes compared along with brain size across 5 primate species DUF1220 shows the most dramatic human-specific copy number increase. The evolutionary increase in DUF1220 copy number parallels the increase in brain size. HumanChimpOrangutanMacaqueMamoset Brain Size (g)1350380390887 Copy # DUF1220272125923530 PPIAL451100 LOC72885552221 FAM72D20000 SRGAP10000 PDE4DIP33411 SEC22B11111 NOTCH2NL11111 HFE211111 TXNIP11111 POLR322222 ANKRD3411111 ANKRD3511111 LIX1L11111 RBM8A11111 GNRHR211111 PEX11B11111 ITGA1011111 NUDT1711111 RNF11511111 CD16011111 PDZK131111 GPR8931111 PRKAB211111 PDIA3P11111 FMO511111 CHD1L11111 BCL911111 ACP611111 GJA511111 GJA811111 LOC64516610000 FCGR121111 SV2A11111 BOLA111111 MTMR1111111 OTUD7B11110 SF3B411111 VPS4511111 PLEKHO111111 ANP32E11111 PRPF311111 C1orf5411111 MRPS2111111 CA1411111 C1orf5111111 APH1A11111

32. DUF1220 Copy Number Versus Brain Size * Neandertal DUF1220 copy number is estimate based on sequence read depth from the Neandertal genome (Green et al 2010). -but correlation is not causation

33. Factors that must be reconciled with model linking 1q21.1 instability, evolutionary adaptation & recurrent disease Evolutionarily rapid DUF1220 copy number increase –Estimate, on average, 28 more DUF1220 domains added to human genome every 1 million years since Homo/Pan split Underlying mechanism must account for continued, recurrent DUF1220 increases Underlying mechanism must account for excess of 1q21.1 disease-associated CNVs containing dosage- sensitive genes Evolutionarily rapid DUF1220 copy number increase –Estimate, on average, 28 more DUF1220 domains added to human genome every 1 million years since Homo/Pan split Underlying mechanism must account for continued, recurrent DUF1220 increases Underlying mechanism must account for excess of 1q21.1 disease-associated CNVs containing dosage- sensitive genes

34. Increased 1q21.1 Instability Increase in DUF1220 Copy Number Evolutionary Advantage (Increase in Brain Size?) Proposed Mechanism Linking DUF1220, Brain Evolution and Disease 1q21.1 duplications Macrocephaly; Autism* 1q21.1 duplications Macrocephaly; Autism* 1q21.1 deletions Microcephaly; Schizophrenia* 1q21.1 deletions Microcephaly; Schizophrenia* *Diseases proposed as “Diametric Opposites” (including brain size), Crespi, Stead & Elliot, PNAS, 2009

35. DUF1220 Model* DUF1220 model proposes that: 1) DUF1220 copy number is directly involved in influencing human brain size, and 2) the evolutionary advantage of rapidly increasing DUF1220 copy number in the human lineage has resulted in favoring retention of the high genomic instability of the 1q21.1 region which, in turn, has precipitated a spectrum of recurrent human brain and developmental disorders DUF1220 model proposes that: 1) DUF1220 copy number is directly involved in influencing human brain size, and 2) the evolutionary advantage of rapidly increasing DUF1220 copy number in the human lineage has resulted in favoring retention of the high genomic instability of the 1q21.1 region which, in turn, has precipitated a spectrum of recurrent human brain and developmental disorders *Dumas & Sikela, Cold Spring Harbor Symposium Quant. Biol., 2009

36. Concluding Thoughts DUF1220 domains shows the largest HLS protein coding copy number increase in the genomeDUF1220 domains shows the largest HLS protein coding copy number increase in the genome –But no one gene made us human –DUF1220 genotyping challenges We know more about our genome than everWe know more about our genome than ever –But there are vast areas of our genome about which we know virtually nothing –No mammalian genome has been completely sequenced

38. Acknowledgements Sikela Lab Laura Dumas Majesta O’Bleness Maggie Popesco Erik MacLaren Andy Fortna Jan Hopkins Jonathon Keeney Jack Davis Jay Jackson Megan Sikela Michael Cox Kriste Marshall Matt Brenton Sonya Burgers Raquel Hink Erin Dorning Park McNair Sikela Lab Laura Dumas Majesta O’Bleness Maggie Popesco Erik MacLaren Andy Fortna Jan Hopkins Jonathon Keeney Jack Davis Jay Jackson Megan Sikela Michael Cox Kriste Marshall Matt Brenton Sonya Burgers Raquel Hink Erin Dorning Park McNair Collaborators Stanford –Jon Pollack –Young Kim Univ. of Kansas - Gerald Wyckoff Univ of Utah –Lynn Jorde Baylor College –Pawel Stankiewicz –Sau Wai Cheng UCSOM –Epidemiology Tasha Fingerlin –Preventive Medicine & Biometrics Anis Karimpour-Fard –Neuroscience Program Rock Levinson John Caldwell Collaborators Stanford –Jon Pollack –Young Kim Univ. of Kansas - Gerald Wyckoff Univ of Utah –Lynn Jorde Baylor College –Pawel Stankiewicz –Sau Wai Cheng UCSOM –Epidemiology Tasha Fingerlin –Preventive Medicine & Biometrics Anis Karimpour-Fard –Neuroscience Program Rock Levinson John Caldwell

39. A Walk Through Our Genome --All regions of the genome are not created equal