DUF1220 Domains & the Search for the Genes that Made Us Human

1. 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

2. 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

3. 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)

4. Chimpanzee Bonobo Orangutan Gorilla

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

6. 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

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 • DUF1220 protein domains • Highly increased in copy number in humans; expressed in important brain regions

8. 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

9. 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 Research11:653-656, 2001

10. Interhominoid cDNA Array-Based Comparative Genomic Hybridization (aCGH) 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).

11. 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)

12. Whole Genome Caryoscope Image of Interhominoid aCGH Data

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

15. 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)

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

18. “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, PLoSBiol. 2004

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

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

21. Orangutan Macaque Bonobo Baboon Gibbon Human Gorilla Chimp Copy Number of DUF1220 (Q8IX62/17-33) Sequences in Primate Species 70 60 50 Q-PCR Predicted Copy Number 40 30 20 10 0

22. 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

23. DUF1220 copy number in Animal Genomes Euarchotanglines Laurasiatheria Afrotheria Metatheria Prototheria A total of 40 genomes were searched, but only the 22 with 4X coverage or higher are displayed. Other Vertebrates

24. DUF1220 Copy Number Statistics in hg19 build 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.

25. 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 Popesco, et al, Science 2006

27. 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 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 *Implies human brain size directly related to the dosage of one or more genes in these 1q21.1 CNVs We note that these CNVs encompass or are immediately flanked by DUF1220 sequences (Dumas & Sikela, Cold Spring Harbor Symposium Quant. Biol., 2009)

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

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

30. 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.

31. 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

32. 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

33. Proposed Mechanism Linking DUF1220, Brain Evolution and Disease 1q21.1 duplications Macrocephaly; Autism* 1q21.1 deletions Microcephaly; Schizophrenia* *Diseases proposed as “Diametric Opposites” (including brain size), Crespi, Stead & Elliot, PNAS, 2009

34. 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 *Dumas & Sikela, Cold Spring Harbor Symposium Quant. Biol., 2009

35. Concluding Thoughts • DUF1220 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 ever • But there are vast areas of our genome about which we know virtually nothing • No mammalian genome has been completely sequenced

37. Sikela Lab Laura Dumas MajestaO’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 PawelStankiewicz SauWai Cheng UCSOM Epidemiology Tasha Fingerlin Preventive Medicine & Biometrics Anis Karimpour-Fard Neuroscience Program Rock Levinson John Caldwell Acknowledgements

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