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 ProgramsUniversity of Colorado School of Medicine

Advanced Genome Analysis CourseUniversity of Colorado School of

Medicine March 5, 2015

Primate EvolutionPrimate Evolution

New World Monkeys (e.g. squirrel monkey,spider monkey)New World Monkeys (e.g. squirrel monkey,spider monkey)

Old World Monkeys (e.g. baboon, rhesus, etc.)Old World Monkeys (e.g. baboon, rhesus, etc.)

GibbonsGibbons

OrangutanOrangutan

GorillaGorilla

HumanHuman

ChimpChimp

BonoboBonoboB/C = ~ 2C/H = ~ 5HC/G = ~ 8HCG/O = ~ 13HCG/O/Gib = ~20Hom/OWM = ~ 25HomOWM/NW = ~ 40

40 MYA

25 MYA

20 MYA

13 MYA

8 MYA

5 MYA

2 MYA

Chimpanzee

Gorilla

Bonobo

Orangutan

More Primates!

---- some things have changed!---- some things have changed!

Human CharacteristicsHuman 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

Reports of “human-specific” genesReports 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

HAR1F Gene

Marques-Bonet, et al Ann Rev Genomics 2009

Molecular mechanisms driving genome evolution

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

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

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)

Fortna, et al, PLoS Biol. 2004Fortna, et al, PLoS Biol. 2004

Human & Great Ape Genes Showing Lineage-Specific Copy Number Gain/Loss

Human

Gorilla Orang

ChimpBonobo

IMAGE:814107IMAGE:261219IMAGE:665496

HB

CG

O

BAC-FISH with clone containing SLC35F5 geneBAC-FISH with clone containing SLC35F5 gene

PLA2G4B/SPTBN5 gene copy number increases in African great apes

0 50 100 140 170 2501p36 1p34 1p31 1p22 1q21 1q23 1q32 1q41Mb

1p1320 210

1

2

0 90 130 170 200 2402p24 2p11 2q14 2q31 2q33 2q37Mb 2q2130 110 2p1650

3

Mb 0 80 160 180 2003p25 3p12 3q13 3q263q2520 130 3p21 50 3q21 3q28

Mb 0 100 1904p16 4q24 4q12 4q3410 80 4p12 50 4q31

4

140

Mb

5

0 100 1905p15 5q23 5q1320 70 5q1150 5q34150130

Mb

6

0 50 1706p25 6q126p21 30 130 10 6p22 40 6q1490 6q22 6q25

Mb

7

0 100 1607p21 7q11 7q21 60 30 7p14 90 7q31130 7q35140 7q22

Mb

8

0 120 1508p21 8q12 40 20 8p12 80 8q21100 8q2460

Mb

9

120 1509p23 9q21 40 30 9p13 80 9q22 100 9q34600

120 14010p15 10q21 10q24 40 2010p11 80 100 10q26500Mb

10

10q25

Mb

11

90 14011p15 11q12 11q13 20 10 11p14 70 11q24500 80 120 11q22 11q14

1 2 3

12

Mb 110 13012p13 12q13 12q14 30 10 12p12 70 12q24500 90 12q21

13

Mb 13q12 13q21 13q33 3013q14 110500 90

14

Mb 14q11 14q31 50 3014q13 700 1009014q32

15

Mb 15q13 700 10015q26 20 40 5015q21 15q2415q22

4 5

5q15

8q22

9q33

14q22

6 7

8

9

10

11

12 13

14

15

16Mb

16p13 700 9016q24 10 20 3016p12 16q22 5016q12

17p13 70 9017q23 10 20 3017q11 17q21 5017q12 17q25

17Mb

18Mb

18p110 10 20 8018q12 5018q21

19Mb

19p13 500 60 10 20 40 19q12 19q13 19p11

20Mb

20p130 10 30 6020q11 5020q13 20

21Mb

0 30 40 5021q22

22Mb

0 3022q11 50 22q13 20 40

XMb

Xp220 50 150Xp11 130Xq21 20 100 70 Xq26 Xq28

YMb

0Yp11 50 20

19q11

16

17

18

19

20

21 22

23

2>_0.5<_

Test/Reference ratio:

1

Human (Homo Sapiens)Bonobo (Pan Paniscus)Chimpanzee (Pan Troglodytes)

Orangutan (Pongo Pygmaeus)Gorilla (Gorilla Gorilla)

3

H

B

C

G

O

6

H

B

C

G

O

9

H

B

C

G

O

13

H

B

C

G

O

Human lineage-specific amplification of AQP7

Human lineage-specific amplification of AQP7

9p22

9q22

Oranutan

Chimpanzee

BaboonMarmosetLemur

HumanBonobo

Gorilla

GibbonMacaqueTest/Reference Ratio:

< 0.4 1 > 2.5

-1.6

-1.4

-1.2

-1

-0.8

-0.6

-0.4

-0.2

0

Hum

an

Bon

obo

Chi

mp

Gor

illa

Ora

ngut

an

Gib

bon

Mac

aque

Bab

oon

Mar

mos

et

Lem

ur

aCG

H l

og

2 F

luo

resc

ent

Rat

io

0

0.2

0.4

0.6

0.8

1

1.2

1.4

Qu

anti

tati

ve R

eal

Tim

e P

CR

C

op

y N

um

ber

aCGH

Q-PCR

r2=0.9532

AQP7AQP7

Human Chromosome 9

SMA SMA Chr5q13Chr5q13

Williams Beuren Williams Beuren Chr7q11.2Chr7q11.2

Prader-Willi Prader-Willi Chr15q11.1Chr15q11.1

DiGeorge DiGeorge Chr22q11Chr22q11

BLAT-Predicted Intronless vs. Intron-Containing HLS Gene Copies in Human, Chimp, and Macaque Genomes

0

5

10

15

20

25

30

35

40

45

50

3214

7047

0930

7813

8559

4438

8432

7612

1223

129

6679

3838

2311

9768

1262

2913

5010

2343

7627

9874

5090

429

7084

2986

8529

8862

3237

9645

1080

4702

6148

8945

6268

4270

4320

7303

9874

1841

7673

4581

1138

8235

8896

9906

1030

854

1031

047

1467

026

1468

074

1474

402

1557

341

1638

749

1641

894

1641

988

1683

035

1699

118

1759

573

1856

246

1874

052

1946

251

IMAGE Clone

Num

ber

of B

LAT

Hits

0

5

10

15

20

25

30

35

40

45

50

Human intron-containingChimp intron-containingMacaque intron-containingIntronless

*

DUF1220Repeat Unit

Popesco, et al, Science 2006

Synonymous and NonsynonymousDifferences Between Aligned Sequences

Synonymous and NonsynonymousDifferences Between Aligned Sequences

Ks = Average number of synonymous changesKa = Average number of nonsynonymous changesKs = Average number of synonymous changesKa = Average number of nonsynonymous changes

T h r P h eA C T T T T

A C C G T TT h r V a l

Nonsynonymous and SynonymousSites in Codons

Nonsynonymous and SynonymousSites in Codons

T h r Ph e

ACT T TT

T h r Ph e

ACT T TT

NNNN

NNNN

1/3 S1/3 SSS

2/3 N2/3 N

What will be the Ka/Ks values for most proteins?

Ka/Ks Distribution

0

200

400

600

800

1000

1200

1400

16000

.00

0.0

8

0.1

6

0.2

4

0.3

2

0.4

0

0.4

8

0.5

6

0.6

4

0.7

2

0.8

0

0.8

8

0.9

6

1.0

4

1.1

2

1.2

0

1.2

8

1.3

6

1.4

4

1.5

2

1.6

0

1.6

8

1.7

6

1.8

4

1.9

2

2.0

0

Ka/Ks value

Nu

mb

er

of

ge

ne

s p

er

bin

Intra-primate comparison mean:0.91Rodent-primate comparison

mean: 0.61

Num

ber o

f gen

es p

er b

in

Ka/Ks Distribution

Ka/Ks Value

• 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)

Genome PDE4DIPTotal

DUF1220NBPFGenes

Human 2 272 23Chimp 3 125 19Gorilla 3 99 15Orangutan 4 92 11Gibbon 3 53 10Macaque 1 35 10Marmoset 1 31 11Mouse Lemur 1 2 1Bushbaby 1 3 2Tarsier 1 1 0Rabbit 1 8 3Pika 1 1 0Mouse 1 1 0Rat 1 1 0Guinea Pig 1 1 1Squirrel 1 1 1Tree Shrew 1 4 3Cow 1 7 3Dolphin 1 4 1Pig 1 3 1Horse 1 8 3Dog 1 3 1Panda 1 2 1Cat 1 3 2Megabat 1 1 0Microbat 1 1 0Hedgehog 1 1 0Shrew 1 1 0

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

* Branch points in millions of years.* Branch points in millions of years.

A Chronology of DUF1220 Domain EvolutionA Chronology of DUF1220 Domain Evolution

O’Bleness, et al, G3: Genes, Genomes, Genetics, 2012

Consensus Tree of Evolutionary Relationships of 429 Primate DUF1220 Sequences

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

CON1 CON2

DUF1220 triplet

HLS1 HLS2 HLS3

CON3

DUF1220 Duplication and Protein Domain Classifications

Chimpanzee Human

DUF1220/NBPF Genome Organization in Chimp & Human

O’Bleness, et al, G3: Genes, Genomes, Genetics, 2012

Pres

tain

ed M

arke

rFr

onta

l Lob

eTe

mpo

ral L

obe

Parie

tal L

obe

Occip

ital L

obe

Cere

bellu

mPl

acen

ta

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.

36kDa5037.525

GAPDH

A

B

Popesco, et al Science 2006

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

A B C

P

denigl

ml

DUF1220 Protein Expression in Adult Human Brain

Popesco et al Science 2006

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

GFAP DAPI

DUF1220AffinityPurified Antibody

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

GFAP

DUF1220AffinityPurified Antibody

DAPI

Noteworthy DUF1220 Copy Number Totals

DUF1220 Copies

Total in Human Genome 272

Total in Chimp Genome (CLS) 125 (23)

Total in Last Common Ancestor of Homo/Pan 102

Total of Newly Added Copies in Human Lineage 167

Total Human-Specific Copies Added via Domain Amplification 146

Total Human-Specific Copies Added via Gene Duplication 21

Avg. Number Added to Human Lineage Every Million Years 28

O’Bleness, et al, G3: Genes, Genomes, Genetics, 2012

Sequences Encoding DUF1220 Domains• Show the largest human lineage-specific increase in copy Show the largest human lineage-specific increase in copy

number of any protein coding region in the genome (160 number of any protein coding region in the genome (160 HLS; >270 total in haploid genome) HLS; >270 total in haploid genome)

• Show signs of positive selection especially in primatesShow signs of positive selection especially in primates• In brain, are expressed only in neuronsIn brain, are expressed only in neurons• Are highly amplified in human, reduced in great apes, further Are highly amplified in human, reduced in great apes, further

reduced in monkeys, single-or-low copy in prosimians and reduced in monkeys, single-or-low copy in prosimians and non-primate mammals, and absent in non-mammalsnon-primate mammals, and absent in non-mammals

• Have increased in human primarily by domain hyper-Have increased in human primarily by domain hyper-amplification involving DUF1220 tripletamplification involving DUF1220 triplet

Key Human-Specific Evolutionary Features of 1q21.1 Region

O’Bleness, et al, Nat Rev Genet, 2012

‡*

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)

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

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.