Understanding human genome Structural evolution through chimpanzee and mouse comparisons

1
Understanding human genome (Structural) evolution
through chimpanzee and mouse comparisons

• Todd D. Taylor, Ph.D.
• Genome Annotation and Comparative Analysis Team
• Computational and Experimental Systems Biology
  Group
• RIKEN Genomic Sciences Center
• taylor_at_gsc.riken.jp
• Bioinformatics and Comparative Genome Analysis
  Course
• Institut Pasteur Tunis - Tunisia
• April 2, 2007

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RIKEN Genomic Sciences CenterYokohama, Japan
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Key projects

• Human
• Chromosome 21 (Nature, May 2000)
• 17 of 33.5 Mb
• Chromosome 18p (Nature, September 2005)
• 16 Mb
• Chromosome 11q (Nature, March 2006)
• 81 Mb
• 4-5 contribution to the Human Genome Project
• Chimpanzee
• Chromosome 22q (Nature, May 2004)
• 33.5 Mb (syntenic to human chr21)
• Chromosome Y (Nature Genetics, January 2006)
• Development of novel methods for gene and
  promoter prediction
• Identifying genes missed by other high-throughput
  methods
• Identification of unique regulatory mechanisms

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Comparative approaches for identification of
functional elements

• Looking for similarities
• Compare with distant species, like mouse
• Regions that are conserved may be important
• Looking for differences
• Compare with close species, like primates
• Regions that are different may be important
• Of course, there are exceptions to every rule!

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Phylogeny of human and its close relatives
5 MYa
Homo
250MYa
350MYa
Hominidae
Hominoidea
Pan
Hominidae
Catarrhini
Gorilla
Anthropoidea
Hominoidea
Eutheria (placentalia)
Primates
Amniota (amniotes)
Pongo
Mammalia
Primates
Gibbons
Old world monkeys
Mammalia
New world monkeys
Prosimians
Lagomorpha
Heterodonty Mammary glands Homoeothermic Hair Plac
entation (in most), amnion, internal
fertilization Sweat and sebaceous
glands Anucleate red blood cells
Rodents
Metatheria
Prototheria
Sauropsida
Reptilia Aves
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Mouse genome mapped on the human genome

• 34 maps to identical sequence in human genome

Hiram Clawson and Kate Rosenbloom (UCSC). 09 June
2006
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Chimpanzee genome mapped on the human genome

• 95 maps to identical sequence in human genome

Hiram Clawson and Kate Rosenbloom (UCSC). 09 June
2006
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Looking for similarities
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Human Chr21 DSCR vs. Mouse Chr16
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Multi-species comparisons
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Potential enhancer elements that are evolutionary
conserved
Nobrega, et al. Science 302, 413 (2003)
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Knocking out conserved sequences
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Effect on Dach1 gene expression pattern
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Looking for differences
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How different are humans arechimps?
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Important differences with humans

• Size
• Intelligence
• Language
• Ageing
• Disease susceptibility
• Cancer
• Schizophrenia
• Autism
• Triplet expansion diseases
• AIDS
• Hepatitis

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What makes us human?Is humanity written in our
genome?
Newton,2002?4??
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Science 295, 131-134 (2002)
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Separation of chromosomes by dual-laser cell
sorting
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BESs mapped to human genome
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BES identity distribution
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Crude differences between human and chimpanzee
genomes

• Number of simple repetitive sequences
• Insertion of Alu and L1 elements
• Unique sequences
• Local duplications
• Translocations
• Inversions
• Fewer CpG Islands predicted in chimp

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Whole chromosome sequencing strategy

• Compare with small representative human
  chromosome (21)
• Clone-based sequencing strategy
• Map chimp BAC-end sequences to human chr. 21
• Screen libraries for additional clones to fill
  gap regions

3 gaps, over 99 coverage
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Whole chromosome comparison
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Larger structural rearrange-ments
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Sequence alteration events per bp
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Distribution of divergence of the autosomes
(whole chimp genome)
Chimpanzee Sequencing Analysis Consortium.
Nature (205) 43769-87
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Base substitution rate

• Overall 1.44
• SINE/Alu 1.81
• LINE/L1 1.38
• CpG islands 2.26
• Simple repeats 4.06

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Correlation between alteration events
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Statistics of HSA21q and PTR22q
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Species-specific repeat expansions
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Emergence of human-specific characteristics
Human-specific characteristics have been acquired
during the 5 million years since the divergence
between Pan and Homo.
Orangutan
Gorilla
Time
Pongo (Orangutan)
Gorilla
Pan (Chimpanzee)
Homo (Human)
5?6MYa
Human(?)
Chimpanzee
Phylogeny of Hominidae
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Cladistic inference
Homo ACGTGTTTGAAATATTACTGATTGTAA Pan
ACGAGTTTGAAATATTATTGATTGTAA Gorilla
ACGTGTTTGAATCATTATTGATTGTAA Orangutan
ACGTGTTTAAATTATTATTGGTTGCAA LCA
ACGTGTTTGAAATATTATTGATTGTAA
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Human-specific large insertions
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Species-specific insertion-deletions
Human
Chimpanzee
Gorilla
Orangutan
positive amplification found for both chimp and
human template DNA
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Example 1 Deletion in Human Lineage
Example 2 Insertion in Human Lineage
1 2 3 4 1 2 3 4 1 1 2
1 2 3 4 1 2 3 4 1 1 2
4200
2900
106
117
106
Example 3 Deletion in Chimp Lineage
Example 4 Allelic Deletion in Chimp Lineage
Pt Hs Gg Pp
Pt Hs Gg Pp
1 2 3 4 1 2 3 4 1 1 2
1 2 3 4 1 2 3 4 1 1 2
2400
4200
1200
1300
154
129
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Human chromosome 21 gene catalog

• 284 genes
• 223 known
• 19 novel CDS
• 25 novel transcripts
• 12 putative
• 5 predicted
• 85 pseudogenes

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Gene catalog comparison

• We lacked information for 6 genes located in
  sequencing gaps
• 6 hsa21 genes are absent from the ptr22 sequence
  (H2BFS, 5 KAP genes from the 21q22.1 cluster)
• 4 hsa21 genes appear to be pseudogenes in chimp
• 3 ptr22 pseudogenes are absent from the hsa21
  sequence
• 1 hsa21 pseudogene has a complete ORF in ptr22

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ORF comparison

• 83 of genes have at least one amino acid
  replacement
• 10 of the potential ptr22 proteins are predicted
  to have a different length
• Amino acid insertion or deletion
• Different start codon
• Different stop codon
• Other, more complex rearrangement

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Amino acid length differences

• Shorter in chimp ADAMTS5

• Longer in chimp C21orf30

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Complex rearrangement TCP10L
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Gene conservation
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Species-specific amino acid replacements

• Human-specific replacements
• KIAA0184
• COL6A2
• HUNK
• AGPAT3
• DSCR3
• PWP2H
• STCH
• SLC5A3
• CHAF1B
• SIM2
• KCNE2
• APP
• C21orf98
• C21orf61
• IFNAR1
• UBASH3A
• TMPRSS3

• Chimp-specific replacements
• BACE2
• TIAM1
• BACH1
• FAM3B
• C21orf33
• ADAMTS1
• C21orf103
• ITGB2
• HLCS
• DNMT3L
• IFNGR2
• PPIA3L
• C21orf59
• MRPL39
• CLDN17
• KRTAP11-1
• CCT8

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Distribution of Ka/Ks ratios
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Distribution of Ka/Ks ratios
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GO categories with highest divergence rates in
hominids
Chimpanzee Sequencing Analysis Consortium.
Nature (205) 43769-87
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Evolutionary transcriptomics
Correralate phenotype with genotype Using
Affymetrix arrays it could be shown thatthe
amount of transcript/gene varies in a
species-specific manner (Enard et al. 2001). -gt
What DNA sequence differences are responsible for
the observed differences in transcript-levels?
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Multiple probes per gene
Transcription start site (TSS)
3UTR
5UTR
Promoter

• Transcriptional control
• RNA stability

Enhancer
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Probes mapped to human chr21
237 genes annotated for chromosome 21 189
represented on the affymetrix A-E arrays
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Gene expression profiling

• 189 annotated genes represented on the Affymetrix
  A-E arrays (Hellmann, Pääbo)

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(No Transcript)
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Primate phylogenetic shadowing?

• Identifying cis-regulatory elements in the human
  genome is a major challenge of the post-genomic
  era
• Promoters and enhancers that regulate gene
  expression in normal and diseased cells and
  tissues
• Inter-species sequence comparisons have emerged
  as a major technique for identifying human
  regulatory elements
• Particularly those to the sequenced mouse,
  chicken and fish genomes
• A significant fraction of empirically defined
  human regulatory modules
• Too weakly conserved in other mammalian genomes,
  such as the mouse, to distinguish them from
  nonfunctional DNA
• Completely undetectable in nonmammalian genomes
• Identification of such significantly divergent
  functional sequences will require complementary
  methods in order to complete the functional
  annotation of the human genome
• Deep intra-primate sequence comparison is a novel
  alternative to the commonly used distant species
  comparisons

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(No Transcript)
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Identification of known and novel conserved
sequences
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Evolutionary conservation of six
primate-conserved sequences
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Non-coding sequences with primate-specific
conservation include three regulatory elements
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Nature (2003) 424788-793
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Proportion of human aligned sequence by category
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Relative contribution of different mutational
events
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Conjoined genes
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Conjoined genes a novel gene class
Fused transcript formed by combining the exons of
two or more distinct genes (child genes)
Child gene A
Child gene B
Conjoined Gene A B
Exon
Intron

• Transcript A-B combines at least one exon
  (complete or partial overlap) from both Gene A
  Gene B
• Usually only supported by a few mRNA/EST
  sequences, and rarely by a CCDS
• Currently, about 32 known cases found by
  searching NCBI Entrez (including 8 from chr 11
  recently submitted by our group)

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Experimental verification
Chr1 SRP9 EPHX1 fusion (1 EST evidence-DA417873)
Alternate splicing and novel exons observed in
fused mRNA
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Conservation of conjoined genes in other
mammalian species
27 Conjoined genes conserved in Chimpanzee
6.5 Conjoined genes conserved in Mouse
Exons considered were part of conjoined gene
mRNAs
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Acknowledgments

• Chimpanzee Chr 22 Sequencing Consortium
• Chinese National Human Genome Center at Shanghai,
  China
• KRIBB Genome Research Center, Daejeon, Korea
• National Yang Ming University Genome Research
  Center, Taipei, Taiwan
• National Institute of Genetics, Mishima, Japan
• RIKEN Genomic Sciences Center, Yokohama, Japan
• GBF, Dept. of Genome Analysis, Braunschweig,
  Germany
• Institute for Molecular Biotechnology, Jena,
  Germany
• Max-Planck Institute for Molecular Genetics,
  Berlin, Germany

• RIKEN
• Yoshiyuki Sakaki
• Tulika P. Srivastava
• Vineet K. Sharma
• Asao Fujiyama
• Masahira Hattori
• Atsushi Toyoda
• Yoko Kuroki
• Yasushi Totoki
• Hideki Noguchi
• Hidemi Watanabe
• Takehiko Itoh (MRI)