Variation in Primate Gene Expression

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Variation in Primate Gene Expression

• Human Genetics Journal ClubNovember 17, 2003

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(No Transcript)
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Overall Plan
Humans, Chimp, Orang, Macaque
M. musculus, M. spretus, M. Caroli
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Evolutionary Control
M. Caroli
M. Spretus
M. musculus
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Sampling Scheme

• U95A
• Brain (x2) 3 H. sapiens, 3 P. troglodytes , 1 P.
  pygmaeus
• Liver (x2) 3 H. sapiens, 3 P. troglodytes , 1 P.
  pygmaeus

• cDNA arrays
• Brain 7 H. sapiens, 4 P. troglodytes , 2 M.
  mulatta
• Liver6 H. sapiens, 5 P. troglodytes , 4 M.
  mulatta
• Blood 10 H. sapiens, 10 P. troglodytes , 10 M.
  mulatta

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Sampling Scheme

• Two Independent samples from adjacent areas
  within liver and brain.
• All subjects died of natural causes. No history
  of mental disease(humans) or brain abnormalities
  on autopsy (primates).
• Samples ascertained within six hours of death.
• Liver samples from different individuals-(humans-
  livechimps/orang-dead)

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Affymetrix vs. cDNA arrays
cDNA
Affymetrix

• 25nt probes
• 12,000 genes (U95A)
• Quartz slide

• 1kb probes
• 17,997 genes
• Membrane-based

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Distance matrix Calculations
Liver
Brain
want to convert ratio to linear distance
measure Taking absolute value eliminates
distinction between /- changes in expression.
Data Consists of 21 pairwise comparisons
(djks) between average expression level of 3
chimps, 3 humans, 1 orangutan
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Affymetrix Data
Bootstrap results(n1,000)
-Bootstrap values for primate branches 99.9
except for Orang-Brain branch-100 of mouse
bootstraps have same branching pattern
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cDNA Array Data
-16 differentially expressed genes were analyzed
by Northern Blot-11/12 genes detected by
Northern were quantitatively and qualitatively
similar to the cDNA array results for all 3
species and both tissue types
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2D Electrophoresis
Human
Chimpanzee
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2D Electrophoresis
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Conclusions-Enard, et al.

• Humans and chimpanzees each have species-specific
  patterns of gene expression that differentiate
  themselves from each other and macaque/orangutan.
• In both primate and mouse systems, expression
  differences acrue preferentially on the lineage
  for which oligos were designed.
• Relative rate of expression change on human
  lineage is higher in brain than liver via oligo-
  (3.8 vs. 1.7) and cDNA(5.5 vs. 1.3) arrays.
• Results corroborated by Northern blot and protein
  analysis

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Similar design Affymetrix Arrays followed by
quantitative RT-PCR, cDNA arrays, and in-situ
hybridization. No parallel mouse
experiments.Goal Identify individual genes
responsible for expression level differences
among primates, and the modal direction of this
deviation (if any).
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Sampling Scheme
Specifically FP, Frontal Pole MFG, medial
frontal gyrus IPL, inferior parietal lobule aIT
anterior inferotemporal cortex STG, superior
temporal gyrus TP, temporal pole.

CreditNeruoanatomy Atlas, University of
Nottinkgham
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Sampling Scheme (Brain)
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Sampling Scheme (Heart)
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U95A Hs/Pt - all probes
Cerebral Cortex-Caceres, et al.
Heart-Caceres, et al.
LiverEnard, et al.
Cerebral Cortex Enard, et al.
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U95Av2 Cortex Results
BULLFROG 230 probe setsDCHIP 89 probe
setsCombined 246 probe sets (plotted above)
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Clusters 1 4
-84 genes with a chimpanzee-specifc hybridization
pattern (38 increased/46 decreased vs hs/rm)
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Clusters 2 3
-143 genes with human-specific expression
patterns.132 increased (cluster 2)11 decreased
(cluster 3)
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Oligo Array Results All Tissues
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Validation RT-PCR
n26 genes of interest selected for analysis of
total RNA from cortex of 3 x (Hs, Pt, and
Mm)amplification of target gene and ß-actin
done in triplicate for each sampleExpression
changes were identified as a 1.3-fold difference
b/w average expression of each speices. no
expression change in 5/26 genes
n16 HsPt
n5 Hs 24
Validation cDNA array
n7,500 human cDNAs (Incyte) spotted in
duplicateCriteria for expression difference
pb/w species 1.364 of expression differences
were due to over-expression in Hs(vs. 92 seen
with oligo array)subset of 64 genes were
consistent between oligo- and cDNA array (33
stat. sig.-Fig1D)
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TWIST/CA2 In Situs
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Conclusions- Caceres, et al.

• Dont use heart tissue controls outside of
  oligo experiment.
• Final list of 169 genes with differences between
  human and chimp (167 agree qualitatively with
  Enard, et al.)
• 77/169 are also significant in heart or liver
  tissue.
• 91/169 are also differentially expressed
  betweenhuman and macaque.
• Perhaps only conclusion we can be sure of is the
  bias towards higher expression in human brain

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Over-expression. Why?

• General level of neuronal activity is increased
  within the brain
• Processes that support this activity are
  increased as well-genes involved in synaptic
  transmission, energy metabolism, protein folding
  (chaperones), and lipid synthesis

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Genes by function
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Distribution of Genes
Correlation Coeff.62
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OMIM entries