Platelet Endo Meeting Feb2009
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Transcript of Platelet Endo Meeting Feb2009
A new procedure for determining the genetic basis of a physiological process in a non-model species,
illustrated by cold induced angiogenesis in the carp
PhD dinner seminarsJohn Herbert
Why angiogenesis due to cold temperature?
It is more difficult for muscles to utilise O2 at lower temperatures
Oxygen solubility vs. deffusion
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5
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35
0 5 10 15 20 25 30 35 40 45 50
degrees C
Sol
./Diff
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O2 solubility
O2 diffusion
What Evidence? Cold induced angiogenesis has been shown in
mammals and vertebrates
25oC
5oC
In the non-model species carp (no genome and little transcriptome ~20K EST sequences), ortholog relationships and microarray re-analyses were used to predict
identity of cold induced angiogenic genes
Model species
Angiogenic genes
Non-model species
Cold induced genes
Functionally linked together
by orthologs
Basic strategy overview
What is an ortholog and how to create this link with 20K
anonymous carp DNA sequences
Bioinformatic Strategy
1) Orthologprediction
3) Cold induced genes
2) Angiogenicgenes
20KESTs
17KContigs
Cold induced angiogenesis genes
How to find orthologs?
Kuzniar A, van Ham RC, Pongor S, Leunissen JA: The quest for orthologs: finding the corresponding gene across genomes. Trends Genet 2008, 24(11):539-551.
BLAST on orthologous groups
BLASTO illustrates limitations with public available software
Zhou Y, Landweber LF: BLASTO: a tool for searching orthologous groups. Nucleic Acids Res 2007, 35(Web Server issue):W678-682
Database from 2007
Singular, just one sequence
Limited filtering options
Use a reciprocal Best Hit approach (BLAST)
• Altenhoff AM, Dessimoz C: Phylogenetic and functional assessment of orthologs inference projects and methods. PLoS Comput Biol 2009, 5(1):e1000262. bidirectional best-hits (BBH)
• Another surprise is the good overall performance of the simple Bidirectional Best Hit approach. Although the method is restricted to 1:1 orthologs, the derived relations show good comparative accuracy in terms of Fitch's definition. Orthologs predicted by BBH also show close functional relatedness.
Reciprocal Best Hit (RBH): Ortholog assignment using
BLAST
Human Carp
• BLAST (heuristic algorithm)• Divides query into words• Searches words against database
sequences• Extends out word matches• An alignment is produced called a
high scoring segment pair• Complexity of words makes a
difference
Choice of BLAST filter of low complexity makes a
difference
Testing BLAST filtering settings
• Moreno-Hagelsieb and Latimer (2008) found soft filtering and Smith-Waterman alignments improved RBH at least 10% in prokaryotic genomes
• We employed a similar approach in this work (eukaryotes). The soft filtering made a a big difference but S-W very little
Eukaryotic Filter comparison
Eurkaryote soft filtering and SW vs. hard filtering and no SW
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soft filter SW soft filter no SW hard filter SW
condition tested
no
rmal
ised
RB
Hs
How to improve on this?
Zebrafish
Another model species
Genome is almost complete
Currently 6 million ESTs
28 thousand Refseq proteins
Carp……….only….
20K ESTs
Diverged 50 MYA
Use zebrafish as stepping stone (SRBH)
Genome duplication
Teleost genome duplication:can be seen with genes important in endothelial
biology
Human chromosome 11
Zebrafish chromosome 18 (on other strand)
Zebrafish chromosome 16
Stepped Reciprocal Best Hit
Zebrafish genome ~complete: 28K Proteins; 6Million ESTs
Seq: AU052068 versus zebrafish and human
SRBH: This was not perfect
Conditional Stepped Reciprocal Best Hit
CSRBH method of choice
Comparison of ortholog assignment methods
0%
20%
40%
60%
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100%
CSRBH SRBH RBH
method used
% o
f su
cces
ful
assi
gn
men
ts
no hits
Faileds
Success
CSRBH52% successful SRBH 49%RBH 41%
Gracey et al. 2004: Coping with cold: An integrative, multitissue analysis of the transcriptome of a poikilothermic vertebrate.
67% of carp cDNAs on the array
67%
33%
on array
missing
17,825 sequences on chip
This data set proved appropriate for meta-analysis to predict cold-induced angiogenic genes in skeletal and heart muscle tissues.
Cold induced genes
Carp gene expression cooling regimen
A) 547 persistently up-regulated genesB) 57 genes up-regulated on initial cooling
567 total non-redundant genes up-regulated
Identifying physiologically relevant genes in human
• 1) In-house method involved collecting genes based on the authors own angiogenesis research, combined with genes on the SABiosciences commercial angiogenic array and those listed in other literature.
• 2) Gene data was downloaded from Genbank: Perl programs, in conjunction with PubMed, were used to find article abstracts that contained angiogenesis keywords
• 3) Gene Ontology process data searched for the same keywords
• 4) Endothelial transcriptomes (Wallgard et al. 2008 and our list) were cross-referenced
• From 3,762 genes found CSRBH, 908 were associated with endothelium or angiogenesis
Combining results found 135 cold induced angiogenesis genes in
carp
Angiogenicgene prediction
908
Cold induced
genes567
Orthologprediction
s3762
135
12 genes picked out from 135 up-reg. angiogenesis genes in response to cold
Granulin (GRN), what the papers say related to
angiogenesis• GRN is not a direct acting angiogenic
factor but has been shown to stimulate VEGF expression in breast carcinoma cells
• GRN is expressed within proliferating ovarian carcinomas blood vessels and interacts with perlecan: regulates tumour angiogenesis
• GRN antibody has been shown to inhibit tumour angiogenesis in human hepatomas implanted into athymic mice
Nucleolin (NCL), what the papers say related to
angiogenesis• Expression of NCL is particularly high on
the surface of ECs in angiogenic blood vessels
• Inhibition of NCL in ECs prevents capillary tube formation and retards EC migration
• MMP9 is associated with NCL in angiogenesis. Hypoxia led to a 3-fold increase in MMP9 protein due to enhanced translational efficiency caused by nucleolin binding to the 3’ UTR of MMP9
• ABs to NCL have been shown to suppress tumour growth and angiogenesis
This bioinformatic strategy works
• CSRBH most efficient method of high-throughput ortholog assignment in non-model species
• Combined with microarray re-analysis and physiological predictions, finds biologically relevant genes in a non-model species
Good news, BMC genomics accepted it just on Friday
Acknowledgements (Thanks)
• Roy Bicknell• Stuart Egginton• Francesca Buffa• Henrik Vorschmitt• Andy Gracey• Andrew Cossins• Molecular Angiogenesis Group• Cancer Research UK funding