Platelet Endo Meeting Feb2009

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  • 1. 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 seminars John Herbert
  • 2. Why angiogenesis due to cold temperature? It is more difficult for muscles to utilise O 2 at lower temperatures
  • 3. What Evidence? Cold induced angiogenesis has been shown in mammals and vertebrates 25 o C 5 o C
  • 4. 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
  • 5. Functionally linked together by orthologs Basic strategy overview What is an ortholog and how to create this link with 20K anonymous carp DNA sequences Model species Angiogenic genes Non-model species Cold induced genes
  • 6. Bioinformatic Strategy 1) Ortholog prediction 3) Cold induced genes 2) Angiogenic genes 20K ESTs 17K Contigs Cold induced angiogenesis genes
  • 7. 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.
  • 8. BLAST on orthologous groups
  • 9. 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
  • 10. 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.
  • 11. Reciprocal Best Hit (RBH): Ortholog assignment using BLAST Human Carp
  • 12.
    • 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
  • 13. Choice of BLAST filter of low complexity makes a difference
  • 14. 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
  • 15. Eukaryotic Filter comparison
  • 16. 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)
  • 17. Genome duplication
  • 18. Teleost genome duplication: can be seen with genes important in endothelial biology Human chromosome 11 Zebrafish chromosome 18 (on other strand) Zebrafish chromosome 16
  • 19. Stepped Reciprocal Best Hit Zebrafish genome ~complete: 28K Proteins; 6Million ESTs
  • 20. Seq: AU052068 versus zebrafish and human SRBH: This was not perfect
  • 21. Conditional Stepped Reciprocal Best Hit
  • 22. CSRBH method of choice
  • 23. CSRBH 52% successful SRBH 49% RBH 41%
  • 24. Gracey et al. 2004: Coping with cold: An integrative, multitissue analysis of the transcriptome of a poikilothermic vertebrate. 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
  • 25. Carp gene expression cooling regimen B) 57 genes up-regulated on initial cooling 567 total non-redundant genes up-regulated A) 547 persistently up-regulated genes
  • 26. 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
  • 27. Combining results found 135 cold induced angiogenesis genes in carp Angiogenic gene prediction 908 Cold induced genes 567 Ortholog predictions 3762 135
  • 28. 12 genes picked out from 135 up-reg. angiogenesis genes in response to cold
  • 29. 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
  • 30. 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
  • 31. 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
  • 32. Good news, BMC genomics accepted it just on Friday 
  • 33. Acknowledgements (Thanks)
    • Roy Bicknell
    • Stuart Egginton
    • Francesca Buffa
    • Henrik Vorschmitt
    • Andy Gracey
    • Andrew Cossins
    • Molecular Angiogenesis Group
    • Cancer Research UK funding