Platelet Endo Meeting Feb2009

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  • 1. A new procedure for determining the genetic basis of aphysiological 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 2at 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. Functionallylinked 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 inducedgenes 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 Genet2008,24 (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 Res2007,35 (Web Server issue):W678-682Database 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 Biol2009,5 (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 ESTs28 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% successfulSRBH 49% RBH 41% 24. Gracey et al. 2004: Coping with cold: An integrative, multitissue analysisof the transcriptome of a poikilothermic vertebrate.17,825sequences on chip This data set proved appropriate for meta-analysisto predict cold-induced angiogenic genes inskeletal and heart muscle tissues.Cold induced genes 25. Carp gene expression cooling regimen B) 57 genes up-regulated on initial cooling 567 total non-redundantgenes 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 Coldinducedgenes 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