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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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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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

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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