functional functional genomics genomics Astrid Lægreid Department of Cancer Research and Molecular Medicine Norwegian University of Science and Technology Microarray Core Facility Norwegian Microarray Consortium 1
outline outline � functional genomics � gene expression � predicting gene function � challenges 2
functional genomics functional genomics biological biological system system screening generation of genome-wide hypoteses biological roles of genes and proteins experimental analysis modeling functions of computational genes and proteins biology 3
genome sequencing genome sequencing human genome � 3 x 10 9 basepairs � ~ 35.000 genes � > 100.000 splice variants genome- -wide screening wide screening genome � how? high-throughput - HTP � what? gene expression, gene-dosage, gene-variation (SNP), protein � with? microarray, mass spectrometry, 2D-gel electrophoresis 4
functional genomics functional genomics biological biological system system screening generation of genome-wide hypoteses biological roles of genes and proteins experimental analysis modeling functions of computational genes and proteins biology 5
chromosome 21 21 chromosome 127 known genes � � � � 98 unknown genes 6
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functional genomics functional genomics biological biological system system screening generation of genome-wide hypoteses biological roles of genes and proteins experimental analysis modeling functions of computational genes and proteins biology 8
from genome genome to to organism organism from genome gene gene copy (mRNA) protein organism 9
from genome genome to to organism organism from genome gene gene copy (mRNA) protein cell organs and tissues organism 10
from genome genome to to organism organism from genome gene gene gene expression expression organism 11
all cell types contain cell types contain the same the same genome genome all ..but differ but differ in gene in gene expression patterns expression patterns…. …. .. gene expression determines what you expression determines what you are…. are…. gene 12
to live is to live is to interact with interact with the environment the environment to challenge environment organism response 13
the organism responds organism responds the by making making new new proteins proteins by challenge environment gene protein response or by stop making making or by stop some of the old of the old ones ones….. ….. some 14
we can can learn learn more by more by measuring measuring we all genes of all expression of genes expression chromosome 1 2 3 4 5 23 gene expression depends expression depends on on gene cell type and type and cell cell state state cell 15
by measuring changes measuring changes in gene in gene expression expression by we can can discover discover genes genes participating participating in a in a we given biological response biological response given challenge chrom 1 2 3 4 5 23 chrom 1 2 3 4 5 23 response 16
measure thousands of of measure thousands genes and proteins proteins genes and in high throughput analyses throughput analyses in high 1 2 3 4 5 23 1 2 3 4 5 23 mRNA profiling protein profiling microarray 2D gel electrophoresis mass spectrometry 17
why measure mRNA? ? why measure mRNA genome gene gene copy (mRNA) protein organism because DNA microarray microarray is the is the because DNA most high throughput method throughput method most high that can can measure measure that gene expression with expression with high high gene sensitivity and and specificity specificity sensitivity 18
functional genomics functional genomics biological biological system system screening generation of genome-wide hypoteses biological roles of genes and proteins experimental analysis modeling functions of computational genes and proteins biology 19
DNA microarray microarray DNA DNA molecules molecules DNA = specific probe microscopic 5.000 - 80.000 probes pr probes pr. . array array 5.000 - 80.000 slide 20
microarray formats microarray formats � cDNA (500-1500 bp) � long oligonuleotides (40-70-mers) � short oligonucleotides (20-25-mers) 21
microarray analysis analysis microarray sample / / 1 sample control 1 control 3 3 scanning scanning RNA laser 1 laser 2 isolation labeling red = ”up” red = ”up” 2 2 hybridization hybridization green = ” = ”down down” ” green 22
microarray analysis microarray analysis 23 Bowtell, Nature Genetics, Supplement, 21:25, 1999
microarray analysis microarray analysis 24 Bowtell, Nature Genetics, Supplement, 21:25, 1999
functional genomics functional genomics biological biological system system screening generation of genome-wide hypoteses biological roles of genes and proteins experimental analysis modeling functions of computational genes and proteins biology 25
biological biological system system generation of hypoteses screening experimenal modelling analysis biological background information 26
biological biological system system generation of hypoteses screening experimenal modelling analysis gene expression biological background information gene function 27
functional classification of genes of genes functional classification from time profiles profiles from time Astrid Lægreid 1 and Jan Komorowski 2 1 Department of Cancer Research and Molecular Medicine Norwegian University of Science and Technology 2 The Linnaeus Centre for Bioinformatics, Uppsala 28
The Transcriptional Transcriptional Program in Program in The the Response Response of Human of Human the Fibroblasts to Serum to Serum Fibroblasts Iyer et al, Science, 283: 83, 1999 8 hours serum treatment 1, protein disulfide isomerase-related protein 2, IL-8 precursor 3, EST AA057170 4, vascular endothelial growth factor 29
fibroblast - 24 h serum - 24 h serum response response fibroblast serum serum samples for microarray analysis 0 1 4 8 24 quiescent proliferating non-proliferating 30
dynamic processes dynamic processes delayed immediate late early immediate intermediate early 0 1 4 8 24 primary secondary tertiary quiescent proliferating non-proliferating 31
molecular mechanisms of of molecular mechanisms transcriptional response response transcriptional serum serum effectors effectors = signal = signal secondary = cellular = cellular transcription response response factors immediate early response factors intermediate/late response genes delayed immediate early immediate early response genes response genes 32
fos - fos - immediate early transcription factor immediate early transcription factor Transpath; biobase.de 33
fos - fos - immediate early transcription factor immediate early transcription factor upstream factors upstream factors Transpath; biobase.de 34
fos - fos - immediate early transcription factor immediate early transcription factor upstream factors upstream factors Transpath; biobase.de 35
fos - fos - immediate early transcription factor immediate early transcription factor upstream factors upstream factors + downstream downstream genes genes + Transpath; biobase.de 36
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co-regulation of genes co-regulation of genes coding for proteins in a network coding for proteins in a network in fibroblast serum-response in fibroblast serum-response pro-endothelin active endothelin inactive endothelin 38
co-regulation of genes co-regulation of genes coding for proteins in a network coding for proteins in a network in fibroblast serum-response in fibroblast serum-response - + furin CALLA/CD10 + pro-endothelin active endothelin inactive endothelin 39
cellular processes cellular processes re-entry stress response cell cycle protein synthesis organelle transcription biogenesis cell lipid synthesis motility 0 1 4 8 24 quiescent proliferating non-proliferating 40
fibroblast serum-response fibroblast serum-response transcriptional program transcriptional program 517 gen-probes differential gene expression 497 unique genes 284 known genes 213 unknown genes 41
Iyer’s analysis of Iyer’s analysis of transcriptional fibroblast serum response fibroblast serum response transcriptional Expression clusters Functional clusters 42
functional classification functional classification from time profiles profiles from time our aim our aim find relationship between gene function - gene expression profile 43
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