Bioinformatics. Transcriptome

Transcription

1 Bioinformatics Transcriptome Université Libre de Bruxelles, Belgique Laboratoire de Bioinformatique des Génomes et des Réseaux (BiGRe)

2 Bioinformatics Transcriptome Université Libre de Bruxelles, Belgique Laboratoire de Bioinformatique des Génomes et des Réseaux (BiGRe)

3 Measuring the expression of all the genes of a genome derisi et al. (1997). Science 278: In 1997, derisi and co-workers develop a method to measure the level of transcription of all the genes of a genome. The method allows to compare the concentrations of mrna of each gene between two experimental conditions Green channel: reference Red channel: test The intensity of a spot indicates the average concentration of the corresponding mrna in the two samples. The color of a spot indicates regulation: Red: up-regulated in the test, relative to the reference condition Green: down-regulated

4 DNA chip technology Cell culture, tissue,... RNA extraction Synthesis of fluorescent cdna Sample 1 Sample 2 RNA cdna RNA cdna Brightness Quantity Color Specificity yellowish reddish greenish not specific sample 1 - specific sample 2 - specific DNA chip Source: derisi et al., Science 1997

5 Scanning result slide from Peter Sterk

6 Complete microarray Source:DeRisi et al. (1997) Science, 278(5338), derisi et al. (1997). Science 278:

7 DNA chips raw measurements Raw measurements Red intensity Red background Green intensity Green background Intensity background = level of expression Red in experimental conditions Green in control

8 DNA chips useful metrics The level of regulation is represented by the ratio r = red " red.bg green " green.bg r >1 r < 1 up-regulated down-regulated The log-ratio provides a more convenient statistic (we will see why during the course) log 2 is even more convenient because the scale is intuitive # red " red.bg & R = log 2 % ( $ green " green.bg' R < 0 down-regulated R > 0 up-regulated R > 1 regulated by a factor of 2 R > 2 regulated by a factor of 4 R > w regulated by a factor of 2 w

9 Time series At each time point, the expression level is compared to the control (log-ratio) Example: Nitrogen depletion ORF Gene 30 min 1 hour 2 hours 4 hours 8 hours 12 hours 1 days 2 days 3 days 5 days YAL001C TFC YAL002W VPS YAL003W EFB YAL004W YAL004W YAL005C SSA YAL007C ERP YAL008W FUN YAL009W SPO YAL010C MDM YAL011W YAL011W YAL012W CYS YAL013W DEP YAL014C YAL014C YAL015C NTG Source: Gasch et al (2000) Molecular Biology of the Cell 11:

10 Examples of experimental conditions Presence/absence of a metabolite gal vs glucose Transcription factor mutants Yap1p over-expression TUP1 deletion Massive environmental changes rich versus minimal medium diauxic shift (7 time points during the shift) Cell differentiation sporulation mating type Cell cycle

11 Temporal profiles of expression derisi et al measured the level of expression of all the genes at 7 time points during the diauxic shit. The figure shows groups of genes show similar expression profiles, Some of these groups contain genes with similar function (e.g. coding for ribosomal proteins) Some of these groups have a common regulatory element in their promoter (e.g. stress response element). derisi et al. (1997). Science 278:

12 Cell cycle In 1998, Spellman and colleagues measure the expression of all yeast genes during the cell cycle. They detect 800 genes showing periodical fluctuations of expression. These genes can be sorted according to the peak of expression, in order to group genes induced during the different phases of the cell cycle (G1, S, G2, M). Spellman et al. (1998) Molecular Biology of the Cell 9:

13 Gene expression data: hierarchical clustering Alpha cdc15 cdc28 Elu MCM CLB2 SIC1 MAT CLN2 Y' On the image, genes are clustered according to expression profiles, using Michael Eisen s software cluster (Eisen et al., PNAS 1998: 95, ). Strengths The profiles and the clusters are visible together Familiar to biologists (frequently used for phylogeny) Weaknesses Isomorphism: each node of the tree can be permuted vertical distance between genes does not reflect the real distance Where to set the cluster boundaries? The tree does not reflect the combinatorial aspect of regulation MET Spellman et al. (1998). Mol Biol Cell 9(12),

14 Gasch (2000) - gene response to environmental changes Gasch et al. (2000) measure the transcriptional response of yeast genes to various environmental changes 173 microarrays ~6000 genes per microarray

15 Classification of cancer types Microarrays are also used to select genes which will serve as molecular signatures to classify cancer types. These genes can then be used to establish a diagnostic for new patients. Golub et al. (1999). Science 286: