genetic regulatory networks and...
TRANSCRIPT
How many genes?
wikimedia.org/ Botaurus stellaris/
wikimedia.org/Alberto Salguero
wikimedia.org/Luc Viatour
Questions
• 5,000-50,000 genes
– What does each gene do?
– How do they interact? (what kind of network do they form)
• Traditional approaches: gene-by-gene
– not practical for large genomes
– 30-75% of genes have no obvious effect when mutated
• Why no obvious effect?
– most genes do nothing?
– lab vs. real world?
– robust networks?
Gene regulatory networks
• Can think of genes as being linked through regulatorynetworks
transcription factor 2 transcription factor 3
transcription factor 1
RNAse
Downstream effectors
• How can this network be discerned?
– One way is by looking at gene expression
Microarrays: genome-wide transcript levels
• tens of thousands of genes
• would like to be able to examine all at once
• microarrays make this possible
• take advantage of specificity of DNA hybridization to
have a unique “probe” for each gene
• use photo-lithography for manufacturing
The Dimensions of a GeneChip55
””
55””
Up to ~6,500,000 Up to ~6,500,000
featuresfeatures // chipchip
1.28cm1.28cm
1.28cm1.28cm
5!m5!m
5!m 5!m
*** ***
Millions of identical Millions of identical probesprobes // featurefeature
http://www.affymetrix.com/corporate/outreach/lesson_plan/educator_resources.affx
CTAAGAGC
GATTCTCG
C : GT : AA : TA : TG : CA : TG : CC : G
Hybridization of matching DNA or RNA sequences Complementary DNA:DNA or DNA:RNA
sequences hybridize
http://www.affymetrix.com/corporate/outreach/lesson_plan/educator_resources.affx
Hybridization
Probe Array
Tagged RNA Target
Hybridized Array
FluorescentStain
Detect
http://www.affymetrix.com/corporate/outreach/lesson_plan/educator_resources.affx
Details of a gene chip
http://www.affymetrix.com/corporate/outreach/lesson_plan/educator_resources.affx
Hybridization of RNA to probes on chip
http://www.affymetrix.com/corporate/outreach/lesson_plan/educator_resources.affx
Specificity of hybridization
http://www.affymetrix.com/corporate/outreach/lesson_plan/educator_resources.affx
Specificity of hybridization
http://www.affymetrix.com/corporate/outreach/lesson_plan/educator_resources.affx
Fluorescence indicates amount of
RNA in sample
http://www.affymetrix.com/corporate/outreach/lesson_plan/educator_resources.affx
Expression array after scanning
http://www.affymetrix.com/corporate/outreach/lesson_plan/educator_resources.affx
How can microarrays help us build GRNs?
• Co-expression or Relevance Network
– measure gene expression across multiple samples
• after perturbation
• time course
• different individuals
• mutants
– Create correlation matrix
– Edges connect genes with correlation > threshold
Good Review: Markowetz, F. & Spang, R. Inferring cellular networks--a review. BMC Bioinformatics 8 Suppl 6, S5 (2007).
Alternative: Graphical Gaussian Models (GGMs)
• Make edges based on partial correlations
• correlations between each gene pair are conditioned on
all other genes in network
• challenges:
– number of genes >> samples
– sparse regression, other
A
CB
D E1. Schäfer, J. & Strimmer, K. An empirical Bayes approach to inferring
large-scale gene association networks. Bioinformatics 21, 754-64
(2005).
2. Opgen-Rhein, R. & Strimmer, K. From correlation to causation
networks: a simple approximate learning algorithm and its application to
high-dimensional plant gene expression data. BMC Syst Biol 1, 37(2007).
Adding direction
• It is important to have directional edges
• one approach is to use time-series data
• Examine (partial) correlation between
– each gene at t=i and the matrix of
gene expression at t=i-1,i-2…
use vector autoregressive (VAR) process
• If genes B and C at t=2 correlated with
gene A at t=1, then can infer direction
A
CB
D E
1. Opgen-Rhein, R. & Strimmer, K. Learning causal networks from systems biology time course data: an
effective model selection procedure for the vector autoregressive process. BMC Bioinformatics 8 Suppl 2,
S3 (2007).
2. Jie Peng, UC Davis.
Adding direction
• An alternative approach is experimental:
– determine which promoters are being bound by
transcription factors
www.chiponchip.org
Adding direction
• If gene A is a transcription factor and binds to the
promoters of B and C, then we can add direction to the
edges
A
CB
D E
Adding direction--using genetics
• If the genotype at gene A correlates expression of gene B then canadd direction.
• an extension of “genetical genomics”
A
CB
D E
100600225150500200expression
B
redblueredredblueredgenotype
A
Review: Li J, Burmeister M. Genetical genomics: combining genetics
with gene expression analysis. Hum Mol Genet. 2005 Oct 15;14 Spec
No. 2:R163-9
Flower Development
• How well do we understand the GRN controlling
flowers?
• How robust is the GRN to perturbation?
Flower development model
• Base topology on prior data
• Develop set of logical rules for interactions
Flower development model
• Model reproduces experimental gene expression
profiles
• Model is robust to starting conditions
• Model is robust to many single parameter changes
• Model predicts novel interactions which can be
experimentally tested.
Example: Circadian rhythms
• Most organisms display circadian rhythms
• controlled by biological clocks with ~ 24 hour
periodicity.
• How are biological clocks constructed?
clock model 2
• For each edge include ordinary differential equations (ODEs) that
describe reaction rates
• estimate ODE parameters by solving for best fit to experimental
data.
• run simulation to compare model to reality
single loop model does not fit data
simulated (dashed) and
experimental (solid) datarhythms persist in lhy mutant
Light = Food
Animals can walk to better foraging
Plants must grow to better foraging
As a consequence, plant development is extensivelyregulated by the environment
A complex network regulates growth
Brassino-steroid
light
ethylene auxin
clock
GA
Growth
Nozue, K., Maloof, J.N. (2006) Diurnal regulation of plant growth. Plant Cell Environ 29, 396-408.
How is growth controlled?
• Hypothesis: transcriptional regulation is involved in gating of dark-induced elongation.
• Experiment: Expression profiling to find genes whose expressioncorrelates with growth.
Nozue K, Covington MF, Duek PD, Lorrain S, Fankhauser C, Harmer SL, Maloof JN. (2007) Rhythmic growth explained by coincidence betweeninternal and external cues. Nature 448, 358-361.
838 genes are up-regulated
in growth phase
Top 10
cycling Dof-factor 3 (CDF3)hydrolasegigantea (GI)salt-tolerance protein (STO)phytochrome-interacting factor 4 (PIF4)phytochrome-interacting factor 5 (PIF5)expressed proteindentinGH3-3expressed protein
Nozue K, Covington MF, Duek PD, Lorrain S, Fankhauser C, Harmer SL, Maloof JN. (2007) Rhythmic growth explained by coincidence betweeninternal and external cues. Nature 448, 358-361.