DNA microarrays

1
DNA microarrays to study nuclear receptor
function
Karin Dahlman-Wright The nuclear receptor group
and the BEA (bio-informatics and expression
analysis) core facility, Department of
Biosciences, Karolinska Institutet
2
Outline

• General introduction
• Microarray technology with focus on the
  Affymetrix GeneChip technology
• Problems and promises of microarray technology
• Applications

3
1. General introduction
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How can DNA microarrays be used to study nuclear
receptor function?

• mRNA applications
• Gene expression profiling for the simultaneous
  detection of very large numbers of genes (all)
• Quantitative monitoring of the expression levels
  of very large numbers of genes under the
  influences of genetic, biochemical and chemical
  perturbations can help us to understand the
  function of genes and how they act together to
  regulate and carry out cellular process. The
  ultimate goal is to reconstruct the global gene
  interaction network.
• Custom design chips to detect levels of NRs (and
  associated proteins) in physiology and disease
• DNA applications
• Human genetics-Candidate gene approaches
• Resequencing
• ChIP on ChIP

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Genetic information to cellular function
GENOME
Chromatin access
Transcription initiation
Transcription elongation
Transcription termination
Splicing
RNA editing
Nuclear RNA degradation
Nuclear-cytoplasmic transport
Cytoplasmic mRNA degradation
TRANSCRIPTOME
Ribosome recruitment
Translation initiation
Translation elongation
Translation termination
Posttranslational modifications
Processive cleavage
Transport - export
Protein degradation
PROTEOME
6
THE HUMAN TRANSCRIPTOME (approximate
figures)

• In each given cell, 35,000
  (100,000) genes (mRNAs) exist and could be
  expressed10 - 30,000 genes are actually
  expressed 1 - 10,000 of these are common to
  many or all cells 5 - 20,000 code for
  specialized cell functionsEach gene is
  expressed as 1 to gt10,000 mRNA copies
  Expression levels fall in abundance classes
  schematically tens of mRNAs have
  thousands of copies hundreds of mRNAs have
  hundreds of copies thousands of mRNAs have
  tens of copiesThe total number of mRNA
  molecules is 100,000 - 1,000,000

7
mRNA applications

• Transcriptional dysregulation Find genes with
  altered expression
• Gene network identification Find networks of
  co-regulated genes
• Pathway identification Find pathways affected in
  disease and by pharmacolocical intervention
• Disease classification Find good marker-genes
  for classifying disease
• Transcript discovery Find transcripts from
  genomic sequence
• Functional prediction Predict function for
  uncharacterized genes

8

• 2. Microarray technology with focus on the
  Affymetrix GeneChip technology

9
Experimental design

• How much variation is in the system?
• Understand and minimize variation
• What level of significance is needed?
• More replicates needed for subtle changes
• How many treatments? How many controls?

• Two condition design
• Normal vs. Disease
• Wild type vs. Knockout
• Multivariate design
• Serial analysis
• Normal vs. Disease vs. Treated disease

10
Microarray technologies

• Probes
• In situ synthesized oligonucleotides-AFFYMETRIX
• (Deposited cDNA)
• Deposited oligonucleotides-Spotted chips

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Spotted chips

• Chips are commercially available
• Chips can be spotted in any laboratory
• One chip-Two samples

• Advantages
• Cost?
• Flexibility
• Opportunities for technology development
• Disadvantages
• Validation required
• Lack of standardization
• Need for reference

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• AFFYMETRIX GeneChips
• 25 mer oligonucleotides interrogate genes,
  sequences of interest
• Affymetrix has rules for probe selection based
  upon
• hybridization properties, specificity and
  potential for cross-hybridization
• Around 1,000,000 probes on one array
• Typically a group of 11-20 probes (pairs) all
  for same gene constitute
• a probe set

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GeneChip Probe Arrays
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GeneChip design
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Expression Assay Format
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GeneChip Hybridization
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Hybridized GeneChip Microarray
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GeneChip array expression probes
mRNA
5
3
Probes
TGTGATGGTGGGAATGGGTCAGAAGGACTCTCTATGTGGGTGACGAGGCC
AATGGGTCAGAAGGACTCTCTATGTG
AATGGGTCAGAACGACTCTCTATGTG
Perfect Match probe cells
Mismatch probe cells
Fluorescence Intensity Image
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AFFYMETRIX-Pros and Cons

• Advantages
• Technology is well validated
• Chips are commercially available
• One chip-one sample
• Very standardized protocol
• Disadvantages
• Cost
• Sensitivity
• Flexibility
• One chip supplier
• Restricted technology development

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Then comes the work!

• Data storage
• Normalization
• Visualization
• Analysis

• Identifying differentially expressed genes
• Statistical tests
• Indentifying biological relevance Data base
  searching
• Clustering
• Mapping pathway

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Data analysis
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Annotation 2
Data analysis
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Standardization

• Minimum information about a microarray
  experiment - MIAME
• MIAME aims to outline the minimum information
  required to unambiguously interpret microarray
  data and to subsequently allow independent
  verification of this data at a later stage if
  required.
• This set of guidelines will then assist with the
  development of microarray repositories and data
  analysis tools.
• MIAME is being developed continuously in
  accordance with our understanding of microarray
  technology and its applications.

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MIAMExpress Description
http//www.bioinformatics.iastate.edu/microarray/M
icroArrayGroup10_2002_v2.ppt
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• 3. Problems and promises of microarray technology

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Gene expression profiling. Current problems and
promises Experience from the AFFYMETRIX core
facility
Since Jan 1st, 2001 we have performed
approximately 2500 assays
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Technical problems

• AFFYMETRIX
• Sensitivity
• Specificity
• Cost
• Sample quantity
• Sample quality
• Sample complexity
• Non-sample related parameters

• cDNA
• Additionally
• Chip quality
• Dye bias
• Selection of reference
• Time consuming to adapt
• primary measures to data
• format suitable for further
• analysis.
• It is difficult!

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What effects the experiment?
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Do not pool samples if possible
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Promises

• Sensitivity-Improved
• Specificity-Improved
• Cost-Reduced
• Sample quantity
• Sample quality
• Sample complexity-Isolation of single cells
• Non-sample related parameters
• Statistical tests for microarray data

31
4. Applications
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Gene expression profiling as an approach to
understand the molecular mechanism of estrogen
action

• Provide gene expression profiles for selected
  estrogen target tissues and specify effects via
  ERa and ERb, respectively. This will
• Increase our understanding of the molecular
  mechanism of estrogen action in health and
  disease
• Stratify diagnosis
• Stratify the use of estrogen agonists and
  antagonists with receptor selectivity
• Identify novel estrogen regulated genes which
  will
• Provide novel therapeutic opportunities
• Increase our understanding of the molecular
  mechanism of estrogen action in health and
  disease
• Identify surrogate markers, which can be used in
  drug development and clinically to follow the
  effect of a given drug

33
Gene expression profiling as an approach to
understand the molecular mechanism of estrogen
action

• Signalling networks
• Receptor specificity
• Phenotype characterization

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Identification of candidate gene for further
studies and identification of groups of changed
genes by clustering
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Estrogen and the cardiovcascular system
Background
1. The incidence of hypertension and
cardiovascular disease is significantly
lower in premenopausal women than in men and
postmenopausal women. 2. Cardiovascular
mortality in postmenopausal women receiving
estrogen replacement therapy is about 30-50
lower than in their untreated counterparts. 3.
BERKO mice have elevated blood pressure
The effects of HRT on CAD are currently
controversial
The effects of HRT on the cardiovascular system
need to be elucidated
36
Identification of estrogen regulated genes in the
heart

• 13 week-old C57BL/6 mouse (Ovariectomy
  11week-old)
• Soy-free diet for 1 week
• 100 mg /kg x day
• Subcutaneous injection

Total RNA Isolation
6h or 3 w
Affymetrix GeneChip probe array
Harvest organ
V
E
Mouse 1
Mouse 4
9 Comparisons
Mouse 2
Mouse 5
Mouse 3
Mouse 6
Repeated 2 times
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6 hrs estrogen treatment
Prostaglandin D synthase
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Confirmation of L-PDGS induction
Otsuki, M. et al. Estrogen Induces Lipocalin-Type
Prostaglandin D Synthase Expression in Mouse
Heart through Estrogen Receptor ?. Mol
Endocrinol. 17(9), 1844-1855 (2003)
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Estrogen regulated genes in aorta in vitro 1
0h
6h
24h
V
V
E
E
G
G
E ICI
E ICI
G ICI
G ICI
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Estrogen regulated genes in aorta in vitro 2
V
E
Genes related to inflammation
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Changes in the global expression patterns reveal
interplay between receptors
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ERb modulation of estrogen action 1
Stimulatory Effect of Estrogen in Bone
Stimulatory Effect of Estrogen in Liver
10000
10000
1000
1000
BERKO (over vehicle)
BERKO (over vehicle)
100
100
100
1000
10000
100
1000
10000
WT( over vehicle)
WT( over vehicle)
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ERb modulation of estrogen action 2
Inhibitory Effect of Estrogen in Bone
WT ( below vehicle)
-100
-90
-80
-70
-60
-50
-40
-30
-30
-40
-50
-60
BERKO ( below vehicle)
-70
-80
-90
-100
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ERb modulation of estrogen action 3
Lindberg, M. K. et al. Estrogen receptor beta
reduces estrogen receptor alfa-regulated gene
transcription, supporting a ying-yang
relationship between estrogen receptors alfa and
beta in mice. Mol. Endocrinol. 17 (2), 203-8
(2003)
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Regulation of candidate mRNAs, identification of
candidate gene for further studies
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Estrogen action and obesity

• Estrogens have been shown to regulate adipose
  tissue in both humans and animals
• Women usually increase the body fat mass after
  menopause when estrogen levels decrease
• Estrogen replacement therapy reduces the fat mass
  
• In rodents, ovariectomy increases adipose tissue
  mass and estrogen treatment reverses this effect
• ERKO, DERKO and ArKO mice have increased fat
  (male and female)
• ERKO male mice did not show increased food
  consumption
• ERKO male mice showed reduced energy expenditure

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Identification of estrogen regulated genes in WAT
(white adipose tissue)

• 13 week-old C57BL/6 mouse (Ovariectomy
  11week-old)
• Soy-free diet for 1 week
• 100 mg /kg x day
• Subcutaneous injection

Total RNA Isolation
10h, 24h and 48 h or 3 w
Affymetrix GeneChip probe array
Harvest organ
V
E
Mouse 1
Mouse 4
9 Comparisons
Mouse 2
Mouse 5
Mouse 3
Mouse 6
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Genes related to metabolism/obesity
Etc......
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LXR? and SREBP-1c mRNA is decreased in WAT after
10 h estrogen treatment
SREBP-1c 10h
LXRa 10h
1,6
D in 3/4
1,4
1,6
D in 3/4
1,2

1,4

1
1,2
Relative mRNA levels
0,8
1
Relative mRNA levels
0,6
0,8
0,4
0,6
0,2
0,4
0
0,2
0
V
E
V
E
V
E
V
E
Real time PCR
Microarray
Real time PCR
Microarray
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Regulation of LXR target genes
Lundholm, L. et al. Gene expression profiling
identifies liver X receptor alpha as an
estrogen-regulated gene in mouse adipose tissue.
J. Mol. Endocrinol., 32, 879-892 (2004)
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• Phenotype characterization and pathway analysis

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ERaKO animals are insulin resistent

• This phenotype is primarily at the level of the
  liver

ERKO
WT
WT
ERKO
WT
ERKO
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Pathway analysis/Increased pathways
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Characterization of phenotype
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ERbKO male muscle phenotype
Male ER?-/- muscles produce somewhat higher
forces during fatigue than male wildtype muscles
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Glenmark, B. et al. Difference in skeletal muscle
function in males vs females role of estrogen
receptor b. Am J Physiol Endocrinol Metab. In
press
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Thanks to
Marie Lindberg Sofia Moverare Claes
Ohlsson Division of Endocrinology Sahlgrenska
University Hospital Göteborg
Johan Liden Zahra Mirzaie David Brodin Marika
Rönnholm AFFYMETRIX core facility
Hui Gao Lovisa Lundholm Maria Nilsson Michio
Otsuki
Galina Bryzgalova Suad Efendic Akhtar
Khan Department of Molecular Medicine
Karolinska Hospital
Birgitta Glennmark Department of Hand Surgery,
Stockholm Söder Hospital, Håkan
Westerblad Department of Physiology and
Pharmacology Karolinska Institutet
Min Liang Bengt-Olof Nilsson 2Department of
Physiological Sciences Lund University
Jan-Åke Gustafsson