Transcript Gene Expression

Genomics, Bioinformatics & Medicine
http://biochem158.stanford.edu/
Gene Expression
http://biochem158.stanford.edu/Gene%20Expression.html
Doug Brutlag
Professor Emeritus of Biochemistry & Medicine
Stanford University School of Medicine
Doug Brutlag 2011
Leveraging Genomic Information
Novel Diagnostics
DNA Microchips & Microarrays
Gene Expression - RNA
Proteomics - Protein
Understanding
Metabolism
Cell Signaling
Differentiation
Disease
Doug Brutlag 2011
Gene Regulatory Mechanisms
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Transcriptional Mechanisms
– Type of promoters & RNA polymerase
– Control of Transcription
– Transcription Factors and TFBS
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RNA processing
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Capping
Splicing and Alternative Splicing
Poly-Adenylation
RNA export to cytoplasm
RNA degradation rates
• Micro RNAs (miRNAs) inhibit translation and degrade mRNA
• Silencer RNAs (siRNAs or RNAi) degrading mRNA
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Epigenetic Mechanisms
– DNA methylation
– Histone modifications
• Acetylation
• Methylation
• Phosphorylation, etc.
– Chromatin remodeling
Doug Brutlag 2011
Eukaryotic Gene Structure
miRNA Target
Doug Brutlag 2011
Multiple Enhancer Sequences
© Garland Publishing
Doug Brutlag 2011
Enhanceosome: Exploring the Structure
http://www.rcsb.org/pdb/education_discussion/molecule_of_the_month/download/Enhanceosome.pdf
ATF-2/cJun
IRF
NF
Kappa-B
Doug Brutlag 2011
Enhanceosome: Integrating the Signal
http://www.rcsb.org/pdb/static.do?p=education_discussion/molecule_of_the_month/pdb122_2.html
HAT =
Histone Acetyl
Transferase
Doug Brutlag 2011
Oct3/4-Sox2 bound to Enhancer
http://www.pdb.org/pdb/explore/explore.do?structureId=1GT0
Doug Brutlag 2011
Repressor Bound to Silencer Sites
Doug Brutlag 2011
Gene Expression Regulatory Network
© Current Opinions in Genetics & Development
Doug Brutlag 2011
Hematopoiesis
http://www.biocarta.com/pathfiles/h_stemPathway.asp
Doug Brutlag 2011
IL7 Regulatory Pathway
http://www.biocarta.com/pathfiles/h_il7Pathway.asp/
© BioCarta
Doug Brutlag 2011
Growth Hormone Receptor Pathway
http://www.biocarta.com/pathfiles/h_ghPathway.asp/
© BioCarta
Doug Brutlag 2011
DNA Microarrays & DNA Chips
Accelerate Gene ExpressionAnalysis
• Parallel Analyses
– Analyze entire genomes instead of single genes
– Analyze expression of entire genome
– Analyze genetic polymorphisms (SNPs)
• Miniaturization
• Automation
Doug Brutlag 2011
Diagnosis Using DNA Arrays
http://www.affymetrix.com/
© Affymetrix
Doug Inc.
Brutlag 2011
Affymetrix Arrays
http://www.affymetrix.com/
Any collection of 25mers (1,200,000) can be synthesized in 100
steps
© Affymetrix
Doug Inc.
Brutlag 2011
Light Directed Oligonucleotide Synthesis
http://www.youtube.com/watch?v=ui4BOtwJEXs&feature=related
Light
(deprotection)
Mask
Glass
25-mer
Repeat
GeneChip
Microarray
© Affymetrix
Doug Inc.
Brutlag 2011
Automated DNA Chip Synthesis
http://www.affymetrix.com/
© Affymetrix
Doug Inc.
Brutlag 2011
Photolithography Masks
http://www.affymetrix.com/
© Affymetrix
Doug Inc.
Brutlag 2011
Hybridization & Detection
labeled probe
© Affymetrix
Doug Inc.
Brutlag 2011
Cystic Fibrosis Array
http://www.affymetrix.com/
© Affymetrix
Doug Inc.
Brutlag 2011
Microarrayer in Pat Brown’s Lab
http://cmgm.stanford.edu/pbrown/
Doug Brutlag 2011
High Precision DNA Printing
Arrayit Corporation
Doug Brutlag 2011
Mechanical Spotting Microarrays
http://www.arrayit.com/
Arrayit Doug
Corporation
Brutlag 2011
DNA Chips are used to
Measure Gene Expression
MicroArray analysis of
whole genome gene expression
Clustering of genes based on
their expression pattern
Searching for conserved sequence
motifs regulating the expression
Doug Brutlag 2011
Megacluster of Yeast Gene Expression
Doug Brutlag 2011
Human Gene Expression Signatures
Genes
T Cells Signaling
DNA Damage
Fibroblast Growth
Biological Conditions
B Cells Signaling
CMV Infection
Anoxia
Polio Infection
Cell Signaling IL4
Growth Hormone
Doug Brutlag 2011
Finding Transcription Factor Binding Sites
Upstream Regions
Co-expressed
Genes
GATGGCTGCACCACGTGTATGC...ACGATGTCTCGC
Pho 5
CACATCGCATCACGTGACCAGT...GACATGGACGGC
Pho 8
GCCTCGCACGTGGTGGTACAGT...AACATGACTAAA
Pho 81
TCTCGTTAGGACCATCACGTGA...ACAATGAGAGCG
Pho 84
CGCTAGCCCACGTGGATCTTGA...AGAATGACTGGC
Pho …
Transcription
Start
Doug Brutlag 2011
Finding Transcription Factor Binding Sites
Upstream Regions
Co-expressed
Genes
GATGGCTGCACCACGTGTATGC...ACGATGTCTCGC
CACATCGCATCACGTGACCAGT...GACATGGACGGC
GCCTCGCACGTGGTGGTACAGT...AACATGACTAAA
TCTCGTTAGGACCATCACGTGA...ACAATGAGAGCG
CGCTAGCCCACGTGGATCTTGT...AGAATGGCCTAT
Doug Brutlag 2011
Finding Transcription Factor Binding Sites
Upstream Regions
Co-expressed
Genes
ATGGCTGCACCACGTTTATGC...ACGATGTCTCGC
CACATCGCATCACGTGACCAGT...GACATGGACGGC
GCCTCGCACGTGGTGGTACAGT...AACATGACTAAA
TTAGGACCATCACGTGA...ACAATGAGAGCG
CGCTAGCCCACGTTGATCTTGT...AGAATGGCCTAT
Pho4 binding
Doug Brutlag 2011
Discovering Transcription Factor
Binding Sites is Difficult
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Binding sites are short (5-15 base pairs)
Not highly conserved (as little as 50%)
Located in long intergenic regions (>10kb)
Not always present (false positive genes)
Doug Brutlag 2011
Three Algorithms
• BioProspector
– Presented in 2000
– Extends Gibb’s sampling (stochastic method)
– For any cluster of sequences
• MDscan
– Deterministic approach
– Enumerative
– Very fast
– For sequences with some ranking information
• MotifCut and MotifScan
– Graph-based
– Does not use PSSMs
– Novel and sensitive
Doug Brutlag 2011
cDNA Diagnostics Using DNA Microarrays
Doug Brutlag 2011
72 Breast Cancers Classified by
451 Gene Expression Assays
451 Genes
72 Patients
Sorlie et al., PNAS 98,Doug
10869-10874.
Brutlag 2011
Breast Cancers Classified by
451 Gene Expression Assays
Sorlie et al., PNAS 98, 10869-10874.
Doug Brutlag 2011
ERB-B2 in Signal Transduction & Oncology
http://www.biocarta.com/pathfiles/h_her2Pathway.asp
Doug Brutlag 2011
Herceptin binds to ERB-B2 (HER-2)
Blocking Cell Growth
Herceptin =
anti-HER2
Fab Fragment
HER2 (Erb-2)
Human Epidermal
Growth Factor
Receptor
Doug Brutlag 2011
Regulation of transcriptional activity by PML
http://www.biocarta.com/pathfiles/h_pmlPathway.asp
© BioCarta
Doug Brutlag 2011
Diagnosis &
Gene Expression
Doug Brutlag 2011
PathWork Diagnostics
http://www.pathworkdx.com/
Predicting Site of Origin for
Cancers of Unknown Primary
© Pathwork Diagnostics
Inc.
Doug Brutlag
2011
PathWork Oncology Suite:
Site of Origin
• 1844 tumors tested one at a time versus all 18 tissues of origin
• Retrospective study on well characterized patient samples
• Uses PathChip (functionally similar to Affymetrix HU-133A
GeneChip)
– 604 specimens used for training
– 636 specimens used for test
– 604 specimens in reserve for final validation
• Reproducibility from lab to lab
• Performance based on sensitivity (> 70%) & accuracy (> 95%)
© Pathwork Diagnostics
Inc.
Doug Brutlag
2011
Production Data Flow
Sample
Clinical
Laboratory
 Receive sample
 Process specimen,
mRNA extraction
and microarray
hybridization
 Submit data file to
PathWork for
processing
Microarray data file
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 Deliver report to
oncologist
Receive microarray
data file
Check file and data
quality
Run model against
microarray data file
Generate report file
Deliver report file back
to clinical lab
PDF Report
Doug Brutlag 2011
CONFIDENTIAL
43
© Pathwork Diagnostics
Inc.
Doug Brutlag
2011
CONFIDENTIAL
44
© Pathwork Diagnostics
Inc.
Doug Brutlag
2011