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Caenorhabditis elegans Functional Genomics
Sheldon McKay
January 22, 2004
Introduction
•
C. elegans as a model organism
•
Functional genomics
Gene knockout project
•
Goals
•
Methods
•
Progress
Gene expression project
•
Goals
•
GFP-promoter fusions
•
SAGE
C. elegans as a model organism
• A 1mm long nematode worm
• Short generation time and large numbers of progeny.
• A metazoan with differentiated tissues and
comprehensively studied anatomy and
developmental program
• Sequenced genome
C. elegans genome
•
•
•
•
‘Essentially complete’ as of December 1998
Contains ~100 million bp on 6 chromosomes
Predicted to contain ~20, 000 genes.
~ 55% of these genes are similar to genes from
other organisms.
• ~ 20% associated with mutationally defined
genetic loci
Genetics with a sequenced genome
• Knowing the sequence isn’t everything.
• We need to identify functions for as many uncharacterized
genes as possible
• Identification of a mutant phenotype in a gene of
previously unknown function can help in assigning a
function
Forward vs. Reverse Genetics
• In classical genetic analysis, we start with a mutant phenotype,
genetically map the gene(s) responsible to chromosomal
locations then, hopefully, find the gene and study the DNA
sequence
• Reverse genetics: We start with a DNA sequence believed to
encode a gene. We then attempt to learn about the gene’s
function through expression analysis and perturbations of its
normal function with tools such as RNAi and mutational
analysis
C. elegans Functional Genomics Projects:
Examples
• Large-scale EST sequencing
• ORFeome
• Microarrays
• RNAi
• Gene Knockout Project
C. elegans Gene Knockout Project
Objectives:
• Study gene function by
determining the null phenotype
• Make knockouts for all genes,
with an emphasis on mammalian
orthologs
Approach:
• Use PCR to detect, isolate and
sequence deletion alleles
PCR Screening for deletions
Problems
• The approach is biased towards large deletions
• Sensitivity is low in complex populations
• Targeting is imprecise
The Poison Primer Technique
1o PCR
2o PCR
NO PRODUCT
The Poison Primer Technique
1o PCR
2o PCR
Deletions recovered with poison primers
Exon
Intron
Normal
Deleted
Average deletion size: 486 bp
Precision knockout of a gene within a gene
Exon
Intron
Normal
Deleted
C. elegans Gene Expression Project
C. elegans Gene Expression Project
Objectives:
Build on the knockout project and
other large-scale functional genomics
projects
Study gene expression patterns, with
an emphasis on human orthologs
Approaches:
• High resolution image analysis of
gene expression with GFP/Promoter
fusions
• Serial Analysis of Gene Expression
(SAGE) and microarray analysis of
gene expression of life stages and cell
types
Goals:
Understand patterns of gene expression
through the course of development and in
particular cell types and tissues
Identify known and novel cis-regulatory
elements and their role in transcriptional
regulation at the gene and network levels
Understand gene expression patterns and
protein interaction networks in the
context of space and time in a developing
organism
Green Fluorescence Protein Fusion
Studies of Gene Expression
Isolating Potential Promoter Regions
PCR primers
Gene model
SOCKEYE -- BCCA Genome Sciences Centre
PCR-based Promoter GFP fusion
(Hobert, Biotechniques 32:728-30)
Promoter
GFP
Promoter
GFP
Neurons
C13F10.4 -- contains similarity to Listeria
monocytogenes Probable DNA-directed RNA polymerase
delta subunit (RNAP deltasfactor).; SW:RPOE_LISMO
Neurons
C13F10.4 -- contains similarity to Listeria
monocytogenes Probable DNA-directed RNA polymerase
delta subunit (RNAP deltasfactor).; SW:RPOE_LISMO
Neurons
C13F10.4 -- contains similarity to Listeria
monocytogenes Probable DNA-directed RNA polymerase
delta subunit (RNAP deltasfactor).; SW:RPOE_LISMO
protein of unknown function -- expressed in neurons
A case-study of tissue-specific upstream
regulatory elements
M03F4.3 7-pass G-coupled transmembrane recepter
Expressed in head, gut, vulva, tail
Use PCR-stitching technique to dissect the
Tissue-specific Control Elements
Vulva, Tail
Head, Gut
Serial Analysis of Gene Expression
SAGE
Objectives:
• Determine temporal and spatial
gene expression patterns
Approaches:
• Construct life-stage and tissue
specific SAGE libraries
• Use GFP markers to isolate
tissue specific cell populations
via FACS
SAGE: Procedure
Digest with
“Anchoring enzyme”
NlaIII
Isolate mRNA,
RT to cDNA
Digest with “Tagging
enzyme”
BsmFI
Sequence
Ligate tags
http://www.sagenet.org/home/Description.htm
SAGE Data Analysis
• Tag abundance  transcript abundance
• Abundant tags = Abundant transcripts
• Identify interesting tags, find out which genes
they belong to
Statistical analysis of tag frequencies
DISCOVERYspace -- BCCA Genome Sciences Centre
Mapping SAGE tags to genes
NlaIII
AAAAAAA
Mapping SAGE tags to genes
Many predicted genes are not confirmed by Expressed
Sequence Tags (ESTs) and do not include 3’ Untraslated
3’-most
regions (UTRs)
CATG
Predicted:
5’ UTR
3’ UTR
Actual:
3’-most
CATG
The virtual transcriptome
Predicted Gene Models
EST Data
Transcripts with
EST support
Genomic DNA Sequence
No EST Data
UTR length
distribution
Estimate UTR length
For unconfirmed gene models
Transcripts without EST support
Conceptual mRNAs
NlaIII
5’
AAAAAAAAAA
6
5
4 3
600000
2
1
Real 3’ UTR
500000
Position
Tags
1
2
3
4
5
6
7
8
9
10
400000
300000
200000
Total
Real
Fake
615706
514979
100727
53897
18734
35163
21028
7578
13450
16070
7974
8096
20213
8968
11245
6756
2163
4593
6068
2777
3291
6309
3259
3050
4080
1711
2369
1245
566
679
Estimated 3’ UTR
100000
0
1
2
3
4
5
6
7
Position of NlaIII site
8
9
10
3’
NlaIII
2
Predicted Gene Models
EST Data
Transcripts with
EST support
Genomic DNA Sequence
Experimental SAGE tags
No EST Data
UTR length
distribution
Map tags
Transcripts without EST support
Digest in silico
Adjust 3’ UTRs
Theoretical SAGE tags
Adjust gene models
Conceptual mRNAs
Relating Gene Expression to Development
Source: http://nema.cap.ed.ac.uk/Caenorhabditis/C_elegans_genome
C. elegans Life Cycle
Adult
Embryo
L4
L1
L3
L2
~3.5 days
SAGE data: embryo
Sage_summary -- BCCA Genome Sciences Centre
Tags/100K
Developmental Series: Top 12 collagen genes
egg
larval
adult
old
Stress Response and Ageing
Adult
Embryo
L4
L1
dauer
L3
L2
~3.5 days
daf-2
• Insulin-like growth factor receptor
• Constitutive dauer formation at 25C; reversible by shift to
15C.
• Increased lifespan at 20C;
• Increased thermotolerance, UV resistance.
• Most alleles hypersensitive to dauer pheromone.
Transcribed Telomeric Sequence (tts-1)
• Most abundant trasncript in a SAGE comparison of
dauer larvae and a normal mixed population (Jones,
et al, 2001)
• corresponds to a previously unknown non-coding
gene
• implicated in dauer larvae induction or maintenance
tts-1 expression patterns
2500
2000
dauer
longevity
1500
starvation
1000
500
0
daf-2
Relating gene expression to anatomy
Micro-dissection of gut tissue
Gonad-less mutant (worm’s insides mostly gut)
Nick the worm’s outer integument and the body contents are
everted due to internal positive pressure
Dissect gut away from the body
Construct SAGE libraries from gut and whole worm mRNA
Microdissected gut tissue
Whole Worm vs. Gut
2500
Aspartyl protease
2000
Vitellogenin
1500
Cytochrome C oxidase
1000
500
0
Whole
Gut
Relating gene expression to
organogenesis and early
development
Isolating specific cells types
Identify GFP constructs expressed in the tissue/cell of
interest
• Make transgenic strain
• Isolate eggs with NaOCl treatment
• Digest with chitinase/trypsin
• Disrupt embryos with physical shearing
• Keep individual cells alive in culture
• Sort via fluorescence activated cell sorter
• Construct SAGE libraries
GFP labeled embryonic intestinal cells
DIC Image
GFP Fluorescence Image
The elt-2 promoter linked to GFP acts a reporter for developing
embryonic gut cells. Strain kindly provided by Dr. Jim McGhee.
FACS sorted embryonic intestinal cells
DIC Image
GFP Fluorescence
Image
We have used an embryonic intestinal promoter linked to GFP as a
reporter for developing embryonic cells. These cells have been
purified after FACS sorting. They can be plated and allowed to
differentiate or used immediately for SAGE studies.
Embryonic Gut Cells
Whole Embryo vs. Isolated Cell Types
900
800
700
600
500
400
300
200
100
0
Whole
Gut
Muscle
SAGE libraries
Summary
Stage
Tissue
Embryo 14 bp tags
whole
Embryo 21 bp tags
whole
L1 starved
whole
L1 normal
whole
L2
whole
L3
whole
L4
whole
Young adult
whole
6 day Adult(fer-15)
whole
1 day Adult(fer-15;daf-2)
whole
6 day Adult (fer-15;daf-2)
whole
10 day Adult(fer-15;daf-2)
whole
Adult (glp-4)
dissected gut 138346
Adult (glp-4)
whole
Embryo (myo-3::GFP)
FACS sorted gut
Embryo (myo-3::GFP)
FACS sorted muscle
Mixed stage
whole
Dauer larvae
whole
Meta library (14 bp tags)
Meta library (21 bp tags)
Tag
Total
133825
220032
116363
109994
130209
127924
141878
119222
110306
101939
100737
116336
14386
117529
81393
58147
175995
65828
1806431
359572
Genes
Unique
25885
44992
19494
17532
24658
24039
25701
23128
19861
16960
14004
19183
4892
19140
19649
16967
37894
18136
130112
62893
8187
8929
6429
6705
7264
7667
8046
6302
6758
5159
4687
5594
6974
6069
4850
9222
5373
14661
9887
Ongoing projects:
• SAGE/Affy Transcription profiling of:
– developmental stages
– ageing
– tissue differentiation
• SAGE-based genome annotation
– gene discovery
– alternative splicing
– 5’ SAGE
• Gene expression clustering
• Cis-regulatory element discovery
Research Team
University of British Columbia
Don Moerman
Heidi Kai
Adam Warner
Erin Halfnight
Rebecca Newbury
Nicholas Dube
Francis Ouellette
Simon Fraser University
David Baillie
Robert Johnsen
Lily Fang
Emily Ha
Allan Mah
Domena Tu
John Tyson
Zhongying Zhao
Martin Jones
Kathleen Huang
Robert Hollebakken
Dion Li
Victor Jensen
BCCA Genome Sciences Centre
Sheldon McKay
Peter Huang
Kim Wong
Courtney Mills
Igor Ostrovsky
Peter Ruzanov
Scott Zuyderduyn
Richard Varhol
Erin Pleasance
Greg Vatcher
Steven Jones
Marco Marra
Jaswinder Khattra
Jennifer Asano
Susanna Chan
Shaun Coughlin
Noreen Girn
Helen McDonald
Pawan Pandoh
Rob Holt
George Yang
Jeff Stott
University of Calgary
Jim McGhee
UBC/Genome BC
Don Riddle