COPENHAGEN BIOCENTER

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COPENHAGEN BIOCENTER
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Mammalian cells

• Screens
• Transcriptosome
• Proteome

BIOLOGY
COMPARATIVE GENOMICS
HYPOTHESES

• HTP technologies
• shRNA libraries, cDNA libraries
• Tissue microarrays
• Yeast Two Hybrids
• Proteomics

BIOLOGY
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Transcriptosomes

• Cell cycle events in synchronized cell cultures
• Cell cycle exit/entry in response to
  extracellular signaling
• DNA damage checkpoint

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Synchronized tissue cultures


Measure regular cyclic changes under standard
growth conditions double thymidine block and
nocodazole/aphidicolin samples for RNA 12 or 24
time points after release from block
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Entry into the Cell Cycle


Transcriptional profiling of quiescent cells
entering the mammalian cell cycle
Science 283, 83-87, 1999
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DNA damage

Monitoring the effect of DNA damage-induced
transcriptionally changes with the idea of
identifying novel genes implicated in DNA damage
and potentially involved in tumor formation

• Types of DNA damage
• Single-stranded (UV)
• Double-stranded (IR)

• Problems with available studies
• Use of cancer cell lines
• Arrays presenting too few genes
• Low effect on known DNA damage induced genes
  (e.g. P21)
• Focus on either the G1 or G2 checkpoint
• Too few biological and technical replicates

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Experimental set-up


Cell lines
p53 deficient human diploid fibroblasts (Tig3)
normal human diploid fibroblasts (Tig3)
UVC (25 J/m2)
?-irradiation (10GY)
non stressed
Treatment
harvest cells after 0 6h
Analysis
microarray
FACS
expression p53,p21,Puma (qPCR, immunoblotting)
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p53 deficient human fibroblasts
TP53
WT
shRNA p53
CDKN1 (p21)
WT
shRNA p53
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Activation of p53 target genes
CDKN1 (p21)
?-Irradiation
UVC
PUMA
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Transcriptosome
Human Genome U133 Plus 2.0 Array
Cluster analysis
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Genes downregulated by UVC partially IR
p21
Genes upregulated by UVC IR
FAS
MDM2
p53-dependent
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Genes upregulated by UVC in WT cells
Genes upregulated by UVC
Genes upregualted by UVC downregulated In IR
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Significantly regulated genes
Wt UVC/ wt IR (6673 probeset)
p53kd UVC/ wt IR (3187 probeset)
517
3832
2530
122
189
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93
P53kd none/ wt none (422 probeset)
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Some functional classification
3832 significantly regulated genes UVC and
IR-induced
Apoptosis 2 Stress signaling 1.2 Cell
cycle 5.3 Cell growth 9.8
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Validation experiments 1
Cdc25A
CDKN1
Cytochrome P450
Fas
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Validation experiments 2
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DNA damaged induced expression of Polycomb group
genes
Cbx5
Cbx4
UVC
0h 3h 6h 12h 0h 3h
6h 12h
0h 3h 6h 12h 0h 3h
6h 12h
Wt
p53 kd
Wt
p53 kd
IR
0h 3h 6h 12h 0h
3h 6h 12h
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DNA damaged induced repression of thePolycomb
group protein EZH2
Bracken et al., submitted
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Mammalian cells

• Screens
• Transcriptosome
• Proteome

BIOLOGY
COMPARATIVE GENOMICS
HYPOTHESES

• HTP technologies
• shRNA libraries, cDNA libraries
• Tissue microarrays
• Yeast Two Hybrids
• Proteomics

BIOLOGY