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Extinction Loss of tissue-specific gene expression in
somatic cell hybrids• 1966- Loss of cell pigment in somatic
hybrids- Davidson and Yamamoto
• 1970s- Albumin extinction- time course/reversability- Petit and Weiss
• 1984- Identification of extinguisher locus TSE-1- Killary and Fournier
• 1986-Identification of extinguisher locus TSE-2- Chin and Fournier
• 1993- Cloning of TSE-1- Jones and Fournier
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Extinction in Somatic Cell Hybrids
• Kinetics- Occurs within hours of fusion
• Level of repression- 500-1000X
• Global loss of tissue-specific gene expression
• Bidirectional
• Transcriptional
• Reversible upon chromosome segregation
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RAT1 fibroblast
PEG Fusion
Somatic Cell Hybrid
X
FTO2B hepatoma
Hat, Ouabain
Generation of Somatic Cell Hybrids
Heterokaryon
Propagation
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Hybrid Hybrid
Hepatoma x fibroblast hybrids
Hepatoma Fibroblast
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Global shut-off of liver-specific genes in cell hybrids is reversible
hepatoma fibroblast hybridUnstable
hybrid
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What are the loci that direct extinction?
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DONOR
Colcemid
24 hr
Cytochalasin B
Centrifugation
Neo-markedChromosome
PEG Fusion
Recipient
G418
Microcell hybrid
Micronucleate cell
Microcell-mediated chromosome transfer
Human HepG2
Hepatoma
(Neo marked)
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Chromosome analysis of microcell hybrids
Mouse chromosme 11 in rat hepatoma cells
FISH
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What are the loci that direct extinction?
• On human chromosome 17
• Down-regulates three liver-specific genes- all are cAMP inducible
– Tyrosine amino transferase (TAT)
– Phosphoenolpyruvate carboxykinase (PEPCK)
– Argininal succinase (AS)
• Downregulation reversed by cAMP induction
• Encodes the regulatory subunit of protein kinase A
Tse-1 mapped by microcell transfer
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Mapping of Tse-1
Tse-1 (aka R1
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Tse-1 (R1a) is underexpressed in liver cells, thus fusion with fibroblast brings levels up to normal
ON
OFF
Mechanism
Liver
Fibroblast and hybrids
Regulatory subunit
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Is Tse-1 a true extinguisher?
• Probably not-
–Downregulates ony 10-fold (TAT and AS genes) to 100 fold (PEPCK gene) instead of 1000-fold in cell hybrids
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What are the loci that direct extinction?
Are there other Tse-1-like genes that contribute to extinction?
• Tse-2 (on human chromomse 2) represses albumin gene only
• Other extinguisher loci have not been identified in any system
Conclusion?• Extinction is a polygenic trait
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Using Robertsonian translocations to map extinguisher loci
Noprthern analysis of a rat hepatoma containing Robertsonian translocations
Result- Nothing much
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What are the cis-acting targets of extinction?
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Tissue-specific factors are absent in cell hybrids
Pituitary
Lymphocytes (B-cells)
Hepatocytes
Pancreas (-cells)
GHF1
TATA
Growth Hormone Gene
OTF-2
TATA
Immunuglobulin Heavy Chain Gene
TATA
IEF1
Insulin Gene
HNF1
TATA
a1-Antitrypsin Gene
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1AT is down regulated at least 3000-fold in hybrids
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2 kb
I
The 1-antitrypsin gene is liver specific
II III IV V
HNF1HNF4
Enhancers-660 +1
Human Ch. 14
1-AT
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HNF4
HNF1
GATA6
C/EBP
HNF3
??
Liver-specific genes
Combinatorial activation of liver-specific genes
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Promoter deletion analysis identifies critical liver-specific sequences in the a1AT promoter
Pro
mo
ter
acti
vity
a1AT promoter deletion constructs
hepatoma
fibroblast
hybrid
HNF1HNF4
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1-AT promoter is highly active in the presence of HNF1in rat fibroblasts
+ HNF1
CAT
-261 +44
Control- TK-CAT
+ + +-- -
1AT
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Both HNF1 and HNF4 binding are required for full promoter activity in hepatoma cells
Pro
mo
ter
acti
vity
a1AT promoter deletion constructs
HNF1HNF4
WT
CAT
-261 +44
1AT
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RAT1fibroblast
PEG Fusion
Somatic Cell HybridRAT1
X
FTO2B hepatoma
HNF1
Hat, Ouabain
cHNF1
X
Hsa14
Can HNF1 expression prevent a1AT extinction?
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HNF1
HNF1 fails to prevent extinction of 1ATHybrids
cellsParentcells
Note - No1 AT
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HNF4 + HNF1 fails to prevent a1AT extinction
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HNF4
Activator?
HNF1
1-antitrypsin Promoter
C/EBPF
C/EBP CPF
HNF4
Activator?
HNF1
1-antitrypsin Promoter
C/EBPHNF1
C/EBP HNF1HNF4
__
__
__
Lack-of-activation Model Dominant Repression Model
__
Extinction Models
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HNF4
Activator?
HNF1
1-antitrypsin Promoter
__
__
__
OrExtinguisher Loci
HNF4 prevents extinction of HNF1, but not downstream genes
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Active Repression
Loss of activation
Extinction Models
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Active Repression:
Extinction Models
Recruitment to heterochromatin
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Active Repression:
Extinction ModelsAltered chromatin remodeling
Ubiquitous genes are “bookmarked”
Tissue specific genes are require special remodeling
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Active Repression:
Extinction ModelsNuclear localization-dependent
Tissue-specifc genes localized in interior of nuclear domains in hybrids
Transcribed B-cell specific genes
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1-antitrypsin locus
II III IV V
I
2 kb
Putative Extinction Targets
HNF4
HNF1
Summary
1
2
Extinction of 1AT gene expression appears to occur at many levels
repression of HNF4 gene transcription repression through the 1AT promoter sequences
repression through internal 1AT DNA sequences
1
2
3
3