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Extinction
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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 TSE1- Killary and Fournier
1986-Identification of extinguisher locus TSE2- Chin and Fournier
1993- Cloning of TSE-1- Jones and Fournier
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
Generation of Somatic Cell Hybrids
PEG Fusion
X
RAT1
fibroblast
Propagation
Hat, Ouabain
FTO2B
hepatoma
Heterokaryon
Somatic Cell Hybrid
Hepatoma x fibroblast hybrids
Hepatoma
Hybrid
Fibroblast
Hybrid
Global shut-off of liver-specific genes in cell
hybrids is reversible
hepatoma
fibroblast
hybrid
Unstable
hybrid
What are the loci that direct
extinction?
Microcell-mediated chromosome transfer
Microcell
hybrid
Micronucleate
cell
DONOR
Colcemid
24 hr
Human HepG2
(Neo marked)
Cytochalasin B
PEG Fusion
Centrifugation
Neo-marked
Chromosome
Recipient
Hepatoma
G418
Chromosome analysis of
microcell hybrids
FISH
Mouse chromosme 11 in rat hepatoma cells
What are the loci that direct
extinction?
Tse-1 mapped by microcell transfer
• 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
Mapping of Tse-1
Tse-1 (aka R1a)
Liver
Regulatory subunit
Mechanism
Tse-1 (R1a) is
underexpressed in
liver cells, thus
fusion with
fibroblast brings Fibroblast and hybrids
levels up to normal
ON
OFF
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
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
Using Robertsonian translocations to map extinguisher loci
Result- Nothing much
Noprthern analysis of a rat hepatoma containing Robertsonian translocations
What are the cis-acting targets
of extinction?
Tissue-specific factors are absent in cell hybrids
Pituitary
GHF1
TATA
Growth Hormone Gene
Hepatocytes
HNF1
TATA
a1-Antitrypsin Gene
Lymphocytes (B-cells)
OTF-2
TATA
Immunuglobulin Heavy Chain Gene
Pancreas (-cells)
IEF1
TATA
Insulin Gene
a1AT is down regulated at least
3000-fold in hybrids
The a1-antitrypsin gene is liver specific
Human Ch. 14
2 kb
a1-AT
I
II
III
HNF4 HNF1
-660
Enhancers
+1

IV
V
Combinatorial activation of liver-specific genes
??
GATA6
HNF4
HNF1
C/EBP
HNF3a
Liver-specific genes
Promoter deletion analysis identifies critical
liver-specific sequences in the a1AT promoter
Promoter activity
HNF4 HNF1
hepatoma
fibroblast
hybrid
a1AT promoter deletion constructs
a1-AT promoter is highly active in the presence of HNF1
in rat fibroblasts
+ HNF1
- - + +
a1AT
-261
CAT
+44
- +
ControlTK-CAT
Promoter activity
Both HNF1 and HNF4 binding are required
for full promoter activity in hepatoma cells
a1AT
-261
CAT
+44
WT
HNF4
HNF1
a1AT promoter deletion constructs
Can HNF1 expression prevent a1AT extinction?
cHNF1a
PEG Fusion
X
Hat, Ouabain
RAT1
fibroblast
FTO2B
hepatoma
HNF1a
RAT1
Somatic Cell Hybrid
X
Hsa14
HNF1 fails to prevent extinction of a1AT
Parent
cells
HNF1
Note - No a1 AT
Hybrids
cells
HNF4 + HNF1 fails to prevent
a1AT extinction
Extinction Models
__
Activator?
__

HNF4
Activator?

HNF4
__
HNF1
HNF1
__
C/EBP

C/EBP
F
F
C
P
a1-antitrypsin Promoter
Lack-of-activation Model
C/EBP
C/EBP
HNF4
HNF1
HNF1
a1-antitrypsin Promoter
Dominant Repression Model
HNF4 prevents extinction of HNF1, but
not downstream genes
__
Extinguisher
Loci
Activator?
Or

__
HNF4
HNF1a
__
a
a1-antitrypsin Promoter
Extinction Models
Active Repression
Loss of activation
Extinction Models
Active Repression:
Recruitment to heterochromatin
Extinction Models
Active Repression: Altered chromatin remodeling
Tissue specific genes
are require special remodeling
Ubiquitous genes
are “bookmarked”
Extinction Models
Active Repression: Nuclear localization-dependent
Transcribed B-cell specific genes
Tissue-specifc genes localized in interior
of nuclear domains in hybrids
Summary
Extinction of a1AT gene expression appears to occur at many levels
1
repression of HNF4 gene transcription
2
repression through the a1AT promoter sequences
3
repression through internal a1AT DNA sequences
Putative Extinction Targets
1
2
3
HNF4
2 kb
HNF1
II
I
a1-antitrypsin locus
III
IV
V