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

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

RAT1 fibroblast

PEG Fusion

Somatic Cell Hybrid

X

FTO2B hepatoma

Hat, Ouabain

Generation of Somatic Cell Hybrids

Heterokaryon

Propagation

Hybrid Hybrid

Hepatoma x fibroblast hybrids

Hepatoma Fibroblast

Global shut-off of liver-specific genes in cell hybrids is reversible

hepatoma fibroblast hybridUnstable

hybrid

What are the loci that direct extinction?

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)

Chromosome analysis of microcell hybrids

Mouse chromosme 11 in rat hepatoma cells

FISH

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

Mapping of Tse-1

Tse-1 (aka R1

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

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

Noprthern analysis of a rat hepatoma containing Robertsonian translocations

Result- Nothing much

What are the cis-acting targets of extinction?

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

1AT is down regulated at least 3000-fold in hybrids

2 kb

I

The 1-antitrypsin gene is liver specific

II III IV V

HNF1HNF4

Enhancers-660 +1

Human Ch. 14

1-AT

HNF4

HNF1

GATA6

C/EBP

HNF3

??

Liver-specific genes

Combinatorial activation of liver-specific genes

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

1-AT promoter is highly active in the presence of HNF1in rat fibroblasts

+ HNF1

CAT

-261 +44

Control- TK-CAT

+ + +-- -

1AT

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

RAT1fibroblast

PEG Fusion

Somatic Cell HybridRAT1

X

FTO2B hepatoma

HNF1

Hat, Ouabain

cHNF1

X

Hsa14

Can HNF1 expression prevent a1AT extinction?

HNF1

HNF1 fails to prevent extinction of 1ATHybrids

cellsParentcells

Note - No1 AT

HNF4 + HNF1 fails to prevent a1AT extinction

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

HNF4

Activator?

HNF1

1-antitrypsin Promoter

__

__

__

OrExtinguisher Loci

HNF4 prevents extinction of HNF1, but not downstream genes

Active Repression

Loss of activation

Extinction Models

Active Repression:

Extinction Models

Recruitment to heterochromatin

Active Repression:

Extinction ModelsAltered chromatin remodeling

Ubiquitous genes are “bookmarked”

Tissue specific genes are require special remodeling

Active Repression:

Extinction ModelsNuclear localization-dependent

Tissue-specifc genes localized in interior of nuclear domains in hybrids

Transcribed B-cell specific genes

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