Molecular Regulation of Hepatic Gene Expression

2/10/03G. BullaBIO4751Advanced Molecular and Cellular Genetics

Liver pathogenesis- Acute and Chronic

• Viral infection- Hepatitis B, C, ……..

• Trauma

• Alcohol-induced

• Drug-induced

•Fibrogenesis•Cirrhosis

www.livercancer.com/

Gall Bladder (peekaboo)

Diaphragm

•Largest gland in the body (1.5 kg)•Receives 25% of cardiac output

Very early events in liver development

Cardiac mesoderm secretes fibroblast growth factors 1, -2 and -8 to cause forgut endoderm to develop into the liver

FGFs

Jung...Zaret. 1999. Science 284:1998

Major liver-enriched transcription factors

FamilyHNF1HomeoboxHNF3 Forkhead (Drosophila)HNF4 Steroid/thyroid

HNF6 Zinc FingerC/EBPLeucine ZipperGATA-6 Zinc Finger

Hepatic gene regulation is primarily at the level of transcriptional regulation

HNF= Hepatocyte Nuclear FactorC/EBP= CAAT/Enhancer Binding Protein

Anterior-posterior axis

Primitive streak

Endoderm differentiation

Foregut endoderm migration into mesenchyme

Organ formation

Liver

Hepatic Determination

Onset of liver gene expression

Amplification of liver gene expression

HNF3

HNF3

HNF3, HNF4

HNF1, c-jun

C/EBP

Transcription factors in early liver development

Phenotypes of transgenic knockouts

HNF1 phenylalanine hydroxylase off; phenylketonuria (liver)wasting disease- renal proximal tubule dysfuction (kidney)

HNF3 wasting disease; die day 3 post birth

HNF3 embyonic lethal

HNF3 minimal phenotype- 80 reduction in 1AT and TAT

HNF4 embryonic lethal (day 3)

DBP no phenotype

C/EBPdie 8 hr post birth- hypoglycemia; adipocyte abnormalities

C/EBPMild-Susceptible to listeria infection

NF-B (p65) embryonic lethal ( liver apoptosis)

HNF4

HNF1

GATA6

C/EBP

HNF3

??

Liver-specific genes

Combinatorial activation of liver-specific genes

• Hepatoma Variants- partial to complete loss of hepatic gene expression

Models for Hepatic Gene Regulation

• Primary hepatocytes- Fail to divide

• Cell Hybrids- complete loss of hepatic gene expression

• Hepatoma- express most hepatic genes

• Animal models- including knockout transgenics

Models for Hepatic Gene Regulation

• Hepatoma Variants- partial to complete loss of hepatic gene expression

• Cell Hybrids- complete loss of hepatic gene expression

• Primary hepatocytes- Fail to divide

• Hepatoma- express most hepatic genes

• Animal models- including knockout transgenics

*

Rat Hepatoma

Hepatoma Variants

Somatic Cell Hybrids

Chromosome Segregation

x Fibroblast

OFF

Liver-specific Gene Expression

SelectionON

Hepatoma Cells in Tissue Culture

2 kb

I

1-antitrypsin locus

II III IV V

HNF1HNF4

Enhancers-660 +1

Human Ch. 14

1-AT

Fado-2 Fg-14 H11, M29,...Variants

HB11, MB29Backselectants

1. 1AT-aprt2. 1AT-gpt

APRT -

1AT -GPT -

1AT +GPT +

APRT +APRT -

1AT +GPT -

HAT DAP,6TX

AAT

1AT +GPT +

APRT +

Generation of Hepatoma Variants

ON OFF ON

HNF1HNF4

Enhancers-660 +1

HNF1-mediated rescue Hepatic Gene Expression in H11 Hepatoma Variant Cells

HNF4

Activator?

HNF1

Liver-specific genes

H11 Cells H11b1-1, -16HNF4HNF11AT

---

HNF1Neo

HNF4HNF11AT

+++

G418

Rescue of hepatic gene expression with by HNF1

Hepatic Gene Expression in H11 Hepatoma Variant Cells

1-AntitrypsinAlbuminAldolase B-fibrinogen-fibrinogen-fibrinogenPEPCK

Genes unaffected in H11 cellsAlcohol dehydrogenaseTyrosine aminotransferaseArgininosuccinate synthetaseHNF3C/EBPTransferrin

Genes rescued by HNF1in H11 cells

HNF4

Activator?

HNF1

HS2 M29 H11, M38

BLOCK BLOCK BLOCK

BLOCK

BLOCK BLOCK

HNF1

Liver gene promoter+1

C/EBP

Hepatoma Variant Cells Contain Distinct Defects in Hepatic

Gene Expression

C/EBP

HNF4

Cell Line

DONOR

Colcemid

24 hr

Cytochalasin B

Centrifugation

Neo-markedChromosome

PEG Fusion

Recipient

G418

Microcell hybrid

Micronucleate cell

Microcell-mediated chromosome transfer

Human HepG2

H11 Hepatoma Variants

(Neo marked)

40X 100X

H11(G2)-89

Detection of human chromosomes using FISH

5 25 33 34 35 36 37 41 44 52 53 54 59 66 61 64 65 72 73 74 75 82 85 87 88 89

H11(G2)-

1AT

Tubulin

Fg1

4R

AT

1

Fg1

4

Rescue of 1AT expression in microcell hybrids

90 G418r clones picked (from three separate experiments)

45 survived expansion

38 MAXs 5 MAXr 2 MAXrG-bandChromosome paintingFISHPCR24/24 1AT- 1AT+ 1AT-

Analysis

H11(G2)-

H11(G2)-

1AT

Tubulin

H11(G2)-

Cyclo

34 40 44 52 71 81 87 89 Y

HNF1

H11(G2)-

HNF4

Cyclo

34 40 44 52 71 81 87 89 Y

Fg-14 dilutions

Fg1

4H

11

Fg14

H11

34 40 44 52 71 81 87 89

Rescue of HNF4, HNF1, and 1AT in microcell hybrids

Fg14

H11

D12S137

Human chromosome 12

1ATMAXr

D12S329

D12S61

D12S64

D12S218

D12S371

D12S2088

11

12

13

21

24

12

13

14

p

q

D12S129

D12S221

D12S314D12S62

H11(G2)-

++

-+

++

4052 81718987++

++

+-

-nd

-+

6137

Mapping the HIT locus on Chromosome 12p

U.S.A.- 0.6%Or 1in 166

Illinois- 0.0009%Or 1 in 100,000

Southern Africa- 20-38%Or 1 in 3

Source- U.N AIDS, 2001

Adults living with AIDS in Africa

Major liver-enriched transcription factors

FamilyHNF1HomeoboxHNF3 Forkhead (Drosophila)HNF4 Steroid/thyroid

HNF6 Zinc FingerC/EBPLeucine ZipperGATA-6 Zinc Finger

Hepatic gene regulation is primarily at the level of transcriptional regulation

HNF= Hepatocyte Nuclear FactorC/EBP= CAAT/Enhancer Binding Protein

M440M280

HNF4 -interaction regionAA 280-440

Ligand binding domain

AA 130-368HNF-1 interaction region

HNF4

HNF1

Zinc finger Activation domain

Activation domainHomeo domain

HNF4

HNF1ApoC-III

+-

• Rat HNF1 is over-expressed in H1A and H1B

• Human HNF1 is repressed in H1A and H1B

• ApoC-III is also repressed in H1A and H1B

• Thus, HNF1 may repress itself and HNF4-mediated activation of ApoC-III

Ktisaki and Talianidis, 1997. Science 277:109HNF1 mediated repression of HNF4 action

CATH4 H4 H4

+ HNF1 + + +-- -

Acetylated

Un-acetylated

CAT assay

Lyse cells, mix with 14C-acetyl CoA, extract and apply to thin layer chromatography

migration

Chloramphenicol

Chloramphenicol

HNF4HNF4HNF4

• All HNF-4 responsive promoters are repressed in H1A and H1B cells

• The control RSV promoter is unaffected • Thus, HNF1 represses HNF4 activity

Fig. 2- Does HNF1 repress HNF4 action?

CAT

HNF4-responsivePromoter

• HNF4, not HNF1, binds to the “A” site of the HNF1 promoter

• HNF1 does not interact with HNF4

Fig. 3- How does HNF1 repress it’s own transcription?

Gel shifts

“supershift” assay

Supershift

HNF4

HNF1

HNF-4 binding site

Ligand binding domain

HNF4

Activation domain

…But perhaps HNF1 really does interact with HNF4 (although weakly).

Fig. 4- How does HNF1 repress HNF4-mediated transcription?

G4-CAT CAT

GAL4 binding site

GAL4 DBD

HNF4 Act. Domain

TATA

M440M280

HNF1

HNF1?

…But perhaps HNF1 really does interact with HNF4 (although weakly).

Thus, HNF1 directly inhibits the HNF4 activation domain

M440M280

HNF4 -interaction regionAA 280-440

Ligand binding domain

AA 130-368HNF-1 interaction region

HNF4

HNF1

• Observe HNF1 interacts with HNF4 at AF2 region (activation region)

Fig. 5- Physical interaction of HNF4 and HNF1- 1st proof

Bead GSTHNF4

+ HNF135S

Assay- GST pull down

Bead GSTHNF4

HNF135S

HNF1

• Thus, HNF1 “pulls” HNF4 activation domain into nucleus

Fig. 5- Physical interaction of HNF4 and HNF1-2nd proof

Transfect HNF4 (227-455) plus• vector alone•HNF1 Full length•HNF1 (440)•HNF1 (280)•GAL4

Experiment

Rollini and Fournier, PNAS 1999, 96:10308HNF4 and HNF1 activates serpin locus

Human chromosome 14

Serpin locus (1AT, CBG,..)

RAT FibroblastR(14n)6

Rat hepatoma F(14n)2

HepatomaVariant

H11

Hepatic phenotype?(HNF4/1/a1AT/CBG) OFF ON OFF

Tools

HepatomaVariant

H11

Can HNF1 or HNF4 remodel a silent locus?

RSV-HNF1

RSV-HNF4

ON

ON

OFF

Fig. 1

EH1 series

EH4 series

Fig. 13.31 Dnase I hypersensitivity of an active gene

Remove protein, Isolate DNA

Treat with DNaseI

Isolate chromatin

Digest with Xba1 or BglII

Southern blot

Agarose gel

MSB= non-expressing cells

Ovalbumin Probe: -globin

DNaseI HS analysis

Hepatoma FibroblastVariant

Fig. 2- Example of DNAseI results

Remove protein, Isolate DNA

Treat with DNaseI

Isolate chromatin

Digest with Xba1 or BglII

Southern blot

Agarose gel

Fig 3. DNaseI results over 130 kb of the serpin locus

HNF1HNF4

Thus, HNF1 and/or HNF4 completely remodel a 130 kb region of DNA to look like that of hepatoma cells

Li, Ning and Duncan 2000. Genes anmd Devel. 14:464

HNF4-/- miceNote- liver specification not changed, but hepatic gene expression is