molecular regulation of hepatic gene expression
DESCRIPTION
Molecular Regulation of Hepatic Gene Expression. 2/10/03 G. Bulla BIO4751 Advanced Molecular and Cellular Genetics. Liver pathogenesis- Acute and Chronic Viral infection- Hepatitis B, C, …….. Trauma Alcohol-induced Drug-induced. Fibrogenesis Cirrhosis. Diaphragm. - PowerPoint PPT PresentationTRANSCRIPT
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