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Molecular Regulation of Hepatic Gene Expression 2/10/03 G. Bulla BIO4751 Advanced Molecular and Cellular Genetics.

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Presentation on theme: "Molecular Regulation of Hepatic Gene Expression 2/10/03 G. Bulla BIO4751 Advanced Molecular and Cellular Genetics."— Presentation transcript:

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2 Molecular Regulation of Hepatic Gene Expression 2/10/03 G. Bulla BIO4751 Advanced Molecular and Cellular Genetics

3 Liver pathogenesis- Acute and Chronic Viral infection- Hepatitis B, C, …….. Trauma Alcohol-induced Drug-induced Fibrogenesis Cirrhosis

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5 Gall Bladder (peekaboo) Diaphragm Largest gland in the body (1.5 kg) Receives 25% of cardiac output

6 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 Science 284:1998

7 Major liver-enriched transcription factors Family HNF1  Homeobox HNF3  Forkhead (Drosophila) HNF4Steroid/thyroid HNF6Zinc Finger C/EBP  Leucine Zipper GATA-6Zinc Finger Hepatic gene regulation is primarily at the level of transcriptional regulation HNF= Hepatocyte Nuclear Factor C/EBP= CAAT/Enhancer Binding Protein

8 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

9 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/EBP  die 8 hr post birth- hypoglycemia; adipocyte abnormalities C/EBP  Mild-  Susceptible to listeria infection NF-  B (p65) embryonic lethal ( liver apoptosis)

10 HNF4 HNF1 GATA6 C/EBP HNF3  ?? Liver-specific genes Combinatorial activation of liver-specific genes

11 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

12 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 *

13 Rat Hepatoma Hepatoma Variants Somatic Cell Hybrids Chromosome Segregation x Fibroblast OFF Liver-specific Gene Expression Selection ON

14 Hepatoma Cells in Tissue Culture

15 2 kb I  1-antitrypsin locus IIIII IVV  HNF1HNF4 Enhancers Human Ch. 14  1-AT

16 Fado-2Fg-14 H11, M29,... Variants HB11, MB29 Backselectants 1.  1AT-aprt 2.  1AT-gpt APRT -  1AT - GPT -  1AT + GPT + APRT + APRT -  1AT + GPT - HATDAP, 6TX AAT  1AT + GPT + APRT + Generation of Hepatoma Variants ON OFF ON  HNF1HNF4 Enhancers

17 HNF1-mediated rescue Hepatic Gene Expression in H11 Hepatoma Variant Cells HNF4 Activator? HNF1  Liver-specific genes

18 H11 CellsH11b1-1, -16 HNF4 HNF1  1AT HNF1   Neo HNF4 HNF1  1AT G418 Rescue of hepatic gene expression with by HNF1 

19 Hepatic Gene Expression in H11 Hepatoma Variant Cells  1-Antitrypsin Albumin Aldolase B  -fibrinogen  -fibrinogen  -fibrinogen PEPCK Genes unaffected in H11 cells Alcohol dehydrogenase Tyrosine aminotransferase Argininosuccinate synthetase HNF3 C/EBP Transferrin Genes rescued by HNF1  in H11 cells

20 HNF4 Activator? HNF1 HS2 M29H11, M38 BLOCK HNF1 Liver gene promoter +1 C/EBP Hepatoma Variant Cells Contain Distinct Defects in Hepatic Gene Expression C/EBP HNF4 Cell Line

21 DONOR Colcemid 24 hr Cytochalasin B Centrifugation Neo-marked Chromosome PEG Fusion Recipient G418 Microcell hybrid Micronucleate cell Microcell-mediated chromosome transfer Human HepG2 H11 Hepatoma Variants (Neo marked)

22 40X 100X H11(G2)-89 Detection of human chromosomes using FISH

23 H11(G2)-  1AT Tubulin Fg14 RAT1 Fg14 Rescue of  1AT expression in microcell hybrids 90 G418 r clones picked (from three separate experiments) 45 survived expansion 38 MAX s 5 MAX r 2 G-band Chromosome painting FISH PCR 24/24  1AT -  1AT +  1AT - Analysis H11(G2)-

24  1AT Tubulin H11(G2)- Cyclo Y HNF1  H11(G2)- HNF4 Cyclo Y Fg-14 dilutions Fg14H11 Fg14 H Rescue of HNF4, HNF1, and  1AT in microcell hybrids Fg14 H11

25 D12S137 Human chromosome 12  1AT MAX r D12S329 D12S61 D12S64 D12S218 D12S371 D12S p q D12S129 D12S221 D12S314 D12S62 H11(G2) nd Mapping the HIT locus on Chromosome 12p

26 U.S.A.- 0.6% Or 1in 166 Illinois % Or 1 in 100,000 Southern Africa % Or 1 in 3 Source- U.N AIDS, 2001 Adults living with AIDS in Africa

27 Major liver-enriched transcription factors Family HNF1  Homeobox HNF3  Forkhead (Drosophila) HNF4Steroid/thyroid HNF6Zinc Finger C/EBP  Leucine Zipper GATA-6Zinc Finger Hepatic gene regulation is primarily at the level of transcriptional regulation HNF= Hepatocyte Nuclear Factor C/EBP= CAAT/Enhancer Binding Protein

28 M440 M280 HNF4 -interaction region AA Ligand binding domain AA HNF-1 interaction region HNF4 HNF1 Zinc finger Activation domain Homeo domain

29 HNF4 HNF1 ApoC-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, Science 277:109 HNF1 mediated repression of HNF4 action

30 CAT H4 H4 H4 + HNF Acetylated Un-acetylated CAT assay Lyse cells, mix with 14C- acetyl CoA, extract and apply to thin layer chromatography migration Chloramphenicol HNF4

31 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-responsive Promoter

32 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

33 Ligand binding domain HNF4 Activation domain …But perhaps HNF1 really does interact with HNF4 (although weakly).

34 Fig. 4- How does HNF1 repress HNF4-mediated transcription? G4-CATCAT GAL4 binding site GAL4 DBD HNF4 Act. Domain TATA M440 M280 HNF1 ? …But perhaps HNF1 really does interact with HNF4 (although weakly).

35 Thus, HNF1 directly inhibits the HNF4 activation domain M440 M280 HNF4 -interaction region AA Ligand binding domain AA HNF-1 interaction region HNF4 HNF1

36 Observe HNF1 interacts with HNF4 at AF2 region (activation region) Fig. 5- Physical interaction of HNF4 and HNF1- 1 st proof Bead GST HNF4 + HNF1 35 S Assay- GST pull down Bead GST HNF4 HNF1 35 S HNF1

37 Thus, HNF1 “pulls” HNF4 activation domain into nucleus Fig. 5- Physical interaction of HNF4 and HNF1-2 nd proof Transfect HNF4 ( ) plus vector alone HNF1 Full length HNF1 (440) HNF1 (280) GAL4 Experiment

38 Rollini and Fournier, PNAS 1999, 96:10308 HNF4 and HNF1 activates serpin locus Human chromosome 14 Serpin locus (  1AT, CBG,..) RAT Fibroblast R(14n)6 Rat hepatoma F(14n)2 Hepatoma Variant H11 Hepatic phenotype? (HNF4/1/a1AT/CBG) OFFONOFF Tools

39 Hepatoma Variant H11 Can HNF1 or HNF4 remodel a silent locus? RSV-HNF1 RSV-HNF4 ON OFF Fig. 1 EH1 series EH4 series

40 Fig 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

41 DNaseI HS analysis HepatomaFibroblastVariant Fig. 2- Example of DNAseI results Remove protein, Isolate DNA Treat with DNaseI Isolate chromatin Digest with Xba1 or BglII Southern blot Agarose gel

42 Fig 3. DNaseI results over 130 kb of the serpin locus HNF1 HNF4 Thus, HNF1 and/or HNF4 completely remodel a 130 kb region of DNA to look like that of hepatoma cells

43 Li, Ning and Duncan Genes anmd Devel. 14:464 HNF4-/- mice Note- liver specification not changed, but hepatic gene expression is


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