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Molecular Regulation of Hepatic Gene Expression

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.

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Molecular Regulation of Hepatic Gene Expression

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

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

  3. Diaphragm Gall Bladder (peekaboo) •Largest gland in the body (1.5 kg) •Receives 25% of cardiac output www.livercancer.com/

  4. 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

  5. Hepatic gene regulation is primarily at the level of transcriptional regulation Major liver-enriched transcription factors Family HNF1 Homeobox HNF3, b, gForkhead (Drosophila) HNF4Steroid/thyroid HNF6Zinc Finger C/EBP, b, g Leucine Zipper GATA-6 Zinc Finger HNF= Hepatocyte Nuclear Factor C/EBP= CAAT/Enhancer Binding Protein

  6. Transcription factors in early liver development Anterior-posterior axis HNF3 Primitive streak HNF3 Hepatic Determination Endoderm differentiation HNF3, HNF4 Onset of liver gene expression Foregut endoderm migration into mesenchyme HNF1, c-jun Amplification of liver gene expression Organ formation C/EBP Liver

  7. 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 • HNF4embryonic lethal (day 3) • DBPno 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)

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

  9. Models for Hepatic Gene Regulation • Primary hepatocytes- Fail to divide • Hepatoma- express most hepatic genes • Cell Hybrids- complete loss of hepatic • gene expression • Hepatoma Variants- partial to complete • loss of hepatic gene expression • Animal models- including knockout • transgenics

  10. Primary hepatocytes- Fail to divide • Hepatoma- express most hepatic genes • Cell Hybrids- complete loss of hepatic • gene expression • Animal models- including knockout • transgenics Models for Hepatic Gene Regulation • Hepatoma Variants- partial to complete • loss of hepatic gene expression *

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

  12. Hepatoma Cells in Tissue Culture

  13. HNF4 HNF1 +1 -660 • Enhancers  1-antitrypsin locus Human Ch. 14 2 kb 1-AT IV V I II III

  14. APRT + APRT + HNF4 HNF1 GPT + GPT + 1AT + 1AT + +1 -660 • Enhancers  Generation of Hepatoma Variants ON OFF ON 1. 1AT-aprt 2. 1AT-gpt Fado-2 Fg-14 HB11, MB29 Backselectants H11, M29,... HAT DAP, 6TX AAT Variants APRT- APRT - GPT- GPT - 1AT+ 1AT-

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

  16. Rescue of hepatic gene expression with by HNF1a HNF1a + Neo H11 Cells H11b1-1, -16 - + HNF4 G418 HNF4 - + HNF1 HNF1 - + a1AT a1AT

  17. Hepatic Gene Expression in H11 Hepatoma Variant Cells Genes rescued by HNF1in H11 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

  18. C/EBP HNF4 HNF1 Hepatoma Variant Cells Contain Distinct Defects in Hepatic Gene Expression Cell Line M29 H11, M38 HS2 Activator? BLOCK BLOCK BLOCK HNF4 BLOCK HNF1 BLOCK BLOCK C/EBP +1 Liver genepromoter

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

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

  21. Rescue of a1AT expression in microcell hybrids 90 G418r clones picked (from three separate experiments) 45 survived expansion Analysis G-band Chromosome painting FISH PCR 38 5 2 MAXs MAXr MAXr 24/24 1AT- 1AT+ 1AT- H11(G2)- H11(G2)- RAT1 Fg14 Fg14 64 65 72 73 74 75 82 85 87 88 89 5 25 33 34 35 36 37 41 44 52 53 54 59 66 61 1AT Tubulin

  22. Rescue of HNF4, HNF1, and a1AT in microcell hybrids H11(G2)- Fg14 H11 34 40 44 52 71 81 87 89 Y HNF4 H11(G2)- Fg-14 dilutions Cyclo Fg14 H11 34 40 44 52 71 81 87 89 1AT H11(G2)- Fg14 H11 Tubulin 34 40 44 52 71 81 87 89 Y HNF1 Cyclo

  23. + + + + + + 52 40 87 89 71 81 37 61 Mapping the HIT locus on Chromosome 12p H11(G2)- r - MAX + + + nd - - - 1AT + + 13 D12S329 D12S314 p 12 D12S62 D12S61 11 12 D12S371 D12S137 13 D12S64 14 D12S221 q 21 D12S218 D12S129 24 D12S2088 Human chromosome 12

  24. Adults living with AIDS in Africa U.S.A.- 0.6% Or 1in 166 Southern Africa- 20-38% Or 1 in 3 Illinois- 0.0009% Or 1 in 100,000 Source- U.N AIDS, 2001

  25. Hepatic gene regulation is primarily at the level of transcriptional regulation Major liver-enriched transcription factors Family HNF1 Homeobox HNF3, b, gForkhead (Drosophila) HNF4 Steroid/thyroid HNF6Zinc Finger C/EBP, b, g Leucine Zipper GATA-6 Zinc Finger HNF= Hepatocyte Nuclear Factor C/EBP= CAAT/Enhancer Binding Protein

  26. M440 M280 Zinc finger Activation domain Ligand binding domain HNF4 HNF-1 interaction region AA 130-368 Homeo domain Activation domain HNF1 HNF4 -interaction region AA 280-440

  27. Ktisaki and Talianidis, 1997. Science 277:109 HNF1 mediated repression of HNF4 action HNF4 - • 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 + HNF1 ApoC-III

  28. HNF4 HNF4 HNF4 CAT assay H4 H4 H4 CAT Lyse cells, mix with 14C-acetyl CoA, extract and apply to thin layer chromatography Acetylated Chloramphenicol migration Un-acetylated Chloramphenicol - + - - + + + HNF1

  29. Fig. 2- Does HNF1 repress HNF4 action? HNF4-responsive Promoter • All HNF-4 responsive promoters are repressed in H1A and H1B cells • The control RSV promoter is unaffected • Thus, HNF1 represses HNF4 activity CAT

  30. HNF1 HNF4 HNF-4 binding site Fig. 3- How does HNF1 repress it’s own transcription? Gel shifts • HNF4, not HNF1, binds to the “A” site of the HNF1 promoter • HNF1 does not interact with HNF4 Supershift “supershift” assay

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

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

  33. M440 M280 Thus, HNF1 directly inhibits the HNF4 activation domain Ligand binding domain HNF4 HNF-1 interaction region AA 130-368 HNF1 HNF4 -interaction region AA 280-440

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

  35. Fig. 5- Physical interaction of HNF4 and HNF1-2nd proof Experiment • Transfect HNF4 (227-455) plus • vector alone • HNF1 Full length • HNF1 (440) • HNF1 (280) • GAL4 • Thus, HNF1 “pulls” HNF4 activation domain into nucleus

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

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

  38. Isolate chromatin Treat with DNaseI Remove protein, Isolate DNA Digest with Xba1 or BglII Agarose gel Probe: a-globin Ovalbumin Southern blot MSB= non-expressing cells Fig. 13.31 Dnase I hypersensitivity of an active gene

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

  40. 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

  41. HNF4-/- mice Note- liver specification not changed, but hepatic gene expression is Li, Ning and Duncan 2000. Genes anmd Devel. 14:464

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