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Extinction

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

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Extinction

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  1. 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

  2. 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

  3. Generation of Somatic Cell Hybrids Propagation PEG Fusion X Hat, Ouabain FTO2B hepatoma RAT1 fibroblast Somatic Cell Hybrid Heterokaryon

  4. Hepatoma x fibroblast hybrids Hepatoma Fibroblast Hybrid Hybrid

  5. Global shut-off of liver-specific genes in cell hybrids is reversible Unstable hybrid fibroblast hybrid hepatoma

  6. What are the loci that direct extinction?

  7. 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 Hepatoma

  8. Chromosome analysis of microcell hybrids FISH Mouse chromosme 11 in rat hepatoma cells

  9. What are the loci that direct extinction? Tse-1 mapped by microcell transfer • 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

  10. Mapping of Tse-1 Tse-1 (aka R1a)

  11. Liver Regulatory subunit Mechanism Tse-1 (R1a) is underexpressed in liver cells, thus fusion with fibroblast brings levels up to normal ON Fibroblast and hybrids OFF

  12. 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

  13. What are the loci that direct extinction? • Tse-2 (on human chromomse 2) represses albumin gene only Are there other Tse-1-like genes that contribute to extinction? • Other extinguisher loci have not been • identified in any system • Conclusion? • Extinction is a polygenic trait

  14. Using Robertsonian translocations to map extinguisher loci Result- Nothing much Noprthern analysis of a rat hepatoma containing Robertsonian translocations

  15. What are the cis-acting targets of extinction?

  16. GHF1 IEF1 Tissue-specific factors are absent in cell hybrids Pituitary TATA Growth Hormone Gene Hepatocytes HNF1 TATA a1-Antitrypsin Gene Lymphocytes (B-cells) OTF-2 TATA Immunuglobulin Heavy Chain Gene Pancreas (-cells) TATA Insulin Gene

  17. a1AT is down regulated at least 3000-fold in hybrids

  18. HNF4 HNF1 +1 -660 • Enhancers  The 1-antitrypsin gene is liver specific Human Ch. 14 2 kb 1-AT IV V I II III

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

  20. Promoter deletion analysis identifies critical liver-specific sequences in the a1AT promoter hepatoma fibroblast hybrid HNF1 HNF4 Promoter activity a1AT promoter deletion constructs

  21. a1-AT promoter is highly active in the presence of HNF1 in rat fibroblasts - + - - + + + HNF1 Control- TK-CAT a1AT CAT -261 +44

  22. Both HNF1 and HNF4 binding are required for full promoter activity in hepatoma cells CAT a1AT -261 +44 Promoter activity WT HNF1 HNF4 a1AT promoter deletion constructs

  23. Can HNF1 expression prevent a1AT extinction?

  24. HNF1 fails to prevent extinction of a1AT Parent cells Hybrids cells HNF1 Note - No a1 AT

  25. HNF4 + HNF1 fails to prevent a1AT extinction

  26. __ __ __ __ Extinction Models Activator? Activator?   HNF4 HNF4 HNF1 HNF1 F HNF1 F HNF4 C/EBP C/EBP C/EBP C/EBP CP HNF1  1-antitrypsin Promoter 1-antitrypsin Promoter Lack-of-activation Model Dominant Repression Model

  27. __ __ __ HNF4 prevents extinction of HNF1, but not downstream genes Extinguisher Loci Activator? Or  HNF4 HNF1      1-antitrypsin Promoter

  28. Extinction Models Active Repression Loss of activation

  29. Extinction Models Active Repression: Recruitment to heterochromatin

  30. Extinction Models Active Repression: Altered chromatin remodeling Tissue specific genes are require special remodeling Ubiquitous genes are “bookmarked”

  31. Extinction Models Active Repression: Nuclear localization-dependent Tissue-specifc genes localized in interior of nuclear domains in hybrids Transcribed B-cell specific genes

  32. 2 kb 3 3 2 2 1 1 Summary Extinction of a1AT gene expression appears to occur at many levels repression of HNF4 gene transcription repression through the a1AT promoter sequences repression through internal a1AT DNA sequences Putative Extinction Targets HNF4 HNF1 II III IV V I a1-antitrypsin locus

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