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Next lectures: Differential Gene expression

Next lectures: Differential Gene expression. Chapter 5 and websites on syllabus Epigenetic control mechanisms Histone modification DNA methylation Nucleosome disruption “machines” Promoters and enhancers Old and new models of enhancer function Novel transcriptional control sequences.

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Next lectures: Differential Gene expression

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  1. Next lectures: Differential Gene expression • Chapter 5 and websites on syllabus • Epigenetic control mechanisms • Histone modification • DNA methylation • Nucleosome disruption “machines” • Promoters and enhancers • Old and new models of enhancer function • Novel transcriptional control sequences

  2. DNA methylation • Eukaryotic DNA methylation occurs on the 5 position of cytosine in the CpG dinucleotide • The major methylation enzyme is DNMT • DNA methylation is seen in plants and animals but NOT in yeast or Drosophila • DNMT knockout mice die in utero

  3. DNMT knockout miceLi, Bestor and Jaenisch (1992) Cell 69:915-926 DNMT is required for post-gastrulation development. These mice do not progress past the 9th day of their 19 day gestation period

  4. DNMT is a “maintenance” methylaseFrom Jaenisch (1997) Trends Genet. 13:323-9 There are, likely, many DNA methyltranferase activities that have yet to be identified. Some are for maintenance and others de novo

  5. Role of DNA methylation in the genome • Genomic stability • Higher mutation rates in dnmt-/- ES cells • Global demethylation in tumor cells • Specific demethylation of oncogene promoters • Transcriptional regulation • Correlation between methylation and closed chromatin structures • Blockade of factor recognition of DNA

  6. Chromatin structure and DNA methylation • Pharmacological evidence • Trichostatin A • Blocks histone deacetylase activity • Prevents DNA methylation dependent repression • Sodium butyrate • Mimicks histone acetylation • Used to “loosen up” chromatin

  7. Mediators of methylation induced gene silencing • Methyl CpG binding proteins are repressive • MeCP2 (Knockout mouse also embryonic lethal) • Has a methyl CpG binding domain and a transcriptional repression domain • Interacts with the mSin3 co-repressor complex which associates with HDAC to repress transcription From Bestor (1998) Nature 393:311

  8. Proposed mechanism: stable repression of gene expression through development From A. Razin (1998) EMBO J. 17:4905-4908 Transcription factors are “transient” while DNA methylation is “not”

  9. How stable is DNA methylation? • Specific DNA de-methylation events have been implicated in gene activation. • Igk gene enhancer (Y. Bergman--Jerusalem) • Growth control genes in tumors (Baylin and Herman--Johns-Hopkins) • DNA de-methylation can be global (as above) or targeted to particular sequences • Santoso, et. al.(2000) J. Biol. Chem. 275:1952 • Schubeler, et. al. (2000) Mol. Cell. Biol. 20:9103

  10. How is DNA methylation regulated? From Ng and Bird (1999) Curr. Opin. Genet. Dev. 9:158-163

  11. “A DNA de-methylase” • Bhattacharya, et. al. (1999) Nature 397:579 • Has a methyl-binding domain (MBD) homologous to that of MeCP2 • Removes ONLY the methyl group from the cytosine without damage to the nucleotide or the DNA backbone • Possibility of dynamic regulation of DNA methylation as is with histone acetylation

  12. Study of DNA methylation • Restriction enzymes isoschizomers with differential ability to cut methylated DNA • Msp I and Hpa II (CCGG) • Hha I (CGCG) • Bisulfite “conversion” followed by PCR • McrBC restriction enzyme (cuts methyl-CpG) • SssI methylase:To artificially methylate DNA • 5-azacytidine:To artificially de-methylate DNA

  13. DNA methylation has been implicated in the following developmental processes: • X-chromosome inactivation (pp. 126-129) • A.D. Riggs • Genomic “imprinting” (p.126, website 5.9) • S.M. Tighlman • Tissue-specific activation of transcription • Allele-specific gene expression • Y. Bergman

  14. Other sequence elements that regulate transcription • Increase gene expression by indirect mechanisms (i.e. not via RNA polymerase) • Suppress or eliminate position-effects • Matrix/Scaffold attachment regions (MAR) • Allow local factor access to sequences • Boundary/Insulator elements • Prevents cooperation of elements on either side of it • Locus control regions (LCR) • Eliminate position-effects via an ill-defined mechanism involving overcoming heterochromatin

  15. MAR/SAR • A-T rich DNA sequences which associate with the nuclear matrix • Hypothesized to define domains of regulatory influence in chromatin • MARs that affect gene expression are often next to defined enhancers (Igm heavy chain) • Improve transgene expression • Limiting influence of integration site • Trafficking gene to regions of nuclear activity

  16. Boundary/Insulator elements • Position-dependent silencers--need to be placed in between a promoter and enhancer • Imposes a cis-obligation on enhancers • Found in genomic locations that suggest a role in separating regulatory influences in the genome • End of the b-globin LCR • In between differentially expressed genes

  17. Boundary/Insulators (continued) • Vertebrate insulators bind the zinc finger protein CTCF which appears to be responsible for their function (Bell, et. al.) • Prototype insulators identified in Drosophila • Scs and Scs’ in the heat shock locus • Gypsy (binds Su(HW) zinc finger protein) • Fab-7

  18. LOCUS CONTROL REGION: Confers high-level, position- independent, copy number-dependent, tissue-specific expression on a linked transgene in chromatin. Action: Contains recognition sequences for many ubiquitous and tissue-restricted transcription factors. Overcomes heterochromatin induced position-effect-variegation by providing an open chromatin domain for a linked transgene 1987: Human -globin locus Grosveld, et. al. 1988: Human CD2 locus Lang, et. al. 1992: Adenosine deaminase Aronow, et. al. 1994: Macrophage lysozyme Bonifer, et. al. 1994: T cell receptor  Diaz, et. al. 1994: Immunoglobulin HC Madisen, et. al. 1995: Human growth hormone Jones, et. al. 1999: l5/VpreB gene locus Sabbatini, et. al.

  19. LCR:Comparison and contrastFrom Li, et. al. (1999) Trends Genet. 15:403

  20. In other words…. • Shared characterisitics of different cis-acting transcriptional control elements • Lineage-specificity: LCR, enhancer • Timing and activation: LCR, enhancer • Facilitating factor access: MAR, LCR • Insulation: boundary, LCR • RNA pol II activity: promoter, enhancer • Needs chromatin: LCR, MAR, boundary**

  21. The first LCR--the globin locusFrom Li, et. al. (1999) Trends Genet. 15:403

  22. HS can be mapped using restriction enzymesFrom Ortiz, et. al. (1997) EMBO J. 16:5037-45

  23. Little is known about the mechanism of LCR activity • Collection of HS, each of which has distinct functions. All are required for LCR activity • Very few proteins (outside of enhancer binding proteins) have been identified which functionally interact with LCRs • HBP-1 (an HMG-Box protein) in the CD2 LCR • EKLF (a zinc finger protein) in the globin LCR

  24. LCRs:Many unanswered questions • Knockout studies have caused controversy over its non-redundant roles in the genome • Molecular basis for its effect on chromatin • Little sequence homology between LCRs • Fraser and Grosveld (1998) Curr. Opin. Cell Biol. 10:361-5 • Kioussis and Festenstein (1997) Curr. Opin. Genet. Dev. 7:614-619 • Festenstein and Kioussis (2000) Curr. Opin. Genet. Dev. 10:199-203

  25. Maintaining gene expression states • Activating and repressive multiprotein complexes first found in Drosophila • Trithorax group proteins are an activating complex: Similarities to SWI/SNF • Polycomb group proteins are the repressive complex: Mammalian homologs found • Do not establish repression, only maintain it. • Reversed by the action of the trithorax proteins

  26. Transcriptional regulation:putting it togetherProposed order of regulatory events • Methylation status/de-methylation • Histone acetylation/de-acetylation • Boundary and matrix attachment regions • Locus control regions • Transcriptional enhancers • Promoters-------> mRNA production • SWI/SNF or Polycomb proteins

  27. Other forms of gene regulationPages 130-137 of Gilbert • Differential mRNA processing • Differential mRNA stability • Selective mRNA translation • Selective mRNA localization/nuclear export • Post-translational modifications • Proteolytic cleavage • Phosphorylation and other small additions

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