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 mice Li, 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” methylase From 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 Transcription factors are “transient” while DNA methylation is “not” From A. Razin (1998) EMBO J. 17:4905-4908

9. How stable is DNA methylation? Specific DNA de-methylation events have been implicated in gene activation. –Ig  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 (Ig  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  -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 locusGrosveld, et. al. 1988:Human CD2 locusLang, et. al. 1992:Adenosine deaminaseAronow, et. al. 1994:Macrophage lysozymeBonifer, et. al. 1994:T cell receptor  Diaz, et. al. 1994:Immunoglobulin HCMadisen, et. al. 1995:Human growth hormoneJones, et. al. 1999: 5/VpreB gene locusSabbatini, et. al.

20. LCR:Comparison and contrast From Li, et. al. (1999) Trends Genet. 15:403

21. 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**

22. The first LCR--the globin locus From Li, et. al. (1999) Trends Genet. 15:403

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

25. 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

26. 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

27. 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

28. Transcriptional regulation:putting it together Proposed 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

29. Other forms of gene regulation Pages 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