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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. Before we begin….. • The material on pages109-116….not new? • Websites: • 5.1 Transcription through nucleosomes • 5.3 Promoter structure (TBPs/TAFs) • 5.4 Families of transcription factors • 5.5 Histone acetylation/chromatin remodeling

  3. A few words about enhancers • Heavily studied since the early 1980’s • Most involve minimal enhancers/promoters or minimal enhancer/heterologous promoter combinations • Most involve studies on plasmid DNA transiently transfected into cells in culture • Most of our models of enhancer activity derive from these kinds of experiments

  4. Example (Fig 5.6 from Gilbert)

  5. Gilbert’s 7 generalizations concerning enhancer function • Most genes require enhancers for activity • Enhancers are the major determinant of differential transcription in time and space • Enhancers can work far from the promoter so multiple signals can be integrated to determine if a gene will be transcribed. Genes can have several enhancers and each enhancer can bind multiple proteins

  6. The 7 generalizations (continued) • Interaction between proteins bound to the enhancer sites and the transcription initiation complex assembled at the promoter is thought to regulate transcription • Enhancers are modular. Particular combinations of factors (rather than any one factor) determines enhancer function

  7. The 7 generalizations (continued) • A gene can have several enhancer elements, each turning it on in a different set of cells • Enhancers can be used to inhibit transcription. In some cases factors that activate the transcription of one gene represses other genes. (Silencers= negative enhancer

  8. My generalization of enhancers • Sequences with enhancer activity bind an enormous array of sequence-specific DNA binding proteins called transcription factors • Transcription factors fall into families with shared structural and functional properties • Enhancers affect transcription efficiency and can do so over great distances of DNA via the binding of transcription factors

  9. General domain structure of many transcrption factors Activation DNA binding dimerization/interaction (example only) Different regions of transcription factor proteins are responsible for discrete functions involved in its regulatory activity

  10. Major families of transcription factors • Homeodomain (helix-turn-helix) (Pax, Hox) • Basic helix-loop-helix (E proteins, MyoD) • Winged helix proteins (HNF-3, Ets) • Basic leucine zipper (fos/jun, C/EBP) • Zinc finger proteins (SP1, CTCF, EKLF) • Nuclear hormone receptors (RAR, RXR, ER, GR, PR) bind steroid hormones

  11. Conserved structures are often DNA binding domains (From Wolffe, Chromatin, 3rd ed.) Some transcription factors contain motifs from chromatin proteins

  12. Motifs shared by transcription factors and chromatin proteins • The “histone fold” • Histone H3 and TAF(II)-40 • Histone H4 and TAF(II)-60 • Histone H2B and CBF (CCAAT binding factor) • Wolffe and Pruss (1996) Deviant nucleosomes: the functional specialization of chromatin. Trends Genet. 12:58-62

  13. Figure 1 from Pruss and Wolffe

  14. Motifs shared by transcription factors and chromatin proteins (continued) • HMG-box • Shared by HMG1 and numerous factors including LEF-1, TCF, UBF, HMG-I/Y • These tend to be DNA-bending proteins that facilitate “enhanceosome assembly” • Winged helix domain • Shared by HNF-3 and Linker histones (H1,H5) • Role in nucleosome spacing/positioning

  15. Structure of the winged helix domain of linker histones and HNF3 (From Wolffe, Chromatin, 3rd Ed.)

  16. HNF3 and the serum albumin enhancer Lab of K.S. Zaret@Brown University • Nucleosomes are randomly positioned on albumin enhancer DNA without HNF3 • HNF3 precisely positions the nucleosome such that it lies under it right at the HNF3 binding site of the enhancer. Adjacent nucleosomes are also positioned as a result • HNF3 has a domain that interacts with linker histone binding sites of the nucleosome core

  17. A closer look • Interaction between proteins bound to the enhancer sites and the transcription initiation complex assembled at the promoter is thought to regulate transcription • Enhancers are modular. Particular combinations of factors (rather than any one factor) determines enhancer function

  18. Example of IFN-beta enhancer(From Wolffe, Chromatin, 3rd Ed.) Illustrates model of an assembled enhancer interacting with pol II

  19. New models of enhancer function • Regulation of nucleosomal positioning • Recruitment of histone acetylase/deacetylase to disrupt nucleosome structure • Prevention of gene localization to centromeric heterochromatin

  20. Reversible histone acetylation • Histones H3 and H4 are acetylated on lysine • Histone acetyltransferases (HAT) • p300/CBP • PCAF/GCN5 • TAF(II)-250 • Histone deacetylases (HDAC) • RPD-3 • Interacts with Sin3 and NcoR co-repressors • Former interacts with Mad/Max family, latter interacts with steroid receptor family members

  21. Role of HAT/HDAC in transcriptional regulation in chromatin (From Wolffe, Chromatin, 3rd Ed.)

  22. Year 2001 model of the IFN-b enhanceosome (From Agalioti, et. al. (2000) Cell 103:667-678

  23. Chromatin “remodeling machines” • SWI/SNF (yeast, mammals) • NURF and CHRAC (Drosophila) • All are multi-subunit complexes • Their activity is ATP dependent (energy) • Cause nucleosome disruption in vitro • Little evidence of targeting specificity

  24. Groudine and Martin • Found that an active enhancer increased the probability of establishing transgene expression, not necessarily the rate of transcription • Searched for a structural correlate of this activity • Not DNA methylation • Not chromatin accessibility • Yes, proximity to centromeric DNA (heterochromatin)

  25. From Francastel, et. al. (1999) Cell 99:259-269

  26. Why care about centromeres? • Silent genes are found “associated” with centromeric heterochromatin • Ikaros family of transcription factors (Zn++ finger) play a role in centromeric localization of inactive genes. • Work from the labs of A.G. Fisher (London) and S.T. Smale (UCLA)

  27. Summary: Enhancers • Enhancers and their associated proteins (transcription factors) are important determinants of gene expression patterns • They affect transcription by many mechanisms • Direct interaction with RNA polymerase • Regulation of nucleosomal positioning • Recruitment of histone acetylase/deacetylase • Prevention of gene localization to centromeric heterochromatin