1. Introduction to Genetic Analysis TENTH EDITION Introduction to Genetic Analysis TENTH EDITION Griffiths Wessler Carroll Doebley © 2012 W. H. Freeman and Company CHAPTER 12 Regulation of Gene Expression in Eukaryotes

2. CHAPTER OUTLINE 12.1Transcriptional regulation in eukaryotes: an overview 12.2Lessons from yeast: the GAL system 12.3Dynamic chromatin 12.4Short-term activation of genes in a chromatin environment 12.5Long-term inactivation of genes in a chromatin environment 12.6Gender-specific silencing of genes and whole chromosomes 12.7Post-transcriptional gene repression by miRNAs

3. The first cloned mammal Dolly, the Finn Dorset lamb in 1996 and her surrogate Scottish Blackface mother Dolly and Bonnie

4. Dolly in Royal Museum of Scotland Ian Wilmut

5. Dolly Dolly Parton

6. globin myosin erythrocyte muscle cell All genes housekeeping All cells have the same genome, but each cell expresses only a subset of all genes Cells differ in gene expression

7. Overview of transcriptional regulation nucleus (membrane) chromatin

8. Gene regulation at multiple levels Transport Localization Modification Complex formation Degradation Many regulatory proteins have to import into nucleus

9. Promoter-proximal elements precede the promoter of a eukaryotic gene

10. Promoter-proximal elements are necessary for efficient transcription Point mutations throughout the promoter region were analyzed for their effects on transcription rates. The height of each line represents the transcription level relative to a wild-type promoter or promoter-proximal element (1.0).

11. Transcription factors need multiple functional domains 1.A domain that recognizes a DNA regulatory sequence (the protein’s DNA-binding site) 2.A domain that interacts with one or more proteins of the transcriptional apparatus (RNA polymerase or a protein associated with RNA polymerase) 3.A domain that interacts with proteins bound to nearby regulatory sequences on DNA such that they can act cooperatively to regulate transcription 4.A domain that influences chromatin condensation either directly or indirectly 5.A domain that acts as a sensor of physiological conditions within the cell Direct or indirect (by interacting with other proteins)

12. Model Organism Yeast Brewer’s yeastBaker’s yeast Saccharomyces cerevisiae Budding yeast

13. The Gal pathway Expressed at low level Induced by galactose Regulated by Gal4

14. Transcriptional activator proteins bind to UAS elements in yeast UAS: Upstream Activation Sequences Binding site for Gal4 Can be far from promoter

15. Transcriptional activator proteins are modular Reporter gene domain swap

16. Transcriptional activator proteins may be activated by an inducer Galactose: inducing signal

17. Transcriptional activator proteins recruit the transcriptional machinery Co-activator: Does not directly bind DNA Specific recognition of target sequence Enhancer can function far away from promoter Enhancer can function upstream or downstream, even far away

18. Transcriptional complexes

19. Combinations of regulatory proteins control cell types Mating type Combinations of binding partners => different binding specificities

20. The structure of chromatin ~150 bplinker DNA

21. A nucleosome is composed of DNA wrapped around eight histones Histone octamer (H2A 2 H2B 2 H3 2 H4 2 ) DNA exposed on the outside

22. The structure of chromatin

23. Euchromatin (loose) Heterochromatin Condensed Repetitive sequences Late replicating Genes silenced

24. Chromatin remodeling exposes regulatory sequences Shifting of nucleosome position Exposes regulatory sequences Linker DNA: sensitive to nuclease Nucleosomal DNA: protected from nuclease digestion Use nuclease sensitivity to determine chromatin state (open/closed) or nucleosome position + SWI-SNF + ATP

25. The SWI-SNF complex for chromatin remodeling Yeast mutant screen sugar nonfermenting (snf) Mating type switch (swi) swi2=snf2 swi2/snf2 (“switch-sniff”) locus SWI-SNF complex

26. Modifications of histone tails results in chromatin remodeling Histone tails are exposed, can be modified Modifies Lysine (K) and Arginine (R) (basic aa) Acetylation: negative charges => repulsion

27. Histone modifications

29. Acetylation of histones Histone acetyltransferase (HAT) Histone de-acetylase (HDAC)

30. Histone modifications Alternative modifications on the same residue Histone code

31. Regulation of gene expression by histone acetylation

32. Histone deacetylation can turn off gene transcription HDAC (corepressor)

33. Inheritance of chromatin states Epigenetic memory: heritable traits (over rounds of cell division and sometimes transgenerationally) that do not involve changes to the underlying DNA sequence. (e.g. chromatin state)

34. Methylation of DNA

35. A model for the inheritance of DNA methylation In mammals, 70-80% of CG are methylated genome-wide. CpG island: clusters around gene promoter hemi-methylated maintenance

36. Enhanceosomes help recruit the transcriptional machinery

37. Enhanceosomes recruit chromatin remodelers Enhancers contain binding sites for many transcription factors, which bind and interact cooperatively.

38. Enhancer-blocking insulators prevent enhancer activation

39. Model for how enhancer-blocking insulators might work

40. Mating-type switching is controlled by recombination of DNA cassettes ds break in MAT made by HO endonuclease => gene conversion silent information regulators (SIR) Sir2 (HDAC)

41. Gene silencing is caused by the spread of heterochromatin w + is expressed in some cells => not a mutation in w gene Clonal => epigenetic memory Position-effect variegation (PEV)

42. Heterochromatin in Drosophila chromosomes ~30% of genome H3K4me2, enriched in euchromatin H3K9me2, enriched in heterochromatin

43. Some genes enhance or suppress the spread of heterochromatin Enhancer Suppresor Su(var)2-5 = HP1 (heterochromatic protein 1) Su(var)3-9 = histone methylatransferase

44. Multiple states of Lysine methylation

45. Heterochromatin may spread farther in some cells than in others

46. Barrier insulators stop the spread of heterochromatin

47. Genomic imprinting

48. Phenotype depends on the parental origin of the genes

49. Inactivation of genes and chrosomomes

50. Genomic imprinting No changes in DNA sequence mouse/human ~100 imprinted genes

51. Genomic imprinting requires insulators DNA methylation imprinting control region

52. Unusual inheritance of imprinted genes

53. Steps required for imprinting Igf2: maternal imprinting (inactive) H19: paternal imprinting (inactive) H19Igf2

54. X inactivation Dosage compensation for X chromosome female: XX male: XY

55. Barr body and Lyon Hypothesis of X inactivation Murray Barr: discoverer Mary Lyon Epigenetic memory Xi: H3K9me, histone hypoacetylation, DNA hypermethylation ~ heterochromatin

56. Xist non-coding

57. Xist RNA covers one of the two copies of the X chromosome RNA fluorescent in situ hybridization (FISH) metaphase chromosomes female fibroblast cell line Xist expression => cis-inactivation

58. A model for X-chromosome inactivation

59. Possible models for the repression of translation by miRNA