1. Organization of DNA Within a Cell from Lodish et al., Molecular Cell Biology, 6 th ed. Fig 6-1 2 meters of DNA is packed into a 10  m diameter cell

2. Appearance of Chromatin Depends on Salt Concentration Physiological ionic strength 30 nm fiber from Lodish et al., Molecular Cell Biology, 6 th ed. Fig 6-28 Low ionic strength Beads on a string

3. The Nature of the Nucleosome from Alberts et al., 3 rd ed., Fig. 8-10 Each nucleosome contains two copies each of H2A, H2B, H3 and H4 147 bp of DNA is would around the histone octamer The linker DNA is 10-90 bp

4. Nucleosomes are Packaged into a 30 nm Fiber from Lodish et al., Molecular Cell Biology, 6 th ed. Fig 6-30

5. Packing of Chromatin Scaffold-associated regions can act as boundaries Condensed chromosomes are visible during metaphase from Lodish et al., Molecular Cell Biology, 5 th ed. Fig 10-24

6. Genes Can be Localized on Drosophila Polytene Chromosomes Polytene chromosomes exhibit a characteristic banding pattern Localization of a gene by in situ hybridization Biotinylated probe was detected by avidin conjugated to alkaline phosphatase AP substrate results in the formation of an insoluble precipitate at the site of hybridization from Lodish et al., Molecular Cell Biology, 6 th ed. Fig 6-44

7. Ecdysone produces a characteristic pattern of puffs in polytene chromosomes of salivary glands Puffs correspond to actively transcribed genes Chromosome Puffs from Alberts et al., 3 rd ed., Fig. 8-23

8. Michael Ashburner

9. The Ashburner Model for Ecdysone Action Culture larval salivary glands with ecdysone and observe the same chromosome puffing pattern as in vivo Cyclohexamide prevents regression of early puffs and induction of late puffs from Thummel, Insect Biochem.Mol.Biol. 32, 113 (2002) This model was proposed in 1974 by observation of chromatin structure

10. Actively Transcribed Genes are Present in Decondensed Chromatin Loss of 4.6 kb Bam HI fragment when the  -globin gene is active and histones are acetylated The 4.6 kb Bam HI fragment is present when the  -globin gene is inactive and histones are deacetylated from Lodish et al., Molecular Cell Biology, 6 th ed. Fig 6-32

11. from Buratowski, Science 322, 1804 (2008) Some Features of Transcription Start Sites Start sites are found in nucleosome- free regions that are resistant to nucleosome wrapping Small RNAs are transcribed in both directions near promoters

12. from Dekker, Science 319, 1793 (2008) Spatial Assembly of Expression Units

13. Histone Tails Histones contain flexible termini that extend from the globular structure of the nucleosome from Lodish et al., Molecular Cell Biology, 6 th ed. Fig 6-31

14. Modification of Histone Tails Posttranslational modifications and histone variants contribute to structural and functional characteristics of chromatin Chromatin structure is a source of epigenetic information The histone code influences chromatin condensation and function and defines actual or potential transcription states The combination of histone modifications constitutes the histone code from Lodish et al., Molecular Cell Biology, 6 th ed. Fig 6-31

15. from Sarma and Reinberg, Nature Rev.Mol.Cell Biol. 6, 139 (2005) Nucleosomal Histones and Their Variants

16. Histone modifications are interdependent Each histone modification has a unique biological role

17. Properties of Acetylated Histones Less positively charged Chromatin is less condensed H4K16Ac prevents formation of 30 nm fiber

18. Control of Gene Expression by Acetylation Repressor recruits a complex that contains a histone deacetylase Activator recruits a complex that contains a histone acetylase from Lodish et al., Molecular Cell Biology, 6 th ed. Fig 7-38 Neighboring histones are acetylated Neighboring histones are deacetylated

19. Acetylation does not always correlate with increased transcription Depends on the particular lysine being acetylated Euchromatin H4 is acetylated at K5 and K8 Heterochromatin H4 is acetylated at K12 Hyperactive male X chromosome H4 is acetylated at K16

20. Histone Acetylation is Involved in Cancer pRb interacts with HDAC Some viral oncoproteins inhibit pRb-HDAC interaction E1A binds to p300/CBP Regulates HAT activity

21. Effect of Histone H3 K9 Methylation from Turner, Cell 111, 285 (2002) SUV39 methylates K9 Methylated K9 recruits HP1 Heterochromatin formation HP1 binds to SUV39 to propagate methylation Methylated K9 or phosphorylated S10 inhibits methylation of K9

22. Effect of Histone H3 K4 Methylation Set9 methylates K4 Inhibits association of NuRD remodeling and deacetylase complex Inhibits association of SUV39 HeK4Me is associated with active genes from Turner, Cell 111, 285 (2002)

23. Drosophila ASH1 methylates H3 on K4 and K9 and H4 on K20 In this context, methylated K9 is not a determinant of silencing Inhibits HP1 binding Facilitates Brahma binding from Turner, Cell 111, 285 (2002) The Effect of Histone H3 K9 Methylation Depends on Other Histone Modifications

24. Action of HMG-box Proteins from Thomas and Travers, Trends Bioch.Sci. 26, 167 (2001) HMG-box proteins bend DNA DNA bending can affect transcription and site-specific recombination

25. Chromatin Immunoprecipitation (ChIP) Use antibody to acetylated histone tail to determine the acetylation state of chromatin Antibody against any DNA binding protein determines the location of the binding site from Lodish et al., Molecular Cell Biology, 6 th ed. Fig 7-37

26. Aging in Yeast Mother cell has a finite lifespan Homologous recombination of rDNA locus generates rDNA circles ERCs accumulate in mother cells and cause aging Inhibition of ERC formation extends longevity

27. Role of Sir2 in Aging Sir complex promotes formation of repressive chromatin structure Sir2 is a NAD-dependent HDAC HDAC activity represses ERC formation Loss of silencing in aging cells Sir2 mutants have shorter lifespans

28. Caloric Restriction Increases Sir2 Activity Caloric restriction increases longevity Extended lifespan is dependent on Sir2 Decreased ERC formation during caloric restriction Increased [NAD + ] during caloric restriction regulates Sir2 function Sir2 may be the sensor of oxidation state of cells to coordinate the pace of aging Sir2 is recruited to sites of DNA damage to maintain integrity of silenced chromatin ERCs are not linked to aging in humans Loss of silencing and misregulated transcription may be a cause of aging