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

4. Chromatin Organization Each layer of chromatin organization reflects aspects of gene regulation from Zhou et al., Nature Rev.Mol.Cell Biol. 12, 7 (2011) Scaffold-associated regions can act as boundaries Condensed chromosomes are visible during metaphase

5. 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

6. 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

7. 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

8. from Jiang and Pugh, Nature Rev.Genet. 10, 161 (2009) Nucleosomes contain 2 copies of H2A, H2B, H3 and H4 147 bp of DNA is wrapped around nucleosome Histone tails emanate from core Some nucleosomes contain histone variants H1 is a linker histone Nucleosome Structure

9. Assembly of Nucleosomes Histone chaperones assemble histones into nucleosomes Histone chaperones prevent non-specific associations of histones with DNA from Das et al., Trends Biochem.Sci. 35, 476 (2010) Histone chaperones prevent formation of deleterious off-pathway intermediates

10. 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

11. 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

12. Histone Modifications Affect Chromatin Structure H3K4 methylation and H3K9 acetylation are hallmarks of active chromatin H3K27 methylation and H3K9 methylation are hallmarks of silent chromatin from Johnstone and Baylin, Nature Rev.Genet. 11, 806 (2010)

13. from Zhou et al., Nature Rev.Mol.Cell Biol. 12, 7 (2011) Histone Modifications Define Functional Elements Each histone modification has a unique biological role Histone modifications are interdependent

14. 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

15. from Jiang and Pugh, Nature Rev.Genet. 10, 161 (2009) Nucleosome position can be mapped by ChIP-seq Cross-link histones to DNA and digest linker Immunoprecipitate Sequence DNA Nucleosome Position Can Be Mapped

16. from Jiang and Pugh, Nature Rev.Genet. 10, 161 (2009) Nucleosome-free region at the beginning and end of genes Nucleosomes have defined locations near the promoter Positioning of Nucleosomes at Promoters Nucleosome position is determined by ATP-dependent trans-acting factors

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. 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

20. Effect of Histone H3K4 Methylation Set9 methylates K4 Inhibits association of NuRD remodeling and deacetylase complex Inhibits association of SUV39 H3K4me is associated with active genes from Turner, Cell 111, 285 (2002)

21. BRCA1 Modifies Pericentric Heterochromatin BRCA1 promotes enrichment of Ub-H2A in pericentric heterochromatin Loss of BRCA1 triggers transcription of satellite-DNA in pericentric heterochromatin Satellite-DNA transcription is sufficient to induce genome instability after loss of BRCA1 from Venkitaraman, Nature 477, 169 (2011)

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

23. from Talbert and Henikoff, Nature Rev.Mol.Cell Biol. 11, 264 (2010) H2A.Z may protect promoters from being methylated H2A.Z is found on either side of a nucleosome-free region Role of H2A.Z

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

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

26. from Jiang and Pugh, Nature Rev.Genet. 10, 161 (2009) Regulation of DNA Accessibility Nucleosome sliding exposes binding sites Chromatin remodelling complexes extract DNA from the nucleosome surface Histone chaperones incorporate histone variants Nucleosome eviction may be necessary for transcription initiation