Organization of DNA Within a Cell

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

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

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

4. Structure of the Chromatin Fiber Chromatin folds into a two start zig-zag tetranucleosomal arrangement from Ausio, BioEssays37, 46 (2014)

5. Nucleosome Structure 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 and facilitates chromatin compaction from Jiang and Pugh, Nature Rev.Genet. 10, 161 (2009)

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

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

8. Histone Acetylation from Tessarz and Kouzarides, Nature Rev.Mol.Cell Biol. 15, 703 (2014) The positive charge of lys residues can form a salt bridge with DNA Histone acetylation removes the positive charge decreasing the binding affinity to DNA and nucleosome stability

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

10. Effect of Histone H3 K9 Methylation SUV39 methylates K9 Methylated K9 recruits HP1 Heterochromatin formation HP1 binds to SUV39 to propagate methylation Acetylated K9 or phosphorylated S10 inhibits methylation of K9 from Turner, Cell111, 285 (2002)

11. 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, Cell111, 285 (2002)

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

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

15. Binding of Histones to Nucleosomal DNA from Ransom et al., Cell 140, 183 (2010) The central region of nucleosomal DNA is organized by H3/H4 heterotetramer The peripheral regions of nucleosomal DNA is bound by H2A/H2B dimers Newly synthesized H3 is acetylated on K56 H3K56ac has a higher affinity for histone chaperones and promotes chromatin assembly H3K56ac is positioned at the entry-exit points on nucleosomes and enhances unwrapping of DNA

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

17. 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, 6th ed. Fig 6-44

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

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

20. Chromatin Immunoprecipitation (ChIP) Fix cells with formaldehyde Isolate and shear chromatin Immunoprecipitate with antibodies to modified histones or binding proteins Identify binding sites by qPCR or Southern blot from Orlando, Trends Biochem.Sci. 25, 99 (2000)

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

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

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

24. Histone Exchange From Venkatesh and Workman, Nature Rev.Mol.Cell Biol.16, 178 (2015) Histone exchange occurs before transcription initiation or during transcription elongation Chromatin remodellers and histone chaperones mediate histone exchange Chromatin modifications facilitate histone exchange H2A.Z-containing nucleosomes are less stable and facilitates nucleosome depletion

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

26. Nucleosome Dynamics During Transcription Initiation A chromatin remodeller removes the nucleosome from the AT-rich promoter region Histone chaperones replace H3-H4 with H3.3-H4 and H2A-H2B with H2A.Z-H2B Retention of H2A.Z over the promoter is ensured by its acetylation preventing its exchange by INO80 H2A.Z prevents methylation of promoter DNA From Venkatesh and Workman, Nature Rev.Mol.Cell Biol.16, 178 (2015)

27. Nucleosome Dynamics During Transcription Elongation From Venkatesh and Workman, Nature Rev.Mol.Cell Biol.16, 178 (2015) Removal of H2A-H2B dimer is sufficient to allow Pol II passage FACT targets ubiquitylated H2A-H2B dimers for removal Nap1 stabilizes hexameric nucleosome

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

29. Spatial Assembly of Expression Units Coordinately expressed genes are associated in complex genomes from Dekker, Science 319, 1793 (2008)