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

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

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

Structure of the Chromatin Fiber Chromatin folds into a two start zig-zag tetranucleosomal arrangement from Ausio, BioEssays 37, 46 (2014)

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 and facilitates chromatin compaction Nucleosome Structure

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

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

Histone Acetylation 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 from Tessarz and Kouzarides, Nature Rev.Mol.Cell Biol. 15, 703 (2014)

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

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

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)

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)

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

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

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 H3K56ac is positioned at the entry-exit points on nucleosomes and enhances unwrapping of DNA H3K56ac has a higher affinity for histone chaperones and promotes chromatin assembly Newly synthesized H3 is acetylated on K56

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

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

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

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

Chromatin Immunoprecipitation (ChIP) from Orlando, Trends Biochem.Sci. 25, 99 (2000) 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 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

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

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

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

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 is necessary for transcription initiation

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

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

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