Organization of DNA Within a Cell

from Lodish et al., Molecular Cell Biology, 6th ed. Fig 6-1

2 meters of DNA is packed into a 10 m diameter cell

Appearance of Chromatin Depends on Salt Concentration

Physiological ionic strength30 nm fiber

from Lodish et al., Molecular Cell Biology, 6th ed. Fig 6-28

Low ionic strengthBeads on a string

Nucleosomes are Packaged into a 30 nm Fiber

from Lodish et al., Molecular Cell Biology, 6th ed. Fig 6-30

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

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

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., 3rd 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, 6th ed. Fig 6-32

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

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

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

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, 6th ed. Fig 6-31

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 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, 6th ed. Fig 7-37

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

Properties of Acetylated Histones

Less positively charged

Chromatin is less condensed

H4K16Ac prevents formation of 30 nm fiber

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, 6th 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)

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)

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

Nucleosomal Histones and Their Variants

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

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

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 chaperonesincorporate histone variants

Nucleosome eviction may be necessary for transcription initiation