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
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
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

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)

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
 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

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
 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

from Jiang and Pugh, Nature Rev.Genet. 10, 161 (2009)

 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)
 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

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
from Lodish et al., Molecular Cell Biology, 6th ed. Fig 6-31 actual or potential transcription states
 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)

 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)

 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
 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)
 Positioning of Nucleosomes at Promoters

Nucleosome-free region at the

beginning and end of genes

Nucleosomes have defined

locations near the promoter

from Jiang and Pugh, Nature Rev.Genet. 10, 161 (2009) 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

Neighboring histones are deacetylated

Activator recruits a complex

that contains a histone acetylase

Neighboring histones are acetylated

from Lodish et al., Molecular Cell Biology, 6th ed. Fig 7-38
Effect of Histone H3 K9 Methylation

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

from Turner, Cell 111, 285 (2002)

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)

Nucleosomal Histones and Their Variants

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

 Role of H2A.Z

H2A.Z is found on either side

of a nucleosome-free region

H2A.Z may protect promoters

from being methylated

from Talbert and Henikoff, Nature Rev.Mol.Cell Biol. 11, 264 (2010)
 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)

 Spatial Assembly of Expression Units

from Dekker, Science 319, 1793 (2008)

 Regulation of DNA Accessibility

Nucleosome sliding exposes binding sites

Chromatin remodelling complexes

extract DNA from the nucleosome surface

Nucleosome eviction may be

necessary for transcription initiation
from Jiang and Pugh, Nature Rev.Genet. 10, 161 (2009)

Histone chaperones
incorporate histone variants