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

The Nature of the Nucleosome

from Alberts et al., 3rd ed., Fig. 8-10

Each nucleosome contains two copies each of H2A, H2B, H3 and H4

147 bp of DNA is would around the histone octamer

The linker DNA is 10-90 bp

Nucleosomes are Packaged into a 30 nm Fiber

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

Packing of Chromatin

Scaffold-associated regions can act as boundaries

Condensed chromosomes are visible during metaphase

from Lodish et al., Molecular Cell Biology, 5th ed. Fig 10-24

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

Michael Ashburner

The Ashburner Model for Ecdysone Action

Culture larval salivary glands with ecdysone and observe the same chromosome puffing pattern as in vivo

Cyclohexamide prevents regression of early puffs and induction of late puffs

from Thummel, Insect Biochem.Mol.Biol. 32, 113 (2002)

This model was proposed in 1974 by observation of chromatin structure

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 Buratowski, Science 322, 1804 (2008)

Some Features of Transcription Start Sites

Start sites are found in nucleosome-free regions that are resistant to nucleosome wrapping

Small RNAs are transcribed in both directions near promoters

from Dekker, Science 319, 1793 (2008)

Spatial Assembly of Expression Units

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

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

Nucleosomal Histones and Their Variants

Histone modifications are interdependent

Each histone modification has a unique biological role

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

Acetylation does not always correlate with increased transcription

Depends on the particular lysine being acetylated

EuchromatinH4 is acetylated at K5 and K8

HeterochromatinH4 is acetylated at K12

Hyperactive male X chromosomeH4 is acetylated at K16

Histone Acetylation is Involved in Cancer

pRb interacts with HDAC

Some viral oncoproteins inhibit pRb-HDAC interaction

E1A binds to p300/CBP

Regulates HAT activity

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 H3 K4 Methylation

Set9 methylates K4

Inhibits association of NuRD remodeling and deacetylase complex

Inhibits association of SUV39

HeK4Me is associated with active genes

from Turner, Cell 111, 285 (2002)

Drosophila ASH1 methylates H3 on K4 and K9 and H4 on K20

In this context, methylated K9 is not a determinant of silencing

Inhibits HP1 binding

Facilitates Brahma binding

from Turner, Cell 111, 285 (2002)

The Effect of Histone H3 K9 Methylation Depends on Other Histone Modifications

Action of HMG-box Proteins

from Thomas and Travers, Trends Bioch.Sci. 26, 167 (2001)

HMG-box proteins bend DNA

DNA bending can affect transcription and site-specific recombination

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

Aging in Yeast

Mother cell has a finite lifespan

Homologous recombination of rDNA locus generates rDNA circles

ERCs accumulate in mother cells and cause aging

Inhibition of ERC formation extends longevity

Role of Sir2 in Aging

Sir complex promotes formation of repressive chromatin structure

Sir2 is a NAD-dependent HDAC

HDAC activity represses ERC formation

Loss of silencing in aging cells

Sir2 mutants have shorter lifespans

Caloric Restriction Increases Sir2 Activity

Caloric restriction increases longevity

Extended lifespan is dependent on Sir2

Decreased ERC formation during caloric restriction

Increased [NAD+] during caloric restriction regulates Sir2 function

Sir2 may be the sensor of oxidation state of cells to coordinate the pace of aging

Sir2 is recruited to sites of DNA damage to maintain integrity of silenced chromatin

ERCs are not linked to aging in humans

Loss of silencing and misregulated transcription may be a cause of aging