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
The Nature of the Nucleosome
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
from Alberts et al., 3rd ed., Fig. 8-10
4
Nucleosomes are Packaged into a 30 nm Fiber
from Lodish et al., Molecular Cell Biology, 5th
ed. Fig 10-21
5
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
6
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, 5th
ed. Fig 10-30
7
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
8
Michael Ashburner
9
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
This model was proposed in 1974 by observation
of chromatin structure
from Thummel, Insect Biochem.Mol.Biol. 32, 113
(2002)
10
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
11
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
12
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
13
Nucleosomal Histones and Their Variants
from Sarma and Reinberg, Nature Rev.Mol.Cell
Biol. 6, 139 (2005)
14
Each histone modification has a unique biological
role
Histone modifications are interdependent
15
Properties of Acetylated Histones
Less positively charged
Chromatin is less condensed
H4K16Ac prevents formation of 30 nm fiber
16
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
17
Acetylation does not always correlate with
increased transcription
Depends on the particular lysine being acetylated
Euchromatin H4 is acetylated at K5 and
K8 Heterochromatin H4 is acetylated at
K12 Hyperactive male X chromosome H4 is
acetylated at K16
18
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
19
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)
20
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)
21
The Effect of Histone H3 K9 Methylation Depends
on Other Histone Modifications
Drosophila ASH1 methylates H3 on K4 and K9 and
H4 on K20
Facilitates Brahma binding
Inhibits HP1 binding
In this context, methylated K9 is not a
determinant of silencing
from Turner, Cell 111, 285 (2002)
22
Action of HMG-box Proteins
HMG-box proteins bend DNA
DNA bending can affect transcription and
site-specific recombination
from Thomas and Travers, Trends Bioch.Sci. 26,
167 (2001)
23
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
24
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
25
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
26
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