Transcript Organization of DNA Within a Cell

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
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
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
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
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
This model was proposed in 1974 by
observation of chromatin structure
from Thummel, Insect Biochem.Mol.Biol. 32, 113 (2002)
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
Some Features of Transcription Start Sites
Start sites are found in nucleosomefree regions that are resistant to
nucleosome wrapping
Small RNAs are transcribed in
both directions near promoters
from Buratowski, Science 322, 1804 (2008)
Spatial Assembly of Expression Units
from Dekker, Science 319, 1793 (2008)
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
from Lodish et al., Molecular Cell Biology, 6th ed. Fig 6-31
The histone code influences chromatin
condensation and function and defines
actual or potential transcription states
Nucleosomal Histones and Their Variants
from Sarma and Reinberg, Nature Rev.Mol.Cell Biol. 6, 139 (2005)
Each histone modification has a unique biological role
Histone modifications are interdependent
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
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
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
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 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)
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
from Turner, Cell 111, 285 (2002)
In this context, methylated K9
is not a determinant of silencing
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)
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