organization of dna within a cell from lodish et al., molecular cell biology, 6 th ed. fig 6-1 2...
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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