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
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
from Zhou et al., Nature Rev.Mol.Cell Biol. 12, 7 (2011)
Scaffold-associated regions can act as boundaries
Condensed chromosomes are visible during metaphase
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
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 Jiang and Pugh, Nature Rev.Genet. 10, 161 (2009)
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
Nucleosome Structure
Assembly of Nucleosomes
Histone chaperones assemble histones into nucleosomes
Histone chaperones prevent non-specific associations of histones with DNA
from Das et al., Trends Biochem.Sci. 35, 476 (2010)
Histone chaperones prevent formation of deleterious off-pathway intermediates
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
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)
from Zhou et al., Nature Rev.Mol.Cell Biol. 12, 7 (2011)
Histone Modifications Define Functional Elements
Each histone modification has a unique biological role
Histone modifications are interdependent
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
from Jiang and Pugh, Nature Rev.Genet. 10, 161 (2009)
Nucleosome position can be mapped by ChIP-seq
Cross-link histones to DNA and digest linker
Immunoprecipitate
Sequence DNA
Nucleosome Position Can Be Mapped
from Jiang and Pugh, Nature Rev.Genet. 10, 161 (2009)
Nucleosome-free region at the beginning and end of genes
Nucleosomes have defined locations near the promoter
Positioning of Nucleosomes at Promoters
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
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
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 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)
from Sarma and Reinberg, Nature Rev.Mol.Cell Biol. 6, 139 (2005)
Nucleosomal Histones and Their Variants
from Talbert and Henikoff, Nature Rev.Mol.Cell Biol. 11, 264 (2010)
H2A.Z may protect promoters from being methylated
H2A.Z is found on either side of a nucleosome-free region
Role of H2A.Z
Action of HMG-box Proteins
from Thomas and Travers, Trends Biochem.Sci. 26, 167 (2001)
HMG-box proteins bend DNA
DNA bending can affect transcription and site-specific recombination
from Dekker, Science 319, 1793 (2008)
Spatial Assembly of Expression Units
from Jiang and Pugh, Nature Rev.Genet. 10, 161 (2009)
Regulation of DNA Accessibility
Nucleosome sliding exposes binding sites
Chromatin remodelling complexes extract DNA from the nucleosome surface
Histone chaperonesincorporate histone variants
Nucleosome eviction may be necessary for transcription initiation
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