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Chromatin Compaction
Level of organization of DNA
Level of organization of DNA
The Problem
• Human genome (in diploid cells) = 6 x 109
bp
• 6 x 109 bp X 0.34 nm/bp = 2.04 x 109 nm
= 2 m/cell
• Very thin (2.0 nm), extremely fragile
• Diameter of nucleus = 5-10 µm
• ∴DNA must be packaged to protect it, but
must still be accessible to allow gene
expression and cellular responsiveness
Solution: Chromosomes
• Single DNA
Molecule and
associated
proteins
• Karyotype
• Chromatin vs.
Chromosomes
HISTONES
• Main packaging proteins
• 5 classes: H1, H2A, H2B, H3, H4.
• Rich in Lysine and Arginine
Eukaryotic chromosomal organization
• Many eukaryotes are diploid (2N)
• The amount of DNA that eukaryotes have varies; the amount of
DNA is not necessarily related to the complexity (Amoeba
proteus has a larger amount of DNA than Homo sapiens)
• Eukaryotic chromosomes are integrated with proteins that help it
fold (protein + DNA = chromatin)
• Chromosomes become visible during cell division
• DNA of a human cell is 2.3 m (7.5 ft) in length if placed end to
end while the nucleus is a few micrometers; packaging/folding of
DNA is necessary
Chapter 12: Organization in
Chromosomes
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Eukaryotic chromosomal organization
• 2 main groups of proteins involved in
folding/packaging eukaryotic
chromosomes
– Histones = positively charged proteins filled
with amino acids lysine and arginine that bond
– Nonhistones = less positive
Chapter 12: Organization in
Chromosomes
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Eukaryotic chromosomal organization
• Histone proteins
– Abundant
– Histone protein sequence is highly conserved among
eukaryotes—conserved function
– Provide the first level of packaging for the chromosome;
compact the chromosome by a factor of approximately 7
– DNA is wound around histone proteins to produce
nucleosomes; stretch of unwound DNA between each
nucleosome
Chapter 12: Organization in
Chromosomes
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Eukaryotic chromosomal organization
• Nonhistone proteins
– Other proteins that are associated with the chromosomes
– Many different types in a cell; highly variable in cell types,
organisms, and at different times in the same cell type
– Amount of nonhistone protein varies
– May have role in compaction or be involved in other
functions requiring interaction with the DNA
– Many are acidic and negatively charged; bind to the
histones; binding may be transient
Chapter 12: Organization in
Chromosomes
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Eukaryotic chromosomal organization
• Histone proteins
– 5 main types
• H1—attached to the nucleosome and involved in
further compaction of the DNA (conversion of 10
nm chromatin to 30 nm chromatin)
• H2A
• H2B
Two copies in each nucleosome
• H3
‘histone octomer’; DNA wraps
• H4
around this structure1.75 times
– This structure produces 10nm chromatin
Chapter 12: Organization in
Chromosomes
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Histones
• Found in all eukaryotic nuclei
• High content of + charged side chains
(lysine and arginine)
• Can exist in different forms due to posttranslational modifications
important in packaging DNA
• DNA tightly bound to a group of small basic
proteins
histones
• Histones constitute ~1/3 of the total mass of
the genetic material
• Chromatin = nucleoproteins + DNA
• 5 types of histones: H1, H2A, H2B, H3 & H4
HISTONES
• NOTE: if histones from different species
are added to any eukaryotic DNA sample,
chromatin is reconstituted. Implication?
• Very highly conserved in eukaryotes in
both
– Structure
– Function
• Protection
• Must allow gene activity
Conservation through time
• H2A, H2B, H3 & H4 highly conserved
among species (H4 of calf thymus and pea
seedlings)
• H1 more variable in different species
• Unit evolutionary period
the time in which the sequence
has changed by 1% after the divergence
of two evolutionary lines
Histones—Degrees of
Conservation
• H4---Only 2 variations ever discovered
• H3—also highly conserved
• H2A, H2B---Some variation between
tissues and species
• H1-like histones
– H1—Varies markedly between tissues and
species
– H1º--Variable, mostly present in nonreplicating cells
– H5---Extremely variable
Variability in Histones
Fig. 8.17 A possible nucleosome structure
Chapter 12: Organization in
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing asChromosomes
Benjamin Cummings.
30
Fig. 8.18 Nucleosomes connected together by linker DNA and H1 histone to
produce
the “beads-on-a-string” extended form of chromatin
H1
Histone octomer
Linker DNA
10 nm chromatin is produced in the first level of packaging.
Chapter 12: Organization in
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing asChromosomes
Benjamin Cummings.
31
Model for Chromatin Structure
• Chromatin is linked together every 200
bps (nuclease digestion)
• Chromatin arranged like “beads on a
string” (electron microscope)
• 8 histones in each nucleosome
• 147 bps per nucleosome core particle with
53 bps for linker DNA (H1)
• Left-handed superhelix
Chapter 12: Organization in
Chromosomes
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First order of DNA
compaction
- Core DNA = 146 bp
- Linker DNA = 8-114 bp (usually 55bp)
- DNA turns 1 and ¾ times around histone octamer.
Electronic micrography observations
• Beads on a string, the 10nm fiber
Experiments using nucleases
• Experiment: Digest chromatin with rat liver nuclease at
low concentration. (or micrococcal nuclease)
• Electrophoresis of the digested chromatin material.
A regular pattern of bands on the gel, approx. every
200 bp
→ Histones distributed evenly on DNA, and at point
which they bind, protect DNA from nuclease
digestion. (nuclease digests double stranded DNA)
Eukaryotic chromosomal organization
• Histone proteins
– DNA is further compacted when the DNA
nucleosomes associate with one another to
produce 30 nm chromatin
– Mechanism of compaction is not understood,
but H1 plays a role (if H1 is absent, then
chromatin cannot be converted from 10 to 30
nm)
– DNA is condensed to 1/6th its unfolded size
Chapter 12: Organization in
Chromosomes
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Fig. 8.20b Packaging of nucleosomes into the 30-nm chromatin fiber
Chapter 12: Organization in
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing asChromosomes
Benjamin Cummings.
39
Eukaryotic chromosomal organization
• Compaction continues by forming looped domains
from the 30 nm chromatin, which seems to compact
the DNA to 300 nm chromatin
• Human chromosomes contain about 2000 looped
domains
• 30 nm chromatin is looped and attached to a
nonhistone protein scaffolding
• DNA in looped domains are attached to the nuclear
matrix via DNA sequences called MARs (matrix
attachment regions)
Chapter 12: Organization in
Chromosomes
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Fig. 8.21 Model for the organization of 30-nm chromatin fiber into looped
domains
that are anchored to a nonhistone protein chromosome scaffold
Chapter 12: Organization in
Chromosomes
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Eukaryotic chromosomal organization
• MARs are known to be near regions of the
DNA that are actively expressed
• Loops are arranged so that the DNA
condensation can be independently
controlled for gene expression
Chapter 12: Organization in
Chromosomes
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Fig. 8.22 The many different orders of chromatin packing that give rise to the
highly
condensed metaphase chromosome
Chapter 12: Organization in
Chromosomes
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DNase I : Digestion of DNA only on one of
its strands
Simple experiment proving DNA is wrapped around the octamer
DNase I cutting sites
- DNase I cuts core DNA only on
portions of DNA which are not
linked to the histones.
- After electrophoresis only 10 bp
fragments are found.
Histone octamer formation
- Two highly conserved histones, H3 and
H4, exist in solution as a specific tetramer
(H3)2(H4)4, which behaves rather like an
ordinary multi-subunit globular protein.
- The same can be said for H2A and H2B.
- The two tetramers form an octamer to
which the DNA binds itself.
Isolating the Core DNA
Second order of DNA
compaction
Secondary Structure
• H1 : essential for the solenoid structure
Secondary Structure: Essential
points
• The Solenoid is stabilized by H1 molecules
• H1 has a globular body that binds to the outward
DNA
• And 2 terminal arms (N- and C-) contact the
adjacent nucleosomes (actually the
correspondent H1 histones that binds to the
nucleosomes)
• 1 tour of solenoid = 6 nucleosomes