Download Class 11

Plant Molecular and Cellular Biology
Lecture 9: Nuclear Genome
Organization: Chromosome
Structure, Chromatin, DNA
Packaging, Mitosis
Gary Peter
9/16/2008
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Learning Objectives
1.
2.
3.
4.
List and explain how DNA is
packaged in the nucleus
Explain how euchromatin and
heterochromatin differ
Explain proposed mechanisms
that maintain heterochromatin
in its state
Explain the structure, functions
of enzymes that mediate
chromatin remodeling and their
proposed mechanisms
9/16/2008
PMCB Lecture 11: G. Peter
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DNA in the Nucleus
is Organized into
Chromosomes
z
Chromosomes –
z
z
One very long linear dsDNA
molecule/chromosome with
z Single copy, Repetitive, and
Highly repetitive sequences
z Centromere sequences
z Two teleomere sequences
z Multiple origins of
replication
Proteins that fold and pack
the long DNA strand into
more compact chromatin
z Histones
z Nonhistone chromosomal
proteins
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PMCB Lecture 11: G. Peter
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Example of Human Genome
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z
z
A human cell contains 2 m of DNA stretched end to end that
must fit into a nucleus that is ~6 uM in diameter
A maize cell contains 2 m of DNA stretched end to end that
must fit in a nucleus that is <10 uM in diameter
Compaction is ~1000 fold for interphase chromosomes and
10,000 fold between dsDNA and mitotic chromosomes
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Packing of DNA
into the Nucleus:
Multiple Levels of
Compaction
3-fold
27 fold
700 fold
~1000 fold
Interphase
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Mitotic
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Evidence for Nucleosomes as the
Basic Unit of Chromosome
Structure
Histone mass = the mass of DNA in chromatin
z Gentle lysis of nuclei and TEM analysis shows that
chromatin is a 30nm wide thread
z Decondensation of chromatin reveals beads on string
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PMCB Lecture 11: G. Peter
z
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Nucleosome Isolation & Organization
z
Unfolded chromatin is digested with
micrococcal nuclease
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z
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Limited digestion leaves histone H1
+ nucleosomal core with an average
of 200bp of DNA
More extensive digestion releases
H1 and yields core particles with
146bp of DNA protected from
nuclease digestion
The 54bp on average is a linker
DNA (Linker varies from 5-80bp)
Nuclesome cores dissociated with
high salt removes the 146 bp DNA
from the octameric histone core
PMCB Lecture 11: G. Peter
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Nucleosome Core Structure:
X-ray Crystal Structure
z
z
z
z
z
Core is a histone octamer with 2
subunits of H2A, H2B, H3 and H4
with DNA wrapped around 1.65
turns in a left-handed coil
Histones are basic proteins rich in
lysine and arginine that make salt
bridges with the backbone
phosphates
Extensive hydrogen bonds (146)
between histones and DNA with
~1/2 forming between amino acids
and phosphates on the DNA
Hydrophobic bonds and salt
bridges also hold the core
together and the DNA
The long amino terminal tails of
each histone extend out from the
central portion of the nucleosome
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PMCB Lecture 11: G. Peter
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Structure of Nucleosome Core
Histones
z
z
z
z
Histones are highly conserved across all
eukaryotic organisms
Histones are small basic proteins (102-135 aa)
rich in lysine and arginine
Each histone contains a region that folds in a
characteristic structure called the histone fold
and a tail region
Tail region is post translationally modified in
various ways to control many aspects of
chromatin structure
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Histone Octamer
Assembly
z
z
Dimers of H3-H4 form
and then two dimers
assemble into a very
stable tetramer
Two H2A-H2B dimers
associate with the H3H4 tetramer
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Nucleosome Packing
into 30nm Fibers
z
z
z
Zigzag and solenoid models
for packing
Histone H1 plays a role by
possibly altering the path of
DNA that exits from the
histone core helping to pull
nucleosomes together
Histone tails may help attach
nucleosomes together
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What is the Fold Compaction
in 30nm Fibers?
z
Assuming that the
30nm chromatin fiber
contains 20
nucleosomes
(200bp/nucleosome)
per 50nm of length
what is the degree of
compaction?
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z
It is compacted 27 fold
in 30nm fibers relative
to extended DNA
z
z
PMCB Lecture 11: G. Peter
dsDNA in 50nm is (20
nucleosomes x 200
bp/nucleosome x 0.34
nm/bp) = 1360nm
1360/50 = 27.2
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Heterochromatin Structure
z
Heterochromatin is highly
condensed and more compact
than the loops of 30nm fibers
z
z
Centromeres, pericentromeres
and telomeres are organized in
facultative heterochromatin
z
z
Heterochromatin contains few
genes
Heterochromatin represses gene
expression
z
z
Remains tightly condensed even
in interphase
Facultative heterochromatin
regions can spread and retract
Histone 3 tails (H3K9Met) and
Lys 27 are methylated and are
underacetylated in
heterochromatin
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Heterochromatin: Centromeres
z
Centromeres contain sequence
elements that are repeated
(>1000X)
z
z
z
Repeat sequences are variable in
number and sequence
composition between species
Centromere sequences are
highly compacted and contain
particularly dense nucleosome
arrangements
Centromere nucleosomes have a
unique histone H3 variant
(CenH3) that together with
centromere specific proteins
combine to form the kinetochore
that attaches the centromere to
the spindle apparatus
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Model for the Organization of a
Chromosome End
z
z
The telomere forms a t-loop
which lacks nucelosomes
In heterochromatin,
unacetylated lysine 16 of
histone H4 is required for the
formation of telomeric
heterochromatin, whereas
acetylation of this lysine
functions as a barrier to the
spread of heterochromatin.
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Model for Higher Order
Euchromatin Structure
z
30nm fibers are folded into
loops of 20,000-100,000 bp
that are attached to a scaffold
through matrix attachment
regions (MARS)
z
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MARS are AT rich DNA sequence
motifs 200-1000 bp in length
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Evidence for Scaffold
z
It appears that interphase and mitotic chromatin
are attached to a scaffold when visualized after
gentle nuclear lysis by TEM negative staining
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Chromatin is Highly Dynamic
z
Interphase chromosomes are in constant flux
controlled by
z
z
small nuclear RNAs, DNA methylation and histone
modification
Chromatin remodeling unfolds 30nm fibers to expose the
regions for other proteins to access and perform
functions such as transcription and DNA replication
z
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Evidence comes from chromosome puff regions in Drosophila
polytene chromosomes and the identification of protein
complexes that remodel chromatin
PMCB Lecture 11: G. Peter
FEBS Letters 567 (2004) 15–19
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Nucleosome Positioning
z
z
z
Nucleosome spacing is irregular
due to the local sequence of DNA
and proteins bound in the vicinity
A-T bases in minor groove make it
more energetically favorable to
bend DNA tightly around the
histone core
Proteins bound to DNA at specific
sites can promote while others
can inhibit nucleosome binding
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Chromatin Remodeling Works at
Multiple Levels
z
Histone H1 controls
30nm chromatin fiber
organization
z
z
Multiple isoforms of H1
and their abundance are
important for cell growth
and proliferation
ATP dependent
chromatin remodeling
works at the level of
nucleosomes
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3-fold
27 fold
700 fold
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Chromatin Remodeling: Dynamic
Repositioning of Nucleosomes
z
Chromatin remodeling
complexes are
multisubunit protein
complexes that
hydrolyze ATP to
change the structure of
the nucleosome core so
that the DNA becomes
less tightly associated
z
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Movement of the H2A &
H2B dimers in the
nucleosome cores may
be the mechanism
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ATP Dependent Chromatin
Remodeling
z
ATP dependent protein remodeling is mediated by multiple large multisubunit
complexes
z
z
z
z
These complexes affect the interaction of DNA with the nucleosomes – opening
the DNA for access by other factors
The SWI/SNF complexes from yeast are required for viability and bind well with
naked DNA
Many sets of different chromatin remodeling enzymes exist
These activities are involved in most all aspects of DNA repair, DNA
transcription, DNA replication
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PMCB Lecture 11: G. Peter
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Modification of Histone NH4 Terminal
Tails Affect the Stability of 30nm Fiber
and Higher Order Structures
z
The NH4 tails of the
histones in the nucleosomal
core are reversibly
z
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z
z
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Acetylated by histone acetyl
transferases
Deacetylated by histone
deacetylases
Phosphorylated by histone
kinases
Dephosphorylated histone
phosphatases
Methylated by methylases
Demethylated by
demethylases
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Position of Postranslational
Modifications on Histone Tails in a
Histone Octamer
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PMCB Lecture 11: G. Peter
Cell, Vol. 116, 259–272, January 23, 2004,
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Histone Code Hypothesis
z
Distinct markings of
histone tails confers
particular “meanings”
by attracting those
proteins involved with
appropriate functions
z
z
Gene expression
should not take place
DNA has been
recently replicated
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PMCB Lecture 11: G. Peter
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DNA Methylation and Chromatin
Organization: Epigenetic Control
in Plants
z
z
z
The DDM1 gene of
Arabidopsis is required to
maintain DNA methylation
levels and is needed for
transposon and transgene
silencing
It also is required for
maintenance of histone H3
methylation patterns
DDM1 is similar to the
SWI/SNF family of ATP
dependent chromatin
remodeling genes
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z
z
DNA methylation patterns
may depend on histone H3
methylation patterns
Epigenetic inheritance of
hypomethylated DNA
occurs
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Telomeres & Telomere Replication
z
z
z
Replication of the ends of
linear DNA molecules are
problematic for the replication
machinery and loss of
sequences from the ends
occurs through multiple cycles
Telomeres are located at the
ends of the chromosomes, and
they have unique repeated
sequences and a 3’
overhanging single stranded
DNA
Telomerase is a DNA
polymerase that completes
replication of telomere
sequences
z
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TRENDS in Genetics Vol.19 No.8 August 2003
Specialized reverse
transcriptase
PMCB Lecture 8: G. Peter
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Telomerase
Cell, Vol. 95, 963–974, 1998
z
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A ribonucleoprotein complex that adds repeated
DNA nucleotides to the end of a 3’OH
The ribonucleotide provides the complementary
bases for synthesis
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Summary
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DNA is folded in very precise ways to fit the
long DNA molecules into a very small space,
but still be able to access the DNA for
replication and the genes for transcription
Chromatin is very dynamic
Some of the mechanisms for regulating
chromatin reorganization are now being
dissected
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