Download Molecular Biology of the Cell

TIGP
Molecular and Cellular Biology
DNA, Chromosomes and Genome
(Chapter 4)
Textbook: Molecular Biology of the Cell
Alberts et al. Sixth Edition
Instructor: Chung-Ju Rachel Wang
Sept 21, 2015
Outlines
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THE STRUCTURE AND FUNCTION OF DNA
CHROMOSOMAL DNA AND ITS PACKAGING IN
THE CHROMATIN FIBER
CHROMATIN STRUCTURE AND FUNCTION
THE GLOBAL STRUCTURE OF CHROMOSOMES
Introduction
Genetic material is
packed in a set of
chromosomes
Introduction
THE STRUCTURE AND
FUNCTION OF DNA
Watson and Crick solved the puzzle of DNA structure
by x-ray diffraction (to determine the 3D atomic
structure). The model was proposed in 1953, that
nicely explains how DNA can be replicated and
inherited as the genetic material.
A DNA Molecule Consists of Two Complementary Chains of
Nucleotides
Polarity: 5’->3’
Antiparallel
A DNA Molecule Consists of Two Complementary Chains of
Nucleotides
Sequence of nucleotides on each strand must be complementary
Two hydrogen bonds
A, G: purine
T, C: pyrimidine
Three hydrogen bonds
A DNA Molecule Consists of Two Complementary Chains of
Nucleotides
the double helix is right-handed with about 10.4 nucleotides per turn
THE STRUCTURE AND
FUNCTION OF DNA
• The Structure of DNA Provides a Mechanism for
Heredity
The Structure of DNA Provides a Mechanism for Heredity
DNA contains a sequence of nucleotides, like the letters of a document written in an alphabetic
script.
Each strand can acts as a template for the synthesis of a new complementary strand.
The Structure of DNA Provides a Mechanism for Heredity
The nucleotide sequence of the human beta-globin
gene. The DNA highlighted in yellow show the
three regions of the gene that specify the amino
acid sequence for the beta-globin protein.
The complete store of information in an organism’s
DNA is called its genome.
Human genome contains 3.2 billion nucleotide
pairs.
THE STRUCTURE AND
FUNCTION OF DNA
• In Eukaryotes, DNA Is Enclosed in a Cell Nucleus
In Eukaryotes, DNA Is Enclosed in a Cell Nucleus
Human fibroblast
Two lipid bilayer membranes
DNA in a eukaryotic cell is sequestered in a nucleus, a compartment is
encircled by nuclear envelope.
CHROMOSOMAL DNA AND ITS
PACKAGING IN THE CHROMATIN FIBER
• Eukaryotic DNA Is Packaged into a Set of
Chromosomes
Human genome (1C): 3.2 x 109 bp
6.4 X 109 bp DNA is distributed in 46 different chromosomes.
If DNA in a single human cell can be laid end-to-end, they would reach 2 meters.
How 2 meters of DNA can fit inside a small nucleus which is only 6 µm in diameter
(equivalent to pack 40km thread in a tennis ball. )
40 Km thread
Eukaryotic DNA Is Packaged into a Set of Chromosomes
Chromosomes Contain Long Strings of Genes
Two sets of chromosomes: one from father and one from mother
“Chromosome painting” technique by DNA hybridization can distinguish each pair of
chromosomes.
Mitosis
23 pairs of homologous chromosomes
Each chromosome consists of a single, long linear DNA molecular along with
proteins that fold and pack the DNA into a compact structure.
Eukaryotic DNA Is Packaged into a Set of Chromosomes
Chromosomes Contain Long Strings of Genes
Karyotype by Giemsa staining.
centromere
The chromosomes for this
staining are at an early stage
in mitosis, when the
chromosomes are
incompletely compacted.
Red knobs indicate positions of
genes that code for the large
ribosomal RNAs.
The banding patterns reflect
variations in chromatin structure.
The band pattern on each
chromosome is unique,
providing the initial means to
identify and number each
chromosome.
Eukaryotic DNA Is Packaged into a Set of Chromosomes
Chromosomes Contain Long Strings of Genes
Aberrant human chromosomes:
In an individual carrying a balanced
chromosomal translocation, the
chromosome painting technique can be
used to detect these chromosomal
rearrangement.
CHROMOSOMAL DNA AND ITS
PACKAGING IN THE CHROMATIN FIBER
• Chromosomes Contain Long Strings of Genes
Chromosomes Contain Long Strings of Genes
A gene is defined as a segment of DNA that contains the instructions for making a
particular protein or even RNA molecule as their final product.
16 chromosomes, high density of genes.
Many genomes of multicellular organisms contain, in addition to genes, a large quantity of interspersed
DNA.
Chromosomes Contain Long Strings of Genes
Two closely related species of deer with very different chromosome numbers.
There is no simple relationship between chromosome number, complexity of the organism, and
total genome size.
CHROMOSOMAL DNA AND ITS
PACKAGING IN THE CHROMATIN FIBER
• The Nucleotide Sequence of the Human Genome
Shows How Our Genes Are Arranged
The Nucleotide Sequence of the Human Genome Shows How Our
Genes Are Arranged
Dark brown:
known genes
Red: predicted
genes
The Nucleotide Sequence of the Human Genome Shows How Our
Genes Are Arranged
About 1300 bp encode a protein.
Little of the genome codes for proteins
CHROMOSOMAL DNA AND ITS
PACKAGING IN THE CHROMATIN FIBER
• Each DNA Molecule That Forms a Linear
Chromosome Must Contain a Centromere, Two
Telomeres, and Replication Origins
Each DNA Molecule That Forms a Linear Chromosome Must Contain a
Centromere, Two Telomeres, and Replication Origins
During interphase, the cell is actively expressing its genes and is therefore synthesizing proteins.
Also, DNA is replicated to produce two closely paired sister DNA molecules. At M phase,
chromosomes condense, the nuclear envelope breaks down, the mitotic spindle forms to separate
sister chromosomes.
Sister chromatids
Each DNA Molecule That Forms a Linear Chromosome Must Contain a
Centromere, Two Telomeres, and Replication Origins
During interphase, chromatin exists as long
threads, so that individual chromosomes cannot be
distinguished.
The highly condensed chromosomes at M phase
are known as mitotic chromosomes. Most easily
visualized.
Each chromosome operates as a distinct structural
unit, and it contains three specialized components
for its functions:
Replication origin
centromere,
Telomeres.
Each DNA Molecule That Forms a Linear Chromosome Must Contain a
Centromere, Two Telomeres, and Replication Origins
CHROMOSOMAL DNA AND ITS
PACKAGING IN THE CHROMATIN FIBER
• DNA Molecules Are Highly Condensed in
Chromosomes
• Nucleosomes Are a Basic Unit of Eukaryotic
Chromosome Structure
Nucleosomes Are a Basic Unit of Eukaryotic Chromosome Structure
The proteins that bind to DNA to form chromosomes are traditionally
divided into histones and non-histone chromosomal proteins.
Nucleosomes Are a Basic Unit of Eukaryotic Chromosome Structure
Nucleosomes as seen in the electron microscope.
Nucleosome discovered in 1974.
A human cell with 6.4 x 109 bp contains ~30 M nucleosomes.
A. Chromatin isolated from an interphase nucleus appears as a thread
about 30nm thick.
B. The unpacked chromatin (beads on string) with nucleosomes and their
linker DNA.
Nucleosomes Are a Basic Unit of Eukaryotic Chromosome Structure
After digested by nucleases that degrade
linker DNA, each nucleosome core particle
consists of 1 complex of 8 histone proteins
and 147 nt DNA.
The linker DNA that separates each
nucleosome core vary in length from a few
bp to about 80.
On average, nucleosome repeat at intervals
about 200 bp.
CHROMOSOMAL DNA AND ITS
PACKAGING IN THE CHROMATIN FIBER
• The Structure of the Nucleosome Core Particle
Reveals How DNA Is Packaged
The Structure of the Nucleosome Core Particle Reveals How DNA Is
Packaged
A disc-shaped histone core
with DNA tightly wrapped in
a left-handed coil of 1.7 turns.
DNA in gray
The Structure of the Nucleosome Core Particle Reveals How DNA Is
Packaged
•
Very conserved, small proteins
(102-135 aa)
•
Share a structural motif (histone
fold), with three alpha- helices
and two loops.
H3-H4 and H2A-H2B form
heterodimers.
•
C. H2A and 2B form dimer
Each core histone contains an N-tail, which is subjected to several forms of modification, and a histone
fold region.
N-terminal tails protrude from the disc-shaped core structure, which is highly flexible.
The Structure of the Nucleosome Core Particle Reveals How DNA Is
Packaged
142 hydrogen bonds are formed
between DNA and histone core, ,
providing the tight association.
More than 1/5 of the amino acid of core
histones are either lysine or arginine,
and their positive charges can
effectively neutralize the negatively
charged DNA backbone.
The minor groove is compressed on the
inside of the turn.
CHROMOSOMAL DNA AND ITS
PACKAGING IN THE CHROMATIN FIBER
• Nucleosomes Have a Dynamic Structure, and Are
Frequently Subjected to Changes Catalyzed by
ATP-Dependent Chromatin Remodeling Complexes
Nucleosomes Have a Dynamic Structure, and Are Frequently
Subjected to Changes Catalyzed by ATP-Dependent Chromatin
Remodeling Complexes
A. Using the energy of ATP hydrolysis,
the remodeling complex can push on the
DNA and loosen its attachment to the
nucleosome core.
Each cycle of ATP binding, ATP
hydrolysis, and release of the ADP and
Pi products moves the DNA on histone
core.
B. The structure of a nucleosome-bound
dimer of two ATPase subunits (green)
that slide nucleosomes in the ISW1
family of chromatin remodeling
complexes.
C. Another large chromatin remodeling
complex contains 15 units, including an
ATPase.
Hydrolyzes ATP to catalyze nucleosome sliding by pulling the nucleosome
core along the DNA, that makes specific DNA sequences available to other
proteins.
Schematics showing two
mechanisms for a dinucleosome
spacing reaction. In scheme (1),
the linker DNA is flexible, and in
scheme (2), the protein chain
between HAND and the ATPase
domain is flexible.
K Yamada et al. Nature 472, 448-453 (2011) doi:10.1038/nature09947
Nucleosomes Have a Dynamic Structure, and Are Frequently
Subjected to Changes Catalyzed by ATP-Dependent Chromatin
Remodeling Complexes
•Cells contain dozens of different
chromatin remodeling complex, that
consists of 10 or more subunits. This
process is carefully controlled.
As genes are turned on and off,
chromatin remodeling complexes are
brought to specific regions of DNA
where they act locally to influence
chromatin structure.
Nucleosome removal and histone exchange catalyzed by ATP-dependent chromatin
remodeling complexes and histone chaperones.
CHROMOSOMAL DNA AND ITS
PACKAGING IN THE CHROMATIN FIBER
• Nucleosomes Are Usually Packed Together into a
Compact Chromatin Fiber
Nucleosomes Are Usually Packed Together into a Compact Chromatin
Fiber
How nucleosome are organized into condensed arrays is still not clear.
A. zigzag model from x-ray crystallography.
The conformation of histones in a tetranucleosome.
It is a possible mode for the 30-nm chromatin fiber.
Nucleosomes Are Usually Packed Together into a Compact Chromatin
Fiber
Histone tails may help to pack nucleosome together into 30 nm fiber in
which the tails of one nucleosome contact the histone core of an
adjacent nucleosome.
Nucleosomes Are Usually Packed Together into a Compact Chromatin
Fiber
The position and structure of histone H1 is shown. The H1 constrains about
additional 20 bp of DNA.
CHROMATIN STRUCTURE
AND FUNCTION
• Heterochromatin Is Highly Organized and Restricts
Gene Expression
• The Heterochromatic State Is Self-Propagating
Genetic inheritance and epigenetic inheritance
• Chromatin structure plays a central role in the development and growth.
Cell memory
Epigenetic information is usually erased during the formation of eggs and sperm.
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Heterochromatin is highly organized and resistant to gene expression
Heterochromatin (highly
condensed form)
Genes there are silenced.
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The Heterochromatic State Is Self-Propagating
The cause of position effect variegation in fly. Heterochromatin (green) is normally prevented
from spreading into adjacent regions of euchromatin (red) by barrier DNA sequences.
In this case, the barrier DNA is removed, heterochromatin can spread into neighboring DNA,
proceeding for different distances in different cells.
The Heterochromatic State Is Self-Propagating
The wild-type White+ gene is moved near heterochromatin, and
White gene’s expression is affected by nearby heterochromatin.
The white- patches represent cell lineages in which the White+
gene has been silenced.
CHROMATIN STRUCTURE
AND FUNCTION
• The Core Histones Are Covalently Modified at
Many Different Sites
The Core Histones Are Covalently Modified at Many Different Sites
The Core Histones Are Covalently Modified at Many Different Sites
The Core Histones Are Covalently Modified at Many Different Sites
•These modifications are reversible.
•Added or removed by enzymes.
•Each enzyme is recruited to specific
sites on the chromatin at defined
times.
•Can further attract specific proteins
for various biological functions, for
example, determining how and when
genes will be expressed.
CHROMATIN STRUCTURE
AND FUNCTION
• Chromatin Acquires Additional Variety Through the
Site-Specific Insertion of a Small Set of Histone
Variants
Chromatin Acquires Additional Variety Through the Site-Specific
Insertion of a Small Set of Histone Variants
These histone variants can replace the regular histones
by ATP-dependent chromatin remodeling enzymes and
histone chaperones.
CHROMATIN STRUCTURE
AND FUNCTION
• Covalent Modifications and Histone Variants Act in
Concert to Control Chromosome Functions
Covalent Modifications and Histone Variants Act in Concert to Control
Chromosome Functions
PHD domain (gray) binds to histone tail (green) H3 lysine 4trimethylation.
B. space-filling model of an ING PHD domain bound to a histone tail.
C. A ribbon model showing how the N-terminal six amino acids in the H3 tail are
recognized. Red lines represent hydrogen bonds.
Covalent Modifications and Histone Variants Act in Concert to Control
Chromosome Functions
The covalent modifications and histone variants
act in concert to produce a “histone code” that
helps to determine biological function.
The reader complex can bind tightly only
to a region of chromatin that contains
several of different histone marks.
Particular combinations of markings on
chromatin to attract additional protein
complexes that execute biological function at
the right time.
Covalent Modifications and Histone Variants Act in Concert to Control
Chromosome Functions
Methylation and acetylation on the same
residue can not exist at the same time.
CHROMATIN STRUCTURE
AND FUNCTION
• A Complex of Reader and Writer Proteins Can
Spread Specific Chromatin Modifications Along a
Chromosome
A Complex of Reader and Writer Proteins Can Spread Specific
Chromatin Modifications Along a Chromosome
How the recruitment of a reader-writer
complex can spread chromatin changes along
a chromosome.
The writer is an enzyme that creates a specific
modification on histone.
After its recruitment to a specific site on a
chromosome by a transcriptional regulatory
protein, the writer collaborate with a reader
protein to spread its mark.
CHROMATIN STRUCTURE
AND FUNCTION
• Barrier DNA Sequences Block the Spread of
Reader–Writer Complexes and thereby Separate
Neighboring Chromatin Domains
Barrier DNA Sequences Block the Spread of Reader–Writer
Complexes and thereby Separate Neighboring Chromatin Domains
(A) The tethering of a region of chromatin
to nuclear pore complex.
(B) The tight binding of barrier proteins to
a group of nucleosomes.
(C) By recruiting a histone modifying
enzymes, barriers can erase the histone
marks that are required for
heterochromatin to spread.
For example, HS4 barrier DNA separates
the active chromatin domain that
contains the beta-globin gene from an
adjacent region (16-Kb) of silenced
condensed chromatin. Deletion of HS4
sequence causes poor expression of
globin genes.
HS4 sequence contains a cluster of
binding sites for histone
acetyltransferases.
CHROMATIN STRUCTURE
AND FUNCTION
• The Chromatin in Centromeres Reveals How
Histone Variants Can Create Special Structures
The Chromatin in Centromeres Reveals How Histone Variants Can
Create Special Structures
In budding yeast, a special centromeric DNA sequence assembles a single
nucleosome in which two copies of an H3 variant histone (CENP-A) replace the
normal H3.
The yeast centromere form kinetochore to capture microtubule.
The Chromatin in Centromeres Reveals How Histone Variants Can
Create Special Structures
Active centromere has CENP-A
histone H3 variant epigenetic mark.
Human centromeres have plasticity.
A series of A-T rich alpha satellite DNA sequences is repeated many thousands of times at each
human centromere (red), and is surrounded by pericentric heterochromatin (brown).
An ancient chromosome rearrangement results in two blocks of alpha satellite DNA. Only one
is stably functional.
CHROMATIN STRUCTURE
AND FUNCTION
• Some Chromatin Structures Can Be Directly
Inherited
Some Chromatin Structures Can Be Directly Inherited
In this model, some of the histone with modifications are distributed to each sister
chromosome after DNA replication.
CHROMATIN STRUCTURE
AND FUNCTION
• Experiments with Frog Embryos Suggest that both
Activating and Repressive Chromatin Structures
Can Be Inherited Epigenetically
Experiments with Frog Embryos Suggest that both Activating and
Repressive Chromatin Structures Can Be Inherited Epigenetically
The well-characterized MyoD gene
encoded a master transcriptional
regulatory protein for muscular gene
promoters, including itself.
The active chromatin surrounding the
MyoD promoter contains the variant
histone H3.3 in a Lys 4 methylate form.
By injecting excess mRNA encoding
normal H3.3 or mutant H3.3,
experiments provide evidence of a
gene-activating chromatin state through
epigenetic control.
CHROMATIN STRUCTURE
AND FUNCTION
• Chromatin Structures Are Important for Eukaryotic
Chromosome Function
In a complex multicellular organism, the cells in different
lineages must specialize by changing the accessibility and
activity of many hundreds of genes. Each cell holds a record of
its past developmental history in the regulatory circuits that
control its many genes. That record, it seems, is partly stored in
the structure of the chromatin.
THE GLOBAL STRUCTURE OF
CHROMOSOMES
• Chromosomes Are Folded into Large Loops of
Chromatin
Chromosomes Are Folded into Large Loops of Chromatin
Lampbrush chromosomes in an
amphibian oocyte.
•A special form of chromosomes,
found in oocytes of most animals
(except mammals).
•Seen at early meiosis stage due to
an active transcription of many
genes.
•DNA are organized into a series
of large chromatin loops,
emanating from a chromosomal
axis.
Early in oocyte differentiation,
each chromosome replicates to
begin meiosis, and chromosomes
form this highly extended structure
containing a total of four replicated
chromatids.
Chromosomes Are Folded into Large Loops of Chromatin
•The genes on the loop are actively
expressed.
•The genes condensed close to axis
are not expressed.
•A given loop always contains the
same DNA sequences
Chromosome axis
Chromosomes Are Folded into Large Loops of Chromatin
Chromosome conformation capture (3C) can determine the
position of loops in interphase chromosomes.
Other methods developed based on 3C
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Chromosomes Are Folded into Large Loops of Chromatin
Each loop contains 50-200k bp DNA
THE GLOBAL STRUCTURE OF
CHROMOSOMES
• Polytene Chromosomes Are Uniquely Useful for
Visualizing Chromatin Structures
Polytene Chromosomes Are Uniquely Useful for Visualizing
Chromatin Structures
•Some insect cells, for example salivary
gland cells in fly larvae have polytene
chromosomes.
•All homologous chromosome copies are
held side by side, linked together by
chromocenter.
•Each polytene chromosome contains a
thousand identical DNA sequences with
dark and light bands with different degree
of chromatin condensation.
THE GLOBAL STRUCTURE OF
CHROMOSOMES
• There Are Multiple Forms of Chromatin
• Chromatin Loops Decondense When the Genes
Within Them Are Expressed
Chromatin Loops Decondense When the Genes Within Them Are
Expressed
H3Lys 9 di-methylation
H3 Lys 9 acetylation (green)
chromocenter.
Pericentric
heterochromatin.
Marks active genes
Decondensed portion of
chromosomes is
undergoing RNA
synthesis.
Chromatin Loops Decondense When the Genes Within Them Are
Expressed
Two homologous chromosome are not in
general co-located.
Chromatin Loops Decondense When the Genes Within Them Are
Expressed
•Gene-rich regions are visualized with a
FISH probe that hybridizes to the Alu
interspersed repeat, which is present in more
than a million copies in the human genome.
•These sequences cluster in gene-rich
regions.
•Heterochromatin regions often are
associated with the nuclear lamina.
Alu signals (green)
Chromatin Loops Decondense When the Genes Within Them Are
Expressed
THE GLOBAL STRUCTURE OF
CHROMOSOMES
• Chromatin Can Move to Specific Sites Within the
Nucleus to Alter Gene Expression
Chromatin Can Move to Specific Sites Within the Nucleus to Alter
Gene Expression
•The position of a gene in the
interior of the nucleus
changes when it becomes
highly expressed.
THE GLOBAL STRUCTURE OF
CHROMOSOMES
• Networks of Macromolecules Form a Set of
Distinct Biochemical Environments inside the
Nucleus
Nuclear subcompartments: Nucleolus
rRNA expression and ribosome assembly and maturation as well as many other specialized reactions.
Networks of Macromolecules Form a Set of Distinct Biochemical
Environments inside the Nucleus
Cajal body: 0.3-1 µm in nucleus, may
related to RNA-related metabolic
processes.
These structures without membrane are composed of selected protein and RNA that
bind together to create networks for multiple biological functions.
Networks of Macromolecules Form a Set of Distinct Biochemical
Environments inside the Nucleus
THE GLOBAL STRUCTURE OF
CHROMOSOMES
• Mitotic Chromosomes Are Especially Highly
Condensed
Mitotic Chromosomes Are Especially Highly Condensed