The Nucleus: Chromosome Structure

1
The Nucleus Chromosome Structure Chromatin

• The Nucleus
• - has inner outer membranes nuclear envelope
• - perforated by nuclear pores elaborate gates
  that
• control transport of small and large molecules
  to/from nucleus
• - connected to endoplasmic reticulum
• Nuclear Lamina
• - composed of proteins called nuclear lamins
• (A, B, C)
• - form intermediate filaments
• - possible attachment point for chromatin
  nuclear RNAs

2
Nucleus the components

• - endoplasmic reticulum (ER) shares nuclear
  membrane (perinuclear space) site where
  ribosomes inject newly-made proteins that are
  destined to be incorporated into membranes
  (e.g., cell surface) or be secreted
• - nuclear pore complex (NPC) highly complex
  proteinaceous pore that regulates the entry/exit
  of proteins, and exit of mRNA
• nuclear lamina provides structural rigidity to
  nucleus and possible sites of attachment for
  chromatin, etc.
• nuclear matrix diffuse nuclear scaffold
  consisting of proteins (e.g. actin)
• - nucleoplasm chromatin/ chromosome-containing
  region
• nucleolus a ribosome-producing sub-compartment
  of the nucleus

3

• The Nucleolus
• - specialized region of the nucleus with densely
  stained chromatin in electron micrographs
• - site of rRNA genes of ribosome assembly
• - rRNA transcripts
• about 80 proteins (from the cytoplasm)
• many RNAs
• small and large ribosome subunits (40S, 60S) are
  assembled and exported to the cytoplasm
  separately
• Chromatin
• - chromatin is the protein DNA complex form
  in which interphase chromosomes exist in the
  nucleus
• (i.e., the physical state of chromosomes during
  interphase)
• - But, during metaphase, chromatin (which is a
  relatively loose spread-out structure in
  interphase nuclei) becomes extensively folded or
  condenses to form the familiar metaphase
  chromosomes

4
Overview of Chromatin Structure
5
Why Chromatin?
Each eukaryotic chromosome contains a continuous
DNA chain along its entire length can be up to
10cm long for human chromosomes ( 3.5 x 108
bp). Therefore the DNA must be organized
in very elaborate ways in order to
package or condense it into a manageable
size, so that it will fit into the
nucleus- accomplished by different levels
of chromatin packaging.
6
Different Levels of Chromatin Organization
Nucleosomes package DNA into the 11(or 10) nm
chromatin fiber-fundamental unit of chromatin
(beads on a string) nuclesomes consist of
histones. 30 nm chromatin fiber or solenoid
created by coiled 11 nm fiber- interphase
chromatin exists as highly condensed solenoid
with inter-spersed extended loops containing
transcribed genes. Metaphase chromosomes
looping/coiling of solenoid DNA into highly
compacted, transcriptionally silent, form of
chromatin- occurs during mitosis.
7
Levels of Chromatin Organization
nucleosome (11 nm Fiber)
30 nm fiber physiologically important structure
DNA
Loops
compacted metaphase chromosome
Loops
?
These are only models at this stage
The loops of 30 nm fibers contain 20,000
100,000 bp (i.e., about the sizes of genes) Note
that other DNA and chromatin-binding proteins
(besides histones) are also involved in
organizing chromatin.
8
Different Levels of Chromatin Organization
Nucleosomes package DNA into the 11 (or 10) nm
chromatin fiber-fundamental unit of chromatin
(beads on a string) nuclesomes consist of
histones. 30 nm chromatin fibre or solenoid
created by coiled 11 nm fiber- interphase
chromatin exists as highly condensed solenoid
with interspersed extended loops containing
transcribed genes. Metaphase chromosomes
further looping/coiling of solenoid DNA into
highly compacted, transcriptionally silent, form
of chromatin- occurs during mitosis.
9
Histones (Components of the nucleosome)
The major protein component of chromatin. -
small, very basic (lys, arg) proteins (to
neutralize the highly charged DNA) - assembly of
histones with DNA requires, a molecular
chaperone, nucleoplasmin Five Types of
Histones H1 Clamp for DNA wrapped around
nucleosome H2A H2B H3 H4 H4 Is very highly
conserved between species (98 identical between
cows and peas) 1 change in 600 million
years H3 Is also very conserved (97 identical)
Found in 2 copies of each in the nucleosome The
DNA is then wrapped around the protein core made
of these proteins -- together they form an octamer
Core Histones
10
HISTONESare highly conserved,small, basic
proteins
H1
Linker histone
H2A
H2B
helix

• Histone acetylation
• is a reversible modification
• of lysines in the N-termini
• of the core histones.
• Result
• reduced binding to DNA
• destabilization of chromatin

Core histones
variable
H3
H4
conserved
N
11
Experimental evidence for nucleosomal packaging
of DNA into 11 nm chromatin fibre
12
Nucleosomal packaging of DNA
In the presence of Histone H1, 175-200 bp DNA is
associated with the nucleosome- but, only 146 bp
is wrapped around the octamer (i.e. if H1 is
removed, 146 bp is observed in nuclease
digests) Histone H1- helps clamp the DNA onto
the nucleosome and participates in higher-order
chromatin folding
13
Histones
dsDNA wraps around the nucleosome two times.
dsDNA
14
Compaction of DNA in Nucleosomes
Each nucleosome serves to compact DNA Compaction
ratio 68 nm / 10 nm 7 (DNA 200 bp /
nucleosome) Length of fully extended 200 bp of
DNA 68 nm However, total condensation of
metaphase chromosomes 1 m ? 100 ?m (104 packing
ratio) - therefore nucleosomes only play a small
part
15
Histone modifications

• Histones are also modified in order to regulate
• chromosome structure compaction
• gene activity
• The main types of modifications are
• Phosphorylation of serines
• Methylation of lysines
• c) Acetylation of lysines neutralizes charges

16
Different Levels of Chromatin Organization
Nucleosomes package DNA into the 11 (or 10) nm
chromatin fiber-fundamental unit of chromatin
(beads on a string) nuclesomes consist of
histones. 30 nm chromatin fibre or solenoid
created by coiled 11 nm fiber- interphase
chromatin exists as highly condensed solenoid
with interspersed extended loops containing
transcribed genes. Metaphase chromosomes
further looping/coiling of solenoid DNA into
highly compacted, transcriptionally silent, form
of chromatin- occurs during mitosis.
17
11 nm fiber is coiled into 30 nm Solenoid-
requires histone H1
Schematic of formation of 30nm chromatin fiber
DNA
Histones
18
Chromatinfibers
11 nm (beads)
30 nm chromatin fiber
highly acetylated core histones (especially H3
and H4)
charged N termini (bind DNA on
neighboring nucleosomes)
Short extended regions can exist between
solenoids
Solenoid

• HIGH level of histone H1
• pull nucleosomes into solenoid

• Reduced level of histone H1

• NO gene transcription in solenoid

• Gene transcription possible

19
Interphase chromosomes mosaics of
solenoids/compacted solenoids and extended
chromatin loops I. Amphibian Lampbrush
Chromosomes consist of elaborately folded 30 nm
fibers and extended loops containing expressed
genes
20
II. Insect Polytene Chromo-somes consist of
gt1000 precisely aligned chroma-tids-contain
condensed and de-condensed regions
21
Heterochromatin versus Euchromatin
Interphase chromosomes consists of two types of
chromatin Euchromatin less condensed 10
consists of active genes Heterochromatin highly
condensed and compacted throughout cell cycle
contains low density of transcribed genes and can
cause gene silencing
22
Heterochromatic regions of insect polytene
chromosomes coalesce into chromocentre
23
Different Levels of Chromatin Organization
Nucleosomes package DNA into the 11(or 10) nm
chromatin fiber-fundamental unit of chromatin
(beads on a string) nuclesomes consist of
histones. 30 nm chromatin fibre or solenoid
created by coiled 11 nm fiber- interphase
chromatin exists as highly condensed solenoid
with interspersed extended loops containing
transcribed genes. Metaphase chromosomes
further looping/coiling of solenoid DNA into
highly compacted, transcriptionally silent, form
of chromatin-occurs during mitosis.
24
Consists of loops/coils of chromatin attached to
a protein network or scaffold
Typical Metaphase Chromosome
-this structure allows the easy separation of
sister chroma-tids and protects the fragile DNA
molecules during mitosis
Primary Constriction contains centromere
FIG. 1
25
The Metaphase Chromosome
-formed by looping and coiling of condensed
chromatin assoc. with H1 phosphorylation
26

• Topoisomerase II is associated with loops (30nm)
  suggests individual loops may be autonomously
  supercoiled
• Allows for independent regulation of
  supercoiling chromatin structure for specific
  regions
• Even higher-order organization is required than
  the higher-order loops for metaphase condensation
• Protein scaffolds appear to be involved
• Regulated (?) by Histone H1 phosphorylation
• - 5 serine residues phosphorylated during
  condensation

27
Summary of Principal Types of Histone
Modifications and Their Roles in Chromatin
Packaging occur in N-terminal tails
1. Acetylation of lysines carried out by
histone acetyl transferases (HATs) acetyl groups
removed by histone deacetylases (HDACs)-
acetylation neutralizes lysine charges and
loosens histone/DNA interactions - promotes
chromatin decondensation 2. Methylation of
lysines carried out by histone methyl
transferases- promotes formation of highly
compacted chromatin e.g. heterochromatin 3.
Phosphorylation of serines carried out by
kinases phosphatases remove phosphates
phosphorylation (especially of histone H1) helps
to pack nucleosomes together and thus tends to
promote higher levels of chromatin compaction
e.g. formation of metaphase chromosomes.
28
Summary
2 nm DNA double helix
11 nm Nucleosome (11 nm fiber)
Interphase chromatin genes ex-pressed in
extended loops (11nm fibers)
30 nm 30 nm Fiber
300 nm Loops I
Condensation into meta-phase chromosomes
700 nm Loops II
1400 nm chromosome
Figure 8.30 Alberts, Mol. Biol. Cell.