Discuss the functional organization of

eukaryotic chromosomes within the interphase

nucleus
Introduction
In the cell, folding drives function. The packaging of DNA into chromosomes
allows a vast amount of information to be stored in a compact environment
whilst allowing rapid access to the information. The distinct morphological
states and folding patterns of DNA allows extra information to be stored in the
form of chemical modification and variations in gene expression.

Chromosomes are packaged in a hierarchical manner in the

interphase nucleus

1. DNA associate with histones to form beads on a string structures called

nucleosomes
2. Nucleosome core particles consist of 146bp of DNA and an octameric
histone core
a. Containing two copies of each core histone (H2A, H2B, H3, H4)
b. Adjacent nucleosomes are connected by linker DNA
c. Associations of linker histone H1 with the linker DNA and the
nucleosome core allows formation of higher order structures
3. Nucleosomes (beads on strings structures) are packed together
(condensed) to form chromatin fiber
4. Chromatin fiber organises into chromosomes upon cell division
a. Chromatin condensation to chromosomes is mediated by
condensins
5. During interphase, DNA exists in its looser chromatin form

Two distinct morphological states of chromatin are seen in

the interphase nucleus

1. Light-microscope studies distinguished two types of chromatin in the

interphase nuclei
a. A highly condensed form: heterochromatin
i.
10% of the genome is packaged this way
ii.
DNA in heterochromatin contains few genes
iii.
When euchromatic regions are converted to a
heterochromatic state, their genes are generally switched
off
iv.
There are different extent of this compactness

2.

3.

4.

5.

v.
Resistant to gene expression
b. Less condensed form: euchromatin
Cells contain two types of heterochromatin
a. Constitutive heterochromatin contains DNA sequences that are
never transcribed
i.
Such as the satellite sequences present at centromeres
b. Facultative heterochromatin contains sequences that are not
transcribed in the cell being examined, but are transcribed in
other cell types
Formation of euchromatin and heterochromatin involves the
post-translational modifications of histone tails
a. These modifications produce altered binding surfaces for effector
proteins that influence chromatin structure
b. Eg. histone H3 that is methylated at lysine residue 9 (H3K9me) is
bound by the HP1 heterochromatin protein, which brings with it
other proteins that act to compact and silence the DNA
c. Eg. histone H3K9 acetylation is an activating modification
i.
Brings in chromatin remodelling enzymes that open
chromatin structure and enable access of transcription
machinery to DNA
d. The number of chromatin types varies according to the organism
studied
i.
Drosophila polytene chromosomes can exist in 3 major
types of repressive chromatin and two major types of
chromatin on actively transcribed genes
Much of the heterochromatin is localised to the periphery of the
nucleus
a. Possibly because once of the principal proteins associated with
heterochromatin binds to a protein of the inner nuclear
membrane
Euchromatin is distributed throughout the nucleus

X chromosome inactivation provides an example of the role

of heterochromatin in gene expression
1. Despite the difference in the number of X chromosomes, the female
and male cell contain equal amounts of proteins encoded by X
chromosome genes
2. A dosage compensation mechanism exists in which one of the two X
chromosomes in female cells is inactivated by being converted to
heterochromatin early in development
3. Therefore only one copy of the X chromosome is available for
transcription in either female or male cells

4. Mechanism of X chromosome inactivation is not fully understood, but

may involve the action of regulatory RNA that coats the inactive X
chromosome to induce conversion to heterochromatin

Recent research using in situ Hi-C found that loops and other
genome folding patterns are an essential part of genetic
regulation

1. Contact map shows which DNA parts are touching each other (thus
suggesting looping)
2. The human genome is subpartitioned into 10,000 loops
a. Each loop is made by taking two pieces of DNA which are far
apart and sticking them together
b. On average 200,000 base pairs long
3. When a gene is at one end of a loop, it is usually activated
a. Gene can be activated by enhancers far away
4. CTCF protein enable the formation of these loops
a. Only binds to particular motif
b. Motif appears on both ends of the loop
c. CTCF pointing direction depends on which of the two DNA
strands CTCF bind to
d. Loops only form when CTCF motifs point towards each other
5. Inside the loop, DNA form a condensed fold, which we call a contact
domain
6. Domains can also be created without loops
7. Typical contact domain is about 200,000 base pairs long
8. The genome is divided into 9000 contact domains
a. Many of which are demarcated by loops

9. All the DNA inside a domain tend to have the same chemical mark
a. Eg. H3K36 trimethylation - on
b. Eg. H3K27 trimethylation - supress
10. Domains with similar histone marks tend to be located in the same
place inside the nucleus

Conclusion
DNA, in forming chromosomes, folds from beads-on-a-string structures
called nucleosomes to chromatin fibers that further condenses. This
organisation allows information to be stored in a small space. The accessibility
of such information is then determined by the morphological state to which
the chromatin conform. Within the nucleus, the DNA code is further
segmented into domains of loops which share similar chemical marks,
allowing related genes to be activated or deactivated rapidly and
simultaneously.

Research
Internal Organization of the Nucleus
https://www.ncbi.nlm.nih.gov/books/NBK9915/
A 3D Map of the Human Genome at Kilobase Resolution Reveals Principles of
Chromatin Looping
http://www.cell.com/cell/abstract/S0092-8674(14)01497-4?_returnURL=http%
3A%2F%2Flinkinghub.elsevier.com%2Fretrieve%2Fpii%2FS0092867414014974%
3Fshowall%3Dtrue

Molecular biology of the cell

Chapter 4: DNA, Chromosomes and genomes

Heterochromatin is highly organised and restricts gene

expression
1. Light-microscope studies distinguished two types of chromatin in the
interphase nuclei
a. A highly condensed form: heterochromatin
i.
10% of the genome is packaged this way
ii.
DNA in heterochromatin contains few genes
iii.
When euchromatic regions are converted to a
heterochromatic state, their genes are generally switched
off
iv.
There are different extent of this compactness
v.
Resistant to gene expression
b. Less condensed form: euchromatin
2. Cells contain two types of heterochromatin
a. Constitutive heterochromatin contains DNA sequences that are
never transcribed
i.
Such as the satellite sequences present at centromeres
b. Facultative heterochromatin contains sequences that are not
transcribed in the cell being examined, but are transcribed in
other cell types
3. Formation of euchromatin and heterochromatin involves the
post-translational modifications of histone tails
a. These modifications produce altered binding surfaces for effector
proteins that influence chromatin structure

b. Eg. histone H3 that is methylated at lysine residue 9 (H3K9me) is

bound by the HP1 heterochromatin protein, which brings with it
other proteins that act to compact and silence the DNA
c. Eg. histone H3K9 acetylation is an activating modification
i.
Brings in chromatin remodelling enzymes that open
chromatin structure and enable access of transcription
machinery to DNA
4. Much of the heterochromatin is localised to the periphery of the
nucleus
a. Possibly because once of the principal proteins associated with
heterochromatin binds to a protein of the inner nuclear
membrane
5. Euchromatin is distributed throughout the nucleus

The heterochromatic state is self-propagating

1. Through chromosome breakage and rejoining, a piece of chromosome
that is normally euchromatic can be translocated into the
neighbourhood of heterochromatin
a. Can occur through genetic accident or experimental artifice
2. This often causes silencing of the normally active genes
3. This phenomenon is called the p
osition effect
a. Spreading of heterochromatic state into the originally
euchromatic region

X chromosome inactivation provides an example of the role

of heterochromatin in gene expression
5. Despite the difference in the number of X chromosomes, the female
and male cell contain equal amounts of proteins encoded by X
chromosome genes
6. A dosage compensation mechanism exists in which one of the two X
chromosomes in female cells is inactivated by being converted to
heterochromatin early in development
7. Therefore only one copy of the X chromosome is available for
transcription in either female or male cells
8. Mechanism of X chromosome inactivation is not fully understood, but
may involve the action of regulatory RNA that coats the inactive X
chromosome to induce conversion to heterochromatin

Chromosomes are arranged in an organised fashion and

divided into discrete functional domains that regulates gene
expression
1. Nonrandom distribution of chromatin within the interphase nucleus
was first suggested by C.Rabl, who proposed that each chromosome
occupies a distinct territory, with centromeres and telomeres attached
to opposite sides of the nuclear envelope
2. This model of chromosome organisation was confirmed by studies of
polytene chromosomes in Drosophila salivary glands
3. Chromosomes are closely associated with the nuclear envelope at many
sites, with their centromeres and telomeres clustered at opposite poles
4. Individual chromosomes occupy distinct territories within the nuclei of
mammalian cells
a. Actively transcribed genes are localised to the periphery of these
territories, adjacent to channels separating the chromosomes
b. Newly transcribed RNAs are thought to be released into these
channels between chromosomes, where RNA processing takes
place

Chromatin in interphase nuclei is organised into looped

domains containing approximately 50 to 100kb of DNA
1. Eg. looped-domain organization of highly transcribed chromosomes of
amphibian oocytes
2. Actively transcribed regions of DNA can be visualised as extended loops
of decondensed chromatin

The effects of chromosome organisation on gene expression

have been demonstrated by a variety of experiments showing
that the position of a gene in chromosomal DNA affects the
level at which the gene is expressed
1. The transcriptional activity of genes introduced into transgenic mice
depends on their sites of integration in the mouse genome
2. This effect of chromosomal position on gene expression can be
alleviated by sequences known as locus control regions
a. Result in a high level of expression of the introduced genes
irrespective of their site of integration
b. Locus control regions only stimulate transfected genes that are
integrated into chromosomal DNA; they do not affect the
expression of unintegrated plasmid DNAs in transient assays

c. Rather than affecting individual promoters, locus control regions

activate large chromosome domains by inducing long-range
alterations in chromatin structure

Separation between chromosomal domains is maintained by

boundary sequences or insulator elements
1. Prevent the chromatin structure of one domain from spreading to its
neighbours
2. Insulators act as barriers that prevent enhancers in one domain from
acting on promoters located in an adjacent domain
3. Like locus control regions, insulator function only in the context of
chromosomal DNA
4. This suggest that they regulate higher-order chromatin structure

Activities such as DNA replication and pre-mRNA processing

may be localised to discrete subnuclear structures or domains
1. Nuclei of mammalian cells contain clustered sites of DNA replication in
which replication of multiple DNA molecules takes place
2. This is demonstrated by immunofluorescent staining with antibodies
against snRNPs and splicing factors
3. Components of the splicing machinery are concentrated in discrete
subnuclear structural domains
a. Components of the splicing apparatus are concentrated in 20 to
50 discrete structures called nuclear speckles
b. They are storage sites of splicing components

Folding drives function, epigenetics is origami

11. Recent research using in situ Hi-C found that loops and other genome
folding patterns are an essential part of genetic regulation
12. Contact map shows which DNA parts are touching each other (thus
suggesting looping)
13. The human genome is subpartitioned into 10,000 loops
a. Each loop is made by taking two pieces of DNA which are far
apart and sticking them together
b. On average 200,000 base pairs long
14. When a gene is at one end of a loop, it is usually activated
a. Gene can be activated by enhancers far away
15. CTCF protein enable the formation of these loops
a. Only binds to particular motif
b. Motif appears on both ends of the loop
c. CTCF pointing direction depends on which of the two DNA
strands CTCF bind to
d. Loops only form when CTCF motifs point towards each other
16. Inside the loop, DNA form a condensed fold, which we call a contact
domain
17. Domains can also be created without loops
18. Typical contact domain is about 200,000 base pairs long
19. The genome is divided into 9000 contact domains
a. Many of which are demarcated by loops
20.All the DNA inside a domain tend to have the same chemical mark
a. Eg. H3K36 trimethylation - on
b. Eg. H3K27 trimethylation - repress
21. Domains with similar marks tend to be located in the same place inside
the nucleus

22. Domains are divided into nuclear subcompartments

23. Domains are segregated into at least 6 subcompartments, each of which
is associated with particular chromatin marks

From the lectures:

Chromosomes are packaged in a hierarchical manner in the

interphase nucleus
6. DNA associate with histones to form beads on a string structures called
nucleosomes
7. Nucleosome core particles consist of 146bp of DNA and an octameric
histone core
a. Containing two copies of each core histone (H2A, H2B, H3, H4)
b. Adjacent nucleosomes are connected by linker DNA
c. Associations of linker histone H1 with the linker DNA and the
nucleosome core allows formation of higher order structures
8. Nucleosomes (beads on strings structures) are packed together
(condensed) to form chromatin fiber
9. Chromatin fiber organises into chromosomes upon cell division
a. Chromatin condensation to chromosomes is mediated by
condensins
10. During interphase, DNA exists in its looser chromatin form