CHROMATIN

CHROMATIN: EUCHROMATIN AND HETEROCHROMATIN

Since our early days at school we have learnt that DNA is the heredity
molecule, it lies within the cell nucleus and contains all the information
that is passed on from parent to daughter cells. However, this DNA is not
loosely arranged in the cell nucleus. In fact, it is bound with nuclear
protein called Histones, and is then organized into a firm, compact
structure called CHROMATIN.

There are specific reasons for packaging DNA into Chromatin with the
help of histone proteins:

1. To protect the DNA from damage or enzymatic attack.

2. To pack the DNA by folding it several times so that the long DNA
can be made compact and accommodated into the tiny cell nucleus.

3. To allow the DNA to be accessed by enzymes for the purpose of

replication or duplication of parent DNA for provision to the
daughter cells.

4. To allow the DNA to be regulated in its function by enzymes, that

control transcription for Protein synthesis. Thus chromatin packing
regulates how and when a gene should express itself.

5. To enable the DNA to be conveniently separated during mitosis so

that each daughter cell gets the correct amount of DNA.

Chromatin is therefore, a complex of macromolecules such as DNA, RNA

and proteins, which is assembled together. The primary protein
components of chromatin are histones that compact the DNA. Chromatin
is only found in eukaryotic cells (more differentiated cells, with well-
defined nuclei). Prokaryotic cells have a different organization of their
DNA, in which the strand of DNA is localized within the nucleoid region
and is termed the prokaryotic chromosome or is called genophore.

The prime reason for packing the DNA into a chromatin assembly is to fit
the molecule in the much smaller volume of the cell nucleus. To
understand this we must consider that an average human cell contains a
very large DNA molecule comprising of about 6.4 billion base pairs of DNA
divided among 46 chromosomes. A single, continuous DNA molecule is
organized into a chromosome; the larger the chromosome, the longer the
DNA it contains. We know that each base pair is about 0.34 nm in length,
hence 6 billion base pairs would constitute a DNA molecule that would be
2 meter long. Now, how can a 2 meter long strand of DNA fit into a cell
nucleus only 10 micrometers (1x 10-5 m) in diameter? The firm packing
and coiling of the DNA reduces its size for this purpose.

Secondly the microenvironment in the cell is replete with nucleases, ions

and other small molecules that would unnecessarily bind to the DNA or
cleave it. The DNA therefore requires a protective shield and at the same
time, this molecule should still be able to carry out its functions. For this
purpose, the DNA should be accessible to specific enzymes for replication.
It should also be available to small transcription factors and enzymes for
transcription. Chromatin is that molecular assembly in which the DNA is
accessible to enzymes and regulatory proteins and yet protected from any
change in the cell. Moreover, if we just try to imagine an incredibly long
string of DNA which is loose within the cell, it would get knotted and
entangled within itself, as well with other molecules in the cell. However,
the organization of the DNA with its nucleoproteins in the form of
chromatin prevents any untoward interaction with the DNA and yet
enables the molecule to carry out its functions smoothly.

The structure of chromatin attains its firm degree of binding from the fact
that the nucleoprotein histones are positively charged, strongly basic
proteins, while DNA due its phosphate groups, is more negatively
charged. The two molecules therefore bind with electrostatic interaction
and other non-covalent forces. The chromatin structure also depends on
several factors. The overall assembly depends on the stage of the cell
cycle. During interphase, the chromatin is structurally loose to allow
access to RNA and DNA polymerases that transcribe and replicate the
DNA. When the DNA does not have to replicate or be transcribed, the
chromatin is more compact and condensed. This enables formation of
chromosomes during phases where the cell has to divide. Hence in a
compact, well-organized structure the DNA is able to move more
systematically to each daughter cell. If the DNA were not compacted and
assembled into distinct chromatin-derived chromosomes, can you imagine
how long, loose strands of DNA could be separated from parent to
progeny cells? Much of the DNA would be entangled into a non-functional,
knotted mass.

According to biochemists, the operational definition of chromatin is the

DNA, protein, RNA complex extracted from eukaryotic lysed interphase
nuclei. Chromatin is the product formed from the combination of DNA and
other types of protein.

DNA, organized as chromatin, is found in two forms along the length of

the chromosomes that reflect the level of activity of the cell:

1. Euchromatin and

2. Heterochromatin

These two forms are distinguished in a cytological manner dealing with

how intensely each form is stained. The euchromatin is less intensely
stained as compared to heterochromatin.

EUCHROMATIN:

When certain dyes, like carminic acetic acid, Giemsa or orceine are used
to stain certain chromosomes, certain regions of the chromosomes, show
up with differences in their staining intensity. The resulting pattern shows
alternately light and darkly stained regions, which are characteristic for
the particular chromosome of a species. During interphase, the
chromosomes are not distinctly assembled. After mitosis has been
completed, most of the chromatin which is in highly compacted mitotic
chromosome form, returns to its diffuse interphase condition.
Approximately 10 percent of the chromatin, however, generally remains
in a condensed, compacted form throughout interphase. This compact,
densely stained chromatin is seen at the periphery of the nucleus.
Chromatin that remains compacted during interphase is called
heterochromatin to distinguish it from euchromatin, which returns to a
dispersed state. The intensity of the nuclear staining becomes feebler
and less uniform than that of the chromosomes seen at metaphase or
anaphase. Heitz in 1929, observed the distinct light and dark stained
zones and he was able to distinguish between heterochromatin and
euchromatin. All the intensely stained domains were termed
heterochromatin while euchromatin referred to the diffuse or lightly
stained regions. Heterochromatin is usually spread over the whole nucleus
and has a granular appearance. It is known today that the
heterochromatic domains are those where the DNA is tightly packed
(strongly condensed) which is the reason for their more intense staining.
The euchromatic domains are less tightly packed and hence pick up less
stain.

Most of the genome within the cell nucleus, that is, 92% of the human
genome is euchromatic. Euchromatin is a lightly packed form of chromatin
(DNA, RNA and protein) that is enriched in genes, and is often, but not
continuously under active transcription. Euchromatin is dispersed and not
readily stainable. It is prevalent in cells that are active in the transcription
of many of their genes while heterochromatin is most abundant in cells
that are genetically less active or not active. Euchromatin comprises the
bands which appear as light-colored bands when stained with Giemsa
stain (G-banding technique) and observed under an optical microscope. In
contrast to these light bands the heterochromatin stains darkly.

Electron microscopic observations have revealed that the basic structure

of euchromatin is an elongated, open, 10 nm microfibril. Though it is
lightly packed in the form of DNA, RNA, and protein, it is definitely rich in
genes and is usually under active transcription. The standard structure of
euchromatin is unfolded, elongated, about the size of a 10 nanometer
microfibril. This minute chromatin functions in the transcription of DNA to
mRNA products. The gene regulatory proteins, including the RNA
polymerase complexes, are able to bind with the DNA sequence due to
the unfolded, non-compact structure of the euchromatin. When these
substances are already bound, the transcription process begins. The
activities of the euchromatin aid in cell survival

HETEROCHROMATIN

In prokaryotes, euchromatin is the only form of chromatin present, there

is no heterochromatin. This suggests that the heterochromatin structure
evolved later along with the nucleus, possibly as a mechanism to manage
the increasing genome size. Heterochromatin thus appears as small,
darkly staining, irregular particles scattered throughout the nucleus or
accumulated adjacent to the nuclear envelope.

On the other hand, heterochromatin is a tightly packed form of DNA. It is

commonly found on the peripheral areas of the nucleus. According to
some studies, there are probably two or more states of heterochromatin.
Inactive satellite sequences are the main constituents of heterochromatin.
Heterochromatin is responsible for gene regulation and protection of
chromosomal integrity. These roles are made possible because of the
dense DNA packing. When two daughter cells are divided from a single
parent cell, heterochromatin is usually inherited, which means that the
newly cloned heterochromatin contains the same DNA regions which
results in epigenetic inheritance and leads to the development of
different levels of gene expression.

Heterochromatin is termed as ‘genetically inert’ indicating that it has little

genetic activity. When a radioactively labeled RNA precursor such as 3H-
uridine is given to cells that are then fixed, sectioned and
autoradiographed, the clumps of heterochromatin remain largely
unlabeled, indicating that they have relatively little transcriptional activity
and hence take up or use very little 3H-uridine. The state of a particular
region of the genome, whether it is euchromatic or heterochromatic, is
stably inherited from one cell generation to the next. Heterochromatin is
said to be late-replicating; it begins replication much later than
euchromatin in the S-phase and continues replicating long after much of
the euchromatin has already replicated its sequences.

Heterochromatin is divided into two classes:

1. Constitutive heterochromatin

2. Facultative heterochromatin

Constitutive heterochromatin:

Constitutive heterochromatin remains in the compacted state in all cells at

all times and, thus, represents DNA that is permanently silenced. In
mammalian cells, the bulk of the constitutive heterochromatin is found in
the region that appears near the ends of the telomeres and centromere
(flanking sequences) of each chromosome and in a few other sites, such
as the distal arm of the Y chromosome in male mammals. Hence it is a
major part of the constitution or structure of the chromosomes and so it
is referred to as constitutive heterochromatin. The DNA of constitutive
heterochromatin consists primarily of repeated sequences, often non-
coding sequences. These regions have relatively few genes. In fact, when
genes that are normally active move into a position adjacent to
heterochromatin (having changed position as the result of transposition or
translocation), they tend to become transcriptionally silenced, a
phenomenon known as position effect variegation. It is thought that
heterochromatin contains components which regulate the expression of
neighbouring genes. The influence of such chromatin can spread outward
a certain distance, affecting nearby genes. The spread of heterochromatin
along the chromosome is apparently blocked by specialized barrier
sequences (boundary elements) in the genome. Constitutive
heterochromatin remains condensed and does not de-condense to go
back to the euchromatin state. It also serves to inhibit genetic
recombination between homologous repetitive sequences. This type of
recombination can lead to DNA duplications and deletions.

Facultative heterochromatin:

Unlike the constitutive variety, facultative heterochromatin is chromatin

that has been specifically inactivated during certain phases of an
organism’s life or in certain types of differentiated cells. It thus appears to
be functional in nature, perhaps condensed or heterochromatinized for a
specific purpose. At given times therefore it de-condenses and reverses to
its euchromatin state.

An example of facultative heterochromatin can be seen by comparing cells

of a female mammal to those of a male. The cells of males have a Y
chromosome and a much larger X chromosome. Because the X and Y
chromosomes have only a few genes in common, males have a single
copy of most genes that are carried on the sex chromosomes. Although
cells of females contain two X chromosomes, only one of them is
transcriptionally active. The other X chromosome remains condensed as a
heterochromatic clump, called a Barr body after the researcher, M.L. Barr,
who discovered it in cells of the female feline species, in 1949. Formation
of a Barr body ensures that the cells of both males and females have the
same number of active X chromosomes and thus synthesize equivalent
amounts of the products encoded by X-linked genes. This phenomenon
was called Dosage Compensation.

The British geneticist Mary Lyon studied X chromosome inactivation and

based on her findings of the inheritance of coat color in mice, proposed
the following theory in 1969, which came to be known as the Lyon
Hypothesis.

1. Heterochromatinization of the X chromosome in female

mammals occurs during early embryonic development and leads to
the inactivation of the genes on that chromosome.

2. Heterochromatinization in the embryo is a random process in the

sense that the paternally derived X chromosome and the maternally
derived X chromosome stand an equal chance of becoming inactivated
in any given cell.

Consequently, at the time of inactivation, the paternal X can be

inactivated in one cell of the embryo, and the maternal X can be
inactivated in a neighboring cell. Once an X chromosome has been
inactivated, its heterochromatic state is transmitted through many cell
divisions, so that the same X chromosome is inactive in all the
descendants of that particular cell.

3. Reactivation of the heterochromatinized X chromosome occurs in

germ cells prior to the onset of meiosis. Consequently, both X
chromosomes are active during oogenesis, and all of the gametes receive
an euchromatic X chromosome.

The Lyon hypothesis was soon confirmed, because although maternally

and paternally derived X chromosomes may contain different alleles for
the same trait, adult females are in a sense genetic mosaics, where
different alleles function in different cells. X-chromosome mosaicism is
reflected in the patchwork coloration of the fur of some mammals.
The random inactivation of X chromosomes discussed here, which occurs
after the embryo implants in the uterus, is actually the second wave of X
chromosome inactivation to occur in the embryo. The first wave, which
occurs very early in development, is not random but rather leads only to
the inactivation of X chromosomes that had been donated by the father.
This early inactivation of paternal X chromosomes is maintained in the
cells that give rise to extra embryonic tissues (eg. the placenta) and is
not discussed in the text. Early paternal X inactivation is erased in cells
that give rise to embryonic tissue and random X inactivation subsequently
occurs.

Euchromatin and hetrochromatin have distinctly different properties. A

comparison of these two forms of chromatin helps to understand the
differences in these two forms of chromatin with respect to their function.

SUMMARY

1. Chromatin makes up the nucleus. It is made up of DNA and protein and

has two forms: euchromatin and heterochromatin.

2. When stained and observed under an optical microscope, euchromatin

is the light-colored band while the dark-colored band is
heterochromatin.

3. Darker staining indicates tighter DNA packaging. Heterochromatin

thus has a more compact DNA packaging than euchromatin.

4. Heterochromatin is termed as genetically inert and has no genes

encoding for proteins, while euchromatin is characterized by coding
genes.

5. Although termed inert, heterochromatin plays a role in the regulation

of gene expression.
6. Heterochromatin is a compactly coiled region while euchromatin is
loosely coiled.

7. Euchromatin contains less DNA while heterochromatin contains more

DNA.

8. Euchromatin is early replicative while heterochromatin is late

replicative.

9. Euchromatin is found in eukaryotes, cells with nuclei, and

prokaryotes, cells without nuclei.

10. Heterochromatin is only found in eukaryotes.

11. The functions of euchromatin and heterochromatin are gene

expression, gene repression, and DNA transcription.