DNA as a Genetic Template

Overview

Two structural features of the DNA molecule provide a basis for the mechanisms of
heredity: the four nucleotide bases and its double-stranded nature. The Watson-Crick
model of double-helical DNA structure, proposed in 1952, drew heavily upon the X-ray
crystallography work of researchers Rosalind Franklin and Maurice Wilkins. Watson, Crick,
and Wilkins jointly received the Nobel Prize in Physiology or Medicine for their work in
1962. Franklin was, controversially, excluded from the prize for reasons that are still
debated.

The Watson-Crick model of DNA, very simply, proposed that DNA is made up of two
strands of nucleotides that twist around each other to form a right-handed helix and that
nucleotide pairing takes place between a purine and a pyrimidine.

The two DNA strands are antiparallel, meaning that the 3’ end of one strand faces the 5’
end of the other. This allows each strand to act as a template for its partner during DNA
replication, producing two new strands of DNA that are exactly complementary to each
other. However, whether or not DNA replication occurred in this fashion was not clear.

The Meselson and Stahl Experiment

Meselson and Stahl grew E. coli for several generations in a medium containing a “heavy”
isotope of Nitrogen, 15N. Over time, the heavy Nitrogen was incorporated into the
nitrogenous nucleotide bases and, thus, into the DNA. After this, the E. coli was placed into
a medium containing a different isotope of Nitrogen, 14N, and grown for several more
generations. After each generation, a DNA sample was isolated from some of the cells,
loaded into a gradient, and centrifuged at high speeds. In a gradient, the DNA will separate
according to its buoyant density (i.e. the density within the gradient where the DNA will
float).

In the 15N medium, a single band of high density was observed in the lower portion of the
centrifuge tube. Immediately after the bacteria were transferred to the “lighter” media, this
single band shifted upwards in the column, indicating a lower density. However,
subsequent generations resulted in two bands: one corresponding to the 14N density and
another in an intermediate location. This result could only be explained by a semi-
conservative mode of replication, thus validating the Watson-Crick model.

Procedure

The Watson-Crick model, proposed in 1953, elucidated two structural features of the DNA
molecule that provide a basis for heredity: its double-stranded nature and the four
nucleotide bases. 

This model proposed that DNA is made up of two strands of nucleotides that twist around
each other to form a right-handed helix.

These strands are anti-parallel in nature - meaning the 3’ end of one strand faces the 5’
end of the other strand.
Each strand of DNA contains a sequence of nucleotides that is exactly complementary to
its partner strand - enabling each strand to act as a template for its partner.

Thus, a cell can replicate its DNA before cell division by separating two strands of DNA and
producing two new strands of DNA that are exactly complementary to each other. 

Additionally, the Watson-Crick model posited that base-pairing takes place between a
purine - either adenine or guanine - and a pyrimidine - either cytosine or thymine. 

Adenine, guanine, cytosine, and thymine comprise the four-letter nucleotide ‘alphabet.’ 

We now know that when these nucleotide letters are strung together into a three-letter
codon during translation, they can code for one of the 20 amino acids - much like letters in
an alphabet can be arranged to spell out words. 

These amino acid ‘words’ are linked into sentences, forming chains that fold into different
proteins. 

Different permutations of codons can result in different genes - much like how different
combinations of words and sentences result in different books. 

The genome is the entirety of the information contained within an organism’s genes and
encompasses all of the RNA molecules and proteins that an organism will produce over its
lifetime.

Differences between genomes result in genotypically and phenotypically distinct organisms

and species.

Chromosome Structure

Overview

A functional eukaryotic chromosome must contain three elements: a centromere,

telomeres, and numerous origins of replication.

The centromere is a DNA sequence that links sister chromatids. This is also where
kinetochores, protein complexes to which spindle microtubules attach, are constructed after
the chromosome is replicated. The kinetochores allow the spindle microtubules to move the
chromosomes within the cell during cell division.

Telomeres consist of non-coding repetitive nucleotide sequences, at their tips. These

sequences are typically similar across species. They usually consist of repeated units of
adenine or thymine followed by multiple guanine nucleotides. Telomeres protect and
stabilize the ends of chromosomes. If a chromosome were to break, it would begin to
degrade at the newly created end, which lacks a telomere.

Origins of Replication and ARSs

Eukaryotic chromosomes must also have numerous origins of replication, which are
sequences of nucleotides that determine where DNA replication begins. While the precise
number of origins of replication in the human genome has yet to be quantified, at least
30,000 would be required in order for replication to occur in a timely fashion. If human
chromosomes contained only one such origin, for example, it would take more than a
month to replicate a single chromosome.

While the importance of origins of replication is established, defining these sequences has
proven difficult. However, some experiments with yeast have identified a few candidates.
When certain chromosomal sequences are added to a yeast cell as an extracellular,
circular DNA molecule, they replicate autonomously. This gives these sequences their
name - autonomously replicating sequences (ARSs). Some ARSs likely correspond to
origins of replication that function within the yeast genome. However, some of these are not
located along a stretch of DNA that is strongly associated with initiation of replication.

In mammals, such as humans, and other more complex eukaryotes, the origin of replication
sequences are poorly defined. This is because they are likely defined by a combination of
nucleotide sequence, associated proteins, and chromatin structure.

Procedure

The DNA-histone complex contained in the nucleus is called chromatin and condenses to
form chromosomes that consist of a single chromatid or sister chromatids - depending upon
the cell cycle stage.

A functional eukaryotic chromosome must have a centromere, a DNA sequence that links
sister chromatids.

The centromere is also where the kinetochores are constructed after the chromosome has
replicated. These protein complexes allow the spindle microtubules to move the
chromosomes around during cell division.

Depending upon the location of the centromere, chromosomes can exist in four major
configurations.

In the metacentric configuration, the centromere is centered, resulting in arms of similar

lengths.

Whereas, in the submetacentric configuration, the centromere is off-center, resulting in

arms of different lengths.

In the telocentric configuration, the centromere is at the very end of the chromosome,
resulting in long, single arms. 

In acrocentric chromosomes, the centromere is located near the end, giving the
appearance of a ‘stalk’ and ‘bulb’.

These various configurations occur naturally, making them useful in identifying specific
chromosomes. For example, the human Y chromosome is acrocentric.     

Each chromatid must also have telomeres, which consist of non-coding repetitive
nucleotide sequences, at their tips. 
The telomeres protect and stabilize the ends of chromosomes. If a chromosome breaks, it
will begin to degrade at the newly created end, which lacks a telomere. 

Finally, a chromosome must have multiple origins of replication, sequences of nucleotides

that determine where DNA replication begins. 

Human chromosomes contain approximately 30,000 origins of replication in order to

expedite the replication process. If a human chromosome only contained one origin of
replication, it would take over a month to replicate a single chromosome. 

When each chromosome replicates, beginning at multiple origins of replication, the

resulting sister chromatids are held together at the centromere with telomeres at their tips. 

Right before cell division, chromosomes are in their most condensed state. This is why
observations of chromosomes are often made at this point in the cell cycle.

The Nucleosome Core Particle

Overview

Nucleosomes are the DNA-histone complex, where the DNA strand is wound around the
histone core. The histone core is an octamer containing two copies of H2A, H2B, H3, and
H4 histone proteins.

The paradox

Nucleosomes, paradoxically, perform two opposite functions simultaneously. On the one

hand, their main responsibility is to protect the delicate DNA strands from physical damage
and help achieve a higher compaction ratio. While on the other hand, they must allow
polymerase enzymes to access DNA bound to the histones for replication and transcription.
The mechanism by which nucleosomes solve these two problems is via partial unfolding of
the DNA from the nucleosomes or histone protein modifications.

Core structure

The histone core proteins share a common structurally conserved motif called the “histone
fold” and have a mobile extended tail region. The histone fold is made up of alpha-helices
and loops. During the histone dimerization, loops of two histone proteins align together,
forming a histone dimer.

Each histone binds to the three consecutive minor grooves of DNA. The alpha-helix and the
N-terminal tail of each histone protein play a crucial role in binding to the DNA. Hence, any
chemical modifications to the histone tail can modify the chromatin assembly and function.
Some of the most common histone modifications include acetylation, methylation, and
phosphorylation.

Histone variants

Histone proteins have various isoforms or variants like H2A.1, H2A.2, H2A.X, H3.3, or
CENP-A. These variants differ in their amino acid sequences and perform distinct
functions. The nucleosomes with histone variants are significantly more mobile than
ordinary nucleosomes. For example, incorporation of H2A.Z into the nucleosome is shown
to activate the transcription.

Procedure

The nucleosome core particle consists of an octamer containing four kinds of histones
encircled by a left-handed coil of DNA. Nucleosome core particles play an important role in
DNA function by controlling DNA compaction and chromatin structure. 

This role is so essential that histones are some of the most highly conserved proteins in
eukaryotes - from peas to cows and other organisms. For example, there are only two
different amino acids out of 102 between the H4 histones of a pea plant and a cow. 

The four kinds of histones that make up the nucleosome core particle, H2A, H2B, H3, and
H4, share some characteristics. First, they are each small - only containing up to 135 amino
acids. 

Second, they share a common structural motif: the histone fold. The histone fold consists of
three α-helices connected by two loops. 

When nucleosome core particles are assembled, histone folds bind to each other first in an
interaction described as a ‘handshake,’ forming two H2A-H2B dimers and two H3-H4
dimers. 

After this, the H3-H4 dimers form a tetramer, which goes on to form the octamer of the
nucleosome core particle with the H2A-H2B dimers. 

The structure of this histone octamer begets extensive interactions between the histones
and the DNA wound around them. 

There are more than 100 hydrogen bonds between the histones and the DNA of the
nucleosome core and many of these are between the amino acid backbones of the
histones and the sugar-phosphate backbone of the DNA.

Finally, many of the amino acids in each core histone are lysine or arginine, which have
positive charges that effectively neutralize the negatively charged DNA backbone.

Nucleosome Remodeling

Overview

Nucleosomes are the basic units of chromatin compaction. Each nucleosome consists of
the DNA bound tightly around a histone core, which makes the DNA inaccessible to DNA
binding proteins such as DNA polymerase and RNA polymerase. Hence, the fundamental
problem is to ensure access to DNA when appropriate, despite the compact and protective
chromatin structure.

Nucleosome remodeling complex

Eukaryotic cells have specialized enzymes called ATP-dependent nucleosome remodeling
enzymes. These enzymes bind to both histone and the wound DNA and can facilitate
nucleosome sliding - a process where the DNA is pushed relative to the histone core, or
partial or complete histone core replacement, altering the composition of nucleosomes and
indirectly affecting the chromatin folding. One of the best-known remodeling complexes is
Swi/Snf, originally identified in yeast.

Mechanism of action

Two models are proposed to explain nucleosome sliding - Twist diffusion and Loop/bulge
propagation. Both these models suggest that DNA distortion propagates over the surface of
the nucleosome.

Twist diffusion model

According to this model, a single base pair is transferred between linked DNA and the DNA
wrapped around the histone core. This base change causes nucleosomal DNA to twist or
untwist to accommodate the gain/loss of base pairs. The twist defect then propagates
around the nucleosome from one DNA segment to the next in a process known as twist-
diffusion. In this manner the histone octamer would shift along with the DNA by the size of
distortion.

Loop/bulge model

According to this model, DNA from the linker region transiently shifts around the
nucleosome, creating a bulge/loop. The loop then travels around the histone, creating or
disrupting the histone-DNA interactions. This way, the nucleosome core slides on the DNA
strand, exposing regions of DNA for genetic activities.

Given the complexity of chromatin folding, both of the models mentioned above may
coexist. However, there are specific questions which both models do not explain, indicating
the actual processes may be even more complex. For example, how is processivity
achieved during sliding? How does each element of the remodeling complex participate in
the process? How do remodelers cooperate with histone chaperones?

The nucleosome remodeling ATPase is involved in several genetic mechanisms, such as

developmental gene expression, rapid transcription in response to environmental cues,
replication of the genome, surveillance of DNA damage, and repair.

Defects in the nucleosome remodeling complex have a wide range of consequences.

During embryonic development, failure of nucleosome remodeling can affect viability, and
cause morphological defects. It may also lead to problems in DNA repair, resulting in
genome instability and cancer.

Procedure

The ordered arrangement of nucleosomes results in compact and protective chromatin

organization, which may obstruct the access to genetic information in several ways. 

First, DNA binding proteins, such as RNA polymerase, cannot readily associate with the
DNA bound to the histone surface. In addition, the DNA wound around the histone is highly
bent, making it unrecognizable by the DNA binding proteins. Finally, non-histone chromatin
proteins may get associated with nucleosomes causing further compaction. 

Therefore, eukaryotic cells have enzymes called ATP-dependent chromatin remodeling

complexes that can temporarily and locally remodel the nucleosomes.

These complexes contain an ATP hydrolyzing subunit that binds both to the histone
proteins and to the DNA wound around it. Hydrolysis of ATP provides the energy required
to disrupt the interaction between the histone core and the DNA.

Energy obtained from multiple rounds of ATP hydrolysis allows the remodeling complexes
to cause nucleosome sliding - a process where the histone is moved along the DNA without
dissociating from it. 

Nucleosome sliding is best explained by the loop-bulge propagation model. Here, the DNA
from the linker region is pushed to the histone core, forming a loop. 

This bulge then propagates around the histone core like a wave, breaking and reforming
histone-DNA interactions. This process shifts the histone core along the DNA, exposing the
hitherto inaccessible DNA sequences. 

Remodeling complexes, in association with histone chaperones, facilitate partial or

complete replacement of nucleosome octamer cores, indirectly affecting the chromatin
folding. For example, the replacement of histone H3 with centromere-specific H3 histones
marks the location of centromere on the chromosome. 

Non-histone DNA binding proteins also influence the nucleosome position and stability.
Some bound proteins may favor nucleosome formation, and other proteins may not allow
nucleosomes to be formed in their vicinity. 

The exact location of nucleosomes therefore depends on the nature of non-histone DNA
binding proteins; and due to the presence of the chromatin remodeling complexes the
composition and arrangement of nucleosomes is highly dynamic.

The Nucleosome

Overview

DNA in a human cell is almost 2m long and it is packed inside a tiny nucleus that is only a
few microns in diameter. The level of compaction of DNA inside the nucleus is astonishing.
It is organized into several sequentially higher levels of compaction to fit into such a tiny
space. The most compact form of DNA is a chromosome that can be seen under a
microscope in a dividing cell.

DNA is wound twice around a protein complex called histone core, that consist of 8 histone
proteins. This complex of DNA and histone protein is called the nucleosome, the
fundamental and functional unit of DNA compaction. Nucleosomes can further coil around
themselves into higher order compaction.
When the DNA is extracted from cells in low salt conditions and examined under a
microscope, it resembles the beads on a string. The string represents the free DNA called
"linker DNA," connecting the bead-like nucleosomes. If the DNA is isolated in physiological
salt conditions (0.15 M KCl), it assumes a fiber-like form with 30 nm diameter that is bound
to H1, a nonhistone protein. The H1 protein tightly binds to both the nucleosome and does
not allow the DNA to slip.

Histones are highly conserved proteins

The amino acid sequences of core histone proteins are highly conserved between distantly
related species. For example, the amino acid sequence of H3 histone between calf thymus
and pea plant has only four amino acid differences.

Nonhistone proteins

Nucleosomes complex is also bound by a small proportion of nonhistone proteins, which

help in maintaining the compaction and organizing long chromatin loops. Nonhistone
proteins are also involved in the regulation of DNA replication and RNA synthesis.

Procedure

Proteins play a key role in determining the physical structure of a chromosome. The most
abundant of these are small, positively charged proteins called histones. This positive
charge allows them to tightly associate with the negatively charged DNA.

During certain stages of the cell cycle, DNA is wound tightly around specific types of
histones, forming structures called nucleosomes. Nucleosomes are often described as
‘beads’ on a ‘string’ of DNA.

A nucleosome consists of a few key elements.  The first of these is an octamer of histone
proteins, two molecules each of H2A, H2B, H3, and H4.

Next, a nucleosome also has a 145 -147 bp length of DNA wrapped around the protein
octamer nearly two times. 

Together, the histone octamer and the DNA wound around it are known as a nucleosome
core particle. 

Each of the histones in the nucleosome core particle has a small, positively charged tail
consisting of 11-27 amino acids. 

The tails extend out from the nucleosome core particle and aid in keeping the negatively
charged DNA and the histones associated. Furthermore, the histone tails can interact with
tails from neighboring core particles, which facilitates DNA packaging.  

A fifth type of histone, H1, plays a key role in nucleosome structure, though it is not part of
the nucleosome core particle. H1 binds to the DNA where it joins and then leaves the
octamer, acting as a clamp and keeping the DNA in place.
Finally, the nucleosome also encompasses the stretch of linker DNA adjacent to the
nucleosome core particle. The linker DNA that separates each core particle can vary in
length, from about 30 to 40 base pairs, between cell types. 

While the terms nucleosome and nucleosome core particle are often used interchangeably,
the nucleosome actually refers to the nucleosome core particle and the adjacent linker
DNA. 

Altogether, nucleosomes are capable of reducing a long DNA molecule into a chromatin
thread that is about one-third of its original length.

Chromatin Packaging

Overview

Each human somatic cell contains 6 billion base-pairs of DNA. Each base-pair is 0.34 nm
long, which means that each diploid cell contains a staggering 2 meters of DNA. How is
such a long DNA strand packed inside a nucleus measuring only 10 - 20 microns in
diameter? 

The chromatin

In combination with specialized DNA binding protein called Histones, the DNA double helix
forms a compact DNA: protein complex called chromatin. The chromatin itself is further
compacted into higher-order structures. The highest level of compaction is achieved during
the cell cycle's metaphase, where the chromatin condenses to form the chromatids of a
chromosome.

Nucleosomes

Nucleosomes are the basic functional and repeating unit of chromatin. A nucleosome
consists of 8 histone proteins wound around by 147 base pairs of DNA. Under electron
microscopy, the chromatin appears as a structure resembling beads on a string due to
nucleosomes' presence along its length. This packaging shortens the fiber length by seven-
fold.

Solenoid model

The nucleosomes are further coiled into 30 nm fibers, so-called because of their diameter
of approximately 30 nm. Such a compaction is explained by a widely accepted hypothesis -
the solenoid model. A solenoid refers to the structure of a wire coiled on a central axis. This
model proposes that nucleosomes are arranged in a left-handed helical conformation with
six or more nucleosomes per turn. One of the non-core histone proteins, H1, plays an
essential role in nucleosome compaction; in its absence the chromatin fiber turns into
irregular clumps of nucleosomes.

Chromatin packaging is an active area of research. The new emerging data has allowed
scientists to view chromatin and nucleosomes not as highly defined structures, but rather
as a continuum of various inter-convertible conformations at all chromatin packaging
stages.
Procedure

Every human diploid cell contains about 2 meters of DNA compressed inside a tiny nucleus
of just a few microns in diameter. The arrangement and coiling of DNA inside the nucleus is
therefore highly organized, and tightly regulated.

First, the chromosomal DNA is associated with histone proteins to form a structure called
the chromatin. The basic structural and functional unit of chromatin is called a nucleosome.
The association of the DNA into nucleosomes shortens the DNA length sevenfold. 

Next, a non-core histone protein called H1 binds to each nucleosome. The H1 histone
changes the DNA path as it exits the nucleosome, helping to further compact the complex.

These nucleosomes are then stacked on top of each other, generating a shorter and thicker
fiber with a diameter of 30 nm, known as 30-nm fibers.  

The arrangement of nucleosomes into the 30-nm fiber is explained by a widely accepted
Solenoid model. The model proposes that nucleosomes are arranged in a left-handed
helical conformation with six or more nucleosomes per turn. This shortens the DNA length
by a further 50-fold.

Any chromatin region that is not being actively transcribed or replicated exists in the 30-nm
fiber form. On the other hand, the chromatin regions that are actively being accessed exist
in an extended beads-on-a-string form. 

The 30 nm fibers are coiled further to form loops of around 300 nm length. These fibers are
then compressed into 250 nm wide coils.

Later during the metaphase of the cell cycle, the chromatin fibers form highly condensed
structures called chromosomes. 

The overall compaction ratio of DNA into the chromosome is approximately 1:10000. Once
the cell divides, the chromosomes uncoil again.

Chromosome Replication

Overview

Before a cell can divide, it must accurately replicate all of its chromosomes, including the
DNA and its associated histone and non-histone proteins.  This process begins at
numerous origins of replication during the S phase of the cell cycle in each of a cell’s
chromosomes simultaneously. Certain nucleotides can act as origins of replication, but
these sequences are not well defined - especially in complex, multi-cellular, eukaryotic
species. The length of DNA that spans an origin of replication and its two respective termini
of replication, where adjacent replication forks will eventually fuse, is called a replicon. DNA
replication progresses in this fashion, cluster of origins at a time, until it reaches the
telomeres, which have their own specialized replication process. At this point, when the cell
is in M phase and ready to divide, the cell’s chromosomal mass has effectively doubled. 
While DNA replication progresses, new histone proteins are synthesized and new histone
core particles are formed. These proteins are just as important to chromosome function as
DNA since histones are critical to the physical structure of the chromosome. 

Origins of Replication

DNA replication begins at certain nucleotide sequences called origins of replication. These
sites interact with specialized initiator proteins that begin the process of DNA separation
and replication. Defining and quantifying these sequences has proven difficult, especially in
complex, multi-cellular, eukaryotic species. For example, the large size of the human
genome would necessitate tens of thousands of origins of replication throughout all of the
chromosomes. However, counting these sites is difficult because there is no clear
consensus on specific origin sequences. It is likely that origins of replication are defined by
a combination of nucleotide sequences, various proteins, and chromatin structure. 

Replication of histones

Histones are proteins that are responsible for packaging DNA into chromatin and then into
chromosomes. Thus, histones are critical to the physical structure and function of the
eukaryotic chromosome. During chromosome replication, new histones must also be
synthesized in order to package the new DNA into nucleosomes. As the replication fork
moves forward, the new and old histones re-assemble randomly on the daughter cells. 

Procedure

During the S phase of the cell cycle, the process of chromosome replication begins
simultaneously in each of a cell’s chromosomes at numerous origins of replication. 

An origin of replication is a nucleotide sequence that interacts with specialized initiator

proteins, called the origin recognition complex, at sites where DNA replication begins. 

Defining these origins of replication is difficult. Given the large size of the human genome,
for example, an estimated tens of thousands of origins would be necessary to replicate the
entire genome in a timely fashion. 

In humans, no clear consensus on specific origin sequences has been reached. Instead, it
is likely that origins are defined by a combination of nucleotide sequences and chromatin
structure. 

The length of DNA that spans an origin of replication and its two respective termini of
replication, where adjacent replication forks eventually fuse, is called a replicon. 

Replication does not occur simultaneously in all of the replicons, however. Different cell
types have their own specific timing for initiation of replication, typically in clusters of
origins. 

DNA replication progresses in this fashion throughout the chromosome until it reaches the
telomeres, which have a unique replication process involving the enzyme telomerase, a
reverse transcriptase. 
At this point in the cell cycle, right before cell division - or M phase, a cell will have
effectively doubled its chromosomal mass. 

In addition to DNA, histone proteins are critical to the physical structure and function of the
eukaryotic chromosome. Thus, during chromosome replication, histone synthesis must
happen simultaneously with DNA replication. 

The replication forks radiating outwards from the origins of replication disrupt the histone
octamers into their subunits. 

The newly synthesized histone subunits and old histone subunits are re-assembled into
octamers and distributed onto daughter DNA strands. Thus, the histone replicates in a
semi-conservative fashion where both old and new histones are segregated randomly to
the newly synthesized daughter strands.

Position-effect Variegation

Overview

In 1928, a German botanist Emil Heitz observed the moss nuclei with a DNA binding dye.
He observed that while some chromatin regions decondense and spread out in the
interphase nucleus, others do not. He termed them euchromatin and heterochromatin,
respectively. He proposed that the heterochromatin regions reflect a functionally inactive
state of the genome. It was later confirmed that heterochromatin is transcriptionally
repressed, and euchromatin is transcriptionally active chromatin.

Difference between euchromatin and heterochromatin

Euchromatin is a lightly stained, gene-rich, and loosely-bound chromatin region. It is usually

dispersed in the nucleus. The histones of euchromatin are extensively acetylated, which
allows loose chromatin compaction.

In contrast, heterochromatin is a darkly stained, repeat-rich, gene-poor, and compact

chromatin. It is mostly seen at the nuclear periphery, often as clumps. The histones of
heterochromatin are methylated, which enables a compact chromatin structure.

Position-effect variegation

Chromosomal rearrangements may position euchromatin genes next to heterochromatin.

Such gene rearrangements can result in gene silencing by virtue of being placed near
heterochromatin, rather than a change in the gene itself. This phenomenon is called
"position-effect variegation (PEV)." Hence, the juxtaposed gene becomes silent in some
cells where it is normally active, resulting in a variegated phenotype. The phenomenon of
PEV is well studied in Drosophila.

The formation of heterochromatin depends on the histone H3 methylation followed by the

association with nonhistone proteins such as Heterochromatin Protein 1 or HP1. Usually,
heterochromatin and euchromatin are separated by a buffer region with many repeat-rich
regions. PEV indicates that heterochromatin, once formed, can spread beyond the buffer
region into the adjoining chromatin. In humans, HUSH complex methylates histones and
contributes to the spreading of heterochromatin and hence, position effect variegation.
Procedure

In a eukaryotic cell, DNA associates with various proteins to form a tightly packed, highly
condensed structure called chromatin. Chromatin is divided into two types, depending upon
the level of compaction.

Heterochromatin, concentrated in the centromeres and telomeres, is highly condensed,

gene-poor, and infrequently transcribed into RNA. 

In contrast, euchromatin, which constitutes a majority of the chromosomal material, is less

condensed, gene-rich, and actively transcribed. 

Heterochromatin and euchromatin are separated by barrier DNA sequences. These

sequences prevent the spread of heterochromatin and maintain stable gene expression
patterns. 

During DNA rearrangement events, such as transposition, a piece of euchromatin can be

translocated near a heterochromatin region without any adjacent DNA barrier sequences.

In such events, genes that are typically active are ‘silenced’ or ‘inactivated.’ This
phenomenon is known as the ‘position effect.’ 

For example, in Drosophila, red eye color is encoded by the ‘white’ gene. Occasionally,
‘white’ is moved near a heterochromatin region without any DNA barrier sequences in
between. 

When flies inherit this genotype, during their early embryonic stages when heterochromatin
is first formed, the absence of barrier DNA allows the heterochromatin to spread into the
neighboring euchromatin. 

However, spreading occurs to different extents in different embryonic cells. This variation in
heterochromatin spreading yields cells that exhibit two distinct phenotypes- one which
actively expresses the ‘white’ gene and others that do not. 

Once established, all of that cell’s progeny inherit this state. This results in the mottled, or
mosaic, eyes in the adult flies. 

This phenotype variegation, caused by the gene silencing mediated by a change in the
position of the ‘white’ gene within the chromosome, is called ‘position effect variegation.’ 

First established in Drosophila, this phenomenon has now been observed in many
eukaryotes, including yeasts, plants, and humans.

Histone Modification

Overview

The histone proteins have a flexible N-terminal tail extending out from the nucleosome.
These histone tails are often subjected to post-translational modifications such as
acetylation, methylation, phosphorylation, and ubiquitination. Particular combinations of
these modifications form “histone codes” that influence the chromatin folding and tissue-
specific gene expression.

Acetylation

The enzyme histone acetyltransferase adds acetyl group to the histones. Another enzyme,
histone deacetylase, removes the acetyl group from acetylated histones. The lysine amino
acids at position 4 and 9 of N-terminal histone tail are often acetylated and deacetylated.
Acetylation increases the negative charge of histones. This weakens the DNA-histone
interaction resulting in loosening of chromatin and increased access to DNA. For example,
in the erythroid cells, beta-globin gene is associated with acetylated histones that increase
its expression. In non-erythroid cells where the gene is inactive, it is found to be associated
with nonacetylated histones.

Methylation

The histone tails at the lysine 9 position of histone H3 can be di- or tri-methylated by
enzyme histone methyltransferase. This methylation can initiate the binding of nonhistone
proteins and increase chromatin compaction. Methylation increases the positive charge on
the histones, resulting in increased affinity between negatively charged DNA and histones
and higher chromatin compaction. Repressed chromatin, also known as heterochromatin,
is highly methylated.

The histone codes or modifications are epigenetically inherited, meaning these

modifications are not genetically coded. Hence, these modifications are faithfully passed on
to the next cell during each cell division as an epigenetic memory.

Procedure

A nucleosome contains a protein core that is made up of four histone core proteins: H2A,
H2B, H3, and H4.

In addition to these four standard core histones, eukaryotes also possess a few variants of
each histone, with the exception of H4. 

The amino-terminal tails of the core standard and variant core histones protrude from the
nucleosomes and are highly unstructured and mobile.

These tails, comprising of about 30 amino acids, are subjected to several forms of covalent
modifications such as the acetylation of lysines, phosphorylation of serines, and mono-, di-,
or tri-methylation of lysines. 

The reactions that lead to these modifications are catalyzed by different enzymes such as
methyltransferases, acetylases, kinases. These groups of enzymes are collectively referred
to as the ‘writers.’ 

The reactions that catalyze the removal of these chemical groups are catalyzed by
enzymes such as demethylases, deacetylases, and phosphatases. These enzymes are
collectively referred to as the ‘erasers.’
Amongst the numerous possible combinations of different histone variants and amino-
terminal end modifications, only certain coordinated sets are known to occur. Some of
these combined sets of modifications encode a specific signal for the cell. 

For example, one set of modifications signals DNA damage and the need for repair.
Another signals gene expression, while others signal gene silencing or chromatin
modification like the establishment and spread of heterochromatin. 

This encoding system is referred to as the ‘Histone Code.’ 

The signals encoded in these modifications are decoded by specific regulatory proteins
called ‘readers.’ These proteins and multiprotein complexes contain various small domains,
each of which recognizes a particular histone mark. 

They bind tightly to a region of chromatin that contains several different histone marks and
attract additional protein complexes with catalytic activities. This leads to specific biological
functions such as chromatin modifications, gene expression, and gene silencing

Spreading of Chromatin Modifications

Overview

The histone proteins in the nucleosomes are post-translationally modified (PTM) to

increase or decrease access to DNA. The commonly observed PTMs are methylation,
acetylation, phosphorylation, and ubiquitination of lysine amino acids in the histone H3 tail
region. These histone modifications have specific meaning for the cell. Hence, they are
called "histone code". The protein complex involved in histone modification is termed as
"reader-writer" complex.

Writers

The writer is an enzyme that can cause specific histone modifications. The common writer
enzymes are histone methyltransferases (HMT) and histone acetyltransferases (HATs).
HMTs add a methyl group to histone tail, which increases the chromatin compaction,
inhibits transcription, and helps to differentiate newly synthesized strands from the parental
strand during DNA replication. HATs add an acetyl group to histone tail, which decreases
the chromatin compaction and allows access to DNA.

Erasers

The PTMs to histones are reversible and can be removed by another group of enzymes
called "erasers". Common erasers are histone deacetylase and histone demethylase. They
remove the acetyl or methyl group from the histone and alter the chromatin compaction. 

Barrier proteins

Reader-writer complexes mark the euchromatin and heterochromatin regions on a

chromatin. Acetylation of histone tail lysine marks the euchromatin, whereas methylation
marks the heterochromatin region. It is important to separate the gene-rich euchromatin
from gene-poor heterochromatin, for optimal regulation of gene expression. On a long
chromatin strand, series of euchromatin and heterochromatin are separated by barrier
sequences. These sequences prevent the spread of histone modification by several ways.
For example, barrier proteins can tether chromatin to nuclear pore and prevent the spread
of heterochromatin.

The aberrant activity of writer-eraser enzymes is correlated with several human diseases,
including Alzheimer's, Fragile X syndrome, and cancer. In Fragile X syndrome, gene FRM1
required for normal cognitive development is hypermethylated, which leads to
transcriptional silencing of the gene.

Procedure

Reader enzymes act in collaboration with writer or eraser enzymes to create chromatin
modifications and spread the chromatin changes for a substantial distance along a
chromosome. 

The process, which functions similarly with writer and eraser enzymes, is described here
using the reader and writer combination as an example.  

The process begins when a transcriptional regulatory protein binds to a specific DNA
sequence. After binding, the regulatory protein recruits a writer enzyme to that specific site
on a chromosome. 

The writer enzyme then starts to add marks to the core histones. 

Once marks have been added to one or more neighboring nucleosomes, a multiprotein
complex containing a ‘reader’ enzyme that recognizes these marks, binds tightly to the
newly modified nucleosome.

The binding activates the ‘writer’ enzyme of the multiprotein complex and positions it near
an adjacent nucleosome, enabling it to add a new mark.

Working in concert, the reader and writer enzymes of the complex catalyze a series of
many ‘read and write’ cycles. These cycles spread a chromatin modification, such as
chromatin condensation, along a chromosome.

The boundary between adjacent chromatin domains like euchromatin and heterochromatin,
which have different structures and functions, is marked by specific DNA sequences called
‘barrier sequences.’ 

These barrier sequences act as the binding sites for various barrier proteins, which block
the propagation of heterochromatin into euchromatin by mediating different barrier actions. 

For example, some barrier proteins recruit histone-modifying enzymes which erase the
histone marks that are required for the spreading of the heterochromatin.

Some barrier proteins tightly bind to the group of nucleosomes, covering them up and
thereby making the covered euchromatin resistant to heterochromatin spreading.

Yet another barrier protein tethers a region of chromatin to large fixed sites, such as
nuclear pores, and forms a barrier that stops the spread of heterochromatin.
Lampbrush Chromosomes

Overview

In 1882, Flemming observed lampbrush chromosomes (LBC) in salamander eggs. Later in

1892, Rückert observed LBCs in shark egg cells and coined the term "lampbrush
chromosomes" because they looked like brushes used to clean kerosene lamps.

LBCs are made up of two pairs of conjugating homologous chromatids. Each chromatid
consists of alternatively positioned regions of condensed-inactive chromatin and loosely
placed-active side loops, which can be contracted and extended. The loops resemble the
puffs of polytene chromosomes. The polytene puffs are composed of several parallel
chromatids, whereas the loops of LBC consist of a single, double helix. 

During the diplotene stage of meiosis prophase, LBCs decondense forming large
chromosomes, approximately 30 times larger than regular mitotic chromosomes. The
average length of LBC loops is 10-15 µm. In some cases, loops can be as large as 50-100
µm.  Polymerase II transcribes the largest loops, and the smallest loops are transcribed by
polymerase III.

LBCs are present in the oocytes of lower vertebrae, invertebrates, and birds. LBCs in all
these organisms share similar structures and functions. Comparative genome studies of
LBCs have shown that the length of side loop increases with the C-value, which is the total
DNA content in the haploid set of an organism.

LBCs are studied for over a hundred years, yet, only a general structural idea of LBC is
known. Recently, LBCs are used as model structures to study cytogenetic analysis and
epigenetic regulation of chromatin structure and gene expression.

Procedure

Amphibians, like many other vertebrates, produce female gametes, or ova, which mature
and develop within the ovary. 

At one particular developmental stage, meiotic prophase-I, homologous replicated

chromosome pairs display a distinct morphology- an enormously extended structure, so
gigantic that it can be easily observed under a light microscope

These very unusual chromosomes are called ‘lampbrush chromosomes’ due to their
resemblance to the brushes used in earlier times to clean kerosene lamps. Due to their
enormous size, they are ideal models for studying chromosomes.

These giant chromosomes are organized into a series of large lateral, uncoiled DNA loops
all along the chromosome axis. In addition to these extended loops, dense, thick coiled
loops are observed along the chromosomal axis, which constitute the majority of DNA. 

These two domains also differ greatly in the level of transcriptional activity. The genes in
the lateral loops are actively transcribed, which gives them a fine fibrillar appearance. 

In contrast, the genes on the condensed chromosome axis are generally not expressed. 
Although they were first described in amphibians, these looped chromatin domains are now
believed to occur in the interphase chromosomes of all eukaryotes. 

Due to their small size and fragile nature, the looped chromatin can’t be observed using a
light microscope in most eukaryotes. Their presence has been inferred using modern DNA
technologies such as ‘Chromosome Conformation Capture.'

Polytene Chromosomes

Overview

Polytene chromosomes are giant interphase chromosomes with several DNA strands
placed side by side. They were discovered in the year 1881 by Balbiani in salivary glands,
intestine, muscles, malpighian tubules, and hypoderm of larvae Chironomus plumosus.
Hence, these are also called "Salivary gland chromosomes." These are found in insects of
the order Diptera and Collembola; in certain organs of mammals; and synergids, antipodes
of flowering plants. Polytene chromosomes are also regularly observed in cells of salivary
gland in Drosophila.

There are certain differences between cells with polytene chromosomes and mitotically
dividing cells. First is the absence of cell division after DNA replication that results in the
accumulation of a  large number of chromatids. Second is the failure of DNA strands to
segregate after each round of DNA replication, resulting in several thousand chromatids
arranged side by side. Third is the intact nuclear membrane and nucleolus during
consecutive DNA replication cycles.

Banding patterns

Variation in the chromatin compaction can result in different concentrations of chromatin

along the length of the polytene chromosome. Since the homologous chromosomes have
identical chromatin compaction and are arranged side by side, it results in multiple compact
dark bands called chromomeres. The regions between the bands are called interband or
inter-chromomere regions. Interbands are lightly stained and are made up of decondensed
chromatin. The chromomere patterns are specific for a species; although the number and
size of chromomeres can change during the organism's lifetime.

Puffs

In certain instances, the interband region forms expanded structures called puffs that are
loosely coiled, to allow RNA synthesis. Therefore, the puffs are excellent models to study
the process of transcription. Exceptionally large puffs called “Balbiani rings” are found in
salivary glands of certain species. These have decondensed chromatin that supports a very
high rate of transcription.

Procedure

Typically, when a diploid cell divides, it first duplicates each of its chromosomes. Then, the
cell distributes a copy of each chromosome between the two daughter cells so that each
daughter cell receives a complete set of chromosomes.
Certain cells undergo multiple cycles of chromosome duplication without undergoing any
cell division. Such cells, called polyploid cells, contain multiple sets of each chromosome.

In the salivary gland cells of Drosophila larvae, this process is taken to an extreme. Here,
hundreds to thousands of copies of each chromosome are generated without any cell
division.

This results in the formation of unique, giant chromosomes, called polytene chromosomes.

As thousands of identical DNA sequences lay side by side, like crayons in a box, certain
features in the chromatin become visible. These features exist in ordinary interphase
chromosomes but are difficult to observe. 

When viewed under a microscope, an alternating pattern of dark bands and lighter
interbands can be seen in the polytene chromosomes.

The bands constitute 95% of the DNA, whereas the interbands constitute 5%. 

Where the chromatin in the bands is more condensed and transcriptionally inactive, the
chromatin in the interbands is less condensed and transcriptionally active. 

As Drosophila larvae progress from one developmental stage to another, specific bands
and interbands greatly increase in diameter, producing structures called ‘Puffs’ or ‘Balbiani’
rings.

These puffs arise from the de-condensation of chromatin and are sites of active DNA
transcription. The chromatin in puffs is arranged in looped structures, much like those
observed in ‘lampbrush’ chromosomes.

Histone Variants at the Centromere

Overview

Histone variants are the histone proteins with structural and sequence variations. These
variants may be regarded as “mutant” forms that replace their canonical histone
counterparts in the nucleosomes. Specific post-translational modifications on the histone
variants enable further chromatin complexity and regulate tissue-specific gene expression.
The most common histone variants are from histone H2A, H2B, and linker histone H1
families. However, several variants of histone H3 variants are also increasingly being
studied.

Histone H3 variant: CENP-A

Centrosomal chromatin contains a specialized histone protein called CENP-A which shares
60% similarity with canonical histone H3. It is essential for kinetochore assembly and
subsequent binding of microtubules. It is also hypothesized that CENP-A acts as an
epigenetic mark to maintain centromere identity. The CENP-A is recognized and targeted
to centromere via the CENP-A targeting domain (CATD).
In vitro studies have shown that centromeric nucleosomes form octomers with two copies
of CENP-A along with two copies of H2A, H2B and H4 histones. Nevertheless, new studies
in different eukaryotes have led to several competing models for the structure of CENP-A
containing nucleosomes. According to the hemisome model, the nucleosome contains only
one copy of each histone, forming a tetramer. In addition, recent studies have shown that
CENP-A nucleosomes are cell cycle-regulated. They exist as octamers in S-phase and as
hemisomes during other phases of the cell cycle.

Deregulation of CENP-A functions is linked to chromosome instability and cancer. Several

pieces of evidence indicate overexpression of CENP-A in colon cancer, adenocarcinoma,
testicular germ cell tumors, breast cancer and hepatocellular carcinoma.

Procedure

The centromere is a constricted region on each chromosome where the kinetochore and
microtubules assemble to ensure faithful separation of the chromatids during anaphase of
cell division. 

In most eukaryotes, the centromere is partitioned into two chromatin domains- the
centromere core and the pericentric heterochromatin region.

The centromere core in all eukaryotes contains a variant of core histone H3, called
Centromere Protein A or CENP-A. Together with the three other core histones H2A, H2B,
and H4, the CENP-A forms the centromere-specific nucleosome. 

The functional C-terminal fold domain of the CENP-A is highly conserved among
eukaryotes. However, the N-terminal tail of the protein shows significant variations both in
size and sequence.

The centromere cores in the yeast Saccharomyces cerevisiae contain a single CENP-A

nucleosome attached to a single mitotic spindle; hence, these are referred to as point
centromeres. 

This core centromere region is flanked by approximately 125 bps of AT-rich pericentric
DNA sequences. This region is characterized by nucleosomes containing methylated
histone H3.

The centromere chromatin forms a three-dimensional structure to expose CENP-A

containing nucleosomes for interaction with the kinetochore and microtubules. 

In contrast, the centromere core of human chromosomes has alternatively arranged CENP-
A nucleosomes and regular H3 nucleosomes. These regular H3 nucleosomes are
dimethylated. 

These alternating blocks can span up to 5 Mbs long and largely contain short, repeated AT-
rich DNA sequences called alpha satellite DNA, flanked by pericentric heterochromatin
DNA.

The timing of CENP-A loading to nucleosomes also differs between species. For example,
while in humans, it's loaded between the anaphase and G1 phases; in plants, the loading
occurs in the late G2 phase.
Inheritance of Chromatin Structures

Overview

Epigenetics is the study of inherited changes in a cell's phenotype without changing the
DNA sequences. It provides a form of memory for the differential gene expression pattern
to maintain cell lineage, position-effect variegation, dosage compensation, and
maintenance of chromatin structures such as telomeres and centromeres. For example, the
structure and location of the centromere on chromosomes are epigenetically inherited. Its
functionality is not dictated or ensured by the underlying DNA sequence, but instead by the
chromatin organization and histone variants. Once established, the centromere
organization and functions remain stably inherited through several cell divisions.

Histones are central to epigenetic inheritance

In the nucleosome, both DNA and histones are chemically modified. DNA is methylated at
cytosine residues, and histones are methylated, acetylated, or phosphorylated. Each of
these modifications constitutes a signal called histone code. Recent advances highlight
methylation as a bona fide epigenetic mark, and chromatin complexity as the primary
carrier of epigenetic marks. The presence of histone variants at specific locations and time
increases the complexity of chromatin organization. For example, the histone H3 variant
CENP-A is incorporated into a nucleosome in a DNA synthesis-independent manner,
resulting in an unusually stable nucleosome.

Inheritance of histones

The DNA methylation, deposition of histones on DNA strands, and post-translational

modifications of histones or histone code are connected to replication machinery. PCNA, a
DNA processivity factor, is the vital protein that links DNA replication with the inheritance of
epigenetic marks. At the replication fork, the nucleosomes are displaced such that H2A-
H2B dimers are entirely removed from the replication fork. The parental H3-H4 tetramers
are then distributed to daughter strands followed by the placement of newly synthesized
histone subunits on parental histones to complete the nucleosomes.

Procedure

Although all somatic cells contain the same genetic information, the cells of the liver divide
to form only liver cells, and skin cells divide into new skin cells.

Each tissue type has specific chromatin packaging and histone modifications which result
in distinct gene expression patterns. 

These structural features of chromatin - such as the centromere, the heterochromatin, and 
euchromatin regions - are epigenetically inherited, meaning their characteristics are passed
from mother to daughter cell in addition to the genetic material. 

This means that the tissue specific phenotypes of liver or skin or other specialized cells are
passed on during each round of cell division without the necessity for changes to the
genetic material. 
De novo centromere formation on newly synthesized DNA begins with the binding of
histone H3 variant CENP-A to the AT-rich satellite DNA to form centromere specific
nucleosomes. Once initiated, the structure selectively recruits more CENP-A histones to its
vicinity, in a cooperative manner. 

During DNA replication, the histone octamer ahead of the replication fork is broken down
into two H2A-H2B dimers and H3-H4 tetramer. 

The two H2A-H2B dimers are completely removed from the octamer, whereas, the H3-H4
tetramers are loosely attached to the DNA and distributed randomly to daughter strands. 

Newly synthesized H3-H4 tetramers are then added to both the strands to fill the spaces.
This is followed by the addition of two H2A-H2B dimers - half which are the original
molecules, and the other half are new - to complete the octamer. 

In yeast, following DNA replication, acetylation of histone H3 in the newly synthesized

strand marks the euchromatin, whereas deacetylation of H3 histone establishes the
location of the compact chromatin domains, or heterochromatin. 

Histone H3 methylation results in the condensation of chromatin. After DNA replication, the
methylated histones are randomly distributed on the daughter strands, which then
associate with the enzyme histone methyltransferase, to methylate the histone H3 on newly
synthesized octamers. 

X-chromosome inactivation is another example of the inheritance of chromatin structure.

Female mammals receive two X-chromosomes, and males receive only one. In females,
one of the X-chromosomes is inactivated, in a phenomenon called dosage compensation. 

Here, a long non-coding RNA, XIST, initiates X inactivation by binding to the entire length
of one X-chromosome in the embryonic stage. Subsequently, the chromosome is
maintained in this inactive mode in successive cell divisions, through all somatic cells.

Euchromatin

Overview

The extent of chromatin compaction can be studied by staining chromatin using specific
DNA binding dyes. Under the microscope, the dense-compacted regions take up more dye,
appearing darker, while the less-compact areas take up less dye and appear lighter. Based
on the compaction level, chromatins are classified into two primary forms – euchromatin
and heterochromatin.

Euchromatin is the less dense region of the chromatin and stains lighter. Euchromatin
contains histone H3 extensively acetylated on lysine at the 9th position of the histone tail
region. Histones in the promoter region have methylated lysine 4 and phosphorylation of
position 10. Extensive acetylation reduces the attraction between histones and DNA,
loosening the chromatin. During interphase, the euchromatin can be found dispersed
throughout the cell nucleus. It replicates during the entire duration of the S-phase of cell
division.
Procedure

In eukaryotes, chromatin exists in two primary forms based on its compaction level -
euchromatin and heterochromatin.

During interphase, the euchromatin is dispersed in the nucleus and replicated throughout
the S phase of the cell cycle.

Euchromatin is a gene-rich, less compact, and actively transcribed region of the chromatin.
When fixed and viewed under a microscope, it appears as lightly stained regions because it
can retain fewer stain particles.

In metaphase chromosomes, the lightly stained regions represent the euchromatin.

The histone tail amino acids of euchromatin are extensively acetylated. Acetylation
increases the negative charge on histone proteins, locally reducing the histone-DNA
affinity. This reduces the chromatin compaction, allowing easier access to DNA.

Heterochromatin

Overview

The extent of chromatin compaction can be studied by staining chromatin using specific
DNA binding dyes. Under the microscope, the dense-compacted regions that take up more
dye are called heterochromatin. Heterochromatin is further classified into two forms –
constitutive heterochromatin and facultative heterochromatin.

Constitutive heterochromatin: It is a highly compact region of chromatin that is mostly

concentrated in the centromere and telomere. Unlike euchromatin, the amino acid at 9th
position in histones H3 tail is di- or tri-methylated. This attracts a specialized nonhistone
protein called heterochromatin protein 1 (HP1) to the methylated site. It represents the
repressed region of chromatin. The human chromosomes 1,9,16 and Y chromosome in
human males contain a large portion of constitutive heterochromatin.

Facultative heterochromatin: This region is denser than euchromatin, but, unlike

constitutive heterochromatin, the lysine at the 27th position of histone H3 tail is di- or tri-
methylated (H3K27me3). These regions are characterized by the binding of Polycomb
repressive complexes: PRC1 and PRC2. The PRC2 domain is thought to bind first and
initiate heterochromatin formation. The PRC2 complexes contain histone deacetylases
enzymes that inhibit transcription and cause chromatin repression. In addition, the PRC2
complex also contains the catalytic domain of several histone methyltransferases
generating di-trimethyl lysines. PRC 1 then binds to the methylated nucleosomes and
condenses the chromatin into a compact structure, inhibiting transcription.

Procedure

Recall that, in eukaryotes, chromatin exists in two major forms based on its compaction
level - euchromatin, and heterochromatin.
The heterochromatin is further divided into constitutive heterochromatin, and facultative
heterochromatin.

Constitutive heterochromatin is a repeat-rich, gene-poor, and highly compacted region.

Under a microscope, constitutive heterochromatin appears darkly stained, as the
compaction allows it to take up more DNA binding dye.

A methylated histone tail characterizes constitutive heterochromatin. Methylation increases

the affinity between histones and DNA, thereby increasing the chromatin compaction and
inhibiting access to DNA.

The methylated histones are also bound by a nonhistone protein called Heterochromatin
Protein 1, which facilitates chromatin compaction and spread of constitutive
heterochromatin.

Facultative heterochromatin is a repeat-poor and gene-silent region. Under the microscope,

it also appears darkly stained due to its higher compaction. The key distinction between
facultative and constitutive heterochromatin is that the genes contained within facultative
heterochromatin regions are flexible.

For example, in one cell, the genes in facultative heterochromatin may be repressed, while
in another, the genes in the same locus may be expressed and wouldn't be stored in the
facultative state.

The facultative heterochromatin regions are often bound by a nonhistone protein called
Polycomb repressive complex 2 that can di- or tri-methylate H3 histones and contribute to
transcriptional repression.

X-chromosome inactivation in female mammals is an example of facultative

heterochromatin. Mammalian females have two X chromosomes, and males have only one.

One of the X chromosomes in females comprises highly condensed heterochromatin,

resulting in the repression of all genes present on that chromosome. This ensures that
genes on the X-chromosome of both males and females are expressed at the same level.
Under the microscope, this inactivated X chromosome appears as a Barr body - a dense,
darkly-stained spot at the periphery of the nucleus.

Chromatin Position Affects Gene Expression

Overview

Chromatin is the massive complex of DNA and proteins packaged inside the nucleus. The
complexity of chromatin folding and how it is packaged inside the nucleus greatly
influences  access to genetic information. Generally, the nucleus' periphery is considered
transcriptionally repressive, while the cell's interior is considered a transcriptionally active
area. 

Topologically Associated Domains (TADs)

The 3-dimensional positioning of chromatin in the nucleus influences the timing and level of
gene expression in eukaryotes. For example, the gene promoters are organized physically
separate from their regulatory DNA elements, such as enhancers. These promoter-
enhancer elements need to be brought together to carry out gene expressions. Each
chromatid comprises several such interacting units, termed as Topologically associated
domains (TADs). In some instances, TADs from two chromatids may also interact with
each other.

Chromosome Territories (CT)

Several TADs accumulate to form the chromosomal territories (CT). These spatial
arrangements and distributions make nucleus a heterogeneous body with distinct
biochemical activities. The positioning of genes inside the CT and the positioning of CTs
itself affects gene expression. In humans, the actively transcribed genes tend to localize
towards the periphery of their CT. Noncoding genes tend to localize towards their
CTs interior. For example, in the human female amniotic fluid cell nuclei, the ANT2 gene is
found on the inactive X chromosome. When the ANT2 gene is localized towards the
periphery of CT, it results in its active transcription.

Chromatins are dynamically repositioned inside the nucleus. Even the terminally
differentiated cells that can no longer divide exhibit chromatin or gene repositioning. This
means that the repositioning is not a random event but a coordinated molecular
mechanism.

Procedure

Gene expression is regulated at multiple levels, starting at transcription initiation and

moving all the way through to the translation of mature mRNA into a functional protein. 

However, not all of the enzymes required for gene expression and regulation are distributed
equally in the nucleus. Instead, they are limited to spatially defined foci.  This results in non-
overlapping "territories" in the nucleus, with specific biochemical activity. 

For example, the genes coding for ribosomal RNA present on chromosomes 13,14,15, 21
and 22, also known as nucleolar organizer regions, are clustered in the nucleolus - the
cell's ribosome formation site. 

This means that chromatin can be repositioned to such functionally distinct foci for
coordinated gene expression and regulation. 

However, chromatin can also extend outside its territory, forming an extended loop that can
alter the gene expression pattern.

For instance, the human gene CFTR is located at the nuclear periphery in the cells where it
is silent. However, in cells where the gene is expressed, the chromatin containing this
region is repositioned towards the interior.

Most eukaryotic cells have multiple chromatin fibers with distinct length and compaction
ratio. Therefore, the chromatin positioning also depends on the physical constraints of its
packaging inside the nucleus. 
In cells with spherical nuclei such as lymphocytes, the chromatin is radially positioned with
actively expressed genes towards the interior and the repressed genes at the periphery. 

In cells with nonspherical nuclei, such as fibroblasts, the shorter chromatin fibers tend to
occupy the internal position while the longer chromatin fibers are positioned at the nucleus'
periphery.

Chromatin repositioning is also associated with different types of cancers, where altered
gene expression patterns due to chromatin repositioning can lead to tumor formation. For
example, the repositioning of chromosome 18 from the nuclear periphery to the interior is
observed in the development of cervical and colon carcinomas.