Epigenetics

First Week

DNA
Organization in
Chromosomes
Introduction
• All organisms inherit traits from their parents, which
are encoded in the succession of four bases in nucleic
acids.
• Although the sequence of bases as well as the 3D
structure of the DNA helix contribute to the expression
of traits, it is thought that the DNA sequence facilitates
trait generation by the regulated expression of genes.
• In addition, the DNA itself is organized in a chromatin
fiber that facilitates the imposition of information along
the DNA sequence. Chromatin also supports the
transduction of this epigenetic information into
regulatory processes that, in turn, affect transcription.
 Viral and Bacterial Chromosomes Are Relatively
Simple DNA Molecules

• The chromosomes of viruses and bacteria are much

less complicated than those in eukaryotes.
• They usually consist of a single nucleic acid
molecule quite different from the multiple
chromosomes constituting the genome of higher
forms.
• Bacterial chromosomes are largely devoid of
associated proteins and contain relatively less
genetic information.
• DNA in bacterial chromosomes is found to
be associated with several types of DNA-
binding proteins.
• Two, called HU and H-NS (histone-like
nucleoid structuring) proteins, are small but
abundant in the cell and contain a high
percentage of positively charged amino
acids that can bond ionically to the negative
charges of the phosphate groups in DNA.
• These proteins function to fold and bend
DNA. As such, coils are created that have
the effect of compacting the DNA
constituting the nucleoid.
• Additionally, H-NS proteins, like histones in
eukaryotes, have been implicated in
regulating gene activity in a nonspecific
way.
 Transcription in Eukaryotes Differs
from Bacterial Transcription in Several
Ways
1. Transcription in eukaryotes occurs within the nucleus. Thus,
unlike the bacterial process, in eukaryotes the RNA transcript is
not free to associate with ribosomes prior to the completion of
transcription. For the mRNA to be translated, it must move out
of the nucleus into the cytoplasm.
2. Transcription in eukaryotes occurs under the direction of three
separate forms of RNA polymerase, rather than the single form
seen in bacteria.
3. Initiation of transcription of eukaryotic genes requires that
compact chromatin fiber, characterized by nucleosome coiling,
be uncoiled to make the DNA helix accessible to RNA
polymerase and other regulatory proteins. This transition,
referred to as chromatin remodeling, reflects the Dynamics
involved in the conformational change that occurs as the DNA
helix is opened.
4. Initiation and regulation of transcription entail a more extensive
interaction between cis-acting DNA sequences and trans-acting
protein factors. For example, while bacterial RNA polymerase
requires only a σ (sigma) subunit to bind the promoter and
initiate transcription, in eukaryotes, several general transcription
factors (GTFs) are required to bind the promoter, recruit RNA
polymerase, and initiate transcription. Furthermore, in addition
to promoters, eukaryotic genes often have other cis-acting control
units called enhancers and silencers.
5. In bacteria, transcription termination is often dependent upon the
formation of a hairpin secondary structure in the transcript.
However, eukaryotic transcription termination is more complex.
Transcriptional termination for protein-coding genes involves
sequence-specific cleavage of the transcript, which then leads to
eventual dissociation of RNA polymerase from the DNA template.
6. In eukaryotes, the initial (or primary) transcripts of protein-coding
mRNAs, called pre-mRNAs, undergo complex alterations, generally
referred to as “processing,” to produce a mature mRNA.
Processing often involves the addition of a 5’ cap and a 3’ tail,
and the removal of intervening sequences that are not a part of
the mature mRNA.
DNA Is Organized into Chromatin in
Eukaryotes
• We now turn our attention to the way DNA is organized in
eukaryotic chromosomes, which are most clearly visible as
highly condensed structures during mitosis.
• However, after chromosome separation and cell division,
cells enter the interphase stage of the cell cycle, at which
time the components of the chromosome uncoil and
decondense into a form referred to as chromatin.
• While in interphase, the chromatin is dispersed throughout
the nucleus.
• As the cell cycle progresses, cells may replicate their DNA
and reenter mitosis, whereupon chromatin coils and
condenses back into visible chromosomes once again. This
condensation represents a length contraction of some
10,000 times for each chromatin fiber.
Chromatin Structure and
Nucleosomes
• As we have seen, the genetic material of viruses
and bacteria consists of strands of DNA or RNA
relatively devoid of proteins.
• In contrast, eukaryotic chromatin has a substantial
amount of protein associated with the
chromosomal DNA in all phases of the cell cycle.
The associated proteins can be categorized as
either positively charged histones or less positively
charged nonhistone proteins.
Histones
• The histones play the most essential structural
role. Histones contain large amounts of the
positively charged amino acids lysine and arginine,
making it possible for them to bond
electrostatically to the negatively charged
phosphate groups of nucleotides.
A basic model for chromatin structure was worked out in
the mid-1970s. The following observations were
particularly relevant to the development of this model:
• 1. Digestion of chromatin by certain endonucleases,
such as micrococcal nuclease, yields DNA fragments
that are approximately 200 base pairs in length or
multiples thereof. This enzymatic digestion is not
random, for if it were, we would expect a wide range of
fragment sizes. Thus, chromatin consists of some type
of repeating unit, each of which protects the DNA from
enzymatic cleavage except where any two units are
joined. It is the area between units that is attacked
and cleaved by the endonuclease.
• 2. Electron microscopic
observations of chromatin
have revealed that chromatin
fibers are composed of linear
arrays of spherical particles.
Discovered by Ada and Donald
Olins, the particles occur
regularly along the axis of a
chromatin strand and resemble
beads on a string. These
particles, initially referred to as
v-bodies (v is the Greek letter
nu), are now called
nucleosomes. These findings
conform to the above
observation that suggests the
existence of repeating units.
• 3. Studies of the chemical association between
histone molecules and DNA in the nucleosomes of
chromatin Show that histones H2A, H2B, H3, and
H4 occur as two types of tetramers, (H2A)2 . (H2B)2
and (H3)2 . (H4)2. Roger Kornberg predicted that
each repeating nucleosome unit consists of one of
each tetramer (creating an octomer) in association
with about 200 base pairs of DNA.
• 4. When nuclease digestion time is extended, some
of the 200 base pairs of DNA are removed from the
nucleosome, creating what is called a nucleosome
core particle consisting of 147 base pairs. The DNA
lost in the prolonged digestion is responsible for
linking nucleosomes together. This linker DNA is
associated with the fifth histone, H1.
 ~200 bp

digest linker DNA

147 bp
• In the nucleus, the chromatin fiber seldom, if ever,
exists in the extended form.
• Instead, the 11-nm-diameter fiber is further
packed into a thicker structure, initially called a
solenoid, but now referred to as a 30-nm fiber. This
thicker structure, which is dependent on the
presence of histone H1, consists of numerous
nucleosomes coiled around and stacked upon one
another, creating a second level of packing. This
provides a sixfold increase in compaction of the
DNA.
• It is this structure that is characteristic of an
uncoiled chromatin fiber in interphase of the cell
cycle. In the transition to the mitotic chromosome,
still further compaction must occur. The 30-nm
structures are folded into a series of looped
domains, which further condense the chromatin
fiber into a structure that is 300 nm in diameter.
• These coiled chromatin fibers are then compacted
into the chromosome arms that constitute a
chromatid, one of the longitudinal subunits of the
metaphase chromosome.
Chromatin Remodeling
• When present in several levels of compaction within
the chromatin fiber, DNA is inaccessible to interaction
with other important DNA-binding proteins.
• To accommodate these protein–DNA interactions,
chromatin must be induced to change its structure, a
process now referred to as chromatin remodeling.
• To allow replication and gene expression, chromatin
must relax its compact structure and expose regions of
DNA to these proteins, and there must also be a
mechanism for reversing the process during periods of
inactivity.
• There are unstructured
histone tails that are not
packed into the folded
histone domains within the
core of the nucleosomes but
instead protrude from it. For
example, tails devoid of any
secondary structure
extending from histones H3
and H2B protrude through
the minorgroove channels
of the DNA helix.
• The significance of histone Several of these potential chemical modifications are now
recognized as important to genetic function. One of the
tails is that they provide most well- studied histone modifications involves
potential targets along the acetylation by the action of the enzyme histone
chromatin fiber for a variety acetyltransferase (HAT). The addition of an acetyl group to
of chemical modifications the positively charged amino group present on the side
that may be linked to chain of the amino acid lysine effectively changes the net
charge of the protein by neutralizing the positive charge.
genetic functions, including Lysine is in abundance in histones, and geneticists have
chromatin remodeling and known for some time that acetylation is linked to gene
the possible regulation of activation.
gene expression.
Histone Phosphorylation/Methylation
Two other important chemical modifications are the methylation
and phosphorylation of amino acids that are part of histones.
These chemical processes result from the action of enzymes called
methyltransferases and kinases, respectively. Methyl groups can be
added to both arginine and lysine residues in histones, and this
change has been correlated with gene activity. Phosphate groups
can be added to the hydroxyl groups of the amino acids serine and
histidine, introducing a negative charge on the protein. During the
cell cycle, increased phosphorylation, particularly of histone H3, is
known to occur at characteristic times. Such chemical modification
is believed to be related to the cycle of chromatin unfolding and
condensation that occurs during and after DNA replication. It is
important to note that the above chemical modifications
(acetylation, methylation, and phosphorylation) are all reversible,
under the direction of specific enzymes.
 Dna Methylation vs Histone Methylation

Interestingly, while methylation of histones within

nucleosomes is often positively correlated with
gene activity in eukaryotes, methylation of the
nitrogenous base cytosine within polynucleotide
chains of DNA, forming 5-methyl cytosine, is usually
negatively correlated with gene activity.
Methylation of cytosine occurs most often when the
nucleotide cytidylic acid is next to the nucleotide
guanylic acid, forming what is called a CpG island.
We must conclude, then, that methylation can have a
positive or a negative impact on gene activity.
 Heterochromatin
In the early part of the
twentieth century, it was
observed that some
parts of the
chromosome remain
condensed and stain
deeply during
interphase, while most
parts are partially
uncoiled and do not
stain. In 1928, the terms
euchromatin and
heterochromatin were
coined to describe the
parts of chromosomes
that are uncoiled and
those that remain
condensed, respectively.
• Heterochromatic areas are genetically inactive
because they either lack genes or contain genes
that are repressed. Also, heterochromatin
replicates later during the S phase of the cell cycle
than does euchromatin. The discovery of
heterochromatin provided the first clues that parts
of eukaryotic chromosomes do not always encode
proteins. For example, one particular
heterochromatic region of the chromosome, the
telomere, is thought to be involved in maintenance
of the chromosome’s structural integrity, and
another region, the centromere, is involved in
chromosome movement during cell division.
• The presence of heterochromatin is unique to and
characteristic of the genetic material of eukaryotes. In
some cases, whole chromosomes are
heterochromatic. A case in point is the mammalian Y
chromosome, much of which is genetically inert. And,
the inactivated X chromosome in mammalian females
is condensed into an inert heterochromatic Barr body.
• When certain heterochromatic areas from one
chromosome are translocated to a new site on the
same or another nonhomologous chromosome,
genetically active areas sometimes become
genetically inert if they now lie adjacent to the
translocated heterochromatin.
• The position of a gene or group of genes relative to all
other genetic material may affect their expression.
Eukaryotic Genomes Demonstrate Complex Sequence
Organization Characterized by Repetitive DNA
• In addition to single copies of unique DNA
sequences that make up genes, many DNA
sequences within eukaryotic chromosomes are
repetitive in nature. Various levels of repetition
occur within the genomes of organisms. Many
studies have now provided insights into repetitive
DNA, demonstrating various classes of sequences
and organization.
• Three main categories of repetitive
sequences(Which we will discuss):
1. Heterochromatin found to be associated with
centromeres and making up telomeres
2. Tandem repeats of both short and long DNA
sequences
3. Transposable sequences that are interspersed
throughout the genome of eukaryotes.
Centromeric DNA Sequences
• The separation of homologs during mitosis and
meiosis depends on centromeres, described
cytologically in the late nineteenth century as the
primary constrictions along eukaryotic
chromosomes. In this role, it is believed that the
repetitive DNA sequences contained within the
centromere are critical to this role.
• The minimal region of the centromere that
supports the function of chromosomal segregation
is designated the CEN region.
Middle Repetitive Sequences:
VNTRs and STRs
• Although middle repetitive DNA does include some duplicated genes (such as
those encoding ribosomal RNA), most prominent in this category are either
noncoding tandemly repeated sequences or noncoding interspersed sequences.
• No function has been ascribed to these components of the genome.
• An example is DNA described as variable number tandem repeats (VNTRs).
These repeating DNA sequences may be 15–100 bp long and are found within
and between genes. Many such clusters are dispersed throughout the genome,
and they are often referred to as minisatellites.
• The number of tandem copies of each specific sequence at each location varies
from one individual to the next, creating localized regions of 1000–20,000 bp
(1–20 kb) in length. The variation in size (length) of these regions between
individual humans was originally the basis for the forensic technique referred to
as DNA fingerprinting.
• Another group of tandemly repeated sequences consists of di-, tri-, tetra-, and
pentanucleotides, also referred to as microsatellites or short tandem repeats
(STRs). Like VNTRs, they are dispersed throughout the genome and vary among
individuals in the number of repeats present at any site. For example, in
humans, the most common microsatellite is the dinucleotide (CA)n, where n
equals the number of repeats. Most commonly, n is between 5 and 50. These
clusters have served as useful molecular markers for genome analysis.
Repetitive Transposed Sequences:
SINEs and LINEs
• Still another category of repetitive DNA consists of
sequences that are interspersed individually
throughout the genome, rather than being tandemly
repeated. They can be either short or long, and many
have the added distinction of being transposable
sequences, which are mobile and can potentially move
to different locations within the genome. A large
portion of the human genome is composed of such
sequences.
• SINEs and LINEs represent a significant portion of
human DNA. SINEs constitute about 13 percent of the
human genome, whereas LINEs constitute up to 21
percent.
Middle Repetitive Multiple-Copy
Genes
• In some cases, middle repetitive DNA includes
functional genes present tandemly in multiple copies.
For example, many copies exist of the genes encoding
ribosomal RNA. Drosophila has 120 copies per haploid
genome. Single genetic units encode a large precursor
molecule that is processed into the 5.8S, 18S, and 28S
rRNA components. In humans, multiple copies of this
gene are clustered on the p arm of the acrocentric
chromosomes 13, 14, 15, 21, and 22. Multiple copies of
the genes encoding 5S rRNA are transcribed separately
from multiple clusters found together on the terminal
portion of the p arm of chromosome 1.
The Vast Majority of a Eukaryotic Genome
Does Not Encode Functional Genes
• Given the preceding information concerning
various forms of repetitive DNA in eukaryotes, it is
of interest to pose an important question: What
proportion of the eukaryotic genome actually
encodes functional genes?

• In humans, it appears that the coding regions of

the estimated 20,000 functional genes occupy only
about 2 percent of the total DNA sequence making
up the genome.