Summary Epigenetics and Gene Editing
Chapter 4: DNA, Chromosomes, and Genomes
CHROMOSOMAL DNA AND ITS PACKAGING IN THE CHROMATIN FIBER
The Nucleotide Sequence of the Human Genome Shows How Our Genes Are Arranged
A striking feature of the human genome is how little of it (only a few percent) codes for proteins.
Nearly half of the chromosomal DNA is made up of mobile pieces of DNA that have gradually inserted
themselves in the chromosomes over evolutionary time, multiplying like parasites in the genome. A
second notable feature of the human genome is the large average gene size - about 27,000
nucleotide pairs. Only about 1300 nucleotide pairs are required to encode a protein of average size
(about 430 amino acids in humans). Most of the remaining sequence in a gene consists of long
stretches of noncoding DNA that interrupt the relatively short segments of DNA that code for
protein. The coding sequences are called exons; the intervening (non-coding) sequences in genes are
called introns. The majority of human genes thus consist of a long string of alternating exons and
introns, with most of the gene consisting of introns. In contrast, the majority of genes from
organisms with concise genomes lack introns. This accounts for the much smaller size of their genes,
as well as for the much higher fraction of coding DNA in their chromosomes. In addition to introns
and exons, each gene is associated with regulatory DNA sequences, which are responsible for
ensuring that the gene is turned on or of at the
proper time, expressed at the appropriate level,
and only in the proper type of cell. In humans, the
regulatory sequences for a typical gene are spread
out over tens of thousands of nucleotide pairs.
These regulatory sequences are much more
compressed in organisms with concise genomes.
In addition to 21,000 protein-coding genes, the
human genome contains many thousands of
genes that encode RNA molecules that do not
produce proteins, but instead have a variety of
other important functions. The nucleotide
sequence of the human genome has revealed that
the archive of information needed to produce a
human seems to be in an alarming state of chaos.
Each DNA Molecule That Forms a Linear Chromosome Must Contain a Centromere, Two
Telomeres, and Replication Origins
To form a functional chromosome, a DNA molecule must be able to do more than simply carry genes:
it must be able to replicate, and the replicated copies must be separated and reliably partitioned into
daughter cells at each cell division. This process occurs through an ordered series of stages,
collectively known as the cell cycle, which provides for a temporal separation between the
duplication of chromosomes and their segregation into two daughter cells. Briefly, during a long
interphase, genes are
expressed and chromosomes
are replicated, with the two
replicas remaining together as
a pair of sister chromatids.
Throughout this time, the
chromosomes are extended
,and much of their chromatin exists as long threads in the nucleus so that individual chromosomes
cannot be easily distinguished. It is only during a much briefer period of mitosis that each
chromosome condenses so that its two sister chromatids can be separated and distributed to the
two daughter nuclei. The highly condensed chromosomes in a dividing cell are known as mitotic
chromosomes. This is the form in which chromosomes are most easily visualized. Each chromosome
operates as a distinct structural unit: for a copy to be passed on to each daughter cell at division,
each chromosome must be able to replicate, and the newly replicated copies must subsequently be
separated and partitioned correctly into the two daughter cells. These basic functions are controlled
by three types of specialized nucleotide sequences in the DNA, each of which binds specific proteins
that guide the machinery that replicates and segregates chromosomes.
One type of nucleotide sequence acts as a DNA replication origin, the location at which duplication
of the DNA begins. Eukaryotic chromosomes contain many origins of replication to ensure that the
entire chromosome can be replicated rapidly. After DNA replication, the two sister chromatids that
form each chromosome remain attached to one another and, as the cell cycle proceeds, are
condensed further to produce mitotic chromosomes. The presence of a second specialized DNA
sequence, called a centromere, allows one copy of each duplicated and condensed chromosome to
be pulled into each daughter cell when a cell divides. A protein complex called a kinetochore forms at
the centromere and attaches the duplicated chromosomes to the mitotic spindle, allowing them to
be pulled apart. The third specialized DNA sequence forms telomeres, the ends of a chromosome.
Telomeres contain repeated nucleotide sequences that enable the ends of chromosomes to be
efficiently replicated. Telomeres also perform another function: the repeated telomere DNA
sequences, together with the regions adjoining them, form structures that protect the end of the
chromosome from being mistaken by the cell for a broken DNA molecule in need of repair. In yeast
cells, the three types of sequences required to propagate a chromosome are relatively short and
therefore use only a tiny fraction of the
information-carrying capacity of a
chromosome. Although telomere
sequences are fairly simple and short in all
eukaryotes, the DNA sequences that form
centromeres and replication origins in
more complex organisms are much longer
than their yeast counterparts. A human
centromere can contain up to a million
nucleotide pairs and is thought to consist
of a large, regularly repeating protein-
nucleic acid structure that can be inherited
when a chromosome replicates.
DNA Molecules Are Highly Condensed in Chromosomes
All eukaryotic organisms have special ways of packaging DNA into chromosomes. Chromosome 22
measures only about 2 μm in length in mitosis, representing an end-to-end compaction ratio of over
7000-fold. Tis remarkable feat of compression is performed by proteins that successively coil and fold
the DNA into higher and higher levels of organization. Although much less condensed than mitotic
chromosomes, the DNA of human interphase chromosomes is still tightly packed. Each chromosome
condenses to an extreme degree in the M phase of the cell cycle. Much less visible, but of enormous
interest and importance, specific regions of interphase chromosomes decondense to allow access to
specific DNA sequences for gene expression, DNA repair, and replication - and then recondense when
these processes are completed. The packaging of chromosomes is therefore accomplished in a way
that allows rapid localized, on-demand access to the DNA.
,Nucleosomes Are a Basic Unit of Eukaryotic Chromosome Structure
The proteins that bind to the DNA to form eukaryotic
chromosomes are traditionally divided into two
classes: the histones and the non-histone
chromosomal proteins, each contributing about the
same mass to a chromosome as the DNA. The complex
of both classes of protein with the nuclear DNA of
eukaryotic cells is known as chromatin. Histones are
responsible for the first and most basic level of chromosome packing, the nucleosome. When
interphase nuclei are broken open very gently and their contents examined under the electron
microscope, most of the chromatin appears to be in the form of a fiber with a
diameter of about 30 nm. If this chromatin is subjected to treatments that
cause it to unfold partially, it can be seen under the electron microscope as a
series of “beads on a string”. The string is DNA, and each bead is a
“nucleosome core particle” that consists of DNA wound around a histone
core. The structural organization of nucleosomes was determined after first
isolating them from unfolded chromatin by digestion with particular enzymes
(called nucleases) that break down DNA by cutting between the nucleosomes.
After digestion for a short period, the exposed DNA between the nucleosome
core particles, the linker DNA, is degraded. Each individual nucleosome core
particle consists of a complex of eight histone proteins - two molecules each
of histones H2A, H2B, H3, and H4 - and double-stranded DNA that is 147
nucleotide pairs long. The histone octamer forms a protein core around which
the double-stranded DNA is wound. The region of linker DNA that separates
each nucleosome core particle from the next can vary in length from a few
nucleotide pairs up to about 80. On average, nucleosomes repeat at intervals
of about 200 nucleotide pairs. The formation of nucleosomes converts a DNA
molecule into a chromatin thread about one-third of its initial length.
The Structure of the Nucleosome Core Particle Reveals How DNA Is Packaged
The high-resolution structure of a nucleosome core particle, revealed a disc-shaped histone core
around which the DNA was tightly wrapped in a left-handed coil of 1.7 turns. All four of the histones
that make up the core of the nucleosome are relatively small proteins, and they share a structural
motif, known as the histone fold, formed from three α helices connected by two loops. In assembling
a nucleosome, the histone folds first bind to each other to form H3-H4 and H2A-H2B dimers, and the
H3-H4 dimers combine to form tetramers. An H3-H4 tetramer then further combines with two H2A-
H2B dimers to form the compact octamer core, around which the DNA is wound. The interface
between DNA and histone is extensive: 142 hydrogen bonds are formed between DNA and the
histone core in each nucleosome. Nearly half of these bonds form between the amino acid backbone
of the histones and the sugar-phosphate backbone of the DNA. Numerous hydrophobic interactions
and salt linkages also hold DNA and protein together in the nucleosome. More than one-fifth of the
amino acids in each of the core histones are either lysine or arginine (two amino acids with basic side
chains), and their positive charges can effectively neutralize the negatively
charged DNA backbone. These numerous interactions explain in part why
DNA of virtually any sequence can be bound on a histone octamer core.
The path of the DNA around the histone core is not smooth; several kinks
are seen in the DNA, as expected from the nonuniform surface of the core.
The bending requires a substantial compression of the minor groove of the
DNA helix. Certain dinucleotides in the minor groove are especially easy to
compress, and some nucleotide sequences bind the nucleosome more
, tightly than others. This probably explains some cases of very precise
positioning of nucleosomes along a stretch of DNA. The sequence
preference of nucleosomes must be weak enough to allow other factors
to dominate, inasmuch as nucleosomes can occupy any one of a number
of positions relative to the DNA sequence in most chromosomal regions.
In addition to its histone fold, each of the core histones has an N-
terminal amino acid “tail,” which extends out from the DNA–histone
core. These histone tails are subject to several different types of
covalent modifications that in turn control critical aspects of chromatin
structure and function. As a refection of their fundamental role in DNA function through controlling
chromatin structure, the histones are among the most highly conserved eukaryotic proteins. This
strong evolutionary conservation suggests that the functions of histones involve nearly all of their
amino acids, so that a change in any position is deleterious to the cell. Eukaryotic organisms also
produce smaller amounts of specialized variant core histones that differ in amino acid sequence from
the main ones. These variants, combined with the surprisingly large number of covalent
modifications that can be added to the histones in nucleosomes, give rise to a variety of chromatin
structures in cells.
Nucleosomes Have a Dynamic Structure, and Are Frequently Subjected to Changes
Catalysed by ATP-Dependent Chromatin Remodelling Complexes
Kinetic experiments show that the DNA in an isolated nucleosome unwraps from each end at a rate
of about four times per second, remaining exposed for 10 to 50 milliseconds before the partially
unwrapped structure recloses. Thus, most of the DNA in an isolated nucleosome is in principle
available for binding other proteins. For the chromatin in a cell, a further loosening of DNA-histone
contacts is clearly required, because eukaryotic cells contain a large variety of ATP-dependent
chromatin remodelling complexes. These complexes include a subunit that hydrolyzes ATP. This
subunit binds both to the protein core of the nucleosome and to the double-stranded DNA that
winds around it. By using the energy of ATP hydrolysis to move this DNA relative to the core, the
protein complex changes the structure of a nucleosome temporarily, making the DNA less tightly
bound to the histone core. Through repeated cycles of ATP hydrolysis that pull the nucleosome core
along the DNA double helix, the remodeling complexes can catalyze nucleosome sliding. In this way,
they can reposition nucleosomes to expose specific regions of DNA, thereby making them available
to other proteins in the cell. In addition, by cooperating
with a variety of other proteins that bind to histones
and serve as histone chaperones, some remodeling
complexes are able to remove either all or part of the
nucleosome core from a nucleosome - catalyzing either
an exchange of its H2A-H2B histones, or the complete
removal of the octameric core from the DNA. As a
result of such processes, measurements reveal that a
typical nucleosome is replaced on the
DNA every one or two hours inside the
cell. Cells contain dozens of different
ATP-dependent chromatin remodeling
complexes that are specialized for
different roles. Most are large protein
complexes that can contain 10 or more
subunits, some of which bind to specific
modifications on histones. The activity
of these complexes is carefully
controlled by the cell. As genes are
