BIO353.

01 2022/23-1

BIO353.01 - Lecture 3: The structure of Chromosomes

In this section we will take a look at the structure and organization of chromosomes. Each
chromosome is a single, very long DNA molecule and eukaryotic genomes typically consist of multiple
chromosomes. If these log molecules were simply crammed into the nucleus, they would run the risk
of breaking apart, which could interfere with their function but also causes problems during DNA
replication and cell division. Therefore, DNA is not naked but organized on and around proteins to
organize chromosomes and to protect the stability of DNA. The proteins organizing DNA also provide
additional ways of regulating DNA function, such as differential gene expression in different cell types
or the activation/inactivation of transcriptional programs during specific conditions or at distinct time
points during development.

Slide 2
We have already seen that prokaryotes usually have a single circular chromosome that contains the
genomic DNA. In addition, prokaryotes often contain smaller circular DNA elements that are called
plasmids. Plasmids are not (always) essential for the life of a prokaryote under normal environmental
conditions but often provide advantages, such as resistance to antibiotics or the ability to utilize
additional energy resources, when the environmental conditions change. Molecular biologists often
use plasmids to grow and harvest specific DNA sequences for molecular cloning purposes.
Eukaryotes, on the other hand, usually have multiple linear chromosomes that contain the genomic
DNA. Yet, certain organelles, such as mitochondria and plastids in plant cells also contain their own
DNA in the form of circular DNA molecules. Mitochondria and plastids are derived from
endosymbiontic a-protobacteria, thus these circular DNA molecules are the remnants of circular
bacterial genomes. However, many genes have been transferred to nuclear chromosomes in the
course of eukaryotic evolution.

Slide 3
The DNA molecules that make up an organism’s genome are often quite large relative to the space
that they are confined to. The genomic DNA of E. coli is 4.7 Mb in size, which corresponds to a length
of about 1.600 µm (4.700.000 bp x 0.034 nm/bp = 1.598 µm). In contrast, an E. coli cell is only about 2
µm long and 1 µm wide. Thus, if you want to fit the genomic DNA into the bacterial cell, you would
need to fold it 800 times. The DNA double strand is 2 nm wide, thus the folded DNA would also be 1.6
µm high if folded as a single sheet. Thus, there is a lot of DNA to be packed into a bacterial cell and it
will have a hard time fitting without any level of organization or compaction. In addition, if the DNA was
naked, it would be less protected against mechanical and other stresses and break easily.

Slides 4 / 5
We have already seen one way of compacting DNA, which can be achieved by supercoiling. The
images show a series of increasingly supercoiled states of the same plasmid. You can appreciate that
the most supercoiled plasmid also has the most compact appearance and probably takes up the least
space inside the cell. Supercoiling also provides additional advantages for a cell, such as storing
unwound DNA, which can be easily accessed by DNA-binding proteins or during transcription and
replication.

Slides 6 / 7
The E.coli chromosome is organized into a large number of loops and coils to achieve the necessary
level of compaction. However, as we will also see for eukaryotic chromosomes, there is always a trade
off between compaction and accessibility of genomic DNA. The image on the left shows an electron
micrograph of the E. coli nucleoid squeezed out of a cell. You can identify a large number of long
loops that emerge from a central structure and then return back to it. The tips of these long loops also
appear heavily supercoiled. The structure in the center of the nucleoid looks different (darker, denser)
and is made up by different proteins that organize the chromosome as shown on the right. The names
of these proteins are not really important for now but it is important to understand what they are doing
to compact DNA without running the risk of generating a tightly constricted knot that cannot be
disentangled and in which DNA is inaccessible to transcription factors or DNA/RNA polymerases.
Some of these proteins bend the DNA, such as IHF, and this makes sense at the tips of the loops to
form hairpins (there are other functions if IHF, which we will see during phage infection of bacteria).
Other proteins may organize loops at their base or stabilize loops along the way. These proteins are
collectively called the nucleoid-associated proteins (NAPs) and serve important functions for the
efficient storage and use of DNA in bacteria. We will see that related proteins do similar things in
eukaryotic genomes. In particular, the SMC (structural maintenance of chromosomes) proteins are
also found in eukaryotes and serve an important function, not just in the organization of chromosomes
but also dynamically during gene regulation. We will return to this aspect of the function in later
chapters.

Slides 8 / 9 / 10
Eukaryotes face the same problem as prokaryotes and the space that is available to store the typically
much larger amount of DNA is even tighter. Human DNA of a single nucleus is about 2.17 m long (see
slide 10 for the calculation). A typical eukaryotic nucleus has a diameter of 6 µm into which the 2.17 m
of DNA has to fit. This is equivalent to putting a 21 km rope of 20 µm thickness into a tennis ball.

Slides 11 / 12
We often think of the genomic DNA during interphase as a bowl of spaghetti in which each
chromosome is one strand of spaghetti that is randomly distributed across the plate. However, more
recent insight into nuclear architecture revealed that individual chromosomes occupy distinct territories
within the nucleus. More like a ball made of a single spaghetti strand next to another spaghetti ball.

Slide 13
Individual chromosomes can be made visible inside the nucleus through a technique that is called
chromosome painting. It is based on hybridization of genomic DNA with differently labeled probes that
are unique for the different chromosomes. This can be visualized under a fluorescence microscope
and every chromosome appears in a unique color. The image on the left shows the fluorescence
picture of a chromosome-painted nucleus and you can see that different chromosomes occupy rather
compact and unique (largely non-overlapping) nuclear spaces. The image on the right is a cartoon of
the same nucleus in which the different chromosomes are labeled.

Slide 14
Thus, the genomic DNA in a nucleus can be broken down into distinct chromosome territories that
appear like pieces of a three-dimensional puzzle of the nuclear ball structure. In addition, the position
of different segments of each of the chromosome (e.g. chromosome ends, centrosomes, euchromatin,
heterochromatin) is also not random within each territory. Regions that are actively expressed (they
are called A compartments for reasons we will understand better in later chapters) tend to be located
more towards the inside of the nucleus, whereas regions that are not expressed (the B compartment)
are located more towards the surface of the nucleus an are attached to the nuclear envelope.
Expression of genes is correlated with open, easily accessible chromatin, repression with closed
chromatin, which is denser and less accessible. This is also the difference (in a drastic
oversimplification of matters) between eu- and heterochromatin, which we will look into in more detail
at the end of this lecture.

Slide 15
Another macroscopic level of organization of chromosomes occurs with the distribution of some of the
general but important sequences that can be found on every chromosome, such as the telomeric ends
and the centromeres at which the mitotic spindle attaches. Both regions do not contain expressed
genes, are heterochromatic, and cluster on the surface of the nucleus in the B compartment.
Interestingly, telomeres and centromeres of different chromosomes have a tendency to be located
within the same larger region of the nucleus.

Slide 16
In a summary then, individual chromosomes occupy distinct nuclear territories and A and B
compartment segregate between the inside and the surface of the nucleus. The B compartment is
actively recruited to the cytoskeleton underneath the nuclear envelope, which is rich in the structural
protein lamin. Attraction occurs through lamin/lamin-receptor interaction and is a dynamic process.
Genes which are actively turned on or off many times shuttle between different positions based on
their expression status. Genes that are turned off get recruited to the nuclear lamina, while genes that
are turned on detach from the lamina and leave the lamin-associated domain.
Slide 17
Each chromosome is a mixture of eu- and heterochromatic regions. Active gene expression (mostly)
occurs in decondensed, open euchromatin, whereas gene expression is (usually) silenced/suppressed
in the dense structure of heterochromatin. The order and extent of eu- and heterochromatin regions
can be different for the same chromosome in different cell types of the same organism and is
correlated with the difference in distinct transcriptional programs in different cells. However, certain
regions of all chromosomes are always found to be compacted in heterochromatin. These ‘constitutive
heterochromatic’ regions are the telomeres at the chromosome ends and the centromeric regions.
Regions that are heterochromatic in some cells but not in others (or under different conditions/at
different time points), are called ‘facultative heterochromatin’. Thus, chromosome regions can change
between these two states in a dynamic and controlled way, which is one of the most important
mechanisms for the control of gene expression.

Slide 18
The difference between eu and heterochromatin can even be seen under the microscope when
chromosomes are stained with certain dyes (Giemsa stain in this example). Heterochromatin, because
of its higher density, appears darker than the loose euchromatin. If you follow a single chromosome on
this example, you can see a banding pattern of alternating eu- and heterochromatic regions. The
centromeres (labeled with CEN) are also highly heterochromatic, whereas the nucleolus (NU) is a
region of high transcriptional activity and appears unstained.

Slide 19
The difference between eu- and heterochromatin can also be seen in interphase chromosomes under
the electron microscope. Heterochromatin appears much darker. You can see that many of the dark
regions are associated with the nuclear lamina, similar to what I showed you before. The nucleolus (n)
in the center of the nucleus is also dark but for a different reason. If you compare nuclei between male
and female cells, you can also consistently identify an additional dark spot in the female nucleus,
which is the Barr body (arrows in the bottom figure on the right). The Barr body is a condensed X
chromosome in female cells that is almost completely packed in a heterochromatin state. Because
females have two X chromosomes, one of them is inactivated as a measure of dosage compensation,
i.e. preventing that twice the amount of of X-linked genes is expressed in female cells relative to male
cells.

Slide 20
The centromere is the region on the chromosome around which the spindle microtubules attach during
cell division. Even before the true difference between eu- and heterochromatin was known, some
observations had been made that are a consequence of the density of heterochromatic structures
around the centromeres. Centromeres appear as constrictions in the overall structure of the
chromosome pairs (the center of the X shape) and stain darker with certain dyes (C banding, like in
slide 20 but with less dye to preferentially show the most heterochromatic regions). The centromeres
are also regions of repressed recombination during meiosis (remember Thomas Hunt Morgan from
lecture 1), which is a consequence of their densely-packed DNA structure.

Slides 21 / 22
Centromeres are specific DNA sequences found once on each chromosome at which the mitotic
spindle attaches during cell division.

Slide 23
It is essential that each chromosome has exactly one centromere. If a chromosome would have two
centromeres, microtubules from different spindle poles could attach to the same chromosome and
eventually tear it apart during anaphase. Chromosomes that do not have any centromere would not be
directed to any of the two daughter cells and eventually be distributed randomly or not at all, resulting
in aberrant numbers of chromosomes (= aneuploidy).

Slide 24
Even though they are closely related in function, the centromere and the kinetochore are not the same
thing. The centromere refers to a specific section of a chromosome, thus it is a DNA structure with a
defined sequence. The kinetochore describes the protein complexes that recognize and bind to the
centromere DNA and to which the spindle microtubules attach. The kinetochore consists of two parts,
the inner and the outer kinetochore, which bind to the centromere and the spindle, respectively.
Slides 25 / 26
Because the centromere serves such a crucial function in every cell of every organism, one would
expect that the centromere is a perfectly structured DNA region with a defined sequence or
arrangement of sequence elements that are identical between chromosomes of the same cell but also
between different organisms. Surprisingly, however, this is not the case, and centromeres differ largely
between organisms but also between chromosomes. In the yeast S. cerevisiae (Brewer’s yeast), the
centromere is a short sequence of 125 bp with a distinct signature of DNA elements (centromere DNA
element, CDE I – III) to which certain proteins can bind. Interestingly, in the closely related fission
yeast (S. pombe), the centromere is about 500 – 800 times larger and has a much less defined
organization.

Slide 27
The centromeres of vertebrates are even larger and span extensive regions of the chromosomes that
can be up to several Mb in size. This is really astonishing, especially since all of these very diverse
sequences essentially do the same job in a cell and as the yeast example shows, only 125 bp are
really necessary to establish a functional centromere.

Slide 28
In humans the centromeres are very large regions that are made of repeated DNA sequences.
Several related short sequences of 171 bp that share about 50% sequence identity between each
other form monomers (differently colored arrows) that aggregate into larger structures, so called
higher order repeats (HOR; rainbow structure on top). These HORs are anywhere between 1 and 3 kb
in size and share 90% of sequence identity between each other. They are also arranged into arrays,
the alpha satellite arrays, that range between 200 kb and 5 Mb in size on different chromosomes. The
cores of HORs are flanked by somewhat random arrangements of monomers at each side, which is
densely packed and referred to as the pericentromeric heterochromatin.

Slide 29
This slide shows an overview comparison of the centromeres of the 24 different human chromosomes.
First, you can see that centromeres do not always fall into the center of the chromosome but can be
quite eccentric and are located close to the telomeres in some chromosomes. Human centromeres
are either roughly in the middle of the chromosome (M: metacentric), shifted towards one
chromosome arm (sM: sub-metacentric, or close to one end (Ac: acrocentric. You can also see that
the centromere size is unique for each chromosome and that the range of sequence covered is very
variable, so is the number of different HORs that form the centromere. There is no obvious
relationship between the size of the centromere and the overall size of the chromosome on which it is
found.

Slide 30
Here you can see the relationship between the centromere (a DNA structure) and the kinetochore (a
massive protein complex). The inner kinetochore is made from several proteins that are called
centromeric proteins (CENP-B to CENP-X). some of these proteins interact with DNA. A specific
centromeric protein (CENP-A) is part of nucleosomes (DNA-organizing proteins consisting of histones)
around the centromere. All of the proteins of the inner kinetochore structure are required for the
interaction with centromeric DNA, while the outer kinetochore includes proteins that make a link
between the inner kinetochore and the spindle microtubules.

Slides 31 / 32
There are essentially three different types of centromeres based on how much DNA they include.
When they are small as in S. cerevisiae, they are called point centromeres and only a single
microtubule can attach to the structure. Larger centromeres, such as the human structures described
in the previous slides, are called regional centromeres. Yet, there are certain species, in which the
entire chromosome can function as a centromere (holocentromere; e.g. the roundworm and common
laboratory model C. elegans).

Slide 33
An important class of proteins that organize chromosomal DNA and which are involved in DNA looping
in eukaryotes are the structural maintenance of chromosome (SMC) proteins (compare to the
nucleoid-associated proteins introduced earlier). Proteins that belong into this group are cohesin and
condensin, which both have function during compaction of metaphase chromosomes and
chromsosome segregation in anaphase. They are also important in the organization of interphase
chromosomes but are less abundant during these times. The SMC proteins are dimers that form a
ring-like structure that opens around a hinge and can close around two DNA molecules. The opening
and closing of SMC proteins depends on ATP binding and hydrolysis, respectively, and the complexes
work much like a bike lock to tether two DNA double helices together.

Slide 34
The images at the bottom show electron micrographs of open and closed SMC proteins.

Slide 35
Cohesin is a protein that is important in holding duplicated sister chromatids together during
replication. It is essential that the sister chromatids stay together as a pair before mitosis to ensure
even segregation into the two daughter cells.

Slide 36
An important change that occurs during the cell cycle is that the relatively loose organization of
interphase chromosomes changes to form condensed and bulky metaphase structures. Metaphase
chromosomes adopt the characteristic X-shaped configuration raising the question as to what holds
metaphase chromosomes together and gives them their shape.

Slide 37
Ulrich Laemmli and James Paulson investigated this question by depleting chromosomes of histone
proteins, which are abundantly associated with DNA during all phases of the cell cycle. They found
that the center of the chromosomal axis contains a meshwork of proteins, which can be seen as the
darker, web-like structures that are surrounded by DNA loops in the right image.

Slide 38
When they separated the proteins on a gel, they found that most of the non-histone proteins
associated with DNA are SMC proteins, along with Topoisomerases. Removal of SMC proteins
resulted in fuzzy-looking and less organized chromosome structures (condensin off).

Slides 39 / 40 / 41
Besides the organization of nuclear DNA that we looked at earlier in this lecture, there is also a
hierarchical organization of the DNA itself. The DNA double strand itself is 2 nm in diameter. This DNA
associates with nucleosomes to form chromatin that looks like beads (= nucleosomes) on a string (=
DNA). Nucleosomes are roughly 11 nm in diameter, thus this arrangement is called the 11 nm fiber.
DNA organized on nucleosomes adopts a higher order three-dimensional structure by folding back
and forth with a diameter of 30 nm. This 30 nm fiber than folds into multiple loops to form the
chromosome structure that is organized on the protein skeleton.

Slides 42 / 43
These images demonstrate the loop structure of DNA again in two different views (lateral, vertical) but
it is obvious that the loops are organized in a radial fashion and are anchored to the protein core.

Slide 44
Even though cohesin and condensin have very similar structure, they contribute to different aspects of
DNA compaction and coherence. Cohesin largely holds the two sister chromatids together following
DNA replication, whereas condensin largely holds DNA of the same chromosome together.

Slide 45
During anaphase of the cell cycle, cohesion is actively cleaved to release the sister chromatids, while
condensin stays intact to keep the individual chromosomes condensed during their migration towards
the spindle poles.

Slide 46
The process of sister chromosome cohesion is slightly different during mitosis and meiosis. During
meiosis the homologous chromosome pairs align to form tetrads. These structures are largely held
together through cross over events (= interchromosomal recombination). Cohesin only contributes little
to the adhesion between homologous chromosome pairs and does not get cleaved completely during
the first meiotic division. Complete cohesin cleavage occurs during the second meiotic division, which
closely resembles mitosis but with only half the number of sister chromatid pairs.

Slides 47 / 48
Both the 11 and the 30 nm fibers depend on the presence and arrangement of nucleosomes, which
we will discuss next. The higher order structures depend largely on SMC proteins but also additional
proteins (e.g. Topoisomerase, CNTF, YY1) that we will meet later during this course. The function of
Topoisomerase II is obvious. Since it can pass DNA double strands through other DNA double
strands, it has an essential role in loop formation and maintenance. CNTF is essential in defining
which DNA sequences form the stem of a loop and will associate closer together in the nucleus. Thus,
CNTF brings DNA regions that are far apart from each other on the same chromosome closer together
to form structures that are called TADs (topologically-associated domains). TAD structures and intra-
TAD loops are important organizing principles that define gene expression and chromatin states
(i.e.eu- vs heterochromatin). We will talk more about this in later chapters.

Slide 49
Nucleosomes organize the DNA into the 11 and 30 nm fiber as we have seen above. They are
structures formed by a nucleosome core particle and the DNA that wraps around it. The core particle
consists of histone proteins that interact with the DNA to attach it tightly to the nucleosome. We will
see that histone proteins of neighboring nucleosomes also interact with each other and thereby can
change the accessibility of DNA. Each core particle organizes exactly 147 bp of DNA and neighboring
nucleosomes are connected through linker DNA of variable length. The minimum length is around 20
bp linker DNA but can be much larger in some cases.

Slide 50
There are a total of 5 members of the histone protein family. The H2A, H2B, H3, and H4 histones form
the nucleosome core particles, while the H1 histone attaches to the assembled core particle and the
linker DNA. H2A, H2B, H3, and H4 are therefore also called the ‘core histones’ and H1 the ‘linker
histone’. All histone proteins have in common that they contain a large percentage of basic amino
acids, such as lysine and arginine, in their structure. These amino acids carry positive charges at
neutral pH and interact with the negative backbone of the DNA to tightly bind it to the core particle.
The core histones all have a common protein domain structure with a conserved histone fold domain
that includes 3 a-helices linked by flexible loops. The N-terminal domains are more variable but have
in common that they contain a large number of amino acids that can be subject to post-translational
modifications, such as methylation, phosphorylation, and others.

Slide 51 / 52
The nucleosome core particles are protein complexes composed of two H3/H4 dimers and two
H2A/H2B dimers. The dimers form by interaction of the middle helix of the histone fold domain. Both
the H3/H4 and the H2A/H2B dimers form tetramers and two of these tetramers form the octamer
structure of the nucleosome. The result is an overall protein that has the appearance of a flat disc in
which H3/H4 double dimers form one half and the H2A/H2B double dimers the other (top and bottom
half in the example shown; follow the colors to see better). The flexible N-terminal histone tails stick
out from the nucleosome structure and point into all directions. The core particle binds to DNA by
interaction of one of the other histone fold domains of each histone protein. This interaction results in
wrapping of the DNA 1.65 times around the nucleosome structure in a left-handed toroid (= negative
supercoiling).

Slides 53 / 54 / 55
Early research was largely focused on the relationship between nucleosomes and DNA. An
informative experiment was to digest nucleosome-bound DNA with a small amount (= low activity) of a
DNA-digesting enzyme (DNase) and to run these fragments on a gel. The enzyme can only cut the
DNA between two nucleosomes but the DNA that is wrapped around the nucleosome itself is
protected and does not get cut. The procedure often created DNA fragments that were multiples of
200 bp, suggesting that the distance between two nucleosomes is somewhere in that range. The idea
is that the amount of enzyme is so low that it cuts DNA rarely and randomly makes only one or no cut
between adjacent nucleosomes. The resulting DNA fragments correspond to one (200 bp), two (400
bp), or any other number (n x 200 bp) of nucleosomes. However, when more enzyme is used, only
one band could be detected, because now the enzyme is not limiting anymore and cuts between all
nucleosome. However, the size of the resulting DNA band is smaller than the 200 bp and runs around
147 bp. In this scenario, the DNase could digest all the accessible DNA within the linker DNA but not
the DNA associated with the core particle. The conclusion from these experiments was that the core
particle binds 147 bp of DNA and that the average linker size is around is 53 bp.

Slide 56
This basic principle is conserved in all organisms. The average linker length may be different but if you
do the math and subtract the linker length from the nucleosome repeat length, you will always end up
with 147 bp. This length is defined by the structure of the histone proteins, which are highly conserved
in evolution and therefore function identically even in vastly different organisms.

Slide 57
H2A and H2B proteins spontaneously form dimers by interacting through the middle helix of the
histone fold domain. H3 and H4 do the same but the dimers also spontaneously form tetramers
through their C-terminal helices.

Slide 58
Nucleosomes form through the interaction of an H3/H4 tetramer with DNA. Then, two H2A/H2B dimers
are loaded successively into the structure to complete the nucleosome. There are specific helper
proteins (histone chaperones) that assist in histone dimer loading (and exchange) to DNA.

Slide 59
The complete structure appears similar to a flat disk with the histone-fold regions in the center of the
structure. The N-terminal tails are flexible and move freely around the DNA/histone complex and are
sensitive to protease digestion.

Slide 60
This slide depicts a more detailed view on the atomic structure of the DNA / histone complex from
different viewpoints.

Slide 61
The H3/H4 tetramer (top, blue and dark green) binds to around 60 bp in the center of the DNA that
wraps around the nucleosome and the first and last 13 bp that enter and leave the nucleosome. The
H2A/H2B tetramer also associates with 60 bp of DNA from the left and right sequences of the middle
of the DNA. If you do the math, this amounts to 146 bp and suggests that each and every base pair of
the DNA that wraps around the nucleosome is tightly and directly associated with histones.

Slide 62 / 63
I have already introduced the difference between the major and minor groove of DNA and we have
seen that the minor groove looks fairly similar regardless of the actual DNA sequence. Thus, the
information content of the minor grove is largely sequence unspecific. Therefore, it is not surprising
that histones almost exclusively interact with the minor groove. Thus, the nucleosome can bind to any
DNA no matter what the sequence actually is, while at the same time sequence-specific DNA-binding
proteins can interact with the major grove (some, not all of them).

Slide 64
As I already mentioned, the tails are not hidden in the structure but present themselves to the outside
of the nucleosome (shown in grey). They emerge from the core of the disk but also from between the
organized DNA.

Slide 65
We started this chapter by considering compaction of DNA. The question then is, what do
nucleosomes contribute to DNA compaction? On this slide you can see a viral DNA (these viruses live
in eukaryotic cells and can substitute for a mini-chromosome in this experiment) with and without
nucleosomes (deproteinized). The compaction that is achieved through histone proteins is very
obvious.

Slide 66
In these early experiments, researchers also investigated the effect of ionic strength on compaction in
the presence of histone proteins. They could see that the DNA/histone structure is somewhat dynamic
and adopts its most compact form when the ion concentration was high. Of course, cells do not
change their ion concentration to regulate DNA compaction but the experiment suggests that such
regulation is possible. We will see that the histone tails can be modified by post-translational
modifications (addition of methyl, acetyl, or phosphate groups) to achieve a similar dynamic regulation
of DNA compaction and accessibility.

Slide 67
A first indication that histones change DNA compaction came from analysis of the contribution of the
H1 histone to the overall structure. H1 is not directly a part of the core particle but binds to the
nucleosome proteins and the DNA in the center of the core particle as well as the DNA that enters the
nucleosome. Essentially, H1 acts like sticky tape that attaches an additional number of base pairs to
the nucleosome and changes the angle at which the DNA meets the core particle.

Slides 68 / 69
The presence of histone H1 changes the appearance of the chromatin and forces it to change from
the 11 nm to the 30 nm fiber. This is largely due to the fact that the angle between the DNA that meets
the nucleosome and the core particle is more restrained, which forces the overall structure to become
more zig-zag like. There are different models as to what the exact organization of nucleosomes and
DNA is like in the 30 nm fiber (solenoid vs zig-zag) but most likely, it is either or and depends on other
influences.

Slide 70
The freely moving tails of neighboring nucleosomes can interact with each other to affect the
shape/compactness of the overall structure and to hold neighboring nucleosomes together more
tightly. This interaction can be regulated and can result in changes in the local compaction of DNA.
This change is at the heart of the difference between eu- and heterochromatin. Essentially,
heterochromatin is DNA that is organized on nucleosomes that are stuck together more tightly through
their histone tails, while nucleosomes interact less with each other in euchromatin.

Slide 71
The genes coding for histones are somewhat unusual in many aspects. First of all, they did not
change significantly even over very long evolutionary times. The protein sequences of H4 in the cow
and the pea differ only in two amino acids, which are synonymous substitution (i.e. the two amino
acids have related properties). Thus, H4 has not really changed much (or at all) in the past 1.6 billion
years of evolution since the plants split from animals, a level of conservation that is not observed in
any other gene.

Slide 72
Normally there is only one gene coding for a specific protein or variants of it. Sometimes genome
duplication results in a second copy of the gene that can produce a protein with related function.
Genes coding for histones are multicopy genes that are found on 7 of the 24 different human
chromosomes and often appear in clusters with multiple copies for each gene (on chromosome 6 in
this case; the numbers indicate the numbers of genes coding for the particular histone).

Slide 73
A typical eukaryotic gene contains introns and the spliced mRNA ends with a poly A tail. Histone
genes are intronless and the mRNA ends with a stem loop, somehow resembling transcription
termination in some prokaryotes. This streamlined structure allows for rapid transcription and
translation of histone proteins. Essentially the amount of histone protein needs to be rapidly duplicated
along with the DNA during replication and cell division.

Slide 74
Histone genes appear in clusters with variable number of gene copies. In addition, certain variants
exist that have rather specific functions. An important H3 variant is CENP-A, which has functions in
the centromere (more later) and H2A.X, which plays a role in DNA repair.

Slide 75
In this experiment you can see two nuclei that were labeled for the unphosphorylated and
phosphorylated form of H2A.X in normal and DNA-damaged nuclei (etoposide). You can see that both
H2A.X occupancy of DNA and phosphorylation of H2A.X increase when DNA is damaged. We will
look more into that when we talk about DNA repair mechanisms but phosphorylated H2A.X serves as
a mark around DNA damage sites and recruits the DNA repair machinery to these sites. You can
appreciate that some DNA damage may exist even in normal nuclei, which undergo constant
maintenance and repair of broken DNA.

Slide 76
Earlier in this lecture, we had a look at the centromere structure of different organisms and were
somewhat surprised by the fact that centromere sequence can be so variable despite the important
conservation of its function.

Slides 77 / 78
Essentially, the CENP-A (= centromeric protein A) variant of histone H3 can substitute for H3 in some
nucleosomes. Like H3, it forms dimers with H4 and tetramers as higher order structures. A small
difference may be that DNA is not so tightly bound to the nucleosome core at the beginning and the
end of the DNA, which gives the nucleosomes a somewhat looser configuration and more flexibility.

Slide 79
CENP-A is almost exclusively found in nucleosomes around the centromere. In the point centromere
of the yeast S. cerevisiae, a single CENP-A-containing nucleosome is found that is associated with the
conserved CDE sequence. In contrast, regional centromeres contain multiple CENP-A nucleosomes
interspersed with regular nucleosomes that contain H3 instead of CENP-A. Thus, occupancy with
CENP-A nucleosomes describes centromeres better than specific DNA sequences. In fact, the
centromere is an epigenetically propagated structure that is defined by CENP-A nucleosomes.
Regions that contain CENP-A will maintain this histone variant after DNA replication and forcing
occupancy of CENP-A at other regions of a chromosome establishes a second centromere.

Slides 80 / 81
CENP-A has a flexible tail that interacts with other proteins of the inner kinetochore and most (not all)
of the interaction between the kinetochore and centromere DNA occurs through CENP-A.

Slide 82
Nucleosomes provide compaction and protection of chromosomes, which is an advantage but, of
course, tightly packed DNA is less accessible than open and undecorated DNA. The question then is,
what defines chromatin states and how can chromatin states be changed when the expression of
genes is changed within a cell.

Slide 83
Nucleosome occupancy of DNA generally has an impeding effect on transcription. In this gel image,
you can see an in vitro transcription assay (= in the test tube) and the effect of histones / nucleosomes
on transcription. The black bands indicate the amount of transcript (mRNA) that can be made from the
same gene/promoter in the presence of different concentrations of histones. We have seen that one
core particle associates with roughly 200 bp of DNA (147 bp + linker DNA). The numbers given for
core histones indicate that ratio. Thus, a number of 1 means a stoichiometry of 1 complement of core
histones for every 200 bp of DNA. You can see that increasing the histone concentration to that level
strongly inhibits transcription.

Slide 84
The same is true for histone H1. If the concentration of H1 is increased to a ration of slightly more than
one H1 molecule per nucleosome, transcription is suppressed.

Slide 85
How can these opposite effects of nucleosomes now be understood in the context of DNA inside a
cell? On one hand they provide protection and compaction, on the other hand, they make DNA less
accessible. You can compare the nucleosome system to these archiving shelves, which allow you to
store a lot of information but in order to access information that is hidden deep inside the archive (=
chromosomal DNA), you have to slide some of the shelves out of the way. The same thing happens
with nucleosomes. To make specific DNA regions accessible, the nucleosomes need to be moved
along the DNA molecule or evicted from the DNA.
Slide 86
One way to look at the problem is that nucleosomes and DNA-binding proteins (e.g. transcription
factors, RNA polymerase, etc.) compete for access to DNA. If for instance two protein binding sites on
the DNA are wrapped around a nucleosome, the binding by the protein (e.g. the transcription factor)
may be hindered or impossible. However, nucleosomes may spontaneously roll around the DNA
(actually, the DNA rolls around the nucleosome) and one of the two binding sites may be exposed. If a
protein binds, the nucleosome cannot roll back and eventually continues to roll away from the binding
site so that the second site also becomes nucleosome free and an additional protein can bind to DNA.
While nucleosomes may slide spontaneously along the DNA, often the rolling process is driven by
specific enzymes that are called nucleosome remodeling complexes.

Slide 87
These nucleosome remodeling complexes can, in principle, do three different things. They can roll
nucleosomes and make binding sites accessible in an energy-consuming process as we have seen on
the previous slide. They can also eject whole nucleosomes, which has the same effect on exposing
binding sites on the DNA but can also result in opening (strand separation) of DNA because, as we
discussed earlier, nucleosomes store negative supercoiling. Other remodeling complexes may
facilitate the exchange of histone dimers, as we have seen for H2A.X that is incorporated around sites
of DNA damage. Not all complexes have all three functions or are active in all contexts.

Slide 88
We have previously discussed the concept of eu- and heterochromatin. Nucleosome remodeling
complexes play important roles in changing regions of chromosomes between these two states. Thus,
chromatin is more dynamic than assumed and changes in chromatin structure is an important
mechanism in controlling gene expression. An important question than is what determines the
recruitment of the remodelling complexes to specific DNA sequences under a given circumstance.

Slide 89
Table 8-6 lists some of the more important nucleosome remodeling complexes and their properties.
The SWI/SNF complex (also called BAF or PBAF) for instance can slide nucleosomes and exchange
dimers (here called transfer). They also have protein domains (bromo-, chromodomains), which
recognize specific posttranslational histone modifications, such as phosphorylation, acetylation, or
methylation, as we will see later.

Slide 90 / 91
If we revisit the experiment that was shown on slide 65 and look with a little more detail, we can see
that certain DNA regions can be entirely free of nucleosomes. These nucleosome-free regions can
occur in two different ways. Either nucleosomes have some (even low) preference for specific DNA
sequences or something else is going on. It could be that some DNA regions are occupied by
proteins, which prevents nucleosomes from forming around (or rolling into) these protein-bound
sequences. The nucleosome-free stretches that you can see in this experiment are the replication
origins of the SV40 viral DNA, which is bound by proteins.

Slide 92
Some DNA sequences, however, are more prone to accommodate nucleosomes than others. Not all
base pairings have the same effect on DNA and may locally distort the 3-dimensional shape of the
linear structure. AT-rich sequences bend the DNA molecule inwards, GC-rich structures have the
opposite effect. Thus, DNA with alternating AT- and GC-rich sequences will already adopt a bent
curvature that can easily accommodate a nucleosome and my make it more difficult for the
nucleosome to roll around freely.

Slide 93
Nucleosomes can also be prevented from binding to DNA by steric hindrance. If, for instance, two
proteins are already bound to DNA and the distance between those two binding sites is shorter than
the 147 bp that wrap around a nucleosome, the nucleosome cannot fit into this sequence. There are
also proteins that anchor nucleosomes into specific positions by simultaneously binding to a specific
DNA sequence and to the core particle.
Slides 94 - 98
How can the distribution of nucleosomes around specific DNA regions be analyzed and investigated
experimentally? We already said that the nucleosomes prevent DNA cleavage enzymes (e.g. DNase
or micrococcal nuclease = MNase) from cutting the DNA strands. If an excess of enzyme is used, the
linker DNA between two nucleosomes will be broken down into a large number of very small
fragments. The DNA bound to nucleosomes will create larger fragments of a minimum size of 147 bp.
The fragments can be sorted by size and the large fragments that correspond to nucleosome positions
can be aligned to the genome. Regions that always contain a nucleosome in every cell will create
more of the large fragments, whereas regions with variable nucleosome positions will create randomly
sized fragments if the sample is prepared across many individual cells.

Slide 99
If we investigate nucleosome occupancy around a typical eukaryotic gene, we can see that the
nucleosomes are not randomly distributed along the gene sequence. Nucleosomes that cover early
sequences in the coding region of the gene, usually occur in fixed positions and are ‘phased’. The
nucleosome distribution becomes more random at later positions within the gene body. Nucleosome-
free regions occur around the promoter just upstream of the transcription start site (TSS) and at the
end of the gene. These regions are covered by proteins, for instance the general transcription factors
around the promoter that prevent nucleosome occupancy. In actively transcribed genes fixed
nucleosomes (the +1 and -1 nucleosomes) can be found downstream and upstream of the RNA
polymerase binding site. Because these nucleosomes are fixed, the neighboring nucleosomes also
appear fixed (= phased).

Slide 100
The nucleosome distribution around the TSS has significance. It is usually found in expressed genes
(red curve) but not in genes that are not expressed and is part of the finer details of gene regulation.
Many promoters that are not actively transcribed are also occupied by RNA polymerase and show
fixed/phased nucleosomes. This indicates that genes are poised to initiate transcription when
additional signals are present inside the cell.

Slides 101 / 102

Because nucleosomes prevent DNA-cutting enzymes from interaction with DNA, nucleosome-fee
regions are more sensitive to DNA-digesting enzymes, such as DNase I. These hypersensitive
regions, if they occur outside of gene coding sequences, often have important regulatory function. The
genes that code for the alpha and beta subunits of hemoglobin are good examples for that.
Remember that hemoglobin is a tetramer made from two alpha and two beta subunits. Yet, there are
multiple alpha and beta genes in the genome that occur in clusters. For instance there are multiple
beta isoforms (epsilon, gamma, delta, beta). The genes that are transcribed to express these isoforms
change over development. In the early embryo the epsilon gene is expressed, followed by gamma,
and beta after birth.

Slide 103
The sequence upstream of the gene cluster contains 5 DNase-hypersensitive sites that only occur in
blood cells, thus, those cells that actually express hemoglobin, but not in other cell types (non-myeloid
cell line). The occurrence of smaller bands is indicative of the presence of hypersensitive sites. These
sites are located in an important regulatory sequence, the locus control region, which is a complex
enhancer that regulates the coordinated expression of beta isoforms.

Slides 104 / 105

So far, we have only considered the core particle of the nucleosomes. Yet, all histone proteins also
have extended N-terminal tails that are rich in amino acids that can be modified by prost-translational
modifications. The second slide gives an overview of the lysine (K), serine (S), threonine (T), and
arginine (R) residues that can be phosphorylated, methylated, acetylated, or ubiquitinylated. Not all of
these modifications occur at the same time and the specific modification pattern serves as a histone
code that has important regulatory function.

Slide 106
To give you an example, some lysine residues can be acetylated but the identity of the acetylated
residues is important and acetylation at these sites can have different meaning, For instance,
acetylation of lysine 5 (= K5) and 12 on histone H4 marks newly synthesized protein, whereas
acetylation of K8 and 16 is enriched in histone H4 that is part of nucleosomes positioned around the
transcription start site.

Slide 107
Essentially the same is true for methylation. Methylation of K4, K36, and K79 is usually found in
nucleosomes associated with actively transcribed genes, while K9 and K27 methylation is usually a
repressive mark. The modification pattern of histone tails, thus, can serve as a marker for a particular
chromatin state (expression, repression, position along the gene, and others). The important question
then is, whether the histone marks are a consequence of the chromatin state or if they contribute to
changing chromatin behavior.

Slides 108 / 109 / 110

Specific histone marks can also occur during particular phases in the life of a cell, such as during cell
division. Serine 10 on histone H3 becomes phosphorylated during chromosome compaction and
subsequent cytokinesis and is often used as a marker for actively dividing cells.

Slides 111 / 112

Different enzymes/proteins generate (writers), remove (erasers), or recognize (readers) specific
histone marks. There are also enzymes that recognize specific histone marks and generate similar
signatures on neighboring nucleosomes (reader/writer).

Slide 113
It is important to distinguish histone/chromatin modifying proteins from chromatin remodelling
complexes, which have different function. Chromatin modifiers create or remove histone modifications,
whereas remodeling complexes change nucleosome structure along DNA.

Slide 114
The modification marks are generated and can also be removed by specific enzymes. Histone acetyl
transferases (HATs) transfer acetyl groups, which can be removed by histone deacetylases (HDACs).
Similarly histones can be phosphorylated by histone kinases and dephosphorylated by histone
phosphatases. Not every HAT or histone kinase can act on each of the potentially available residues
within the histone tails but different enzymes exist that have substrate (amino acid residues on specific
histones in this case) specificity. The transfer of chemical groups not only changes the structure of the
amino acid but also changes the electrical properties of the histone tails. Acetylation changes the
positively charged amino group of lysine into an uncharged residue. Similarly phosphorylation adds a
negative charge to the amino acid. This change in electrical properties, by itself, can have profound
effect on nucleosome behavior.

Slide 115
Methylation by histone methyl transferases (HMTs) does not change the charge on the methylated
lysine and the residue remains positively charged. Different from the other modifications, however,
lysine can be mono-, di, or trimethylated. You may have guessed already, the number of methyl
groups attached to lysine matters and is another layer of complexity of the histone code and specific
lysine methylases eventually only catalyze one of the methylation steps.

Slide 116
The same is true for arginine, which can also be mono- and di-methylated without changing the
positive charge. Differently from lysine, however, the two terminal nitrogens of the guanidino group
can be methylated separately resulting in different methylation patterns.

Slide 117 / 118 / 119

What is the effect of changes in the histone marks. In its unmodified form, the positively charged tails
of the histones associate with the negatively charged backbone of the associated DNA and also
between different nucleosomes. This results in a tighter attachment of the DNA onto the nucleosome
abut also a higher compaction of neighboring nucleosomes. Acetylation eliminates some of the
positive charges and the histone tails detach. Thus, as an effect, the DNA is less tightly bound to the
histones and the nucleosomes are sticking to each other less tightly. This is an important step to make
the histone bound DNA more accessible. The acetylated residues on the histone can be recognized
by other proteins / protein complexes through bromodomains. Thus, specific protein complexes (e.g.
nucleosome remodeling complexes) can be recruited to acetylated nucleosomes.
Slide 120
Methylation does not have such a profound effect on the flexibility of the histone tails but the specific
marks can be detected (= read) by other protein complexes that contain chromo-domains that interact
with methylated residues on histone tails. Again, these chromodomain proteins can be part of larger
protein complexes that change nucleosome occupancy and DNA compaction.

Slide 121
Thus, the occurrence of specific posttranslational histone modifications is an important early step in
the transition between eu- and heterochromatin. Chromatin remodeling complexes can be recruited to
specific nucleosomes through bromo- and chromodomains and either promote condensation or
decondensation of the chromatin structure.

Slides 122 / 123

I already mentioned that not all modifications of the same type (e.g. methylation, acetylation) are
mediated by the same enzymes (e.g. HMTs or HATs) but that these enzymes have specificity for
individual modifiable residues. The table lists some of the most important histone-modifying enzymes.
You can see that some have specificity for a specific histone, others have specificity for a particular
lysine on a specific histone. You can also see that some of these enzyme complexes contain bromo-
or chromodomains themselves. These enzymes can also read specific histone modification and either
add their specific modification on top of an existing one or they recognize the histone marks that they
created themselves and act on a neighboring nucleosome to propagate the mark from one
nucleosome to the next.

Slides 123 – 128

It is very common these days to analyze chromatin signatures (i.e. the specific histone modifications)
to understand how genes are regulated. In this example two neighboring genes are shown. The
STAT4 gene is actively transcribed in this cell type, whereas the MYO1B gene is inactive. If you look
at the distribution of some of the histone marks, you can see that the pattern is fundamentally different
between these linked genomic regions (slide 118). Lysine 4 (K4) on histone H3 (together denominated
as H3K4) is mono- (H3K4m1), di- (H3K4m2), and tri-methylated (H3K4m3) around the expressed
gene but not the inactive one. Histones around the active gene are also methylated on H3K36 and
H4K20. Thus, those methylation patterns are marks for active chromatin (slide 119). Di- and tri-
methylation of H3K27 are repressive marks. Surprisingly, mono-methylation of the same lysine is an
active mark (slide 120). The same is true for H3K9.

Slide 129
This summary shows the same histone marks centered around the transcription start site and the
gene is to the right. Some modification patterns are more symmetrical, whereas others are skewed
into one or the other direction. The different histone marks correlate with the level of expression
(different colors)

Slides 130 / 131

There is not much apparent logic to the meaning of specific modifications, except that acetylation and
mono-methylation usually represents active chromatin and that di- and tri-methylation of some
residues signals repression but not of others. Specific histone modifications can either occur around
the gene promoter, the gene body, or both.

Slides 132 – 135

To change a chromosome region containing one or several gens from a condensed, repressed state
(heterochromatin) into an open, actively transcribable state (euchromatin) acetylation of histones often
is the first step. This results in a less tight attachment of the DNA to nucleosomes and breaks
interaction between neighboring nucleosomes. As a consequence, the DNA changes from the 30 to
the 11 nm fiber or from eu- to heterochromatin. This enables binding of proteins to DNA sites that
were previously hidden inside the compact structure and inaccessible. These proteins can recruit
chromatin remodeling complexes. Chromatin remodeling complexes can also be recruited by the
specific histone marks through bromo-/chromosomains. The remodelers then slide the nucleosomes
to loosen up the DNA even more, which enables binding of transcription factors or other DNA-binding
proteins and subsequently RNA synthesis.
Slides 136 / 137
These events are illustrated here for the IFN-gamma locus. In the repressed state the gene is bound
by nucleosomes. Transcription factors (colored ovals) that bind to the genes enhancer create a
platform for the recruitment of GCN5, which is a histone acetyl transferase (HAT). A specific kinase is
also recruited that phosphorylates some of the residues. The new pattern in the histone tails recruits
the SW/SNF (BAF) remodeling complex, which slides the nucleosome away from the transcription
start and, thereby, enables binding of TFIID to the TATA box in the promoter.

Slide 138
Regulation of genes by the retinoic acid receptor illustrates a different scenario for regulation. The
receptor binds to specific DNA sequences in the promoter of genes if no ligand (= retinoic acid) is
bound. When bound at the DNA, it recruits HDAC1, which de-acetylates histones with the effect that
the gene is repressed. Binding of the ligand removes the receptor form the DNA and the HDAC
activity is lost.

Slide 139
In summary, chromosomes are patterned by alternating stretches of eu- and heterochromatin that is
defined by specific histone modification patterns. As we have seen, some of these marks are self-
propagating, meaning that the presence of a specific histone modification recruits enzymes that
transfer the same modification onto neighboring nucleosomes. How then, are the boundaries between
eu- and heterochromatin maintained inside a cell and how is the spread of histone marks prevented?

Slides 140
Barrier elements play an important role in preventing chromatin marks from spreading. Two different
modes of action can be distinguished. Large barrier elements can change the distance between
neighboring nucleosomes. Thus histone-modifying enzymes cannot reach over the barrier element to
modify the next available histone. Barrier elements can also position specific histone modifiers that
constantly reestablish the preferred histone mark.

Slide 141 - 144

The loss of an insulator also explains the famous position effect variegation of Drosophila eye color
that you learned about in BIO252. The white gene, which is responsible for red pigmentation of the
eye is located close to the chromosome end. Telomeres are typically in a heterochromatic state and
an insulator normally prevents spreading of the heterochromatin marks to the white gene. A
spontaneous inversion changed the arrangement and the telomeric heterochromatin is not separated
from the white gene by an insulator anymore. Subsequently the heterochromatin can spread and
inactivate the white gene. This event happens independently in every cell and results in a mosaic
pattern of red (euchromatin) and white (heterochromatin) cells.

Slides 145 / 146

To complicate matters, the different histone marks also influence each other. In this example the
relationship between methylation of H3K79 or H3K4 and ubiquitinylation of H2BK123 were
investigated. Both, deletion of the ubiquitinylation enzyme (rad6) or mutation of lysine 123 on H2B
prevented H3K4 and H3K79 methylation. Other relationships exist as shown in the diagram.

Slides 147 / 148

So far, we have seen that the histone marks are important regulatory switches that control the function
and behavior of DNA. An important question is whether and how these marks are
propagated/maintained during cell divisions. Do both sister chromosomes share identical histone
marks or do they segregate randomly between the two chromosomes? The latter scenario would lead
to a loss of specific heterochromatin regions. For instance only one of the sister chromosomes would
have a centromere because, as we have seen, they are epigenetic structures defined by specific
histones (CENP-A).

Slide 149 / 150

This example illustrates the fate of the sister chromosomes. During fertilization in Drosophila, the male
chromosomes are repressed by H3K27 tri-methylation, shown here by specific antibody labeling in
green. After the first cell division both daughter cells contain maternal and paternal chromosomes and
each cell harbors a full complement of methylated sperm-derived chromsomes. This suggests that
chromatin marks are inherited by some sort of epigenetic memory.

Slides 151 - 152

During replication the nucleosomes are fully or partially disassembled and the remnants are equally
distributed to the two sister chromosomes. It is sufficient to remove H2A/H2B for the replication
machine to synthesize through DNA and the H3/H4 tetramer often stays associated with the DNA.
Thus, the specific marks on these histones are found on both sister chromosomes, however, at half
their original density. New nucleosomes are formed in the sites between the remaining nucleosomes
and the marks can spread to and be reconstituted on the newly formed nucleosomes.

Slide 153
Now it also makes sense that most and many of the more important histone marks are found on H3
and H4, while H2A and H2B only carry a few residues that can be modified. H3/H4 stays associated
with the DNA during replication, whereas the H2A/H2B dimers are evicted.

Slides 154 - 156

Newly replicated DNA has initially only half of the nucleosome complement and new nucleosomes are
assembled to fill in the gaps. The existing marks, that are still present on the H3/H4 dimers that were
derived from the template DNA are then propagated to neighboring nucleosomes.

Slide 157 / 158

Histones are loaded onto DNA through the help of specific helper proteins, the histone chaperones.
The CAF-1 chaperone associates with the sliding clamp and ensures that H3/H4 is loaded to newly
synthesized DNA to initiate the assembly of a full nucleosome by the other chaperones.

End of transcript – shf (10.10.22)