Download THE STRUCTURE OF CHROMATIN

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THE STRUCTURE OF CHROMATIN
Textbook: Read the pages dealing with chromosome structure, histones, and nucleosomes.
Chromosomes are composed of essentially equal masses of DNA and protein. This
combination of DNA and protein was called chromatin by the early microscopists because it
stained the same color (chromo) as the chromosomes with various stains. Chromatin is still a
useful term, as long as we do not consider it to be just some amorphous substance. Chromatin is
very structured. We cannot understand how the nucleus “works” unless we understand this
structure. (When genes are active in a chromosome then of course transcription is occurring.
Then the chromosome, and thus chromatin, will also be associated with RNA if we analyze its
chemical composition.)
The structure of the interphase chromosome
(1) Each interphase chromosome contains one DNA double helix. (Unless it has
passed through S-phase and then it has two double helices, joined at the centromere
region. At this stage one can say that each chromatid has one DNA double helix.)
(2) A large proportion of the protein in chromatin consists of the proteins called
histones. There are 5 major histone molecules.
(3) The histone molecules are basic (positively charged) proteins, which is why they
associate so well with the negatively charged double helix.
(4) It is the positively charged R-groups of lysine and arginine that are most
responsible for making histone positively charged. Please know the structure of the
amino acid lysine:
(5) In the early 1970s, electron microscopists showed (with isolated and thinly “spread”
chromatin) that the primary structure of a eukaryotic chromosome appeared as
“beads on a string”. Treatment of the chromatin preparation with micrococcal
DNAase (modern terminology is DNase) resulted in separated “beads” The DNA
wrapped around the histones is protected from nuclease (DNase) attack so the
“beads” (nucleosomes) remain intact. But the linker DNA gets cut up into its
constituent deoxyribonucleotides.
(6) The beads were given the term nucleosomes.
(7) Analysis of the nucleosome showed them to be composed of: (a) 2 “turns” of DNA
double helix around (b) 8 histone molecules. The 8 histones are said to from an
octamer (oct = 8, mer = parts).
(8) There were 2 molecules each of the following 4 types of histone molecule; 2A, 2B,
3, and 4.
Note: There is a diagram on the effect of DNAase (point #5 above) on the very last page of this set
of notes.
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This is an electron micrograph of isolated chromatin
from a human body (somatic cell), perhaps a liver cell.
The chromatin has been spread extensively and then
stained with lead (Pb++) acetate. Notice the very
obvious “beads on a string”.
What about the so-called “higher order” structure of the chromosomes? That is, how is
the “beads on a string” structure further condensed, in an organized way, so the chromosomes can
fit into an interphase nucleus?
(1) The nucleosomes are “pulled together” by the addition of another type of histone molecule
(histone 1), to the outer surface of the nucleosome, and various non-histone proteins to the
“linker” region of the DNA. The latter is not shown in the diagram below. The “beads on a string”
chromatin “fiber” is about 10 nm in diameter.
HISTONE 1
LINKER REGION
(2) This compacted “beads on a string” structure is now twisted to form a thicker “fiber” that is
30 nm in diameter. Hence this fiber is about 3X thicker than the plasma membrane (which
has a diameter of about 10 nm).
(3) This 30 nm diameter fiber is then folded (pleated) into loops when it is bound to various
non-histone scaffold proteins.
(4) Depending upon the exact condensation state of the heterochromatin there can be further
“packing” events.
(5) To form a mitotic chromosome another set of proteins called condensins is required.
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It is interesting to note that the tightest packing of chromatin that occurs seems to be in the
“head” of sperm cells. In this case nucleosomes are not used in the initial condensation of the
DNA. After all, the 8 histone molecules of the nucleosome form quite a large “spool”! Instead, in
the sperm nucleus the DNA helix is first condensed by the use of organic molecules called
polyamines, specifically the polyamine called spermine
DIFFERENTIATION
SPERMATID – HAS
HETEROCHROMATIN
AND EUCHROMATIN;
HAS NUCLEOSOMES
Needless to say, the sperm chromatin is completely inactive!
SPERM – HISTONES ARE
REPLACED BY THE
MUCH SMALLER
POLYAMINES; DOES NOT
HAVE NUCLEOSOMES
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A closer look at nucleosomes and histones
I have said that it is now clear that gene regulation in eukaryotes begins at the level of
chromatin structure (less condensed euchromatin vs. more condensed heterochromatin) and
location (genes in a transcription factory or not). Today a lot of work is being done on the
chromatin remodelling that allows both changes in condensation and, presumably, changes in
location of stretches of chromatin in the cell (e.g. inside or outside transcription or replication
factories). To understand how chromatin remodelling occurs we need to look in more detail at the
structure of nucleosomes.
The important feature of this diagram is the presence of
the histone tails that protrude from the nucleosome. There are
8 histone molecules in a nucleosome so there are 8 protruding
tails. The artist has, unfortunately, shown only 4 histone
molecules in the nucleosome but 8 histone tails. There are 4
other histone molecules completely hidden by the 4 you can
see.
Certain of the amino acids of the histone tails can be specifically modified by enzymes in the
nucleus. A family of different histone methyltransferases can add up to 3 methyl groups to the Rgroups (in polypeptides these are also called “residues” or “side chains”) to the amino acid lysine.
There are also DNA methyltransferases, which add methyl groups to cytosine in DNA, that are
very important. But we are not going to consider the effects of DNA methylation.
A family of different histone acetyltransferases (HATs) can add acetyl groups
(essentially acetate) to lysine. Various histone kinases can add the terminal phosphate of ATP to
the amino acid serine. A kinase is an enzyme that can add the terminal phosphate of ATP to a
molecule. Kinases are named according to what molecule (or molecules) they can add the
phosphate to. For example glucose kinase (or glucokinase) and histone kinase.
The patterns of modification to the histone tails are very complex. But the significance of
some of the histone tail modifications is already known. For example, acetylation of certain amino
acids of the histone tails can cause chromatin to become less condensed, and thus more available to
transcription or replication..
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But it actually depends upon which specific amino acids of the histone tails are being
acetylated. And upon the tails of which of the 4 types of histone are being acetylated! And upon
what is happening to other amino acids in the histone tails; are they being acetylated, methylated,
or phosphorylated! So the histone tails are acting computer-like, summing up the total pattern of
amino acid modification in the histone tails. The diagram below is not one to learn (!) but just to
consider;
This diagram shows the possible ways in which the amino acids in the tails of only one
of the 4 types of histone molecule in the nucleosome can be modified. The other three types can be
modified differently. Of course, the pattern of amino acid modification depends on the conditions,
so think of how many possible permutations and combinations of modified amino acids there can
be at a given time for just one nucleosome. This is why researchers talk about the histone code.
The term histone code refer sto all possible permutations and combinations of modified
amino acids there can be on the histone tails of nucleosomes and what each of these possibilities
means in terms of nucleosome function. Most of the “meanings” of the histone code remain
unknown. The very simplified simplified idea is shown below.
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Methylation of histone usually turns a gene off.
Acetylation of histone usually turns a gene on.
Phosphorylation -- we're not sure what that does.
Although, as stated above, it is not clear what effect the phosphorylation of serine in the
histone has on their function, one can localize the areas in the nucleus where the tails of
nucleosome histones are phosphorylated! This is because antibodies can be made that specifically
bind to phophorylated histone compared to non-phosphorylated histone.
The red denotes phosphorylated histone
(using a fluorescent antibody against
phosphorylated histone). The green
denotes mitochondria (fluorescent
antibody against cytochrome oxidase).
Notice how phosphorylated histones are
localized in specific areas of the nucleus.
Certainly there are many nucleosomes, in
the dark areas, that are not phosphorylated.
When the various amino acids in the histone tails are modified it causes different proteins to
bind to these modified regions. And then other proteins bind to these proteins as if in a “scrum”. It
is the binding of these proteins that causes the changes in chromatin.
Gene silencing and cell “memory”
The cells of the very early embryo are said to be totipotent; that is they can differentiate
into any of the cells required by the adult organism. After a certain number of divisions (mitoses),
however, they lose this ability to so easily differentiate into all of the various cell types
characteristic of the adult organism. Some cells, the stem cells, retain the ability to differentiate
into cells of a certain related type e.g. blood cells or intestinal epithelial cells. Such cells are said to
be pluripotent. Differentiation involves the turning off and on of sets of genes. Once a group of
cells is destined to differentiate into related types of cells in the nervous system it makes sense to
permanently turn off their ability to become intestinal epithelial cells. This is a cost saving in terms
of regulatory effort and ensures that cells of the nervous system never accidentally turn into
intestinal epithelial cells! This permanent gene silencing involves the modification of the
histone tails, especially methylation and de-acetylation. This permanent, at least permanent in
terms of the cells as they exit in the organism as opposed to those on a petri plate, involves
chromatin condensation i.e. heterochromatin formation. The genes involved are said to have been
silenced. It is interesting that daughter cells retain a “memory” of this silencing of the appropriate
genes. An extreme case of this inheritable gene silencing is that of the inactivation of one of the Xchromosomes in mammalian females (to from the Barr body).Once one of the X-chromosomes in
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a cell is inactivated, at about the 5000 cell stage in the the developing embryo, all the future cell
generations of that cell have the same X-chromosome inactivated. This presumably results from
the inheritance of methylated nucleosomes. When sister chromatids are made, in S-phase, not only
does each chromatid receive one old strand of the initial DNA double helix (by the process of
semi-conservative DNA replication) they also inherit half of the old histones!
Reversibilty of histone tail modifications
While I have emphasized in the above paragraph that some chromatin changes caused
histone tail modification can be very stable, even from cell generation to cell generation, other
changes are quite readily reversible. There are specific demethylases, deacetylases, and
phosphatases that can reverse the effects of the methyltransferases, acetylases, and kinases,
respectively. It is usual in cell control systems to have such antagonistic sets of enzmyes.
Epigenetics
It is clear from what we have seen that genes activity in eukaryotes is influenced by the
structure of chromatin at a higher structural level than the direct effects on the gene promoters. We
will discuss gene regulation at the promoter level before we finish talking about the nucleus two
lectures from now. The effects of histone tail modification on gene activity that can be
transmitted from one cell generation to the next, by mitosis or meiosis are said to be
epigenetic (epi = above) effects. For example, when an X-chromosome is inactivated the copies of
that chromosome remain inactivated (for transcription but not for replication!) in all future
genartions of cells derived from the initial cell in which the X-chromosome was initially
inactivated. This inactivation depends upon histone tail modification and not gene regulation at the
promoter level. The same histone based inactivation of chromatin, but at a much less global level
than in X-chromosome inactivation, seems to be involved in the cellular inheritance of various
diseases.
Key words:
In additon to these notes on “The structure of chromatin” I will be giving out the structures of
lysine, acetylated lysine, methylated lysine, and phosphorylated serine. I will want you to be able
to draw these structures.
histone
histone methyltransferase
histone acetyltransferase
histone tail
nucleosome
linker region
post-translational protein modfication
micrococcal DNAase (or “nuclease”)
histone 2A, 2B, 3, and 4 (the so-called “core histone”)
histone 1 (the “compaction” histone)
octamer
anino acid residue (= R-group)
condensins
polyamine
spermine
spermatid
sperm
histone kinase
kinase
histone code
chromatin re-modelling
cell memory
epigenetics
X-chromosome inactivation
Barr body
S-phase
semi-conservative replication of DNA
chromatin
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The DNA wrapped around “non-activated” nucleosomes is not susceptible to degradation
by DNAases (“nucleases”).
LOCATIONS OF ATTACK BY MICROCOCCAL NUCLEASE
FREE
DEOXYRIBONUCLEOTIDES
FROM THE DIGESTION OF
LINK DNA