Biology Worthy of Life
An experiment in revivifying biology
This article has been largely superseded by “Getting Over the Code Delusion”, and “How Our Genes Come to Expression”, as well as some of the articles you will find under relevant headings in the topical index for the Biology Worthy of Life project. This article was first published in NetFuture #176, May 28, 2009. By clicking on the shaded rectangles at the end of many scientific terms, you can immediately read a definition of the terms in a separate window. This requires JavaScript to be enabled in your browser.
The decades following the 1953 discovery of the double helix were a time
when everything important seemed to hinge on the fixed and definitive
"genetic code"
. The researcher's task was to work out explicitly a
prescriptive logic already contained in the code. It was not a time when
molecular biologists were likely to preface their discussions of gene
expression
with statements like this:
DNA is a living molecule, writhing, twisting and bending in response to the physical forces applied to it by genetic processes (Kouzine and Levens 2007).
Nor was it a time when gene packaging materials and a diverse bestiary of
regulatory factors would have been described, with today's peculiar emphasis,
as participants in a "delicate 'dance' in time and space", a "regulatory
ballet", "an intricate dance of associations", and a "chromatin
choreography". Nor had there yet occurred that strange increase we now
observe in the use of terms such as "balance", "tension", "context", and
"plasticity", together with an exponential explosion during the first
decade of the twenty-first century in the appearance of the word "dynamic"
in reference to the chromosome
, chromatin
remodeling proteins
, nucleosomes
, and the
spatial organization of the nucleus — all pointing (in the words of
two biologists) toward the "highly choreographed" events regulating gene
expression, a "three-dimensional pavane . . . that controls our genome"
(Fraser and
Engel 2006).
Or, again, as recently as 1996, when the yeast genome was fully decoded and all its secrets supposedly laid bare, who would have expected to read this a decade further on:
Even for a [yeast] genome that has been studied intensively since it was sequenced
10 years ago, a glimpse into the complexity of its transcriptional
architecture makes this genome appear like novel territory (Lior et al. 2006).
How could the relatively simple yeast genome remain novel ten years after the complete unveiling of its structural sequence? And as for the choreography of the cellular "dance", isn't this a mere metaphor? Surely the authors quoted above did not really mean to say that a dance controls the genome! After all, the decisive logic of the genomic code is supposed to be what ultimately controls everything else.
Or so it has been thought. But the pace of discovery today is almost blinding, and it seems to be a time for the loosening of old thought patterns. The appeals to genomic plasticity, balance, tension, and context, the language of dynamism and artistic movement — these are not mere signs of a collective weakness for poetic invention, but rather are evoked by the phenomena under consideration. There is, in fact, nothing to prevent the contemporary molecular biologist from thinking along the following lines:
The organism must be rooted in something more than an abstract, fixed, and unchanging logic. It is a dynamic material presence, and requires a materially effective genesis. If DNA
and all the other contents of the living cell provide the physical foundation for the organism as a whole — for the seeing eye, the beating heart, the graceful and compelling movement of the ballet dancer — then why shouldn't their own, molecular performance be at least as artful and complex as that which they support on a larger scale? Why shouldn't their gestures be as meaningful and expressive as those of the organs whose development they so effectively underwrite? Is there any reason to think that the life animating the cell and its DNA should be any more reducible to a static logical sequence — should be any less subtle or capable or dynamic — than eye, heart, and ballerina? Why should we project tiny, neatly programmed mechanisms into the cell when the organisms we see don’t at all look or behave like such mechanisms?
Of course, these thoughts, especially if expressed in this way, would still today appear rather eccentric. There are, as we will see, logic-centered and mechanistic habits of thought that fiercely oppose giving embodied (and therefore observably aesthetic) form and movement their due.
But nevertheless, the landscape on which we all must move with our ideas,
outworn or forward-looking as they may be, is inexorably changing. It's a
landscape upon which, less than a decade after the height of the fever
induced by the Human Genome Project, the author of a review in a major
biological journal could write that genome mappings and
genomic comparisons of species "shed little light onto the Holy Grail of
genome biology, namely the question of how genomes actually work" in
living organisms (Misteli 2007).
And so today whoever would take a researcher to task for eccentric thinking might have a harder time of it than usual. In surveying contemporary molecular biology, the least anyone can say is, "Things are getting very interesting!"
If you arranged the DNA in a human cell linearly, it would extend for about two
meters. How do you pack all that DNA into a cell nucleus about ten
millionths of a meter in diameter? According to the usual comparison it's
as if you had to pack 24 miles (40 km) of extremely thin thread into a
tennis ball. Moreover, this thread is divided into 46 pieces (individual
chromosomes
) averaging, in our tennis-ball analogy, over half a mile
long. Can it be at all possible not only to pack these into the ball, but
also to keep them from becoming hopelessly entangled?
Let's begin visualizing the situation in a little more detail.
Imagine you have a rope consisting of two strands spiraling around each
other in the manner of a double helix. Imagine
further that there are short, fairly rigid rods connecting the two strands
at regular intervals along their entire length. There are many of these
rods — roughly ten along the brief length required for one strand to
spiral completely around the other a single time. Each rod represents two
linked nucleotide bases
, or base pairs
(complementary "letters" of the genetic code
), with one
base bound tightly to one strand of the rope and the other bound to the
complementary strand.
It would be good if the rope, in its double helical form, has been soaked
in a starch solution or some other stiffening agent. This is because DNA,
as a result of its chemical constitution, possesses a degree of natural
rigidity; left to itself, the overall structure resists bending, and one
strand "wants" to wrap around the other a fixed number of times for any
given length of the helical axis. It is
possible to increase this number somewhat (that is, to wind the strands
more tightly around each other) or else to decrease it (unwind the
strands). But in both cases this is to work against the stiffness of the
natural structure, and therefore to create tension that must be
accommodated in one way or another.
You have doubtless seen this accommodation many times. Hold the ends of a
two-stranded rope in your hands and begin to twist one of the ends so as
to tighten the spiraling strands. Before long you will find the rope
coiling into something like a figure eight, and then into ever more
complex forms as you continue twisting, until finally you end up with a
"nest of worms". And much the same happens if you twist in the opposite
direction, as if you were trying to unwind or loosen the spiraling
strands. The coil resulting from a tightening twist is called a positive
supercoil, while a loosening twist produces a negative supercoil1.
The forces involved in these deformations can be very large — as you
will have discovered if you have ever tried to coerce a stiff rope through
multiple stages of coiling by twisting its ends, or even if you have found
yourself wrestling with a recalcitrant garden hose while trying to coil it
neatly. Matter can be very resistant! Analogous forces come into play
with chromosomes as well.
DNA is often in
a negatively supercoiled state to one degree or another — and, as we
will soon see, this is owing to additional reasons beyond the fact that,
in order to fit into its own space in the nucleus, it is coiled, bent,
wound and otherwise structured to an almost unfathomable degree. The
question is how any sort of order is maintained: how is the nest of worms
packaged and managed in every cell of the human body in a manner allowing
the thousands of distinct, individual genes, and perhaps hundreds of
thousands of regulatory sequences
, to come to
harmonious expression within the complex life of the larger cell?
The first step in DNA packaging involves tiny "spools" made of histone proteins
— some thirty million of them in the human genome
, so that
there can be several hundred thousand or more in a single chromosome
. The DNA
double helix
commonly wraps about two times around this spool, continues
on for a short distance, then wraps around a second spool, and so on. The
DNA-enwrapped spool is called a nucleosome
.
This first level of DNA packaging is often described as "beads on a
string". (See third image from left in the figure below.) The DNA and
histone spools, together with numerous other attached proteins and smaller
chemical groups, give an overall, ever-changing form and structure to what
is called chromatin— the
actual material of the chromosome. But with this spooling of the double
helix we have seen only the beginning of the compaction that must occur in
order to fit the chromosome into the cell nucleus. Unfortunately, the
higher-order structures of the compacted chromosome are still little
understood. The spools with their DNA somehow get packed into dense,
three-dimensional arrangements, and this entire arrangement coils further
upon itself beyond anyone's current ability to unravel the details. Such
difficulty, however, rarely hinders the adventurous from offering visual
models. So, for what it is worth, here is a conventional picture showing
several stages in the condensation of a chromosome (it is best viewed at
full screen width):
For credits and permissions, see
https://upload.wikimedia.org/wikipedia/commons/4/4b/Chromatin_Structures.png.
During the cell's normal functioning the chromosome is not as fully
condensed as it is during cell division (the two images at far right).
Nor is it all in one state. Some parts of it — especially the parts
containing many active genes — are in something rather more like the
"beads-on-a-string" form, while other parts may be in the conformation of
the 30-nanometer fiber, and vast regions are in a much more wound-up form.
(Thirty nanometers is 30 billionths of a meter, or about 3 thousandths of
the diameter of a typical cell nucleus.) In general — but with
exceptions — the more compact the chromatin, the less
available are the genes for transcription
.
Another image follows below, this one showing four proposed models —
each viewed from two different angles — for the structure of
chromatin in the 30-nanometer fiber. The models do not show the actual
spools or other proteins, but only the DNA. (The DNA
is given as a simple "wire", without any representation of the two helical
strands.) However, you can see how one spool could be positioned inside
each double spiral of DNA. Then, given the scale of the image, you would
need to picture this arrangement extending linearly for enormous
distances, even if only a small part of a chromosome were represented.
![]() Graphics by Julien Mozziconacci (https://en.wikipedia.org/wiki/File:ChromatinFibers.png) |
Linker DNA — the short, connecting lengths of DNA between spools
— is shown in bright yellow, and the wrapped DNA is flesh-colored.
The different models are based on different assumptions about the total
number of base pairs
from the start of one spool to the start of the next one
— that is, the length of wrapped DNA plus linker DNA. These lengths
are the numbers shown in the figure. You can bring the upper and lower
images into proper relation if you imagine each of the upper images
rotated ninety degrees around a horizontal axis so as to bring the
brightly colored (blue, pink, green, or gold) double spiral of the upper
image into the position shown in the lower image. Finally, the white
"lumps" in the figure represent linker histones
, which hold
the DNA to the spool and help to stabilize the entire array.
Perhaps none of this helps us greatly to understand how the
extraordinarily long chromosome,
tremendously compacted to varying degrees along its length, can maintain
itself coherently within the functioning cell. But here's one relevant
consideration: there are enzymes called topoisomerases
, whose task
is to help manage the forces and stresses within chromosomes.
Demonstrating a spatial insight and dexterity that might amaze those of us
who have struggled to sort out tangled masses of thread, these enzymes
manage to make just the right local cuts to the strands in order to
relieve strain, allow necessary movement of individual genes or regions of
the chromosome, and prevent a hopeless mass of knots.
Some topoisomerases cut just one of the strands of the double helix, allow it to
wind or unwind around the other strand, and then reconnect the severed
ends. Other topoisomerases cut both strands, pass a loop of the
chromosome through the gap thus created, and then seal the gap again.
(Imagine trying this with miles of string crammed into a tennis ball
— without tying the string into knots!) I don't think anyone would
claim to have the faintest idea how this is actually managed in a
meaningful, overall, contextual sense, although great and fruitful efforts
are being made to analyze isolated local forces and "mechanisms".
Before we try to bring the picture a little more alive, there's one small exercise that may help us. Many window shades have a looped cord for adjusting the light. If you slip your finger through the loop at the bottom and then twist it around in one direction many times, you will get our familiar double-stranded helix. Now, while keeping firm hold of the loop at the bottom, insert a pencil between the strands near the near the middle of the cord's length and then force the pencil downward. You will observe that the stands become progressively more tightly wound beneath your pencil, until it can move no more. At the same time the cord above the pencil becomes more loosely wound. Alternatively, you can let go the loop at the bottom, in which case the cord will spin around as the pencil descends.
This is relevant to the chromosome because when a gene is transcribed, its two
double helical strands need to be separated, or "unzipped", as the
transcribing enzyme
moves along. How, then, does the chromosome accommodate
the twisting forces imposed by this local "unzipping" of its two strands?
You might expect the chromosome to spin like the cord with a pencil moving
down it. Certainly there is some such movement. But if it were to
proceed in an unconstrained manner, as with the released window shade
cord, the entire chromosome ahead of the transcribing enzyme
would have to make about 2850 complete turns during
transcription of an average-sized gene (Lavelle 2009), which means
rotating at several turns per second. Clearly, given the length, the
mass, and the complex bending and looping forms of the chromosome, and
given the extremely thick "soup" of macromolecules in the cell nucleus,
such movement would be greatly impeded.
Furthermore, the ends and many points within the chromosome are typically
"fastened down", as we will see later, so there isn't all that much
freedom of movement. Of course, when you move the pencil down between the
two strands of the window cord, you could allow the pencil itself to spin.
This would leave the helical structure of the cord mostly unchanged
outside the immediate vicinity of the pencil. However, in the cell our
"pencil" — the transcribing enzyme — is part of a very large
molecular complex. In addition, it is associated with a cumbersome set of
proteins for disassembling nucleosomes ahead of its
transcribing activity, reassembling them behind, and performing various
other tasks. And it is attached to the ever-lengthening strand of RNA
that it is
itself producing. So it faces limits upon its mobility similar to those
of the chromosome.
The upshot of it all is that there are many complex movements, highly
constrained and absorbed in varying ways by the different resistant
elements of the complex structures involved. In general, positive
supercoiling occurs ahead of the transcribing enzyme's "unzipping"
action and negative supercoiling behind it. Topoisomerases play their
role in managing both the stresses and the overall conformation of the
chromosome "tangle", as do many other poorly characterized players in the
sculptural drama of form and force that is the chromosome.
I have so far described the packaging of human DNA as a mere
technical challenge. That's a big problem. The tensions and movements,
the bending and unbending, the coiling and uncoiling, are much more than
the expression of mechanical forces aimed at chromosome
condensation. It was quite wrong of me to begin by asking you to imagine
twisting a rope since, after all, there is no one — no specific
agent — in the cell nucleus performing this task. The chromosome is
not a passive, limp object moved only from outside. Interacting with its
surroundings, it is as much a living actor as any other part of its living
environment. Maybe instead of a rope, we should think of a snake,
coiling, curling, and sliding over a landscape that is itself in continual
movement.
The chromosome, in other words, is doing something. It is engaged in a highly effective spatial performance. It's movements are not simply the result of its being packaged and kept out of trouble, but rather are well-shaped responses to sensitively discerned needs. These movements bear decisive significance for the life of the cell and organism as a whole. Far better to picture the chromosome as both a sensing and muscular presence than as a rope.
To begin with, the mechanical stresses induced by transcription are now
known to contribute broadly to gene regulation
. "The organization of global transcription is tightly
coupled to distribution of supercoiling
sensitivity
in the genome"
(Blot 2006). Increases in twist (positive supercoiling)
are associated with chromatin
folding and
gene silencing
in the supercoiled region, whereas decreases of twist
(negative supercoiling) are associated with "acquisition of
transcriptional competence" (Travers and Muskhelishvili 2006). Moreover,
"negative supercoils are dynamic. The slithering and branching of the
interwound strands allow DNA
to act like a chaperone
, promoting
the long-range assembly and disassembly of protein-DNA complexes" (Deng et
al. 2005) — complexes that play a vital role in gene regulation.
Each type of cell has its own characteristic patterns of supercoiling,
which is doubtless related to the fact that it also has its own
distinctive patterns of gene expression
. Christophe Lavelle of the Curie Institute in France
summarizes the recent research findings this way:
As DNA is rotating inside the polymerase
[transcribing enzyme], positive and negative supercoiling is induced downstream and upstream
, respectively. Transcriptionally generated torsion, rather than a mere waste product to be disposed of by topoisomerases, has instead recently been shown to propagate through the chromatin fiber and trigger local DNA alterations, detected as a regulatory signal by molecular partners. (Lavelle 2009)
To illustrate the regulatory possibilities: researchers at the National
Cancer Institute in Bethesda, Maryland, found that negative supercoiling upstream
(behind) a transcribing enzyme was sufficient to cause a local,
nonstandard conformation of the double helix
, which in
turn enabled recruitment of regulatory proteins sensitive to such changes
in structure (Kouzine et al. 2008).
So the chromosome's twisting and writhing is not merely arbitrary; it is sculpturally significant movement, carrying meaning for the chromosomal stretches along which it is communicated.
But there are many other dimensions of the chromosome's spatial
performance. Each chromosome has its own preferred territory within the
nucleus and its preferred neighbors, which also differ from one cell type
to another. These territories "are dynamic and plastic structures" that
"can be dynamically repositioned" (Schneider and Grosschedl 2007). Since
living conditions are close, the neighbors matter. A chromosome's
territory appears to be shaped rather like an irregular potato or a
sponge. There is at least some socializing between adjacent chromosomes,
with protrusions of one territory penetrating into the hollowed-out
portions of the next territory and even of more remote territories (Ling
et al. 2007). So not only are distantly separated portions of the same
chromosome brought into intimate contact by the geometry of the sponges,
but loci on separate chromosomes can also be brought into contact.
It happens that both sorts of contact have a great deal to do with gene
expression. On an earlier view, the DNA sequences
regulating a
protein-coding gene were always close to the gene or at least not very far
removed. But in more recent years it's been recognized that some
regulatory sites — "enhancers"
and
"silencers"
and "locus control regions"
— may be located on distant parts of the chromosome,
thousands or hundreds of thousands or even millions of base pairs
away from
the gene being regulated
. Expression is enabled, for example, when the distant
enhancer is brought into physical proximity with the gene or genes it
regulates. (Another remarkable feat of contextually apt physical
coordination!) In connection with this, an activator
protein is
bound to the enhancer and then, perhaps in concert with one or more
co-activators
, may assist in constellating the massive transcription
complex
on the gene's promoter
sequence
.
A locus control region (LCR) is a DNA
sequence that helps to regulate a cluster of related genes.
One research team in the Netherlands, working with mice, examined an LCR
for a set of genes relating to the production of beta-globin (a
constituent of hemoglobin). In fetal liver tissue, where these genes are
highly active, the LCR was found to associate with dozens of genes,
including many involved in beta-globin production. Some of these genes
were tens of millions of base pairs
distant on
the chromosome. Further, in fetal brain tissue, where the beta-globin
genes are inactive, the LCR again associated with many other sites —
but now a completely different set. The researchers concluded:
Our observations demonstrate that not only active, but also inactive, genomic regions can transiently interact over large distances with many loci in the nuclear space. The data strongly suggest that each DNA segment has its own preferred set of interactions. This implies that it is impossible to predict the long-range interaction partners of a given DNA locus without knowing the characteristics of its neighboring segments and, by extrapolation, the whole chromosome. (Simonis et al. 2006)
It's not only where loci on a single chromosome are brought
into contact with each other that they can interact, however. Increasing
numbers of cases are being reported where contact between sites on
different chromosomes plays a crucial role in gene regulation
. In fact, the mouse study just cited demonstrated a number
of such contacts. (A few researchers began to speak of "kissing
chromosomes" — a not very helpful phrase that seems now to have been
dropped from the literature.) Here's a schematic representation of one
case of interchromosomal interaction. (Skip the next three paragraphs if
you don't want to bother with the technical details.)
![]() From Schneider and Grosschedl 2007. |
"T helper" or TH cells are human immune system cells. One type of TH cell (TH1) produces, among other things, interferon (IFN-gamma), while a second type (TH2) produces various interleukins such as IL-4 and IL-5. But before a cell becomes either a TH1 or TH2, it resides in a less differentiated state as a "naive" TH cell (referred to as a "T cell" in the figure). Only after stimulation by an antigen (a substance that provokes formation of antibodies), does the TH cell become either a TH1 or TH2 cell.
If you look at the genes responsible for producing interferon in TH1 and
interleukins in TH2 cells, you find the usual suspects: the interferon
gene on chromosome 10 seems to be regulated by nearby genomic elements,
while the interleukin genes on chromosome 11 are also regulated by local
sites — in particular, by an LCR. But a
research team at the Yale University School of Medicine decided to
investigate the larger spatial picture. They discovered that regulatory
regions
associated with the interferon and interleukin genes,
despite being on separate chromosomes, were physically close together in
naive cells. Upon stimulation of naive cells by an antigen, the
"negotiations" between these regulatory regions somehow determined which
of the genes would be active and which would be repressed — and
therefore whether the cell would become a TH1 or TH2 cell. Following this
determination, the two chromosome regions moved apart.
The illustration (right side) shows the case where a TH2 cell has
resulted. Interferon (IFN-gamma) is not expressed (upper right) because the looping pattern separates the
requisite gene from its enhancer
. On the
other hand, IL-5 is expressed (lower right), because the protein
SATB1, which plays a large role in chromatin
organization
and transcription
regulation, has anchored a series of chromosome loops in
just such a way as to bring the IL-5 gene and Rad50 promoter
into
proximity with the locus control region. (Spilianakis et al. 2005; see
also commentary in Kioussis 2005.)
Of course, as researchers dealing with this sort of thing readily acknowledge, questions abound. What guides particular sites on two different chromosomes to their rendezvous, and what sees to their subsequent separation? One could imagine, in the case of distantly separated sites on the same chromosome, that a regulatory protein binds to the one locus and then "tracks" along the chromosome until it finds the second locus (which it must have some way to recognize as significantly related to the first). But it's not at all easy to picture what it is that selects and brings together many loci on different chromosomes.
As is evident from the case of TH cells, chromosome looping can keep sites
apart as well as bring them together. It not only serves the purpose of
expression, but also of repression
. In a study of red blood cells, a group of scientists from
Children's Hospital in Philadelphia showed that successive stages of cell
maturation were marked by different proteins playing a direct role in
reconfiguring chromosome loops — first for expression of a
particular gene, and later for repression (Jing et al. 2008). In general,
chromosome loops help to make possible the more or less independent
regulation of different gene regions — an important role in an
environment thick with diverse regulatory factors and processes.
But how does a locus on a chromosome "take off"
through the three-dimensional space of the nucleus, uncoiling from a more
condensed state into a thin thread and looping outward from its territory
for considerable distances, as if drawn by an invisible hand toward a
rendezvous with a distant location? One group of researchers positioned a
transcriptional activator
on a particular chromosomal site located close to the
periphery of the cell nucleus. Within 1 - 2 hours, the site migrated to
the interior of the nucleus, following a curvilinear path roughly
perpendicular to the nuclear envelope
. The movement, which was interspersed with several-minute
periods of quiescence, reached a maximum velocity of about 1/10 the
nuclear diameter per minute. These results led the researchers to speak
of "fast and directed long-range chromosome movements" (Chuang et al.
2006).
A fairly recent surprise has been the discovery of actin and myosin in connection with some chromosome movements. These two substances, which play a major role in the contraction of muscles, seem also to provide a kind of "musculature" within the nucleus. Get rid of them, and certain observed movements stop. But little is yet known about how the movement is actually achieved, and even less about how it is directed.
What is now known, however, is that the nucleus is much more than a
linear assembly line for the construction of proteins based on genetic
sequences. It participates in an elaborately organized,
three-dimensional space, and the positioning and movement of both
chromosomes and the regulatory elements within the nucleus have everything
to do with the functioning of the genome
.
But while extraordinary research energies are now directed toward
articulating the undeniable structural organization of the cell nucleus in
its relation to gene regulation, an overall, coherent picture of the organization remains
elusive. The titles of several articles currently lying on my desk point
to the challenge investigators face:
"Dynamics and Interplay of Nuclear Architecture, Genome Organization, and Gene Expression" (Schneider and Grosschedl 2007).
"Dynamic Genome Architecture in the Nuclear Space: Regulation of Gene Expression in Three Dimensions" (Lanctôt et al. 2007).
"The Third Dimension of Gene Regulation: Organization of Dynamic Chromatin Loopscape by SATB1" (Galande et al. 2007).
"Dynamic Regulation of Nucleosome Positioning in the Human Genome" (Schones et al. 2008).
"Dynamic Organization of Gene Loci and Transcription Compartments in the Cell Nucleus" (Spudich 2008).
"Nuclear Functions in Space and Time: Gene Expression in a Dynamic, Constrained Environment" (Trinkle-Mulcahy and Lamond 2008).
You will have noted the repeated juxtaposition — spatial organization on the one hand, dynamism on the other. How does one capture organization that is dynamic and ever-shifting? The question only becomes more acute when we look at a few additional aspects of nuclear organization, as currently described in the literature:
Chromosome Domains. Chromosomes, as we have
seen, participate in the highly structured space of the nucleus. But that
is not all. They themselves are structured along their length, being
subdivided by various means and in ever-changing ways into chromosome
domains. We've already seen the organization of the chromosome into
densely compacted regions (known as heterochromatin
) and less condensed, more active regions
(euchromatin
). The boundaries between such regions are not always
well-defined. Simply by residing close to a region of heterochromatin, a
gene that otherwise would be very actively transcribed
might be
only intermittently expressed
, or even silenced
altogether.
Where somewhat more cleanly separate regulation of neighboring loci is
important, special DNA sequences
called
insulators
can help prevent the "leaking" of influence from one region
of the chromosome to the next.
Chromosome domains are also established by the twisting forces (torsion)
communicated more or less freely along bounded segments of the chromosome.
(The boundaries might be defined, for example, by the tethering points of
chromosome loops.) The loci within such a region share a common torsion,
and this can attract a common set of regulatory proteins. The torsion
also tends to correlate with the level of compaction of the chromatin
fiber, which in turn correlates with many other aspects of gene
regulation. And even on an extremely small scale, the twisting (by
linker histones
) of the short stretches of DNA between nucleosomes
— or
the untwisting brought about by the release of the histones — is
presumed to drive the folding or unfolding of the local chromatin
(Travers and
Muskhelishvili 2006). All this reminds us that gene regulation is defined
less by static entities than by the quality and force of various
movements.
There are still other ways that the chromosome reveals itself as a dynamic, complexly structured context. Genes expressed in the same cell type or at the same time, genes sharing common regulatory factors, and genes actively expressed (or mostly inactive) tend to be grouped together. One way such domains could be established is through the binding of the same protein complexes along a region of the chromosome, thereby establishing a common molecular and regulatory environment for the encompassed genes. But it's important to realize that such regions are more a matter of tendency than of absolute rule. A few examples from a summary by Elzo de Wit and Bas van Steensel at the Netherlands Cancer Institute illustrate the situation:
** In yeast, "25% of the genes that display a cell-cycle-dependent expression pattern were located next to a gene with the same expression pattern".
** The fruit fly genome "contains a few hundred clusters of 10-30 adjacent genes
of which most have a similar expression pattern".
** In mustard plants, "5-10% of all genes are within coexpressed chromosomal regions".
** And "the human genome contains megabase-sized [million-"letter"-sized] regions where most genes tend to be expressed at high expression levels, alternating with large regions that harbor genes that are predominantly expressed at low levels" (de Wit and van Steensel 2009).
These rough tendencies do not enable precise predictions, but yet the tendencies are really there; they point toward meaningful organization, even though the observed "rules" are less the determiners of that organization than they are the continually modified products of it. This is exactly what you would expect in any living context, where a larger unity — the unity that leads us to refer quite naturally to a living being — shapes the activity of local parts and processes to its own intention.
The "Pull" of the Nuclear
Periphery. There is a fibrous network — the "nuclear lamina"
— located primarily at the inside face of the nuclear envelope.
In vitro studies (what used to be referred to as "test-tube
studies") have shown that several proteins of this network can interact
with chromatin. And now, as the Dutch biologists summarize, it has been
found that there are more than 1300 lamina-associated chromosome domains
(LADs) in the human genome
that do indeed preferentially locate themselves at the
nuclear periphery. De Wit and van Steensel (2009) mention studies showing
that the artificial anchoring of a chromosome
locus to the
nuclear lamina "can cause partial downregulation of some (but not all)
genes surrounding the anchoring sequence
".
There seems to be a general rule that the chromosomes and chromosome
territories located toward the periphery of the nucleus are less
transcriptionally active and also less gene-dense. Conversely, the nuclear
interior shows higher rates of activity and greater gene density.
Researchers can activate
genes near the nuclear envelope and then watch them as they
move toward the interior. Likewise, they can silence
genes in the
interior and watch them relocating to the periphery. Nevertheless,
transcription does occur in the outlying regions, and silent regions of
chromosomes reside in the interior. And, as always, multiple dimensions
of regulation work together. For example, the radial positioning of genes
seems to be connected to specific histone modifications
of the sort we looked at in "Twilight of the Double Helix"
— although it's a
matter of rough rather than absolutely consistent correlation (Strašák et
al. 2009).
In an interesting twist, the nuclei of rod cells in mice and other
mammalian species with nocturnal vision reportedly show a distinct radial
organization, but it is the reverse of that described above: the more
condensed, transcriptionally inactive
heterochromatin is positioned centrally, while the less condensed euchromatin
is positioned peripherally (Bártová et al.
2008).
Nuclear Matrix. It is not only the peripheral
nuclear lamina that provides a kind of skeletal structure for organizing
the nucleus. There is, throughout the nuclear space, a still poorly
characterized and elusive "nuclear matrix" — so elusive that its
fundamental nature is still debated. The nuclear lamina can be
considered part of it, and there are many other substances that seem to
play a role, including the SATB1 protein we encountered in connection with
looping chromosomes, a topoisomerase
, actin
, and even
the DNA transcribing
and replication
enzymes. Many of the proteins that associate with
chromatin
, affecting its form and compaction, are considered to be
components of the nuclear matrix. In other words, the nuclear matrix is
not simply a passive structure that objects can attach to. It consists of
active agents — and, in our current context, that means agents of
gene regulation
.
The human genome contains an estimated 30,000-80,000 "matrix attachment
regions" (MARs) — relatively short DNA sequences susceptible of
being anchored to the nuclear matrix (Ottaviani et al. 2008). These
anchoring points can contribute to the formation of the loops we've been
talking about. Some MARs are more or less permanently attached to the
matrix and may be associated with higher-order chromatin
compaction
and the repression
of genes not required in a particular cell type. Others
only transiently attach to the matrix and are thought to play a major role
in the management of gene expression
. The configuration of attachments at any given moment
shapes the overall chromosome architecture, and the consequent looping
patterns effectively insulate some regions from regulatory factors while
exposing others. In sum:
Our understanding of how the genome functions in the context of the nucleus has been propelled by indisputable evidence that distinct genomic sites bind to regulatory proteins at the nuclear matrix. The emerging picture is that these genomic anchors regulate transcription
and replication
by dynamically organizing chromatin in three-dimensional space. (Ottaviani et al. 2008)
By this time I'm sure you recognize the need to ask (upon hearing that
genomic anchors "regulate transcription"): What is it that regulates the
anchors? How do they know when and where and for how long to participate
in their anchoring task? The point, which is one of our enduring themes
in these articles, is that there is no single, controlling level of gene
regulation, subordinating the rest of the organism to itself. Noting that
"the existence of regulatory cross-talk between spatially interacting loci
opens up a new dimension in the study of gene regulation", Christian
Lanctôt et al. (2007) go on to remark that "not only does [this
cross-talk] constitute an additional level of complexity in the search for
regulatory elements in the genome, it also
implies that chromatin
mobility itself, and therefore the ensuing long-range
gene-gene interactions, might be a target of regulation".
How could it be otherwise within a harmoniously functioning organism? I suspect it's quite safe to say that every aspect of the cell is in one way or another a target of regulation, and at the same time takes on some of the role of regulator. Or better (since that last statement reduces the word "regulation" to something close to nonsense): every part participates in the whole organism and is informed by the whole. Of course, in the current state of biology this remark, too, will strike many as nonsense. One could reply by asking whether it's any more nonsensical than all the usual talk of "regulation", but a more positive approach would be to take up the question of holism, as we will do later in this series.
Nuclear Compartments and Organelles. Loops are created
when separate points on a chromosome are brought
together and at least temporarily bound at the same location.
Multiple-loop structures result when a number of different loci fraternize
in this way. This raises the question: in what sense are the regions
where these gatherings occur "real places"? That is, what structural
identity do they possess beyond the fact that they happen to be sites
where active chromosomal loci have gathered?
In one sense, the answer is easy. In order for genes to be active, there
must be transcribing enzymes and many other factors related to transcription
. So it
stands to reason that these centers of activity are distinctively
constituted. High-resolution surveys of the cell nucleus do in fact show
many such places, which have come to be called transcription "factories"
(a rather prejudicial term). Estimates for their number range from 500 to
10,000 (Trinkle-Mulcahy 2008), and it has been conjectured that, on
average, some eight transcribing enzymes are present in each center of
activity.
One group of researchers, describing how distantly separated genes in red
blood cells "colocalize to the same transcription factory at high
frequencies", go on to summarize the situation: "active genes are
dynamically organized into shared nuclear subcompartments and movement
into or out of these factories results in activation or abatement of
transcription. Thus, rather than recruiting and assembling transcription
complexes, active genes migrate to preassembled transcription sites"
(Osborne et al. 2004). The implication, noted by the authors, is that
"mechanisms regulating recruitment of genes into factories would be
expected to have a fundamental role in gene expression".
The transcribing enzymes in (or, rather, at the outer surface of) the active
transcription centers, according to the emerging view, do not themselves
move along the genes; they "reel in" the genes they are transcribing. In
this way they act as critical structural elements for maintaining the
loops, which come and go as the various enzymes and regulatory factors
bind and release them (Carter et al. 2008).
But, still, uncertainty remains about how much "there" is really there in
the transcription centers. To what degree do enduring structures exist
apart from the organized "structure" of the ongoing processes of
transcription? There is presumably something there, but it's
proven subtle and difficult to pin down. The matrix attachment regions of
chromosomes, which presumably play a role in bringing genes to
transcriptional centers, are being identified, but it remains to find
anything in the way of a very fixed and definite structure for them to
attach to. The "structure", such as it is, seems to be as much process as
product.
There are yet other nuclear compartments relating to gene expression, but we will not pursue them here.
The intricately formed activity of the nucleus varies from one cell type
to another and from one stage of an organism's development to another. It
both shapes and mirrors the distinctive character of the individual cell.
But this character is not some abstract essence detached from whatever
else is going on in the organism at a particular moment. We can only
assume that, whether the cat we are looking at is stalking or eating or
sleeping or raising its fur in a confrontation with an enemy, the
expressive differences we can recognize in these activities would be
matched by expressive differences at every level of the cat's life,
including the level of gene transcription and nuclear
organization — if only we were capable of reading the cell with the
same qualitative attention we devote to the outward behavior of the cat.
If the cat is raising its fur, then the skin, muscle, and other cells must
in some sense be "raising their fur" as well.
In other words, the chromosome movements
we've looked at are always part of the larger activity of the organism.
It's not just that a locus of the chromosome moves from point A to point B
in order to connect with a group of other loci; this process in
turn takes place in order to achieve equally significant
performances at higher levels of observation, whether it's a matter of the
cell's response to a nearby lesion or to starvation or to the organism's
emotional state. The activities in the cell nucleus are part of an
overall organic picture, and the scientist will do well to remember
occasionally how remote is the detailed knowledge of the sort I've
outlined above from any coherent and contextual understanding of what's
going on.
This is presumably why biologists Amy Hark and Steven Triezenberg (2001), speaking of the variety of protein complexes affecting chromatin structure and gene expression, point to "a web of functional interactions that might be viewed as either elegantly integrated or hopelessly tangled". Hopelessly tangled, that is, if we do no more than lose ourselves in tracking isolated "causal factors" and "effects"; elegantly integrated if we can somehow rise to a more pictorial and qualitative grasp of what clearly is in fact a unified whole.
Perhaps we have the most incentive to seek such a wider understanding when we confront disease. There is no doubt, for example, that the phenomena investigated by the epigeneticist bear heavily on cancer, even if there is little effort as yet to read the cell as an expressive whole. Certainly research into particular "mechanisms" is proceeding at full tilt. Referring to how the microenvironments of the nucleus bring together the various gene-regulatory signals in all their necessary combinations, one team of researchers reviews the implications for cancer diagnosis and treatment:
Solid tumours, leukaemias, and lymphomas show striking alterations in nuclear morphology as well as in the architectural organization of genes, transcripts
, and regulatory
complexes within the nucleus . . . . These cancer-related changes disrupt several levels of nuclear organization that include linear gene sequences, chromatin
organization and subnuclear [compartments] . . . . Modifications in chromatin remodelling complexes
, the persistent association of regulatory proteins with gene loci, and DNA methylation
epigenetically modulate genome
accessibility to regulatory factors for the physiological control of cell fate.... (Zaidi et al. 2007).
The researchers add that the effects of therapeutic treatment hang in the
balance of these complex interactions, since even a patient's sensitivity
to radiation and chemotherapy depends on the "composition, assembly and
architectural organization of regulatory machinery within the cancer cell
nucleus". The hope, finally, is that the "functional relationship between
nuclear organization and gene expression" will bring advances in "tumor diagnosis" and "therapeutic
responsiveness".
That hope may sooner or later be fulfilled. But the scale of the challenge looks hard to underestimate!
In any case, the fluid spatial organization of the nucleus and the
movement of chromosomes within it clearly play a vital role in bringing about the
right "marriages" between participants in the intricate playing out of
genomic expression — and also in avoiding the wrong marriages. The
evidence suggests, according to UK geneticists Peter Fraser and Wendy
Bickmore, that "the dynamic spatial organization of the nucleus both
reflects and shapes genome
function . . . . We now have a picture of a genome that is
'structured', not in a rigid three-dimensional network, but in a dynamic
organization [that] clearly changes during normal development
and
differentiation"
(Fraser and Bickmore 2007).
We began by asking ourselves how the cell condenses two meters of DNA into a
nucleus ten millionths of a meter in diameter. The question is justified,
but we can see by now that it's hardly a mere matter of avoiding a snarled
state so that an autonomous logic of transcription
can proceed
along its fated way. The adroit dynamics and deft sculpturing of
chromosomes and an entire galaxy of proteins are as much the "whole point
of the show" as any fixed code. The logic of transcription itself is, at
least in part, a disciplined art of movement. The next time you find
yourself picturing heredity as the transmission of fixed, determinative
elements from parent to offspring, you might pause to ask yourself how
such statically imagined elements could determine the art of movement that
also comes to expression in successive generations.
There is, after all, as much cause and effect, as much determination of
outcomes, as much logic and reason, in the compaction and twisting,
the movement and re-shaping, as there is in any other aspect of the cell
nucleus, even if the dynamism is fluid and irreducible to digital terms.
The chromosome performs an unceasing dance and — crucially —
the ever-shifting pattern of the dance lends its form and organization
to the expression of genes. Perhaps that is why a pair of geneticists
could write — very wisely, I think — that trying to define the
chromatin complex "is like trying to define life itself" (Grewal and
Elgin 2007).
If we ignore the artful movement, it's not because we find in it little meaningful expression of the cell's nature, but only because we have a difficult time translating it into the familiar and preferred terms of science. But that's a limitation of our science, not of the cell. We already have enough evidence to say that the movement, as movement, must be at least as deft and graceful, and at least as well-calculated, as any Olympic gymnast's.
Do the genes control the cell and hand down instructions? Whatever reason there may be to view the matter from that angle, there's at least as much reason to think of the dance of chromosomes as controlling the genes. Which individual genes can be expressed and how much; which "signaling" functions of the cell are brought to bear on any particular gene; which large stretches of the chromosome are prepared for longer-term expression and which are put into "cold storage" — all this is not so much digitally enunciated as gestured by the entire context. And the choreography continually varies, summoning genes to participate in the power of its higher-order artistry.
It is not too much to say that the cell presents us with forms constantly modulated by the cellular environment and beyond — living sculptures, shape-shifting in response to a music we have not yet inquired about, let alone learned to hear.
(You will find the latest versions of the currently available parts of this series at the website, "From Mechanism to a Science of Qualities".)
1. Terms such as "twist", "writhe", and "coil" are given precise technical definition by topologists — definitions I have made no effort to honor strictly here.
Please Note: With a view toward the needs of the readership, I have preferred to cite review articles, where they are available and, in general, have made little effort to reflect in my citations the priority claims of the various investigators of any particular phenomenon. Public (online) accessibility of papers and ease of access to the relevant information are primary criteria for my selection — qualified, of course, by the limits of my own familiarity with the literature.
Bártová, Eva, Jana Krejci, Andrea Harnicarová et al. (2008). "Histone Modifications and Nuclear Architecture: A Review", Journal of Histochemistry and Cytochemistry vol. 56, no. 8, pp. 711-21. doi:10.1369/jhc.2008.951251
Blot, Nicolas, Ramesh Mavathur, Marcel Geertz, et al. (2006). "Homeostatic Regulation of Supercoiling Sensitivity Coordinates Transcription of the Bacterial Genome", EMBO Reports vol. 7, no. 7, pp. 710-5. doi:10.1038/sj.embor.7400729
Carter, David R. F., Christopher Eskiw, and Peter R. Cook (2008). "Transcription Factories", Biochemical Society Transactions vol. 36, pp. 585-9. doi:10.1042/BST0360585
Chuang, Chien-Hui, Anne E. Carpenter, Beata Fuchsova, et al. (2006). "Long-range Directional Movement of an Interphase Chromosome Site", Current Biology vol. 16 (Apr. 18), pp. 825-31. doi:10.1016/j.cub.2006.03.059
Costelloe, Thomas, Jennifer FitzGerald, Niall J. Murphy, et al. (2006). "Chromatin Modification and the DNA Damage Response", Experimental Cell Research vol. 312, pp. 2677-86. doi:10.1016/j.yexcr.2006.06.031
Deng, Shuang, Richard A. Stein, and N. Patrick Higgins (2005). "Organization of Supercoil Domains and Their Reorganization by Transcription", Molecular Microbiology vol. 57, no. 6 (September), pp. 1511-21. doi:10.1111/j.1365-2958.2005.04796.x
De Wit, Elzo and Bas van Steensel (2009). "Chromatin Domains in Higher Eukaryotes: Insights from Genome-wide Mapping Studies", Chromosoma vol. 118, pp. 25-36.
Fraser, Peter and Wendy Bickmore (2007). "Nuclear Organization of the Genome and the Potential for Gene Regulation", Nature vol. 447 (May 24), pp. 413-7. doi:10.1038/nature05916
Fraser, Peter and James Douglas Engel (2006). "Constricting Restricted Transcription: The (Actively?) Shrinking Web", Genes and Development vol. 20, pp. 1379-83. doi:10.1101/gad.1438106
Galande, Sanjeev, Prabhat Kumar Purbey, Dimple Notani, et al. (2007). "The Third Dimension of Gene Regulation: Organization of Dynamic Chromatin Loopscape by SATB1", Current Opinion in Genetics and Development vol. 17, pp. 408-14. doi:10.1016/j.gde.2007.08.003
Grewal, Shiv I. S. and Sarah C. R. Elgin (2007). "Transcription and RNA Interference in the Formation of Heterochromatin", Nature vol. 447 (May 24), pp. 399-406. doi:10.1038/nature05914
Hark, Amy T. and Steven J. Triezenberg (2001). "Chromatin and Transcription: Merging Package and Process" (review of Chromatin Structure and Gene Expression, edited by Sarah C. R. Elgin and Jerry L. Workman), Cell vol. 105, no. 3 (May 4), pp. 321-3. doi:10.1016/S0092-8674(01)00352-X
Jing, Huie, Christopher R. Vakoc, Lei Ying, et al. (2008). "Exchange of GATA Factors Mediates Transitions in Looped Chromatin Organization at a Developmentally Regulated Gene Locus", Molecular Cell vol. 29, no. 2 (Feb. 1), pp. 232-42. doi:10.1016/j.molcel.2007.11.020
Kioussis, Dimitris (2005). "Gene Regulation: Kissing Chromosomes", Nature vol. 435, no. 7049 (June 2), pp. 579-80. doi:10.1038/435579a
Kouzine, Fedor and David Levens (2007). "Supercoil-driven DNA Structures Regulate Genetic Transactions", Frontiers in Bioscience vol. 12 (May 1), pp. 4409-23).
Kouzine, Fedor, Suzanne Sanford, Zichrini Elisha-Feil, et al. (2008). "The Functional Response of Upstream DNA to Dynamic Supercoiling in Vivo", Nature Structural and Molecular Biology vol. 15, no. 2 (Feb.), pp. 146-54. doi:10.1038/nsmb.1372
Lanctôt, Christian, Thierry Cheutin, Marion Cremer, et al. (2007). "Dynamic Genome Architecture in the Nuclear Space: Regulation of Gene Expression in Three Dimensions", Nature Reviews Genetics vol. 8, no. 2 (Feb.), pp. 104-15. doi:10.1038/nrg2041
Lavelle, Christophe (2009). "Forces and Torques in the Nucleus: Chromatin under Mechanical Constraints", Biochemistry and Cell Biology vol. 87, pp. 307-22. doi:10.1139/O08-123
Ling, Jian Qun and Andrew R. Hoffman (2007). "Epigenetics of Long-Range Chromatin Interactions", Pediatric Research vol. 61, no. 5, Pt. 2, pp. 11R-16R. doi:10.1203/pdr.0b013e31804575db
Lior, David, Wolfgang Huber, Marina Granovskaia, et al. (2006). "A High-resolution Map of Transcription in the Yeast Genome", PNAS vol. 103, no. 14 (Apr. 4), pp. 5320-5. doi:10.1073/pnas.0601091103 https://www.pnas.org/content/103/14/5320.full.
Lomvardas, Stavros, Gilad Barnea, David J. Pisapia, et al. (2006). "Interchromosomal Interactions and Olfactory Receptor Choice", Cell vol. 126 (July 28), pp. 403-13. doi:10.1016/j.cell.2006.06.035
Misteli, Tom (2007). "Beyond the Sequence: Cellular Organization of Genome Function", Cell vol. 128 (Feb. 23), pp. 787-800. doi:10.1016/j.cell.2007.01.028
Osborne, Cameron S., Lyubomira Chakalova, Karen E. Brown, et al. (2004). "Active Genes Dynamically Colocalize to Shared Sites of Ongoing Transcription", Nature Genetics vol. 36, no. 10 (Oct.), pp. 1065-71. doi:10.1038/ng1423
Ottaviani, Diego, Elliott Lever, Petros Takousis, et al. (2008). "Anchoring the Genome", Genome Biology vol. 9, article 201 (Jan. 22). doi:10.1186/gb-2008-9-1-201
Schneider, Robert and Rudolf Grosschedl (2007). "Dynamics and Interplay of Nuclear Architecture, Genome Organization, and Gene Expression", Genes and Development vol. 21, pp. 3027-3043. doi:10.1101/gad.1604607
Schones, Dustin E., Kairong Cui, Suresh Cuddapah, et al. (2008). "Dynamic Regulation of Nucleosome Positioning in the Human Genome", Cell vol. 132 (Mar. 7), pp. 887-98. doi:10.1016/j.cell.2008.02.022
Simonis, Marieke, Petra Klous, Erik Splinter, et al. (2006). "Nuclear Organization of Active and Inactive Chromatin Domains Uncovered by Chromosome Conformation Capture-on-chip (4C)", Nature Genetics vol. 38, no. 11 (Nov.), pp. 1348-54. doi:10.1038/ng1896
Spilianakis, Charalampos G., Maria D. Lalioti, Terrence Town, et al. (2005). "Interchromosomal Associations between Alternatively Expressed Loci", Nature vol. 435, no. 7042 (June 2), pp. 637-45. doi:10.1038/nature03574
Spudich, James A. (2008). "Dynamic Organization of Gene Loci and Transcription Compartments in the Cell Nucleus", Biophysical Journal vol. 95, no. 11 (Dec. 1), pp. 5003-4. doi:10.1529/biophysj.108.139196
Travers, Andrew and Georgi Muskhelishvili (2007). "A Common Topology for Bacterial and Eukaryotic Transcription Initiation?" EMBO Reports vol. 8, no. 2, pp. 147-51. doi:10.1038/sj.embor.7400898
Trinkle-Mulcahy, Laura and Angus I. Lamond (2008). "Nuclear Functions in Space and Time: Gene Expression in a Dynamic, Constrained Environment", FEBS Letters. doi:10.1016/j.febslet.2008.04.029
Zaidi, Sayyed K., Daniel W. Young, Amjad Javed, et al. (2007). "Nuclear Microenvironments in Biological Control and Cancer", Nature Reviews Cancer vol. 7 (June), pp. 454-63. doi:10.1038/nrc2149
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Steve Talbott :: The Chromosome in Nuclear Space