How Our Genes Come to Expression
(It Takes an Epigenetic Village)
This is a preliminary draft of one chapter of a book-in-progress
tentatively entitled, “Evolution As It Was Meant To Be — And the Living Narratives That Tell Its Story”.
You will find
a fairly lengthy article serving as a kind of extended abstract of major
parts of the book. This material is part of the
Biology Worthy of Life
Project. Copyright 2017-2021
The Nature Institute.
All rights reserved. Original publication: January 21, 2021.
Last revision: January 21, 2021.
If your understanding of genetics comes from your newspaper’s science
section, or a popular science magazine, or any other source intended for
the general public, then you will not have been given the remotest glimpse
of what actually goes on with the genes in our bodies. In fact,
geneticists themselves have been known to lament how limited their
knowledge of gene-related activity is, simply because the demands of
professional specialization scarcely allow a wide field of view.
But it turns out that a wide field of view is the one critical
prerequisite for any adequate understanding of genes. Only a broad survey
can illustrate how every gene, like a significant word in a text, receives
its full meaning only through the interweaving and converging influences
issuing from all the elements of its context.
My aim here is to offer such a wider, “epigenetic” view — and to do so in
the briefest space possible. If I succeed, you will begin to sense a
biological landscape that reconfigures many long-standing assumptions, not
only about genetics itself, but also about the character of living
After the discovery of the structure of the DNA double helix in 1953 and
the elaboration of the “genetic code” during the early 1960s, the
of a gene was thought of as the production of a
functional protein corresponding precisely to instructions
gene — instructions that were spelled out in the gene’s sequence
DNA “letters”, or nucleotide bases
. The protein’s production,
based on this sequence, was routinely described as a cut-and-dried, rather
mechanistic affair. The larger picture was sometimes summed up in the
DNA makes RNA, RNA makes protein, and protein makes the organism.
A few key terms may help to flesh out the formula as it was then
understood. (All the special vocabulary is elaborated in the
The first step in gene expression was thought to be the binding of a
protein transcription factor (one of many such factors existing in
the cell) to DNA at or near a target gene. This led to the adjacent
binding of a complex protein called RNA polymerase (often described
as a “molecular machine”), which then transcribed the DNA sequence
of the gene into an RNA molecule closely mirroring the DNA sequence.
Finally, the RNA was exported from the cell nucleus into the cytoplasm,
where it was translated into a specific protein. The translation
was carried out by another complex “molecular machine”, this one composed
of both protein and RNA and known as a ribosome. The sequence of
amino acids in the resultant protein was said to have been coded
for by the sequence of nucleotide bases in the gene. A parallel was
sometimes drawn with Morse code, in which a sequence of dots and dashes
codes for a sequence of alphabetic letters.
The discovery of the entire scheme, which seemed so neat and tidy, was
almost universally welcomed.
But there was already a curiosity. Consider the picture. The production
of a protein from DNA was initiated by a protein transcription factor.
The “molecular machines” doing the work of transcription and translation
consisted, in whole or in part, of proteins. Moreover, it was recognized
that proteins were decisive for the very existence of DNA, as well as its
replication, maintenance, and repair. So not only were proteins required
in order to explain their own synthesis, but they were also required in
order to explain the existence of DNA. At the same time, DNA was clearly
required for the existence of proteins.
You might think the chicken-and-egg problem here would have given the
scientific community pause during its single-minded, twentieth-century
rush toward a gene-centered view of life. Was it really genes that made
the organism, including its proteins? Or was it proteins that made the
organism, including its genes? Or were both points of view terribly
flawed and unbiological, so that we were being asked to rise to a more
living and integral level of understanding where it is impossible to say
that one thing unambiguously “causes” another?
Fast forward to today, and consider just one of the terms mentioned above:
“transcription factor”. A riddle posed by many such protein factors
involves their “promiscuous binding”. Transcription factors, it turns
out, are not targeted to specific DNA sequences by some iron necessity.
Many of them are quite capable of binding at numerous locations throughout
the genome — that is, at far more loci than they are actually found at in
typical assays of living cells. In other words, we have to look for much
more than a definitive, sequence-based targeting logic if we want to
understand how transcription factors activate (or inhibit) specific genes
in this or that specific kind of cell and context.
So the question arises, How does a transcription factor “know” which gene
or genes to interact with? If its specificity — its ability to bind where
it is needed — is not dictated by a simple and determinative match between
its own binding domain and the DNA sequence it binds to, then how do we
make sense of its well-directed activity? Is this sense something like
the logic at work in a humanly devised mechanism? Or is it more like a
living language, where each word has expressive potentials that cannot be
rigorously defined in advance of actual use?
The answer — or, rather, the many answers — are still unfolding today.
The one indisputable truth is that it takes a molecular “village” — a
vigorous and entire cellular context — to establish the correct and
ever-changing relations between a transcription factor and the genes it
helps bring to expression. The old idea that the relations among
transcription factors, genes, and gene products are unambiguous — are
governed by a fixed and easily comprehended necessity — is no longer
Transcription factors and DNA engage in a complex play of form
To begin with, not just the DNA sequence, but also the moment-by-moment
sculptural form, or conformation, of a DNA locus affects the binding
potential of a transcription factor. This form is dynamically imposed in
part by the cellular environment, Also decisive are the plastic
conformational potentials of the transcription factor itself. And then
there are the many other essential molecules (”co-factors”) that may not
even have the ability to bind to DNA, but which are nevertheless essential
co-participants, along with transcription factors, in an interactive
community through which a gene, or set of genes, is made ready for
For example, one way a transcription factor can contribute to the
expression of a gene is by bending a short stretch of DNA into a shape
conducing to further interaction. (For a striking illustration of this,
below.) By this means the initial presence of a transcription factor can
make it easier than it would otherwise be for a second protein to bind
nearby. In the case of one gene relating to the production of interferon
(an important constituent of the immune system): “eight proteins modulate
[DNA] binding site conformation and thereby stabilize cooperative
(Moretti et al. 2008).
And so, despite the fact that “DNA is often mistakenly viewed as an inert
lattice” onto which proteins bind in a sequence-specific way
the fact of the matter is altogether different. Proteins and DNA are
caught up in a continual conversation of mutual influence and shifting
form. It becomes obvious, then, that “No simple code combines all the
various determinants of transcription factor binding specificity”
(Slattery et al. 2014).
In other words, a transcription factor’s “recognition” of a DNA binding
site is not a digital, yes-or-no matter, but a community judgment. And
how could it be otherwise, given that no cell in our bodies lives merely
for itself? Our activities always involve vast, cooperating communities
of cells. Every cell is caught up in a larger context of meaning and must
be capable of adapting itself to, and supporting, virtually any of the
infinitely varying activities we find ourselves engaging in.
A living flexibility is therefore crucial. So it is no surprise when one
pair of researchers, studying a group of transcription factors in the
genomes of animals, report “a dazzling array of strategies employed by
[these] transcription factors to control gene expression.” The “emerging,
unifying theme”, they say, is the ability of these transcription factors
“to interact with many diverse partners. This high connectivity is
probably crucial to assemble highly context-specific, transcriptionally
active complexes at selected sites in the genome”
(Bobola and Merabet 2017).
Genes and proteins interact in tangled causal webs
It is hard to take in the full significance of this “high connectivity”,
which is typical of so many biological processes. One way to visualize
the complications is to consider the fact that some transcription factors
can target genes for other transcription factors. And, of course, this
second group of transcription factors might target the genes for still
other transcription factors as well as the genes or regulatory sequences
associated with the first group. We can easily imagine the tangled causal
webs resulting from this kind of inter-connectivity, where causal “arrows”
can eventually circle back to their starting point. Unsurprisingly, there
are entire fields of research today given over to complex gene and
regulatory networks such as this one:
Figure 14.1. A modest regulatory network showing
interactions among a cluster of transcription factors (blue), genes
(green), and microRNAs (miRNAs — purple) under specific conditions in
certain mouse immune cells. (We will discuss microRNAs
Red lines indicate regulation of transcription. Blue lines indicate
post-transcriptional regulation — that is, regulation of RNAs once they
have been transcribed. Only selected relationships are shown; for
example, regulation of the microRNAs (as opposed to their regulation of
other molecules) is omitted from the
Returning to the puzzle of transcription factor “promiscuity”: this word
reflects neither undisciplined profligacy nor uncertainty of function.
Rather, it points to the unbounded, context-specific potentials of
transcription factors. Their contribution to essential cellular
processes, after all, is properly focused and far from promiscuous. They
are caught up within a wisdom that seems to “know” exactly what it is
doing. It’s just that this doing is complex and living — flexible
and adaptive — far beyond what a simple, definitive, one-dimensional
mapping between DNA sequence and a rigidly complementary protein shape
would allow. This flexibility is what allows community-tuned activity in
the larger surround to influence local goings-on in endlessly nuanced ways
— all so as to satisfy the needs of the current context.
It is important to underscore here a fact we have found ourselves coming
up against throughout this book: the tangled causal web we discover in
organisms is not merely a matter of complexity. There are many nonliving
physical contexts so complex that, as a practical matter, we cannot easily
trace precise lines of cause and effect. This is true of eddies in a
great river or in the atmosphere, and it is even true of some kinds of
computer program. And yet no one would doubt in these cases that the
relevant causes could be traced, at least in principle, or that the
tracing would give us what is considered to be a full accounting of what
we were looking at.
But, as I began explaining in
the purposive behaviors of organisms exhibit a kind of coherence and
meaning that is not satisfactorily explained when we look only at
principles of physical causation. The “causal confusion” in the
organism’s case is not due merely to the complexity of the physical
relations, but rather to the fact that explanation must be found at a
“higher” level than physical lawfulness. The significance of what is
going on is recognized only when we consider the insistent coordinating
principles through which physical events are made to serve the needs and
interests of organisms. Because concepts such as “need” and “interest”
are incommensurable with the accepted principles of physical explanation,
they demand recognition as explanatory principles in their own right.
The cell holds DNA in an intimate and instructive embrace
Our brief discussion of genes and transcription factors has, so far, been
hopelessly simplistic. The chromosomes in our cells do not consist of a
naked DNA double helix sporadically bound at particular sequences by this
or that transcription factor. The picture is wholly different. Our DNA
is intimately bound up with a massive, intricate, and dynamic
protein-RNA-small molecule complex that, together with the DNA, is called
chromatin. “Chromatin”, in other words, can pass as simply a name
for the full substance of chromosomes. The proteins in this complex are
as weighty as the DNA itself — and much more active and directive when it
comes to gene expression.
Some of the protein constituents of this chromosomal substance — both the
longer-term and the many transient constituents — can bind directly to
DNA, thereby facilitating, blocking, or modifying the transcription of
this or that gene. But other elements of chromatin, while not directly
bound to DNA, nevertheless contribute crucially to the regulation of gene
expression. Overall, the molecular factors associated with chromatin play
roles such as the following:
- they help to condense or decondense the packing of the DNA (more
tightly condensed DNA tends to be less accessible to activating factors);
- they move chromosomes or parts of chromosomes to different regions
of the cell nucleus (the interior of the nucleus tends to be more
transcriptionally active than the periphery);
- they attach parts of chromosomes to the nuclear envelope (many
factors at or near the envelope bear on gene expression);
- they interweave and (almost miraculously, it might seem) disentangle
chromosomes, while also forming decisively important chromosome loops
(such as those we heard about in
— all so as to form various-sized “communities” of functionally related
- they loosen the two strands of the double helix in some places and
twist them more tightly in others, which can make the difference between a
gene’s accessibility or inaccessibility to transcription factors;
- they alter the electrical characteristics of particular loci (yet
another feature bearing on the expression of affected genes);
- and so on almost without end.
As you may surmise, then, it’s not as if the power to determine gene
expression outcomes is one-sidedly delegated to any genetic sequences. It
is rather as if the result arises in the way a musical performance is
evoked from a jazz orchestra. A distinct locus of DNA certainly offers
its own expressive potentials, but there is no telling — no predicting
solely from an analysis of the sketchy DNA “musical score” — how the locus
may be employed within the improvised, multi-cellular performance leading
from a single fertilized egg cell to the mature human being.
But perhaps we would do better to imagine an exquisitely detailed,
never-ending, self-assured, yet highly improvisational dance involving
billions of molecular dancers within a cell — all coordinated with the
choreography in neighboring cells and with the ongoing story of the
organism as a whole. The performance is a long way from that of a
calculating or information-processing machine.
In any case, the present point is that our DNA is thoroughly “wedded” to —
bound together with — an almost unfathomably intricate arrangement of
The protein and RNA constituents of this chromatin complex are fully as
“information”-rich as the DNA. Genes, as such, cannot do anything,
and certainly cannot transcribe themselves. The doing is in large part a
function of the associated proteins, which, among other things, thereby
participate in their own genesis. Alongside them are many other
molecules, including water molecules
all of whom give collective expression to the purposive coherence of the
cell as a whole.
I have so far offered only a rather vague and general description of the
highly effective embrace in which DNA is held. In later sections we will
look further at some of its key features.
is hard to do
Meanwhile, leaping tall edifices of thought in a single bound, we will
pass over the question how cells “know” which genes need to be expressed
within the current context of a person’s activity and within the virtually
infinite number of uniquely performing cellular niches within our bodies.
We will also avoid asking how any single cell — which can play only a
spatially minute part within an organ such as the liver or within a
process such as wound healing — finds its own proper role in whatever the
current larger performance happens to be. And so, assuming all the
necessary contextualization and direction to be somehow wisely taken care
we will imagine just one cell embarking on a single task: to give
expression to one among its 21,000 or so genes. How might this cell
Our imaginative exercise will necessarily be more than a little
artificial. That’s because we need to think one thing at a time, whereas
in the cell countless mutually entangled things are all happening at once.
But we will try to make the best of it.
You may recall from
that packing DNA into a typical human cell nucleus is like packing about
24 miles of very thin, double-stranded string into a tennis ball, with
the string divided into 46 separate pieces, corresponding to our 46
To locate a modest-sized protein-coding gene within all that DNA is like
homing in on a half-inch stretch within those 24
Or, rather, two relevant half-inch stretches located on different pieces
of string, since most of our cells have two copies of any given gene,
residing on different chromosomes. Except that sometimes one copy differs
from the other and one version is not supposed to be expressed, or one
version needs to be expressed more than the other, or the product of one
needs to be modified relative to the other. So part of the job may be to
distinguish one of those half-inch stretches from the other, and to act
differently in the two cases. “Decisions” everywhere, it seems.
As a functional unit, a gene must participate in a performance appropriate
to its context, and the highly distributed activity responsible for its
function must be cobbled together by the cell according to the needs of
the moment. There is no predefined path to follow once the cell
has located the “right” half inch or so of “string”, or once it has done
whatever is necessary to bring that locus into proper relation with other
chromosomal loci participating in, and essential to, a joint performance.
One issue has to do with the fact that there are two strands of the double
helix, and (in a chemical sense) these complementary strands “point” in
opposite directions. In humans, protein-coding sequences can occur on
both strands. Likewise, transcription (of both protein-coding and
regulatory sequences) occurs on both strands, which is to say that the
transcribing enzyme (RNA polymerase) can move in either direction along
the double helix. The direction chosen — that is, the strand along which
the RNA polymerase will move — depends on the meaning within the current
context of the sequences that exist at the current locus. Somehow,
acting within and guided by its present context, RNA polymerase must have
the “good sense” to choose the appropriate activity from among the various
And even when the cell would initiate transcription in one particular
direction, it must “choose” the exact point in the genetic sequence at
which to begin. Different starting points can yield functionally distinct
results. “Many studies focusing on single genes have shown that the
choice of a specific transcription start site has critical roles during
development and cell differentiation, and aberrations in …
transcription start site use lead to various diseases including cancer,
neuropsychiatric disorders, and developmental disorders”
(Klerk and ’t Hoen 2015).
Intertwined with all the preceding issues is the cell’s task of assembling
a pre-initiation complex (PIC). This variable arrangement of
regulatory elements typically sets the stage for the transcriptional
activity to follow.
is a cartoon figure that merely names some of the protein PIC constituents
that arrange themselves on DNA (shown as a black line) near locations
where gene transcription is to begin. You needn’t concern yourself with
names and meanings, beyond the general description I am offering now.
Figure 14.2. The pre-initiation complex (cartoon
The cell’s narrative at this point could hardly be more dramatic — or more
subtle. The largest oval in Figure 14.2, named “Mediator”, is a massive
molecule consisting variably of up to 30 protein subunits
arranged in modules and interacting in numerous ways among themselves, as
well as with other PIC constituents and “visiting” molecules. Depending
on context, Mediator can vary endlessly in both subunit composition and
function. Its effects upon gene expression are many, and still only
shows the known interaction partners for the Mediator subunits in just one
cell type — mouse neural stem cells. The figure omits the numerous
interactions among the Mediator subunits themselves. It also omits the
interactions among the molecules shown in the surrounding circle. And,
perhaps most importantly, it omits the interactions those molecules have
with still others not shown in the diagram. For it is just a fact that
each of these molecules shown in the outer circle could be made the center
of its own diagram. Reflecting on this can usefully remind us of what it
means to say that all biological activity in a cell, no matter how
micro-focused our vision, turns out upon broader inspection to be an
almost impossibly intricate and coordinated activity of the whole.
And, of course, Mediator is just one element of the PIC. Each of the
other elements has its own story to tell. The entire PIC was once
regarded as a rather mechanical, routine, and mostly unvarying assembly of
“parts” whose unproblematic duty was to initiate gene transcription in a
standard way. But, of course, that was to overlook how thoroughly every
aspect of gene expression must vary if it is to serve the needs of
a living being. The PIC is now seen to be an infinitely modifiable,
highly dynamic complex, responding both to the immediate DNA context and
to influences arriving from distant reaches of the cell. Its overall
“decision-making” role, which can differ from one gene to the next, is
hardly the functioning of a routinely analyzable mechanism.
It doesn’t require of the reader a technical penetration of these figures
to get a sense for the kind of thing that is going on — especially if one
keeps in mind that we are talking, not about rigid machinery of the sort
we are familiar with in our daily lives, but rather about molecular
interactions within a highly fluid context where machine-like
constraints to forcibly channel the interactions are altogether absent.
DNA (red) in the grip of the tata-binding protein
I will mention here just one other element of the pre-initiation complex.
Figure 14.5 shows DNA (in a wholly artificial, simplistic, and impossibly
rigid, concrete representation) being “gripped” by the tata-binding
protein (TBP), shown in blue. TBP is also seen as the crescent-moon shape
at the bottom of
The protein “clasps” the DNA in an intimate and rather tortuous manner — a
clasp that might remind one of the forcible interaction between two human
A severe bend of about eighty degrees is thereby applied to the double
helix. This bend, which also tends to pull the two strands of the helix
apart, is a general prerequisite for the assembly and activity of the rest
of the PIC. As always, the cell is doing something sculptural, not
narrowly informational in the usual sense.
As we heard at the outset, the (protein) enzyme that transcribes DNA into
The enzyme certainly does not work alone, however, and its task is by no
means automatic. To begin with, its critical interactions with various
elements of the pre-initiation complex help determine whether and exactly
where transcription will begin. Then, after those “decisions” have been
made, RNA polymerase moves along the double helix transcribing the
sequence of genetic “letters” into the complementary sequence of an RNA.
Throughout this productive journey, which is called elongation, the
RNA polymerase still keeps good and necessary company. Certain molecular
co-activators modify it during its transit of a gene’s sequence, and these
modifications not only enable transcription elongation to begin, but also
provide binding sites for yet other proteins that will cooperate
throughout the transcription journey. The collective interaction here, as
in the activities discussed above, can vary in many details from one
context to another — all in order to contribute to a meaningful
that could hardly repeat itself in exactly the same way.
The table below offers some perspective on the number and variety of
protein factors influencing elongation. You need not puzzle over the
details. A quick browse of this incomplete listing (as of 2013) will give
you at least an inkling of the kind of intricate complexity the cell must
organize in order to carry out transcriptional elongation. As always, it
is important to realize that each of the factors listed here enters the
picture out of its own world of regulation. At the molecular level of the
organism we are always looking at ever-widening circles of interaction,
without limit. It’s just a question of how narrowly we choose to focus
our attention — and how much of the context we consequently block from
. Don’t Read This Table!
(Just feel it.)
Some factors regulating RNA polymerase elongation
Kwak and Lis 2013
||Related factors and notes
||Generates nucleosome-free region and promoter structure
|General Transcription Factors
||Generates promoter structure for pausing
||Increases elongation rate
||Rescues backtracked RNA polymerase II
||RNA polymerase III
||Stabilizes RNA polymerase II pausing
||Stabilizes RNA polymerase II pausing and facilitates elongation
|Positive elongation factor
||Phosphorylates NELF, DSIF, and RNA polymerase II CTD for pause
||Increases elongation rate
||Increases elongation rate
||Contains P-TEFb and ELL
||Directly recruits P-TEFb
||Directly recruits P-TEFb
||Recruits P-TEFb via SEC
||Facilitates P-TEFb recruitment, counters NELF/DSIF
||Methylates RNA 5’ end to complete capping
|Premature termination factors
||Decaps nascent RNA for XRN2 digestion
||Cleaves hairpin structure for XRN2 digestion
||Torpedoes RNA polymerase II with RNA 5’-3’ exonucleation
||Releases RNA polymerase II from DNA
||Antitermination and stabilizes paused RNA polymerase II
||H2A-H2B eviction and chaperone
||Tracks with RNA polymerase II
||Tracks with RNA polymerase II
||SWI/SNF remodeling in gene body
||Maintains gene body nucleosome organization
||ISWI remodeling at promoter
||Transcription independent nucleosome loss
|Polymerase-associated factor complex
||Loading dock for elongation factors
|Histone tail modifiers
||Acetylates H4K16 and recruits Brd4
||Acetylates H2AK5 and activates PARP
||Acetylates H3 and facilitates nucleosomal elongation
||Also in cytoplasm
||Deacetylates and inhibits spurious initiation in gene
||Methylates H3K36 and regulates acetylation-deacetylation
||Phosphorylates H3S10 and recruits 14-3-3 and MOF
||Monoubiquitinates H2BK123 and facilitates nucleosomal
I will mention here only one aspect of this cooperation of multiple
factors. Transcription is an essentially rhythmical performance, with
various sorts of pauses along the way. (Again, dynamic sculpture, or
dance!) One pause of great significance occurs after RNA polymerase has
just begun transcribing DNA but before it has fully separated from the
pre-initiation complex. The factors that influence whether transcription
will continue at this point — or remain paused for an extended period —
play a large role in the regulation of gene expression.
But once that first pause is ended, the elongation journey often continues
to be marked by a series of further, generally briefer pauses. These have
to do, at least in part, with the need to disengage DNA from its intimate
mutual embrace with certain constituents of chromatin (histone complexes,
about which we will learn more
The polymerase has various assistants to aid in this disengagement, which
may involve disassembly of the protein complexes. Typical of chromatin in
general, these complexes are rich repositories of regulatory information,
so they will need to be reassembled behind the transcribing complex, and
the remarkably nuanced meanings embodied in their composition and
structure will somehow have to be preserved, reestablished, or modified.
So the rhythm of pauses depends, at least in part, on the polymerase’s
helper molecules and on the positioning of certain protein complexes along
the double helix, both of which will vary from one gene to another and
even from one time to another. All this, and not just the so-called
genetic code as such, shapes the functional significance of the DNA
sequence within its chromosomal context. As we will see shortly,
different versions of a protein may be produced, depending on the timing
of the pauses.
Finally — and mirroring all the possibilities surrounding initiation of
gene transcription — there are the issues relating to its termination.
Again, they are far too many to mention here. Transcription may conclude
at a more or less canonical terminus, or at an alternative terminus, or it
may proceed altogether past the gene locus, even to the point of
overlapping what, by usual definitions, would be regarded as a separate
gene farther “downstream”. The cell has great flexibility in determining
what, on any given occasion, counts as a gene, or transcriptional unit.
The last part of the transcribed gene is generally non-protein-coding, but
nevertheless contains great significance. Examining this region in a
single gene, one research team identified “at least 35 distinct regulatory
elements” to which other molecules can bind
(Kristjánsdóttir, Fogarty and Grimson 2015).
Importantly: additional dramatic and diverse regulatory potentials arise
from the customized “tail” that the cell commonly adds to the end of an
mRNA after its transcription from DNA. The regulatory processes
called into play by this tail can affect everything from the stability of
the mRNA to its cellular localization and the efficiency of its
translation into protein. It can even play a role in determining exactly
what protein will ultimately be produced. And the patterns of these added
tails tend strongly to differ from one tissue type to another.
“Decisions” yet again.
Much of this post-transcriptional regulation is accomplished by proteins
and other molecules that bind, not only to the end, but also to the
various regulatory sequences at the head of the RNA transcript. It
all occurs in a context-sensitive manner, where cell and tissue type,
phase of the cell cycle, developmental stage, location of the transcript
within the cell, and converging environmental factors, both intra- and
extra-cellular, may all play a role.
But it’s not only the RNA sequence that provides opportunities for
management by the cell. The three-dimensional, folded structure of
the molecule offers boundless occasion for further regulation. So here,
as with DNA, we find gene expression to be in part a matter of sculptural
performance. And, again, it is not just a matter of static form, but of
movement. According to molecular biologists at the University of Michigan
and Duke University, “RNA dynamics play a fundamental role in many
[There are] many structural maneuvers that occur over timescales ranging
from picoseconds to seconds … These transitions include large-scale
secondary-structural transitions at [greater than tenth-of-a-second]
timescales, base pair/tertiary dynamics at microsecond-to-millisecond
timescales, stacking dynamics at timescales ranging from nanoseconds to
microseconds, and other ‘jittering’ motions at timescales ranging from
picoseconds to nanoseconds. RNAs often harness multiple modes to achieve
(Mustoe et al. 2014)
“Epigenetics” refers to that which is not genetics as such, but rather is
“added to”, or “on top of” genetics. You might therefore think that the
transcription factors, RNA polymerases, and other proteins mentioned
above, which are not themselves genetic elements, would therefore
be treated under the heading of epigenetics. Oddly, however, this has not
been the case. Presumably, the reason is that these factors have for so
long been taken for granted as if they were mere adjuncts to the
“controlling logic” of DNA sequences.
But this never made much sense. What I have tried to suggest in my
descriptions above is that these “mere tools” are more and more being
recognized as participants in a dynamic communal context out of which
alone our genes come to disciplined expression according to the needs of
Now, however, it is time to approach — albeit with painful brevity — what
is generally considered the epigenetic mainstream. After all, we now know
that gene transcription is merely a small part of all the activity shaping
gene expression. The many processes “on top of” transcription are fully
as rich and multifaceted as the various features of transcription itself.
We have already heard about RNA splicing, which we looked at in
“The Mystery of an Unexpected Coherence”. As we learned in that chapter,
cells don’t just passively accept the RNAs that emerge from the
transcription process, but rather “snip” them apart and “stitch” (splice)
some of the pieces back together, while leaving aside other pieces for
purposes both known and unknown. It happens that these operations
typically begin before the RNA is fully transcribed, and the rhythm of
pauses by RNA polymerase during elongation influences which pieces form
the mature transcript.
For the vast majority of human genes the splicing operation can be
performed in different ways, yielding distinct protein variants (often
called isoforms) from a single RNA. It would be hard to find any
major aspect of human development, disease etiology, or normal functioning
that is not dependent in one way or another on the effectiveness of this
liberty the cell takes with the products of its gene sequences.
But RNA splicing is hardly the end of it. Through RNA editing the
cell can add, delete, or substitute individual “letters” of the RNA
Or, leaving the letters in place, the cell can apply over 170 distinct
chemical modifications to
Both the editing and the modifying are major topics in themselves, but not
ones we can linger on here.
MicroRNAs: a large world of tiny regulatory factors
An entire, diversified area of research involves small, non-protein-coding
RNAs. The only ones we will discuss here are known as microRNAs
(miRNAs), which are generally derived through the cleaving and processing
of longer RNAs. A microRNA commonly joins forces with a large protein
complex, called the RNA-induced silencing complex
microRNA guides the RISC to specific mRNAs by means of (sometimes only
rough) base pair complementation. (See
base pair complementarity
in the glossary.) Once a target mRNA is located, the RISC can cleave or
otherwise degrade it, or else block its translation. In this way a
typical microRNA can degrade or tune the amounts of a considerable number
of different mRNAs.
Such degradation is an example of RNA decay in general, for which
there are many different, interwoven pathways in cells. It is easy to
overlook the fact that decay is fully as important — and fully as much in
need of careful regulation — as the production of the RNA in the first
place. During development for example, cell differentiation would be
impossible if the RNAs and proteins appropriate for an earlier form of a
cell could not be recycled. In this way their constituent nucleotides or
amino acids can support synthesis of new RNAs and proteins necessary for
the cell’s forthcoming, more differentiated form. The same general
principle holds for all changing conditions that require fresh responses
from the cell.
MicroRNAs are key fine-tuners of the relative numbers of mRNAs in a cell
under any given circumstances. We can only wonder how they are
“instructed” by the larger context so as to “know” what those relative
numbers ought to be. But we do know some of the means employed.
One of the more recent stories about the role of microRNAs in regulating
gene expression points to a complexity almost beyond all hope of detailed
understanding. Evidence suggests that just about any RNA in the human
body can help to regulate any number of other RNAs, just as it in turn is
regulated by them. This intertwining of fates is due not only to the
competition for resources (an extremely abundant RNA, by monopolizing the
available amino acids in a cell, can make it more difficult for other RNAs
to be translated into protein), but also to the impact of microRNAs.
Here’s how it works:
Many protein-coding RNAs are densely covered with binding sequences for
microRNAs, so that a typical microRNA will find about 200 different RNA
species it can target for decay or modification. This means that if a
particular RNA is being highly expressed — and all the more if it is a
“microRNA sponge” possessing multiple binding sites for a specific
microRNA — it can have the effect of up-regulating other RNAs that are
targets for the same microRNA. It “soaks up” most of the microRNAs that
might otherwise degrade those other targets.
Network of competing endogenous RNAs (orange boxes). Gray arrows show
where specific microRNAs (miR-25, and so on) are known to bind distinct
ceRNAs, and blue arrows indicate possible interactions involving microRNAs
known to be
The RNAs that in this way regulate other RNAs by competing for shared
microRNAs are known as “competing endogenous RNAs” (ceRNAs). Figure 14.6
shows one such interacting network. The ceRNAs are shown in orange boxes,
with directly “competing” pairs located at opposite ends of the arrows.
The microRNAs mediating the competition are listed alongside the arrows.
Let’s consider just two of the RNAs in the figure. PTEN, when translated,
yields a protein that is, among other things a tumor suppressor. (It also
appears to facilitate cell migration, and to play a part in the adhesion
of cells to each other.) PTENP1, on the other hand, is an RNA derived
from a so-called “pseudogene”, assumed to result evolutionarily from a
mutational duplication of the PTEN gene, followed by further
mutations compromising its protein-coding function. Pseudogenes are one
more example of those many DNA elements, once written off as nonfunctional
“junk”, which are now being “caught in the act” playing important roles.
In the present case, we know at least one role for PTENP1. Its RNA may be
incapable of being translated into protein, but it nevertheless shares
many microRNA binding sites with the PTEN RNA. By sequestering those
microRNAs away from PTEN, PTENP1 allows the tumor-suppressor to be
expressed at proper levels. If, on the other hand, the pseudogene becomes
dysregulated for some reason, then microRNAs that would otherwise bind to
PTENP1, end up instead binding to, and repressing, PTEN, which reduces its
tumor-suppressing activity. It has in fact been shown that PTENP1
functioning is selectively lost in human cancers, consistent with its
importance as a microRNA
And yet, the situation is actually much “worse” than is shown above.
MicroRNAs can also regulate other microRNAs, whether by direct targeting
or, indirectly, by targeting transcription factors or regulators of those
other microRNAs. For example, one particular microRNA (known as miR-499)
was shown not only to regulate target genes (via their mRNAs) in the usual
way, but also altered the expression of 11 other miRNAs. These changes
resulted in 969 down-regulated genes, only 7.8 percent of which were
directly targeted by miR-499. In other words, “hundreds of genes may be
altered in expression” via these indirect pathways radiating from a single
(Hill and Tran 2021).
Here we see the same obstacle to any straightfoward causal understanding
that we encountered above regarding transcription factors activating or
repressing other transcription factors. Tracking the mutual, broad-scale,
and often subtle interactions where “everything seems to be affecting
everything else” will presumably challenge researchers for a very long
while. It looks like a classic picture of the unanalyzable holism of all
cellular processes. All the other interwoven aspects of gene regulation
discussed in this chapter, when added together, only add further to the
Some epigenetic processes profoundly implicated in gene expression
transform the DNA sequence itself. That is, they modify the nucleotide
bases (“letters”) of the so-called “genetic code”. One of these processes
is known as DNA methylation, which is widely recognized to be of
extreme importance for gene regulation.
DNA methylation is the addition of a methyl group (with chemical formula
–CH3) to certain DNA bases. There are four different bases in
DNA, and the one most commonly methylated is cytosine. In its methylated
form, this has been referred to as the “fifth base of DNA”. Millions of
bases throughout the genome are selectively and dynamically methylated in
the cells of normal human tissues. The difference between a methylated
and unmethylated base is hardly less significant, in its own way, than the
difference between one base and another. But, unlike the general rule for
the “raw” sequence of DNA bases, the methylation of those bases can be
altered during development and in response to environmental influences.
In this sense, much of our DNA inheritance is not at all the
fixed-once-and-for-all destiny it is so often taken to be. (And, of
course, just about everything else discussed in this chapter makes the
An “attached” methyl group is said to “tag” or "mark" the affected base.
However, words such as “attach”, “tag”, and “mark” are grossly inadequate,
suggesting little more than an annotation in the margin of a text, or a
digital label on an otherwise unchanged entity. But in fact what DNA
methylation gives us is chemical transformation — the metamorphosis of
many millions of letters of the human genome under the influence of
pervasive and incompletely understood cellular processes. And the altered
balance of forces — the modulation of chemical, electrical, and sculptural
qualities of chromosomes — resulting from all these chemically transformed
bases, certainly plays with endless possible nuances into the expression
of our genes.
We have been learning about the extreme consequences of these
metamorphoses. In the first place, the transformations of structure
brought about by methylation can render DNA locations no longer accessible
to the protein transcription factors that might otherwise bind to them in
order to activate nearby genes. On the other hand, by changing the local
physical properties of the double helix, methylation “is observed to
either inhibit or facilitate [DNA] strand separation, depending on
methylation level and sequence context”
(Severin et al. 2011).
This has a direct effect on gene expression — for example, because strand
separation is essential for the work of the polymerase that transcribes
Many proteins that recognize and bind specifically to methylated sites are
then able to recruit other proteins that restructure and functionally
alter the chromatin — for example, condensing it in a manner conducing to
gene repression throughout an entire chromosomal region.
It would be difficult to overstate the pervasive role of this epigenetic
factor in the organism. Stephen Baylin, a geneticist at Johns Hopkins
School of Medicine, says that the silencing, via DNA methylation, of tumor
suppressor genes is “probably playing a fundamental role in the onset and
progression of cancer. Every cancer that’s been examined so far, that I’m
aware of, has this [pattern of] methylation”
(quoted in Brown 2008).
In one study among various others — a study of colorectal cancer tissues —
the researchers identified 1549 genomic regions with methylation patterns
differing from the patterns in similar, non-cancerous tissues
(Wei et al. 2016).
There are often many more methylation anomalies in cancerous tissues than
there are mutated genes.
In an altogether different vein, researchers have found that “DNA
methylation is dynamically regulated in the adult human nervous system”.
Distinctive patterns of DNA methylation are associated with Rett syndrome
(a form of autism) and various kinds of mental retardation. Changing
patterns of methylation also figure in aging, and constitute a “crucial
step” in memory formation
(Miller and Sweatt 2007).
Among many other things, DNA methylation appears to play a key role in
tissue differentiation; in the activation (rather than only the
repression) of gene transcription; and in the regulation of alternative
RNA splicing. And, as by now we might expect, DNA methylation itself is
regulated by processes converging from all corners of the cell and larger
The nucleosome: a
of DNA and protein
Nothing more vividly illustrates the cell’s dynamic and transformational
“embrace” of its DNA than the thirty million or so nucleosomes that
form the main bulk of human chromosomes. Each nucleosome consists of
several histone proteins complexed together in a core particle,
around which various other proteins help to bend and wrap the rather stiff
DNA double helix. The DNA circles the core particle approximately twice
and is (more or less) held in place there, largely by means of
electrostatic forces and hydrogen bonding. It is time to focus on this
remarkable protein-DNA complex — a complex that, for all its centrality,
scarcely figures in the broader public understanding of genetics.
is an electron microscope-derived image published in the journal
Science in 1974, the decade when the nucleosome’s existence was
discovered by a team of researchers at the University of Tennessee and Oak
Ridge National Laboratory. You can see the nucleosomes as “beads” along
the string-like DNA.
Figure 14.7. DNA (black “string”) and nucleosomes (“beads” on the
string), as imaged by an electron
A nucleosome most commonly consists of eight histone proteins (two copies
of each of four histones, known as H2A, H2B, H3, and H4). The two
stretches of linker DNA at the entry and exit points of the
nucleosome, are typically held together by a linker histone (H1).
The latter plays a role, both in influencing how the DNA is bound to the
core particle, and also in managing the packing together of neighboring
(See the cartoon representation in
Figure 14.8. A schematic representation of a
nucleosome, showing the eight histones of the core particle together with
the linker histone (H1) and the encircling
to the challenge of packing all the DNA of a cell into the space of the
nucleus. As it happens, nucleosomes play a large role in this packing.
Depending on their arrangement, which varies with the context, they help
to organize the DNA molecule into a fiber that is said to be anywhere from
(roughly) 1/5 to 1/50 of the uncondensed length. Something like 75
percent of our genome is wrapped up in nucleosomes, and a typical gene
will have scores of nucleosomes within its body. This radically alters
the popular image of a chromosome as a vast, uninterrupted length of the
spiraling double helix.
shows (again in cartoon form) nucleosomes with and without linker
histones, as well as the varying degrees of DNA compaction that can be
achieved with the aid of nucleosomes.
Figure 14.9. Levels of chromatin folding and compaction. Here the
“chromatosome core particle” refers to the nucleosome core particle with
linker H1 added. (However, all such histone-plus-DNA configurations can
still be referred to as “nucleosomes”.) The abbreviation “bp” refers to
nucleotide base pairs, so that “147 bp” and “167 bp” refer to the
approximate length of DNA wrapped around the different core particles,
measured in base pairs. DNA is ever more fully compacted as the
nucleosomes are packed more tightly together. For simplicity, DNA-bound
proteins other than histones are not shown. Also, only histone-DNA
interactions on a single chromatin fiber (chromosome) are depicted here,
not interactions among different
“Ribbon” images of the nucleosome core particle, as in
though highly schematic, are intended to signify certain abstract features
of the histone protein structure. The DNA encircling the histones is
shown, cartoon-like, in purple.
Figure 14.10. A “ribbon” representation of nucleosome
Figure 14.11. Yet a different way to represent the structure of a
nucleosome. See main
And yet again, though still with extreme artificiality in terms of the
visual image, we have representations such as
which are generated using data from sophisticated molecular imaging
techniques. The red, white, and blue stick figure represents the DNA
encircling (about one and two-thirds times) the histone core particle.
Red and blue patches on the core particle represent acidic and basic
areas, respectively. These, via their effect on the distribution of
electrostatic charge over the surface of the histones, have a bearing on
many of the functional aspects of the nucleosome discussed below.
Here it is well to remember one of the primary lessons of
twentieth-century physics: we are led disastrously astray when we try to
imagine atomic- and molecular-level entities as if they were tiny bits of
the stuff of our common experience. It would be far better to think of
the core particle’s “substance”, “surface”, “contact points”, and
“physical interactions” as forms assumed by mutually interpenetrating
forces in their intricate and infinitely varied play.
In particular, as geneticist Bryan Turner of the School of Cancer Sciences
at the University of Birmingham (UK) reminds us, the nucleosomal core
particle “is much more flexible than the crystal structure [which is the
basis for images like
might lead us to believe”, and our current understanding of it “does not
lend itself to simplifying generalisations”
As we will see, the impressive enactments of form and force about the
nucleosome are central to any understanding of gene function.
Every “thing” in biology is really an activity, or is caught up in
activity, and the extraordinarily dynamic nucleosome is no exception. For
example, nucleosomes are the primary feature of chromatin that, as we
noted earlier, must be disassembled, or at least “remodeled”, during gene
transcription, and then restored to a fully functional state after the
transcribing enzyme (RNA polymerase) has passed by.
More generally, the individual histones in a nucleosome can come and go at
an almost alarming rate — with an average exchange time of just a few
minutes for many nucleosomes. And in some situations the histones
exchanged in this way can be different histones — known as “histone
variants” — with each variant exerting its own distinct sort of influence
on gene expression and chromatin dynamics. Individual histones can even
be removed from a core particle altogether, leaving it “incomplete” and
now with seriously altered function.
Further: in the course of its life the cell can, and does, reposition huge
numbers of nucleosomes along the double helix, bringing to bear upon them
a whole galaxy of regulatory interactions. The positioning of nucleosomes
— which may be achieved by protein complexes that slide the DNA around the
core particle — matters at a highly refined level: a shift by as little as
two or three bases (two or three “letters” of the “genetic code”) can make
the difference between an expressed or silenced gene
(Martinez-Campa et al. 2004).
(Individual genes typically contain thousands of bases.)
Still further: not only the exact position of a nucleosome along the
double helix, but also the precise rotation of the helix in its
embrace of the histones is important. “Rotation” refers to which part of
the DNA double helix faces toward a histone surface and which part faces
outward. Depending on orientation, the nucleotide bases will be more or
less accessible to the various gene-activating and repressing factors that
recognize and bind to specific sequences.
This in turn relates to the fact that there are two grooves (the
major and minor grooves) running the length of the double
Proteins that recognize a particular sequence of nucleotide bases
typically do so in the major groove, where the sequence is most readily
Figure 14.12. A schematic representation of the DNA double helix,
showing the major and minor
However, many proteins bind to DNA in highly selective ways that can be
determined by factors other than the exact DNA sequence. For example,
investigations have shown that the minor groove may be compressed so as to
enhance the local negative electrostatic potential. Regulatory proteins
“read” the compression and the electrostatic potential as cues for binding
to the DNA. The “complex minor-groove landscape”
(Rohs et al. 2009)
is indeed affected by the DNA sequence, but also by associated proteins.
Regulatory factors “reading” the landscape can hardly do so according to a
strict digital code. By our musical analogy: it’s less a matter of
identifying a precise series of notes than of recognizing a melodic and
harmonic motif performed by a full orchestra.
You can see, then, why one molecular biologist has referred to the
“bewildering array of molecular mechanisms that have evolved to alter the
physical properties of nucleosomes” and thereby to play a role in gene
Also consider this:
Influences such as DNA methylation, posttranslational modifications of the
core histone proteins, histone variants, [histone gene] mutations and the
level of chromatin compaction may each contribute to a multitude of
additional energy states within the chromatin network. All these factors
can potentially alter intra- and internucleosomal forces and establish a
different or more extended ensemble of nucleosome conformational states,
and therefore further fine-tune the functional activities. This is
consistent with the notion of a heterogeneous population of nucleosomes
within chromatin, all in a dynamic state and able to respond to continuous
changes from environmental ques [sic].
(Joshi et al. 2012)
But our story of nucleosome-based regulation has so far been radically
A tale of tails
We will now look more closely at those parts of the nucleosome where it
may be that the most dramatic story unfolds. Below is an
of Figure 14.10, representing a nucleosome. The eight histones of the
core particle are shown as a ribbon diagram, with the DNA double helix
(schematically depicted in purple) wrapped around it somewhat less than
two times. You will note a number of squiggly “pig’s tails” extending
outward from the core histones. These are the thin, flexible, and mobile
histone tails, ten of which are present in the typical core
particle. There are hundreds of distinct chemical modifications of these
tails (referred to as post-translational modifications), and the
countless resulting patterns of modification within any given nucleosome
or group of nucleosomes are intimately bound up with the expression of
genes. In fact, there is little relating to gene regulation, DNA
replication, chromatin structure and dynamics, or the overall functional
organization of the nucleus that is not correlated in one way or another
with patterns of histone tail modifications.
Learning about these tails, we may be reminded (albeit in a highly
fanciful manner) of both the sensory functions of insect antennae and the
motor functions of limbs. On the “sensory” side, the tails are receivers
of molecular signals coming from all directions in the form of
post-translational modifications. The nucleosome provides a context where
the integrated significance of these signals can be “read off” (to use the
standard phrase) by the gene-regulatory proteins that are sensitive to
them. These readers may then “recruit” (again standard usage) various
other proteins that either help to restructure chromatin in one way or
another, or more directly regulate the expression of genes.
There are in fact many protein “readers” that interact with single
modifications, or with groups of them, or with the asymmetrically modified
tails of a histone pair, or with a histone modification in proximity to a
site of DNA methylation. Every such reader protein acts out of its own
world of biochemical genesis, folding, post-translational modification,
and conformational plasticity, and together these proteins tell an
important part of the story of gene regulation.
Finally, the tails can also act with a kind of brute force as “muscular”
effectors. They can, for example — no doubt depending at least in part on
their various modifications and protein associations — insinuate
themselves into one of the grooves of the double helix, thereby loosening
the DNA from the nucleosomal core particle (and making it more available
for transcription), or else binding it more tightly. In both cases, one
way this is accomplished is by altering the electrical balance between
histone and DNA.
Some of those tails are also thought to establish nucleosome-to-nucleosome
contacts, helping to compact a stretch of chromatin. How and whether this
is done can make genes either more or less accessible for transcription
and various forms of regulation.
Perhaps you can now see why the members of one research team, writing
about histone tail modifications, find themselves reflecting upon
the incredibly intricate nature of the chromatin landscape and resultant
interactions. The biological consequences of [interactions between histone
tail modifications and regulatory proteins] are highly context dependent,
relying on the combinatorial readout of the spatially and temporally
fluctuating local epigenetic environment and leading to a highly
fine-tuned [regulation] of particular genomic sites
(Musselman et al. 2012).
A still closer look
We have progressively magnified our field of view by shifting from the
overall structure of chromatin, to the nucleosome with its histone core,
and then to the individual histone tails. Important principles of gene
regulation operate at each different level. Now, magnifying our view one
last time, we will home in on a single histone tail modification. The
most commonly discussed modifications are the acetylation and
methylation of certain lysine amino acids in the tails, but there
are many other kinds of modification. Here I will focus on the
modification called ubiquitination simply because its gene
regulatory roles do not seem quite as extensive (or just are not as well
investigated) as those performed by some other tail modifications. This
makes their description here a little more manageable.
Monoubiquitination is the “attachment” (a poor word, as I indicated
of a single ubiquitin chemical group to a lysine amino acid of a protein.
In the case of histone tails, this can be done at more than one lysine,
but we will look only at the monoubiquitination of lysine 120 on the tail
of the histone known as H2B, all of which can be designated
H2BK120ub1 (where ‘K’ is the symbol for lysine), but which is
commonly referred to as H2Bub1.
So what is the significance of this modification at a single histone tail
location? Here’s one summary:
H2Bub1 takes part in almost every molecular process associated with
chromatin biology. H2Bub1 has been shown to regulate transcription
initiation and elongation, DNA damage response and repair, DNA
replication, nucleosome positioning, RNA processing and export [from the
nucleus], chromatin segregation and maintenance of chromatin boundaries.
Given the large number of molecular processes regulated by H2Bub1, it is
not surprising that H2Bub1 plays a vital role in some of the most
fundamental biological processes that occur within multicellular
organisms. [Loss of an enzyme responsible for ubiquitination] results in
very early embryonic lethality. Furthermore, aberrant H2Bub1 levels can
affect cell cycle progression, apoptosis [“programmed cell death”], stem
cell differentiation, development, viral infection outcome and
(Fuchs and Oren 2014)
(I draw largely on the paper by these authors in the remainder of this
Of course, H2Bub1 does nothing “in general”; results are always specific
and context-dependent. For example, blocking this modification in a
particular human cell line was found to upregulate some genes,
downregulate others, and leave a great many unchanged. Under some
circumstances, H2Bub1 is particularly needed for the transcription of
relatively long genes. And the modification also plays an important role
in histone “crosstalk”, helping to regulate other crucial modifications
within the same or on different histones.
A search for “effector” molecules that, singly or cooperatively, associate
and interact with the H2Bub1 modification led to the identification of
more than ninety proteins, many with known functions in gene regulation
consistent with those known to be “effects” of H2Bub1. This points us to
what could be a still further extension of our survey, whereby we might
analyze one or more of those proteins. We would then have to trace the
modifications they undergo, and the larger regulatory world in
which they are caught up. But there would be no end of this, since
following up any particular line of inquiry in a cell or organism sooner
or later leads to everything else.
I have made repeated reference to these ever-widening circles of causal
influence. Here I will just momentarily hint at this broader reality in
relation to the histone tail modifications called “methylation” (not to be
confused with DNA methylation). A methyl group is added to various
histone amino acids by enzymes called “methyltransferases”, and is removed
by other enzymes called “demethylases”. The mammalian genome is said to
encode thirty five histone methyltransferases and twenty three
demethylases. This is where the complications enter.
In an article entitled “Controlling the Controllers”, the authors discuss
how these methylating and demethylating enzymes are themselves modified
and regulated by the addition of phosphoryl groups, with “diverse effect”
on enzyme function. Further, the phosphorylation of the enzymes is in
turn “regulated by upstream signalling pathways”. And, still further,
“different histone methyltransferase and demethylase enzyme families are
connected to upstream signalling pathways in different ways”
And so the circles widen. But now we must return to our narrower focus.
It remains to mention only that, with ubiquitination as with so many other
molecular biological investigations, researchers are vexed by an imagined
“need to establish causality more unequivocally”
(Fuchs and Oren 2014)
— a need that never seems fully satisfied as our understanding grows.
This search for unambiguous causes is a fruitless one
because the kinds of causes being looked for don’t exist in organisms.
As for the relations that do exist in organisms, just reflect for a
moment. Think, for example, of the transcription network depicted in
Then think of the networks of hundreds of mutually regulating mRNAs and
microRNAs also discussed above and illustrated in
And now consider the virtually infinite combinations of histone tail
modifications and their endlessly elaborated meanings and pervasive
“crosstalk”. Many other domains of gene regulation have been alluded to
in preceding sections, and untold others could have been mentioned.
And now ask yourself what all this must mean. There seem only two
possibilities: complete bedlam and chaos of causes working at
cross-purposes, or else the play of an encompassing wisdom whose
all-embracing effectiveness and power of coordination we can hardly yet
even begin to conceive.
Movement and rhythm
Few if any details of nucleosome structure and dynamics are fixed and
constant. Nothing illustrates this more vividly than the fact of DNA
breathing on the nucleosome surface. This refers to the partial and
rhythmical unwrapping and re-wrapping of the double helix, especially near
the points of entry and exit on the nucleosome. This provides what are
presumably well-gauged, fractional-second opportunities for
gene-regulating proteins to bind to their target DNA sequences during the
This breathing also relates to the transcriptional pausing by RNA
The polymerase appears able to take advantage of the breathing in order to
move, step by step and with significant pauses, along the genes it is
transcribing. In this way the characteristics of nucleosomes — how the DNA
breathes, and whether it is firmly or loosely anchored to the histones —
can affect the timing and frequency of pauses. And, as we saw earlier, the
rhythm of pauses and movements then affects the splicing and folding of
the RNA being synthesized, which in turn bear on how the RNA can be
regulated as well as the structure and function of the protein molecule
produced from the RNA. A proper music is required for the overall
performance to be successful. So it appears that the references to
“choreography” and “dance” one sometimes encounters in the literature may
be more than mere poetic niceties.
With a different sort of rhythm nucleosomes will sometimes move — or be
moved (as I have remarked before, the distinction between “actor” and
“acted upon” is forever obscured in the living cell) — rhythmically back
and forth along the DNA, shifting between alternative positions in order
to enable multiple transcriptional passes over a gene by RNA polymerase.
Stem cells exhibit what some have called “histone modification pulsing”,
which results in the continual application and removal of both
gene-repressive and gene-activating modifications of nucleosomes. In this
way a delicate balance is maintained around genes involved in development
and cell differentiation. The genes are kept, so to speak, in a finely
poised state of “dynamic and balanced readiness”, so that when the
decision to specialize is finally taken, the repressive modifications can
be quickly lifted, leading to rapid gene expression
(Gan et al. 2007).
This state of suspended readiness in stem cells also seems to be served by
a rhythmical (10 – 100 cycles per second), back-and-forth spatial
movement, or vibration, of chromatin within the cell nucleus. Associated
with “hyperdynamic binding of structural proteins” mediated by
nucleosomes, this vibration is thought to help maintain the largely open
chromatin state characteristic of stem cells. The movement depends on the
metabolic state of the cell and is progressively dampened as the stem cell
differentiates into a specialized cell with substantial portions of its
chromatin in a condensed state
But quite apart from stem cells, it is increasingly appreciated that
nucleosomes play a key role in holding a balance between the active and
repressed states of genes in many cell types. As the focus of a highly
dynamic conversation involving histone variants, histone tail
modifications, and innumerable chromatin-associating proteins, decisively
placed nucleosomes can (as biologist Bradley Cairns writes) maintain genes
“poised in the repressed state”, and “it is the precise nature of the
poised state that sets the requirements for the transition to the active
state”. Among other aspects of the dynamism, there is continual turnover
of the nucleosomes themselves — and of their separate components — a
turnover that allows transcription factors to gain access to DNA sequences
“at a tuned rate”
It is perhaps worth mentioning here that in certain bacteria a 24-hour
(circadian) rhythm correlates with the changing state of DNA supercoiling
— that is, with a tighter or looser twisting of the double helix. It
appears that something similar may be going on in higher animals, where
DNA supercoiling is so closely “wrapped up” with nucleosomes. In these
organisms one of the factors involved in the extremely complex processes
by which genes are regulated in a circadian fashion is the rhythmic
application of histone modifications to selected nucleosomes
(Woelfle et al. 2007),
presumably with direct implications for chromatin structure and DNA
From Static Mechanism to Dynamic Regulator
In an article entitled “Understanding Nucleosome Dynamics and Their Links
to Gene Expression and DNA Replication”, Pennsylvania State University
molecular biologists William Lai and Franklin Pugh concluded their review
of nucleosomes this way:
“Originally viewed as a rather static mechanism of chromatin packaging,
the nucleosome core complex is now well recognized as one of the key
regulatory components of the genome. We also now see that instead of
static protein complexes, nucleosomes are in fact exceptionally dynamic
and that their positioning and composition are crucial for genome
regulation. As such, the study of nucleosome dynamics is essentially the
study of genome regulation. The complex interaction between nucleosome
occupancy and positioning allows the cell to properly regulate
accessibility of various proteins and their complexes to DNA and thus to
regulate gene expression programmes. A variety of regulatory cofactors
such as chromatin remodellers, chaperones and general regulatory factors
operates both independently and synergistically to maintain the precise
organization and composition of nucleosome arrays at specific genomic
loci. This dynamic environment probably exists so that the genome may
respond and adapt quickly to both external stimuli as well as be able to
quickly recover from chromatin-disruptive activities such as transcription
With reference to that last sentence, it needs adding that what “responds
and adapts quickly” to external and internal stimuli is not really the
rather passive genome so much as the entire, all-encompassing regulatory
environment, of which the nucleosome is a neat picture and summary.
The nucleosome, we can fairly say, is a ceaselessly transforming matrix
and organizational hub whose structure and pattern of activity is never
exactly duplicated anywhere in the genome. It is where the infinitely
ramified interface between the larger cell and its DNA comes to its most
focal expression. And that expression turns out to be livingly nuanced
activity, dynamic beyond what anyone imagined during the age of the double
helix as the one-dimensional “secret of life”.
And so, seemingly in the grip of the encircling DNA with its relatively
fixed and stable structure, yet responsive to the ceaselessly varying
flows of life around it, the nucleosome holds a muscular and intelligent
balance between gene and context — a task requiring flexibility and a play
of appropriate rhythm.
Such, then, is the intimate, intricate, well-timed choreography through
which our genes come to their proper expression. And the plastic,
shape-shifting nucleosome in the middle of it all provides an excellent
vantage point from which to view the overall drama of form and movement.
A story mostly untold
We have, in our review, only sparsely sampled the overwhelming number of
causal factors participating in gene expression. The topics not touched
upon — the unmentioned domains of regulatory, or epigenetic, activity
affecting what the cell makes of its genes — may be greater than the sum
of the topics I have briefly alluded to here.
There is, for example, the recently intensifying exploration of the
importance of modifications, not only on the histone tails, but also on
the histone cores. These also are proving relevant to gene expression,
and in complex ways, both direct and roundabout.
We could also have talked about the entire universe of regulation
governing the translation of mRNA molecules into protein after they have
been exported from the cell nucleus into the cytoplasm. The task is
accomplished by complexes of protein and RNA known as “ribosomes”. The
diverse factors the cell gathers together for translation rival those we
see in gene transcription.
And once a protein is generated, there is the problem of its folding (and
re-folding), often with the help of “chaperone” proteins. Many proteins
can potentially fold in an almost unlimited number of ways, yet achieving
the “right” folds is crucial for protein function. We have seen that both
alternative splicing and folding of an RNA can occur (with major
functional implications) during its transcription from DNA.
Similarly, the folding of a protein can begin during its
translation from RNA. Moreover, the folding outcome may be
affected by the innumerable factors playing into the activity of
translation. We do not often find just one thing at a time being
accomplished by any biological process.
Then, still further downstream from gene transcription, there are the
various post-translational modifications (PTMs) that may be applied,
removed, and re-applied to any gene-regulatory protein (transcription
factors, co-activators, co-repressors, chromatin remodelers, and so on),
just as we saw with the histone proteins belonging to nucleosomes. These
again shape the molecule’s function, often in a dynamic, ever-shifting way
as the modifications come and go. Together, the many thousands of
proteins subject to PTMs, and the diverse effects of these modifications,
make for a vast regulatory landscape almost impossible to comprehend. The
resulting regulatory activity is always context-dependent, relating to
larger, governing purposes rather than being the mere effect of a local
We could also talk about what is, in one sense, the most fundamental
biological activity of all — metabolism. After all, every performance of
our body derives in one way or another from the food we eat. Metabolites
and the organization of metabolic processes play critical roles in many
aspects of gene expression related to everything from circadian rhythms to
Or we could talk about how some RNAs, especially non-protein-coding RNAs,
form a “scaffolding” that gives structure to the cell nucleus and
therefore plays a fundamental role in just about all nuclear functions.
Except that words such as “scaffolding” and “structure” can be very
misleading, as two researchers point out in a paper entitled “Role of
Nuclear RNA in Regulating Chromatin Structure and Transcription”. We
should expect, they write, that “any nuclear structure that is assembled
employing RNA cannot be static but [must be] constantly recycling degraded
RNA with newly synthesised ones”. So “the original concept of a static
nuclear matrix must be re-evaluated in terms of a dynamic scaffold”
(Michieletto and Gilbert 2019).
Perhaps the most intense and significant new field of research bearing on
gene regulation in recent years relates to phase transitions in the
cell, and especially in the nucleus. Like ice crystals forming and
dissolving in water held near the freezing point, or like oil droplets in
some other liquid (or like water droplets in oil), complex combinations of
proteins, RNAs, and other molecules can form separated-out liquid or
semi-solid aggregates within the cellular plasm. The dynamic functional
role of these aggregates in bringing molecular communities together at the
right place, in the right amounts, and at the right time is now a prime
topic relating to just about everything discussed in this chapter. Among
other things, the new understanding we are gaining in this field makes a
mechanistic or deterministic interpretation of cellular physiology even
less tenable than it already was.
And if any new field of research ranks second to phase transitions in
importance, it surely must be the one focusing on the role of the
microbiome. The collective DNA sequence of the microorganisms in
our bodies exceeds that of the human sequence. The processess rooted in
this “foreign” DNA can affect our biology, much as can the processes
stemming from our own DNA. And the effects extend to regulation of our
But surely it is time for us to stop. Anyone desiring a glimpse of the
wider range of topics relating to gene expression, might wish to scan the
expanded outline of topics at the beginning of the article, “How the
Organism Decides What to Make of Its Genes
A decisive problem for the classical view of DNA is that a human cell
employs its 21,000 or so genes to generate an estimated 250,000 to 1
million distinct proteins
(Klerk and ’t Hoen 2015).
The activities shaping these abundant outcomes are not strictly determined
by DNA. Rather, they arise from all corners of the cell and larger
organism, just as the outcomes themselves — all those distinct proteins —
are ushered to their proper places in every tiniest niche throughout the
whole. We are always watching integral and unified performances. The
idea that genes are originating causes that make everything else
happen is grotesquely wrong-headed.
Mina Bissell, a researcher who has received many recognitions, has put the
matter this way: “The sequence of our genes are [sic] like the keys on the
piano; it is the context that makes the music”
(Bissell and Hines 2011).
We might add that the raw DNA sequence does not even contain all the keys;
let’s say: just the white keys. The flats and sharps, without which the
music would lose its savor, are provided by DNA methylation, RNA editing,
and so much more.
And Shelley Berger, the Daniel S. Och professor of cell and developmental
biology at the University of Pennsylvania School of Medicine’s Wistar
Institute — after noting that a single histone tail modification “recruits
numerous proteins whose regulatory functions are not only activating but
also repressing”, and that “many of these marks have several, seemingly
conflicting roles” — summarized the situation this way:
Although [histone] modifications were initially thought to be a simple
code, a more likely model is of a sophisticated, nuanced chromatin
“language” in which different combinations of basic building blocks yield
dynamic functional outcomes.
What she says about histone tail modifications could just as well be said,
as we have seen, about the entire universe of gene regulation. We are
looking at a meaningful and thoughtful language through which living
narratives are constructed. In slightly different terms, Berger envisions
histone modifications as participating in “an intricate ‘dance’ of
In the plastic organism, what goes on at the local level is always shaped
and guided by a larger, coherent context — a context that surely has
meaning, but (as in natural languages) never an absolutely fixed grammar
or logic. And, in fact, while overwhelming evidence for a meaningful,
gene-regulatory conversation involving histone modifications has emerged,
there is little to suggest a rigid code — this despite the strong urge by
molecular biologists to find one.
The overall picture of gene expression is one of unsurveyable complexity
in the service of remarkably effective living processes. What all the
foregoing shows is that the whole cell and the whole organism are forever
We have no explanatory coherence so long as we are following individual
chains of molecular causation. The mutually interpenetrating lines of
influence converging upon and issuing from our DNA reveal their full
meaning only when we consider what needs and interests are reflected in
the overall, coordinated pattern of causes — what the organism is doing
The “promiscuity” of binding — that is, binding in the absence of
definitive binding sequences — is a problem relating to protein-nucleotide
interactions in general. For example, 55 percent of RNA-binding proteins
“do not contain any known RNA-binding domain at all”
(Editors of Nature Structural & Molecular Biology 2021).
Figure 14.1 credit:
I will not discuss the RNA portion of chromatin here. But its
importance, which researchers are now struggling to unravel, looks as
though it may rival the diverse functions of the protein portion.
No contemporary biologist has a sound basis for assuming “necessary
contextualization and direction”, because
the idea of wise direction is foreign to the current foundations of
biology. But every biologist, in talking about specific molecular
processes, nevertheless does make the assumption — and makes it for
the simple reason that there is no alternative. We either assume the
wisely guided context or our immediate work becomes meaningless. It loses
its whole point, which is to explain how one or another process
contributes to a function or task — that is, to an effectively
directed, purposive activity
biologists are forever implicitly placing themselves within a theoretical
framework that, from their own standpoint, is indefensible.
By “modest-sized” I mean: about 2000 nucleotide bases in length.
Figure 14.2 credit:
Kazantseva and Palm 2014
Creative Commons cc-by license
Figure 14.3 credit:
Tóth-Petróczy et al. 2008,
editing by Dennis Pietras, Buffalo NY.
cc by-sa 4.0.
Here is one paragraph from a paper on the Mediator complex:
The Mediator is an evolutionarily conserved, multiprotein complex that is
a key regulator of protein-coding genes. In metazoan cells, multiple
pathways that are responsible for homeostasis, cell growth and
differentiation converge on the Mediator through transcriptional
activators and repressors that target one or more of the almost 30
subunits of this complex. Besides interacting directly with RNA polymerase
II, Mediator has multiple functions and can interact with and coordinate
the action of numerous other co-activators and co-repressors, including
those acting at the level of chromatin. These interactions ultimately
allow the Mediator to deliver outputs that range from maximal activation
of genes to modulation of basal transcription to long-term epigenetic
(Malik and Roeder 2010)
Mediator also has tissue-specific aspects:
Adding yet another degree of complexity, members of the same transcription
factor family can target different Mediator subunits to activate
transcription of the same gene, through the same promoter elements, in
different cell types.
(Conaway and Conaway 2011)
Figure 14.4 credit:
Quevedo et al. (2019).
cc by-sa 4.0.
Figure 14.5 credit: courtesy of David S. Goodsell and
RCSB Protein Data Bank.
The Wikipedia article, “Tata-binding protein” (accessed on April 1, 2019),
offers a succinct description of part of this interaction: “When TBP binds
to a [particular sequence] within the DNA, it distorts the DNA by
inserting amino acid
side-chains between base pairs, partially unwinding the helix, and doubly
kinking it. The distortion is accomplished through a great amount of
surface contact between the protein and DNA. TBP binds with the negatively
charged phosphates in the DNA backbone through positively charged lysine
and arginine amino acid residues. The sharp bend in the DNA is produced
through projection of four bulky phenylalanine residues into the minor
groove. As the DNA bends, its contact with TBP increases, thus enhancing
the DNA-protein interaction.”
There are actually three RNA polymerase enzymes in humans: RNA polymerase
I, II, and III. I will be speaking of RNA polymerase II, which
transcribes the great majority of our genes. Also, “RNA” in the following
descriptions will refer either to messenger RNA (mRNA), which can be
translated into protein, or else to RNA more generally. References to
specific non-protein-coding RNAs such as microRNAs (miRNAs) will be
flagged as such.
Just about any functional significance of an RNA — from what protein it
produces, to its stability and cellular localization, to the various
roles of its three-dimensional structure — can be affected by this
editing. One kind of editing (known as A-to-I editing) “is extremely
abundant in primates: over a hundred million editing sites exist in [RNAs
derived from] their genomes”
(Levanon and Eisenberg 2014).
However, biologists have only begun to explore the functional significance
of most of this editing, and there remains among the majority of
researchers today a tendency to dismiss as “random noise” whatever their
current methods and concepts cannot presently illuminate.
Regarding one of these modifications, known as mRNA adenosine methylation
(m6A), Timothy Nilsen, a molecular biologist at Case Western
Reserve University in Cleveland, has written:
A series of papers have appeared in rapid succession, together providing a
wealth of unequivocal evidence for m6A function. But these
findings still have not led to a coherent picture of the number and
variety of functions of the m6A modification.
In the several years since he wrote that, the picture has, bit by bit,
been filled in, and continues to be filled in. But there is a long way to
Figure 14.6 credit: From
Tay, Rinn and Pandolfi (2014).
Figure 14.6 is extremely simple. The authors of the paper from
which the figure is drawn refer to a study of brain cancer (glioblastoma)
where “the analysis was significantly extended beyond the binary ceRNA
associations described in most other studies”, and “the PTEN ceRNA
interactions were found to be part of a post-transcriptional regulatory
layer comprising more than 248,000 microRNA-mediated interactions”.
Of course, anything can be analyzed in one way or another if we narrow our
vision sufficiently and disregard, for example, the purposive
(telos-realizing) aspects of what is going on. The question is
whether analyzing living
activity by breaking it into physically explicable part-processes yields
an explanation or understanding of its telos-realizing character.
Throughout this book I have been pointing out the incommensurability
between a strictly physical analysis of biological phenomena and the
easily recognizable meaning of those phenomena.
Figure 14.7 credit:
Ada Olins and Donald Olins, University of Tennessee/Oak Ridge Graduate
School of Biomedical Sciences.
An example of the functioning of linker histones: “Our results establish
H1 as a critical regulator of gene silencing through localized control of
chromatin compaction, 3D genome organization and the epigenetic landscape”
(Willcockson et al. 2020).
The functions of the linker histone are also indicated by the fact that
“mutations in H1 drive malignant transformation primarily through
three-dimensional genome reorganization, which leads to epigenetic
reprogramming and derepression of developmentally silenced genes”
(Yusufova et al. 2020).
And then there is this: “The biochemical functions of H1 in the regulation
of nuclear DNA metabolism should not be limited to a single,
one-size-fits-all DNA compaction paradigm. Rather, H1 appears to be an
active biochemical player in chromatin and a potent effector of multiple
aspects of chromosome structure and chromatin functions”
Figure 14.8 credit:
Figure 14.9 credit:
Fyodorov et al. 2018.
Figure 14.10 credit:
(cc by-sa 3.0).
Figure 14.11 credit:
Figure 14.12 credit:
Zygote Media Group
(cc by 2.5
Makowski, Gaullier and Luger 2020:
Some transcription factors (TFs) only recognize nucleosomal DNA when
nucleosome “breathing” occurs, that is when the DNA is partially and
temporarily unwrapped from the nucleosome surface … histone
post-translational modifications facilitate DNA breathing. TF binding
facilitates further nucleosome unwrapping by promoting the binding of
additional TFs, and/or in coordination with chromatin remodelers. Some
TFs can bind their cognate motifs on fully compacted nucleosomal DNA and
initiate ATP-independent DNA unwrapping or even histone eviction.
However, outcomes in which TF binding stabilizes nucleosomes are also
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Steve Talbott :: How Our Genes Come to Expression