Biology Worthy of Life
An experiment in revivifying biology
When Francis Crick and James Watson announced in 1953 that they had
discovered the double-helical "secret of
life", they bequeathed to our imaginations an image combining the cool,
efficient, geometrically precise beauty of a crystal, with the compelling
logic of a computer program.
The logic, as it would be pieced together over the next few years, was
simple and elegant. Four chemical groups — nucleotide bases, the distinctive "letters" of a life-engendering code
— were
strung by the millions along both spiraling strands of the double helix
. Each
successive group of three letters lying along a strand was a code naming a
particular amino acid
, and a sequence of many such codes represented, in proper
order, all the amino acids making up a single protein. Thousands of such
proteins, so constructed, were the primary workhorses of every cell,
forming many of its various structures and mediating its countless
chemical interactions. And so the double helix (otherwise known as DNA
), with its
carefully sequenced letters, was the instruction book for assembling a
living organism.
The salient facts of organism assembly in this early picture were likewise
straightforward. The forty-six chromosomes in a human
cell consisted, most essentially, of double helical DNA, and this DNA was
divided into numerous genes
, each of which in turn coded for one protein. By a process
known as "transcription
" and
facilitated by an enzyme, an individual gene gave rise to a kind of mirror
image of itself in the form of a molecule known as "messenger RNA" (mRNA
). This
molecule, also containing a sequence of nucleotide bases, preserved the
gene's coding for a protein. Then another kind of RNA, called "transfer
RNA" (tRNA
), came into play: in conjunction with some specialized
machinery at the site of protein synthesis, tRNA read the code imprinted
upon the mRNA and used it to assemble amino acids into the specified
protein. This latter process was called "translation
".
Perhaps the most compelling detail in this picture was the fact that when a mistake occurred — when a letter of the DNA code was transcribed into the wrong letter of mRNA — an error-correction machinery zeroed in on the mistake and fixed it. Nothing could have illustrated more vividly the directed, computer-like efficacy of the entire process. The scheme was both satisfyingly logical and causally effective. The DNA codes that named a protein simultaneously constituted the master template and initiating machinery for constructing it. The master DNA instruction manual was passed from parent to offspring with remarkable fidelity, and its instructions were executed in such a way that information and control always flowed in a single direction. "DNA makes RNA, and RNA makes protein", as the saying went. Within the individual organism, DNA was a kind of First Cause or Unmoved Mover. As Nobel laureate Max Delbrück put it, DNA "acts, creates form and development, and is not changed in the process" (1971).
In fact, the story was so neat — and, for most researchers, so entirely convincing — that one heard occasional murmurings of regret about the unfortunate lot of future biologists. Wouldn't they be left with the not very stirring task of working out the subordinate details? If the overall logic and the governing causal pathways were already known, at least in principle, what could remain except for nitpicking at ever lower levels of analysis?
And yet, as we now know, the story was crushingly false to life.
Biologists need not have fretted over their sources of career satisfaction
— nor over their employment prospects. It was some forty years
after the discovery of the double helix that one of
the most massively funded research projects in the history of science
mobilized genetic laboratories around the world to tease out the complete
and definitive text of the genomic "Book of Life". It is true that this
Human Genome Project, which many hoped would lead directly to the final
solution of life, again raised questions about a meaningful future for
biologists. But any such worry has again been set aside. For the project
was scarcely completed when the realization struck that the "solution" was
still enigmatically encoded as a raw, undeciphered text. The key to
interpretation, many decided, was an even more ambitious project, the
elucidation of the "proteome" — the tens of thousands of proteins in
the body, with their complex folding patterns and endlessly diverse
functioning. That effort is now under way.
Meanwhile the envisioned keys to life themselves have been growing ever
more diverse, each speaking its own distinctive language, and each looking
like part of a puzzle that keeps growing in scope and complexity faster
than our identification of individual pieces. Take, for example, the
actual stuff of chromosomes, called chromatin, which
consists not only of DNA
but, even more extensively, of proteins that give their own
form and structure to the chromosome. For many years this unruly protein
setting was largely ignored as geneticists focused on the controlling
wizardry of the coding genes. But now numerous laboratories are
uncovering how the continual and intricately choreographed modification
of chromatin affects the activity of genes. Although the researchers'
first impulse was to find another "simple code", it now appears, according
to geneticist Shelley Berger of Philadelphia's Wistar Institute, that "a
more likely model is of a sophisticated, nuanced chromatin 'language' in
which different combinations of basic building blocks yield dynamic
functional outcomes" (2007, p. 407). And so the chromatin interpretation
industry has become one of the largest enterprises within molecular
biology.
But chromatin is hardly the end of it. New and strange names point to
multiplying decipherment challenges. We hear of the "methylome" and
"membranome
", the "histone code"
and "RNA
interference code"
. And, most encompassing of all, there is the "epigenome",
consisting of all the varied cellular processes that bear on the activity
of genes — processes that not only influence whether or not a gene
is transcribed, but can even alter the effective sequence of genetic
letters. These epigenetic
("extra-genetic") processes seem to determine the genetic code at least as
much as they are determined by it. But if this is true, then what has
become of the master controller?
Every biologist today will grant the inadequacy of the story of the 1950s. Many would probably add that it's perfectly natural for our understanding to grow more complex with time — so why harp on the inevitable limitations of those early pioneers who made great discoveries?
But this, I'm convinced, is to miss the dramatic significance of the current revision of our understanding of the living organism. Exactly how that earlier story was false, and with what seismic implications for the foundations of biology, is still scarcely appreciated by the general public — or even by many of those scientists who have been pronouncing the end of the era of the gene.
What's at stake is the nature of biological explanation — our understanding of understanding itself. Particularly at issue are the distortions introduced by a one-sidedly logical-causal habit of thinking — distortions worsened by the continuing failure to enter into the more organic sort of understanding that so many have hoped for over the years and even centuries.
I will have a great deal to say about the character of both logical-causal thinking and organicism. But first we need to ground ourselves in some of the striking revelations stemming from the ongoing research in epigenetics — research that has now, by force of its paradigm-subverting potential, assumed a position front and center in the consciousness of molecular biologists.
Already at the beginning of the double helix era a troubling question
bedeviled all discussions of the DNA sequence as the
Master Logic and First Cause of life. Every human being begins life as a
single cell containing an entire genome. But over the course of
development this cell becomes many radically different kinds of cell in
all the various tissues of the body. From muscle to nerve, from retina to
kidney, from skin to brain, every cell contains the same chromosomes with
the same DNA sequences1. If
these sequences are what determine traits, how do we account for the
dramatically different kinds of cell?
Imagine the situation concretely. You have a single, undifferentiated cell, and then this cell divides and the two daughter cells
enter upon pathways leading to different tissues. During cell division,
the chromosomes
are faithfully replicated
, so that each daughter cell receives the same "instruction
set". How is it that these identical instruction sets proceed to direct
the cells along divergent paths, so that offspring of the one eventually
gain the ability to expand and contract as part of a muscle, while
offspring of the other take on a rigid form together with a specialized
ability to transmit electrical signals? It seems that a cell of the heart
muscle must possess a "self-understanding" decisively different from that
of a brain cell, and this understanding cannot derive solely from its DNA.
The supposed instruction set evidently does not contain all the
instructions.
Actually, this problem was raised by many observers long before the genomic era. For example, the developmental biologist F. R. Lillie, remarking in 1927 on the contrast between "genes which remain the same throughout life" and a developmental process that "never stands still from germ to old age", asserted that "Those who desire to make genetics the basis of physiology of development will have to explain how an unchanging complex can direct the course of an ordered developmental stream" (Lillie 1927, pp. 367-8).
Fundamental though it was, the objection received little attention for several decades. Meanwhile, a central result of the Human Genome Project posed a second problem. Instead of the expected hundred thousand or more genes in the human genome, there turned out to be only twenty-five thousand or so — roughly the number possessed, for example, by a simple, one-millimeter-long, transparent roundworm, Caenorhabditis elegans. If it really is genes that account for the organism in all its complexity, how can it be that a human being and a primitive worm can be accounted for by a similar number of genes? "As far as protein-coding genes are concerned", writes Ulrich Technau, a developmental biologist from the University of Vienna, "the repertoire of a sea anemone . . . is almost as complex as that of a human" (Technau 2008, p. 1184).
The answer increasingly proposed by biologists is that genes are far from
the whole story if you want to understand the organism. Some ninety-nine
percent of human DNA does not consist of genes — that is, does not code
for
proteins. Most of this noncoding
DNA was long
referred to as "junk" and was assumed to be an evolutionary accumulation
of meaningless genetic detritus. As it happens, though, an intriguing
pattern has emerged: noncoding DNA accounts for only 10% of the DNA of a
one-celled prokaryote, 32% in yeast, 75% in roundworms, 83% in insects,
91% in a pufferfish, and 98% in a chicken (Costa 2008, p. 12). In other
words, the more complex the organism, the greater the amount of junk!
The obvious thing to do was to look more closely at this neglected DNA.
And after several years of looking, the reversal of thought has been both
radical and ironic: the "junk" is now hailed as a primary measure of our
evolutionary progress. In concert with the cell as a whole, it helps to
provide the sophisticated coordination of genomic resources
distinguishing the higher organisms from the lower.
This same junk is also thought to contain part of the answer to our first problem — organ differentiation in the presence of a fixed genetic code. The power of differentiation lies, not in the genes, but in the management of them. The junk, it turns out, has a lot to do with this management. Furthermore — and this is where the currently flourishing discipline of epigenetics comes to full flower — the resources for management are found, not only in noncoding DNA, but in processes broadly distributed throughout the cell.
We will look at some of these processes after briefly noting the kind of experimental result that has encouraged researchers to begin exploring the epigenome.
In the mammalian genome chromosomes
normally
come in pairs, one inherited from the mother and the other from the
father. Any given gene occurs twice, with one version ("allele
") located on
the first chromosome of a pair and the other on the second. When the two
alleles are identical, the organism is said to be homozygous
for that
gene; when the alleles are different, the organism is
heterozygous
. For example, there are mice that, in their natural
("wildtype") state, are dark-colored — a color that is partly
dependent on a gene known as Kit. The mice are normally homozygous
for this gene. When, however, one of the Kit alleles is replaced
with a certain mutant
gene, the now heterozygous mouse shows white feet and a
white tail tip.
That result was perfectly natural (if you call such artificial gene
manipulations "natural"). But it is also where the story becomes
interesting. Scientists at the University of Nice-Sophia Antipolis in
France took some of the mutant, white-spotted mice and bred them together
(Rassoulzadegan et al. 2006). In the normal course of things, some of the
offspring were again wildtype homozygous animals — neither of their
Kit alleles was mutant. However, to the researchers' surprise,
these "normal", wildtype offspring maintained, to a variable extent, the
same white spots characteristic of the mutants. It was an apparent
violation of Mendel's law of
inheritance: while the genes themselves were sorted between generations
properly, their effects did not follow the "rules". A trait was displayed
despite the absence of its corresponding gene. Apparently something in
addition to the genes themselves — something epigenetic —
figured in the inheritance of the mice offspring, producing the
distinctive coloration.
Another group of researchers, led by Michael Skinner at the University of
Washington, looked at the effects of the fungicide vinclozolin on
laboratory rats. (Anway et al. 2006; Crews et al. 2007). Banned in
Scandinavia and Europe but allowed on some crops in the U.S., vinclozolin
is an endocrine-disrupting chemical. If pregnant female rats are exposed
to it while their embryos are undergoing sexual organ differentiation, the male offspring develop serious problems as adults
— death of sperm-generating cells, lowered sperm count and motility
and, later, immune abnormalities and various diseases including cancer.
The remarkable thing is that the effects, which were not rooted in changes
to the DNA sequence, were found to be transmitted over
four generations without weakening. That is, acquired characteristics
— deficiencies in embryos brought on by fungicide exposure —
were inherited by offspring who were not subject to the same exposure.
This led Skinner to ask a troubling question: "How much of the disease we
see in our society today is transgenerational and more due to exposures
early in life than anything else?" (quoted in Brown 2008).
The whole business looks rather like vindication for the long-dismissed
Lamarckian doctrine of the inheritance of acquired characteristics, a doctrine
that has indeed been making a comeback of late. But inheritance aside,
puzzling results such as these put the question, "Are genes equivalent to
destiny?" in a new light. In 2007 a team of researchers at Duke
University reported that exposure of pregnant mice to bisphenol A (a
chemical used in many common plastics such as baby bottles and dental
composites) "is associated [in the offspring], with higher body weight,
increased breast and prostate cancer, and altered reproductive function".
The exposure also shifted the coat color of the mice toward yellow —
a change again found to be transmitted across generations despite its not
being linked to a gene mutation. But more to the present point: the
changes brought on by the chemical were negated when the researchers
supplemented the maternal diet with folic acid, a B vitamin (Dolinoy et
al. 2007).
And so the "epigenome" — everything in the cell that bears on gene
expression
— responds to healthy as well as unhealthy influences.
As another illustration of this:
researchers at McGill University in Montreal looked at the consequences of
two kinds of maternal behavior in rats. Some mother rats patiently lick
and groom their newborns, while others generally neglect their pups. The
difference turns out to be reflected in the lives of the offspring: those
who are licked grow up (by the usual measures) to be relatively confident
and content, whereas the neglected ones show depression-like symptoms and
tend to be fearful when placed in new situations.
This difference is correlated with different levels of activity in
particular genes in the hippocampus of the rats' brains. Not that the
genes themselves are changed; the researchers found instead that various
epigenetic modifications of the hippocampus alter the way the genes
work (Weaver et al. 2004). Other investigations have pointed toward
similar changes in the brains of human suicide victims who were abused as
children (Poulter et al. 2009).
Perhaps even more surprisingly, mouse embryos grown by means of in vitro fertilization (IVF) — spending their first several days in a petri dish — showed epigenetic changes resulting in altered gene expression. And now there are reports that humans conceived through IVF have an increased risk of several birth defects. The main suspect is again the epigenome (Kolata 2009).
So what is going on?
All the examples just given show how the environment can play into the organism's genetic performance. They suggest that genes do not bear a fixed meaning, independent of their context. And one aspect of this context currently receiving intense scrutiny at the cellular level has to do with RNA.
Far from simply carrying out orders for the production of proteins, RNA seems to be
involved in wide-ranging cellular functions. Humans possess only about
twenty-five thousand protein-coding genes — genes that give rise to
mRNA
that in turn
yields protein — and these constitute about 1.2% of our DNA
. Yet by one
estimate 93% of the genome produces RNA transcripts
—
transcripts that, except for a tiny percentage, are not templates
for proteins (Zimmer 2008, p. D5). If they are not engaged in producing
proteins, what are these noncoding
RNAs
doing?
They seem to be doing a great deal, although scientists have barely begun
to unravel the story. Take, for example, the mice who retained white
spots on paws and tail despite the loss of the corresponding mutant gene.
When the researchers extracted all the RNA — but not the DNA —
from cells of mutant mice and then injected this RNA into the fertilized eggs of
normal mice, the eggs developed into adults with the mutant
characteristics. It appears, then, that RNA has something to do with the
epigenetic inheritance of the white spots.
But there are numerous different kinds of RNA, and there are even more roles they play in the organism. Further, they are only one kind of element in the overall epigenetic landscape — a landscape whose complexity makes any summary presentation extremely misleading. Nevertheless, here are a few pointers into that complexity:
DNA methylation. Every cell "tags" or "marks" various
sites along a DNA molecule with a small chemical group known as a "methyl
group". These marks, or their absence, can dramatically alter the
expression
of nearby genes, often shutting them down or silencing
them.
Researchers investigating those mice exposed as embryos to bisphenol A
found, among other things, decreased methylation near a key gene affecting
coat color. In humans, distinctive patterns of DNA methylation are
associated with Rett syndrome (a form of autism) and various forms of
mental retardation. 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).
It's not only the local gene that can be affected by methyl marks,
however. The larger pattern of methylation can play a role in
orchestrating gene expression over extended stretches of a chromosome by
recruiting proteins that alter the chromosome's structure. This is
connected with chromatin remodeling, discussed below. And, as we will
also see shortly, noncoding RNAs figure
in DNA methylation.
While some epigenetic changes are heritable through the germ line
, many are
not — and necessarily so. You wouldn't want the epigenome
of a heart
cell or kidney cell — or, more relevantly, a gonad cell — to
find its way unchanged into the fertilized egg. The slate upon which all
the developmental
processes of the adult have been written needs to be wiped
clean in order to clear a space for the next generation. (Or relatively
clean — heritable epigenetic marks are somehow preserved.) As part
of this slate-cleaning, a wave of demethylation
passes along
each chromosome shortly after fertilization and is completed by the time
of implantation in the uterus. Immediately following this, a new
methylation occurs, appropriate for the embryo and giving it a fresh
epigenetic start. When, in mammals, the stage of embryonic methylation is
blocked artificially, the organism quickly dies2.
Histone modification and
chromatin remodeling. You will recall that there is more protein than
DNA in a human
chromosome. The two together constitute chromatin
, an
intricately formed, ever-changing substance whose physical, chemical, and
electrical qualities figure greatly in gene activity. Among the key
proteins are histones
, eight of which join together to form something like a
spool. Such spools occur along the entire length of the chromosome, with
the double helical
DNA wrapping roughly two times around each spool and then
extending, string-like, a short distance before wrapping around the next
spool. (The DNA-histone complex is called a "nucleosome
".) But
normally not much of the DNA is "strung out" in this way. The nucleosomes
commonly pack themselves into dense, three-dimensional arrangements, upon
which are superimposed yet further levels of condensation.
All this is intimately bound up with the transcription of DNA into
mRNA
— that
is, with the expression
of genes. Wherever the chromosome is densely packed, the
enzymes and other substances participating in transcription do not have
easy access to the genes, and therefore gene expression is reduced. And
this is where methylation enters the picture again. Methyl groups can
attach not only to DNA, but also to the histones — and particularly
to the long, filamentary "tails" extending out from the histones. The
methyl groups here, too, affect the expression of local genes. They do
this in part by mobilizing various proteins, which then become associated
with the chromatin and alter its conformation. Some of these chemical
complexes seem to work with each other while others work against each
other. The net result is a "chromatin remodeling"
that may proceed, wave-like, down long stretches of the
chromosome, rendering genes either less or more available for
transcription.
And, for good measure, the whole remodeling process can be facilitated by
DNA methylation. "Thus modification at one level, in this case methylation
on the genomic DNA, may have pronounced effects at other levels of
organization of the chromatin, a theme of growing importance in the field"
(Feil 2008, p. 2).
Other chemical groups beside methyl — groups such as phosphate,
acetyl, and ubiquitin — can also attach to the histones, each with
its distinctive and as yet scarcely traced interactions and effects. But
there are few simple rules. While histone acetylation is generally
associated with higher transcription rates, both methylation and
ubiquitylation may either repress or activate transcription. Similarly,
the phosphorylation of a particular histone site can correlate either with
opening up of the chromatin structure and activated transcription, or
(during cell division) with the closing and condensation of chromatin
— thereby illustrating "the importance of genomic context" (Berger
2007, p. 408). In general, where a methyl, ubiquitin, or other
group attaches to a histone tail, and
how the group associates with other molecules, shapes its role in
gene transcription. Such histone modifications
— not only local modifications, but their global
pattern — can be correlated with cancer and can even aid in
predicting the clinical outcomes of cancer treatments (Seligson et al.
2005).
Chromatin remodeling, however, affects more than gene expression
within the genome
of an
existing organism. It also helps to shape the possibilities for future
genomes. It does this by influencing the location and rates of mutation
throughout
the genome. New evidence suggests that "the physical structure of the
genome can directly influence the rate of mutation down to the
single-nucleotide
level, with far-reaching implications for genome evolution"
(Semple and Taylor 2009). This is one of the ways the long-reigning
doctrine of random variation is currently being undermined — that
is, the doctrine that chance is the supplier of the stuff from which
organisms are fashioned.
RNA interference. Various lines
of research during the 1990s led to the discovery of extremely short RNA
molecules with an extraordinary ability: they could, with great
efficiency, silence particular genes. The frenzy of investigation triggered by
this discovery of "RNA interference" (RNAi) has already yielded what
geneticists are unabashedly referring to as a "revolution" in their
field.
The central molecular players here go by the name of "small interfering
RNA" (siRNA). They are derived from the disassembly of long,
double-stranded RNA — often from incoming viruses. They are truly small
— only about 21-25 nucleotides long — but their short
sequences
are nevertheless long enough to provide a match with just
one particular mRNA
and thereby to target that mRNA. The siRNA, after becoming
part of a larger protein complex called a "RISC"
, repeatedly
locates its target mRNA, whereupon one of the RISC proteins cleaves the
mRNA to pieces. Or else, depending on how perfect the complementarity
of sequences between the siRNA and mRNA turns out to be,
the latter may simply be disabled from translation rather than sliced up.
In neither case is the relevant gene directly silenced, but the
mRNA resulting from it is repressed. This is known as
"post-transcriptional silencing".
The process, however, is far from being as neat as this description might suggest. For example, an entire drama plays out in the production of siRNA from virus RNA or, sometimes, from other, endogenously produced molecules. And, of course, the question of overall function arises: what significance is there in the selection of mRNAs for silencing, and how is this selection managed? There are complications at the target end of the process as well. A given mRNA can be masked from the siRNA by virtue of attached proteins, preventing its destruction. Or, conversely, those proteins may lay it bare for destruction by unfolding it and exposing it to the siRNA's complementary nucleotide sequence.
The still rapidly unfolding story of RNA interference is taking on ever
wider significance. To begin with, it's not only in the cell of origin
that siRNA plays a role. It can migrate to other parts of the body
— and its migration to germ cells might
explain some cases of epigenetic inheritance
. That is, its presence in the germ cell could have much
the same result as the loss or mutation
of a
gene.
It's also been found that siRNAs do not act only post-transcriptionally;
they can cooperate with other players in directly silencing genes.
They do this by participating in various DNA methylation and chromatin remodeling
processes. It appears that, by means of their own short
nucleotide sequences, they target specific regions of the chromosome for
structural modification (Moazed 2009), with implications for gene
expression
in those regions.
And, in yet another surprise, researchers have discovered a role for siRNA
in what they are calling "small RNA-induced gene activation" — the
very opposite of silencing. By targeting a promoter site close
to a particular gene, the siRNA can powerfully increase expression of the
gene.
This last point illustrates an important truth of the living organism: we
dare not assume that the meaning of any substance or any process remains
constant in all contexts. What the discoveries in epigenetics are telling
us is that this is true even of those foremost symbols of immovable
constancy, the genes.
The dramatic significance of RNA interference is indicated by the excitement of those researchers wishing to put it to use. For example, they are already using RNA interference to silence the genes that help speed the deterioration of ripe tomatoes on your kitchen shelf. Involving as it does short, easily synthesized molecules, RNAi "has provided scientists with an incredibly powerful tool . . . . it is possible to selectively inactivate virtually any gene, simply by introducing an appropriate synthetic RNA into the cell" (Jablonka and Lamb 2005, p. 136). Of course, if the entire story of epigenetics tells us anything at all, it is that the word "simply" in this enthusiastic endorsement will not fully justify itself. But hope springs eternal.
Micro-RNA. There is another class of very short RNA not always
clearly distinguished from siRNA
in the
technical literature. It is not derived from viruses, but only (by
various elaborate pathways) from double-stranded RNA
encoded in
the genome
. Its final processing occurs outside the nucleus in the
cell cytoplasm. Like siRNA, it becomes associated with a multiprotein
RISC, locates mRNA molecules, and then disables them in one way or another
— evidently not so much by cleaving them as by preventing their
translation. And, like siRNA, this "micro-RNA" (miRNA) identifies the
target mRNA
based on a complementation between its own sequence
of
nucleotide bases
and that of the target — usually near one end of the
target. However, unlike with siRNA, this match of sequences need not be
very exact, so that a single micro-RNA
can prevent
translation of many different mRNA molecules, effectively silencing
or reducing
the expression
of many genes.
There are at least several hundred micro-RNAs in the human genome, each of which might in this way regulate the activity of hundreds of genes. All together, micro-RNAs, siRNAs, and other classes of small RNAs not discussed here "have the potential to regulate the expression of almost all human genes" (Siomi and Siomi 2009, p. 403). They can serve to activate as well as repress gene activity, and some of them are associated with cancer, while others seem to help prevent it. In the opinion of Whitehead Institute molecular biologist David Bartel, "It's going to be very difficult to find a developmental process or disease that isn't influenced by micro-RNAs" (quoted in Pollack 2008, p. D3).
If we were to look a little more closely, we would find that not only do
small RNAs regulate gene expression, but they in turn are regulated by yet
further systems of "control". For example, proteins can block the
formation of small RNAs from their precursors, or else be required as
assistants in this formation. It can even happen that, through a kind of
mimicry, an mRNA "fools" a RISC
into binding to it, but because of the way the mRNA differs
from the normal target mRNA, the RISC cannot disable it. In this way the
mRNA takes the micro-RNA out of action, resulting in elevated expression
of the actual target mRNA.
The idea of target mimicry introduces unanticipated complexity into the network of RNA-regulatory interactions and raises the possibility that a large number of mRNA-like noncodingRNAs recently identified in humans could be attenuators of the regulation [by small-RNA-protein complexes]. (Siomi and Siomi 2009, p. 403)
Intersecting "networks of regulation" is how this sort of thing is commonly described. One might begin to suspect that, one way or another, almost everything is involved in the regulation of almost everything else — not a very useful observation, perhaps, except so far as it lends pointedness and poignancy to the question, If everything is doing the regulating, what is left to be regulated? Or, if there is no clear distinction between regulator and regulated, maybe we're just not using the right language at all.
Transcription factors, RNA editing, and much more. Even before researchers shifted their attention to the epigenome over the past decade, certain well-established findings were powerfully nudging them toward a less linear-logical, more contextual understanding of the gene. The simplistic early schema — DNA > RNA > protein — has been under the stress of ramifying complications for a long while.
To begin with, there was not only the curious fact that the supremacy of
the logically neat gene required a substantial part of the genome
to be
dismissed as junk; a good part of the real estate within
protein-coding genes also had to be dismissed. That is, the cell as a
whole does a great deal of picking and choosing when it comes to deciding
what really constitutes a gene. The parts of the traditionally defined
gene that survive this process are called "exons", while the segments cast
aside are "introns".
The separation of the exon sheep from the intron
goats occurs
only after the gene is transcribed into an initial form of mRNA known as
"precursor mRNA". Through a splicing
process
influenced by complex signaling within the cell, the introns within this
precursor are culled, and the remaining sections are knitted together.
But none of this is cut-and-dried. The same precursor mRNA can undergo
different splicing patterns ("alternative splicing"), so that particular
protein-coding regions of DNA produce, by one estimate, an average of 5.7
different final transcripts (Zimmer 2008, p. D5). At least 86% of human
genes, it is thought, are subject to alternative splicing (Muers 2008). An extreme case is a gene active in the
inner ear of chickens (with an assumed analog in humans): it has 576
alternatively spliced variants. These variants
code for a protein that has a role in determining the sound frequency to which inner ear cells respond, and the variations in the protein sequence parallel variations in the frequencies to which different cells respond. It seems that having so many versions of the protein enables the chicken to tune its cells and distinguish between the sounds it hears. (Jablonka and Lamb 2005, p. 67)
There's an awful lot of significant management going on here, and it's not all being orchestrated by genes.
Even more contrary to expectation, some of the exons composing the final
mRNA may come
from other genes and even other chromosomes ("trans-splicing"). And,
quite apart from the various types of splicing, there is RNA "editing"
whereby specific nucleotide bases
("letters" of the code) are removed and replaced with
different letters not corresponding to the original DNA
sequence
. Or else
additional bases are inserted in the sequence. Both the editing and
splicing suffered by particular gene transcripts
may
systematically differ in different types of cell, despite the identical
DNA sequences in those cells.
Nor is that the end of it. Once the splicing and editing are completed,
the same mature mRNA can be translated into many
different proteins; the same protein can fold in various ways, which
radically alters its functioning; and this result, whatever it may be, is
the potential subject of countless "post-translational modifications"
through being cut up or having any number of chemical groups added to it.
The folding and post-translational modification, in turn can be influenced
by, among other things, the character of nearby molecules known as
"chaperones
". In other words, the protein end result — or,
rather, the vast range of possible end results — of a particular DNA
sequence can hardly be thought of as determined by a single cause, genetic
or otherwise. Given the endlessly interwoven processes at work, there is
no possible way to conclude with less than this: the cell as a whole has
the final say about what a gene means.
Coming back, finally, to the DNA that was supposed to be masterminding the
entire show: near many genes (or sometimes remote from them) there are
various regulatory DNA sequences that help to
modulate the expression of the genes — for example, enhancers
and
silencers
. Of course, something, or many things, must participate in
the regulating. It turns out that some 2600 proteins in the human body
can, by virtue of their form, bind themselves to DNA. Those that bind to
regulatory sites such as enhancers and silencers are known as
"transcription factors"
. Depending on the transcription factor and the DNA
sequence it binds to, its presence may tend to either activate or repress
gene transcription. Or it may act in cooperation with other proteins
— co-activators
and co-repressors
— that
do not themselves bind directly to DNA but rather aid or hinder the
recruiting of RNA polymerase
, the enzyme
that actually transcribes genes. In a seemingly boundless tapestry of
shifting patterns, many proteins act in concert, so that their effect upon
DNA is a subtle integral of their separate "causal" potentials.
In all these processes, DNA itself, of course, plays its crucial role. The point is
only that there's no one point-of-origin and no causal chain of command,
however circuitous, that by itself provides, or could even conceivably
provide, an adequate and understandable picture of what is going on.
Understanding, as we will see later, requires something more than
logical-causal thinking.
To itemize distinct "mechanisms" in the way I have just now done is to
encourage exactly the sort of isolating perspective that needs to be
overcome. None of these factors and influences can be cleanly separated
from the others. According to Aaron Goldberg and his colleagues at
Rockefeller University's Laboratory of Chromatin Biology, "It is becoming
clear that significant crosstalk exists between different epigenetic pathways".
For example, small RNAs
"often act in concert with various components of the cell's
chromatin
and DNA methylation
machinery to achieve stable silencing". There's much to
sort out, they say, but "the emerging dialectic of epigenetics, including
the marks, writers, presenters, readers, and erasers, promises to be a
rich conversation" (Goldberg et al. 2007, pp. 637-8; never mind the
authors' strange juxtaposition of "machinery" and "conversation").
In a similar vein, geneticist Shelley Berger speaks at some length about
the methylation of a particular histone location.
Originally the mark
was simply thought to have positive effects on
transcription
. But ongoing research has revealed a dizzying array of
outward-rippling interactions between this methylated site and various
other activators
, repressors
,
co-repressors
, and so on. "How", she asks, "can the binding of so many
complexes to one [type of methylated histone site] be explained?"
Compelled toward rather nontechnical language to capture the situation,
she says "it may be that there is an intricate 'dance' of associations,
with these changing places over time". There is a kind of rhythm between
positive- and negative-acting complexes, where "the entire chromatin
context [of the methylated histone] would dictate the overall outcome".
Thus a useful analogy may be that the modifications [of chromatin] constitute a nuanced language, in which the individual marks (the "words") become meaningful only once they are assembled and viewed within their unit array, such as a transcription unit (a "sentence"). To put it simply, the genomic and regulatory context must be considered for the biological meaning to be understood. (Berger 2007, p. 409)
But, as we have seen, the regulatory context seems to extend outward without limit. Nothing less than the dynamics of cell, whole organism, and environment can make sense of any particular tract of DNA — can interpret it and turn it into a fitting expression of its larger context. The genome, perhaps we could say, is not so much an instruction manual as a dictionary of words and phrases together with a set of grammatical constraints. And then, from conception through maturity, the developing organism continually plays over this dictionary epigenetically, constructing the story of its destiny from the available textual (genetic) resources.
1. Actually, while this is the usual way of stating the matter, there are
many cases throughout the animal kingdom where
chromosomes
are not the same in every cell.
The human immune system provides one striking example:
During the maturation of lymphocytes (the white blood cells that produce the antibodies needed to fight infection and destroy foreign cells), DNAsequences
in the antibody genes are moved from one place to another, and are cut, joined, and altered in various ways to produce new DNA sequences. Because there are so many different ways of joining and altering the bits of DNA, vast numbers of different sequences, each coding
for a different antibody, are generated. Consequently, the DNA of one lymphocyte is different from that of most other lymphocytes, as well as from that of other cells in the body. (Jablonka and Lamb 2005, p. 68)
For many other examples, see pp. 68-70 of the cited work. However, the fact that so many different tissues and organs do have the same DNA still raises the question discussed in the main text.
2. Early stages of this slate-cleaning and management of methylation have already begun in the undeveloped egg cells present in the gonads of the female embryo.
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Steve Talbott :: The Twilight of the Double Helix