[Contd. from Part 2]
In the last parts of this series, we discussed natural observations and scientific experiments which establish the reality of epigenetics. To vaguely point to some mechanism that exists in the biological ether can get us only so far. And so from this post onwards, we’ll be entering the actual molecular nitty-gritty of it all and see what fundamental biological entities and processes epigenetic phenomena refer to.
Before we move on to the actual discussion in this part- I have two disclaimers. First, readers may note I don’t have any notes on chapter 3. That’s because chapter 3 is about the basics of genome biology- what the DNA looks like, how it’s transcribed, repaired, and expressed. My blog already assumes its readership to be knowledgeable about these issues. Second, Dr. Carey’s literary ingenuity really shines through in her accounts of the molecular processes that go on in the cell, exactly what we’ll be picking up today. She uses colorful but very to-the-point analogies to form reader’s intuitions about these matters (we’ll be seeing some examples later in this post). So even though we’re skipping chapter 3 notes- even seasoned biology students would get something out of reading the chapter in terms of fine-tuning their visualizations.
Let’s first take stock of what we have established. As Gurdon’s experiments proved, cells don’t lose anything in terms of actual DNA content as they become more specialized. The ball rolling down the epigenetic landscape yet remains the same ball. Which means the cell uses extra-genetic mechanisms to preferentially suppress (or enhance) the expression of certain types of genes depending on the specific differentiation program they’re committed to. Cells in the eye, for example, can’t afford to express the genes for hemoglobin, and so they must turn them off.
This presents two conceptual questions molecular biology must answer. First, what precise mechanisms does the cell use to turn on and off its genes? Second, cells have a rather limited life-span, and almost every cell of our body gets replaced. That means these gene-suppressing (and enhancing) mechanisms must not only exist, but also be faithfully inherited from cell generation to generation. Given the view of DNA being the hereditary molecule in our cells, how can we make sense of this?
Dr. Carey offers a very apt analogy. DNA is often talked about as a book, but she submits that it makes more sense to talk about it as a script for a play. A director’s script usually has all the lines for all the actors. The same script will result in different sorts of plays, if not different plays altogether, depending on how the play is directed. This is done not by changing the script itself- all the actors may have the same basic script, but the director may make specialized notes to each actor’s copy depending on his or her role. So in the script for The Dark Knight, for example, Christian Bale would have the same script as Heath Ledger, but the director’s notes on their scripts would differ. Once these scripts are photocopied, both the script as well as the directorial notes will survive the process.
In this big picture analogy, the director is the cell’s epigenetic mechanisms, the script without the director’s notes is the DNA, the actors are individual cells, and photocopying represents replication. The play, of course, is life.
With this overarching logic in place, let’s see how the cell conducts its plays.
One of the key processes the cell uses to turn off gene expression is slapping a methyl (-CH3) group on the cytosine nucleotide in DNAs. An enzyme by the name of DNA Methyl Transferase (DNMT) carries out this reaction. A methyl group is really small compared to the overall base (15Da compared to the 600Da of a base pair)- the author compares this to sticking a grape to a tennis ball.
The chemical structure of a methyl group (blue) stuck to a nucleotide- see how small it looks when you compare it to the entire impressive DNA structure in the model to the right.
All cytosines are not created equal in their susceptibility to methylation- this is usually done for cytosines that are followed by a guanine (C followed by G, written as CpG). In 1985, the British scientist Adrian Burd discovered CpG motifs are not randomly distributed throughout the genome- rather, they are concentrated around the upstream region of genes where the gene promoters lie. So the hypothesis (that has now been confirmed) was that methylation of these CpG islands on or near gene promoters can switch off, or at least decrease, gene expression. The exact process here involves a protein- MeCP2 (Methyl CpG binding protein 2)- which, as its name suggests, binds to CpG motifs that have been methylated. This protein binding reduces expression by either recruiting other proteins in the cell which are involved in gene repression, or preventing transcription factors from binding (or both).
These epigenetic “marks” can also be stably inherited following a logic very similar to semiconservative replication. In brief, if a newly synthesized DNA molecule has one strand with and the other without epigenetic marks, DNMT corrects the imbalance by marking the newly synthesized (and hence unmarked) strand according to the patterns left on the old strand.
DNA methylation gets us a little close to understanding some of the body’s key epigenetic processes, but it still doesn’t explain everything. Different sorts of cells, for instance, repress different proteins depending on their specialization. So how does the cell know which genes to methylate and repress? How does an eye cell know to methylate and repress a skin cell?
Histone modification is the other key epigenetic process. There are some notable differences between methylation and histone modification- the latter is, for example, much less stable than the former. If DNA methylation are the printed directorial comments on each actor’s script, histone modification is more similar to pencil marks which will can only survive a few rounds of photocopying- in fact, they may be as transient as post-it notes and not be inherited at all. Methylation only represses gene expression, histone modification can increase or decrease it. Methylation is a relatively simple process, somewhat similar to a (gene expression) on-off switch, while the effect of histone modification comes in decrease, reminiscent of a radio dial.
People often think of DNA as naked spaghetti strands suspended in the casserole of the nucleus. If they really were as loose, they couldn’t have been fitted into the cell at all. Rather, DNA is spooled around globular proteins called histones. Histone proteins are organized in clusters of eight- four above and four below, like four ping-pong balls stacked on top of one another. The DNA strand loops around them like a licorice whip around a marshmallow (none of these colorful analogies are mine, just to be straight). It’s impossible for the cell’s transcription apparatus to read the DNA where the coiling is too tight.
The model above shows each individual histone as being completely globular, but that’s not totally accurate- they also have a wiggly chain or tail extending from them. These tails are where epigenetic codes are “written”. Like DNA methylation where a methyl group was added to cytosine, an acetyl group can be added to a particular amino acid (lysine) on the tail as well. Unlike DNA methylation, however, this sort of histone acetylation drives gene expression up.
In fact, also unlike DNA methylation, there are many different ways in which cells slap groups onto histone tails, and each of these ways have their unique effects on gene expression. Different sorts of functional groups attached to histone tails mean different degrees of increase or decrease in gene expression. These correlations between particular histone modifications and consequent effect on gene expression constitute a code or a grammar. The rules here are incredibly difficult to unearth, and that’s what scientists are working on today.
Functional groups attaching to histone tails have a myriad of effects on gene expression
Dr. Carey uses the following innovative illustration to give us a mental image of how the entire phenomenon of histone modification looks like:
Imagine a chromosome as the trunk of a very big Christmas tree. The branches sticking out all over the tree are the histone tails and these can be decorated with epigenetic modifications. We pick up the purple baubles and we put one, two or three purple baubles on some of the branches. We also have green icicle decorations and we can put either one or two of these on some branches, some of which already have purple baubles on them. Then we pick up the red stars but are told we can’t put these on a branch if the adjacent branch has any purple baubles. The gold snowflakes and green icicles can’t be present on the same branch. And so it goes on, with increasingly complex rules and patterns. Eventually, we’ve used all our decorations and we wind the lights around the tree. The bulbs represent individual genes. By a magical piece of software programming, the brightness of each bulb is determined by the precise conformation of the decorations surrounding it. The likelihood is that we would really struggle to predict the brightness of most of the bulbs because the pattern of Christmas decorations is so complicated.
As with DNMT and MeCP2 being the writers and readers respectively of DNA methylation, histone modification also employs a number of enzymes for these purposes. Again, the entirety of the code hasn’t been figured out as of yet, but lack of these reader and writer proteins are often causes of debilitating diseases- speaking to the importance of these modifications.
So to review- the two key was in which cells modify the levels of gene expression are DNA methylation and histone modification. The former is akin to a simple on-off switch, while the latter is much more complex, allowing for sophisticated fine-tuning of gene expression patterns in particular sorts of cells.
As a concluding (somewhat personal) note- learning about these mechanisms is what finally convinced me that when it comes to control of biological processes, there’s no element that’s more primary to another. Much of my undergraduate education seemed to at least implicitly assume DNA/genetic reductionism, so when in graduate school I learned about epigenetic mechanisms- my mind couldn’t process how these could work without DNA precisely telling them what to do. In reality, protein processes in the cell are not exclusively dependent on DNA, or vice versa. DNA and cellular processes always work together in an organism, with no part being in any way secondary or dependent in a unidirectional, asymmetric way.
Some people feel like the enormously complex processes that occur throughout development are similar to how a Rube Goldberg machine operates- a horribly complex and elaborate system with each of its part depending on some other. This analogy holds with one exception- in a Rube Goldberg machine, you need an initial trigger, be it the pull of a chord, the kick of a boot, what have you. That really doesn’t happen in case of life. At each stage of development, gene expression depends on epigenetic marks made by proteins, and the coding of these proteins depend on the genes. The epigenetic marks, in turn, are inherited from prior cells, in which the same process was repeated. You can trace an organism’s life history back this way until you reach the zygote. Surely the zygote is ultimately dependent on the DNA for instruction, non? Actually, here too crucial epigenetic information is inherited from the proteins in the egg. So it’s impossible to point to any part in the cell in the life of an organism- be it the DNA or any protein- and say, “this is the part that started it all”. In reality, it makes no sense to divide life’s processes in this way. All of life is integrated and interdependent with no primacy of any part over the other.