[Contd. from Part 1]
In part 1 of this series, the basic idea of epigenetics was presented as a refutation of DNA/genetic reductionism. We explored this concept by reference to some examples and case studies. There’s a much simpler way to understand why epigenetics is needed for biology to make sense, however. Consider the fact that all of the 50 trillion cells in the human body essentially have the same genome (excluding rare mutations). However, a liver cell is completely different from a heart cell. The cells in your eye seem to have almost nothing in common with the ones in your gum. And yet, at the DNA level, their 3 billion nucleotides are arranged in the exact same away. To top it off, this bewildering variety of cell types in our bodies ultimately originated from just one cell, the zygote.
Clearly, reference to DNA alone can’t explain this phenomenon, and we need to appeal to some innate non-genetic biological mechanisms to explain any of this. Enter epigenetics. This seems like a very straightforward way of making the point we did in part 1.
Thing is, this rather everyday observation wasn’t always available to past scientists. Nowadays it’s common knowledge that all cells have the same DNA. But that took quite a bit of clever experimentation to find out.
John Gurdon’s experiment
When one thinks about the phenomena of cell differentiation- how a single zygote differentiates into so many different types of cells in our body- two explanations conceptually present themselves:
One, as the zygote develops into a particular type of cell- the rods or cones in the eye, say- their DNA itself undergoes change. Only those genes that are responsible for the unique properties of light sensing are preserved in the cell, and the rest are done away with. So on this model, cell differentiation is equivalent to deletion of particular bits of DNA depending on cell type.
Two, during development, the DNA of a cell stays as it is. The zygote and the rods/cones have the same DNA, but extra-genetic biological mechanisms are used to silence the effects of some genes, while preserving the effects of others. On this model, cell differentiation sees no change at the level of the DNA, it’s just the extra-genetic cellular mechanisms that suppress particular types of genes from being expressed.
The English biologist Sir John Gurdon designed an experiment to see which of these hypotheses was true. The basic premise for his experiment was very simple. If cells do lose DNA as they become more specialized (as per hypothesis 1), then that means the DNA derived from a specialized cell shouldn’t be able to do the job of zygote DNA. On this hypothesis, the zygote DNA would have a fuller complement of genes that would be missing from more specialized cells.
Sir Gurdon extracted the nucleus from a developed muscle cell from a toad and introduced it into an unfertilized toad egg. As it happened, the eggs did manage to develop into normal tadpoles. This didn’t have a high success rate, but the fact that even one of these eggs with the DNA extracted from a developed cell could give rise to a fully functional organism meant that no DNA is removed from an adult cell during the process of cell differentiation or development. The “zygote DNA” is no different than “specialized cell DNA”, so the latter can be used to replace the former and yet be expected to work.
To take stock, Gurdon’s experiment didn’t so much as prove anything than it disproved the hypothesis that DNA is lost from cells during development. It’s by this disproof that the second hypothesis above was confirmed.
Waddington’s epigenetic landscape
As it happened, a conceptual framework for understanding cell differentiation was already available by the time Sir Gurdon ran his experiments.
The picture you see above is a model for cell differentiation due to the British Polymath Conrad Waddington. This offers a particularly elegant framework to understand the issue. If the ball in the picture is allowed to roll (because of gravity), it will travel down one of the troughs at bottom. Once it reaches the bottom, it’s impossible for it to naturally “switch tracks” and go to the bottom of some other trough. Gravity would prevent the ball to roll uphill under normal conditions.
The ball, of course, represents the zygote; and the troughs represent specialized differentation pathways. Once the zygote rolls down any particular pathway, it becomes “committed” to that pathway and can’t normally switch tracks. This is why a liver cell can’t become a heart cell, because once the zygote transforms into the former, it can’t normally ignore the restrictions imposed upon it by the differentiation program (think of that as the gravity and inertia working on the ball) and choose to become the latter.
Another important benefit of this landscape model is that it appropriately captures the results of Gurdon’s experiments. Notice that as the ball rolls down particular troughs, it still remains the same ball- it doesn’t morph into something else, a square for example, precluding the possibility of putting it on top of the landscape and rolling it down once more. Same way, the zygote DNA doesn’t undergo change as it commits to any particular differentiation program. That’s why in Gurdon’s experiments it was possible to introduce differentiated cell DNA into an egg (putting the ball on top again) and having it develop (rolling down particular troughs).
Can we roll the ball uphill more efficiently?
At this juncture, it would be useful to introduce some terminology. A totipotent cell is a cell that can transform into any cell type, including the placenta. Only the zygote fits this bill. A pluripotent cell, on the other hand, can also transform into pretty much all cell types with the exception of the placenta. Embryonic stem (ES) cells fit this description- they are near the top of the landscape and can roll down any trough. The cells that roll down the troughs are differentiated or specialized cells, we’ll call them lineage-committed (LC) cells.
Gurdon engineered his LC cells into pluripotency by replacing the DNA of the latter with that of the former. There has to be a more efficient way of getting the job done. If the pluripotent cells are characterized by a certain gene expression profile- meaning if only certain genes are expressed in pluripotent cells- that means by tinkering with the gene expression profile of LC cells, it should theoretically be possible to transform them into ES cells. All that would be required to do is to express the specific gene complement that endows pluripotency to a cell, and that would revert any cell to its ES state. It would be possible to roll the ball uphill. This called for another set of experiments to be performed, and the baton was taken up by Shinya Yamanaka in Kyoto.
Professor Yamanaka and co. started with a complement of 24 genes that were known to be involved in developmental pathways. Their goal was to express these genes into mouse fibroblasts, and by gradually knocking down some genes at a time, find out the minimal set of genes required to induce pluripotency. By this sort of progressive whittle-down of genes, they discovered that only 4 genes being expressed was all it took for a cell to become pluripotent. Once it became committed to a particular lineage, these genes got switched off and more specialized genes started being expressed. Their findings, counterintuitive as it may have seen back then, were later confirmed by other laboratories.
Being able to cultivate embryonic stem cells in this way was a huge technological breakthrough, and talking about them would take us a few more paragraphs. However, this post series is more about theoretical biology than practical or technical relevance of the experiments. This is why I’ve omitted some crucial experimental details (e.g. how the controls were set up, why certain cell types were chosen for the tests etc) only to present what we eventually came to learn from them in terms of basic epigenetics research. To that point, it’s important to recognize that Yamanaka’s experiments offered additional confirmation of Gurdon’s findings- not only were the ES cells not different from the LC cells in terms of their DNA, but the latter can actually be transformed into the former by genetic manipulation. The fact that the ball can be rolled uphill in this way is very clear evidence that the ball at the top of the landscape and the one at the bottom of a trough are one and the same.
Gurdon told us cells manipulate, and don’t erase, bits of the DNA to commit them to particular lineages. Yamanaka gave us more information on how that’s achieved in mechanistic terms.