So, hi everybody. What we are going to do today is to have a discussion with Doctor Andrew Keniry about DNA demethylation and also reprogramming. So, these were two topics that we've covered within the course but, not in great detail yet and yet, they're very active areas of research and Andrew knows an awful lot about both of these areas, given where he did his PhD. So, Andrew, active demethylation was first known to occur because people saw there were periods in development when you would lose methylation at a really rapid rate without having any cell division. And so I guess it was striking for quite a long time that there must be something going on, there must be an enzymatic process going on. >> Sure. That's right. So in the one cell embryo, we can see both potential mechanisms for the loss of DNA methylation because we have both the male and female pronucleus in that one cell and soon after fertilization we see both of those nuclei staining very brightly for DNA methylation. And then, very quickly, before any cell division in the male pronucleus, we see a very rapid loss of DNA methylation, staining for DNA methylation and so, this means that there has been an active process of DNA demethylation. Whereas in the female pronucleus, it happens more slowly over multiple rounds of division, and essentially, the DNA methylation is being lost passively through multiple rounds of cell division, and essentially just being diluted through cell division. >> Right, so I guess that's because DNMT1 is not doing it's job, it's not maintaining the methylation, and in fact DNMT1 is excluded from the nucleus for some of that critical period, during early development. >> That's exactly right. So if DNAMT1 is excluded from the nucleus the mechanism is not there to maintain DNA methylation through cell division and you just get a dilution of the DNA methylation through time. >> So, what's always struck me is that it seems like even from the early 90s, but certainly at least from about 2000, there was very strong evidence that we had active demethylation but it took quite a long time before the enzymes, the TET enzymes that were involved in the active process were discovered. So I guess, why did it end up taking so long? Do you have an opinion as to why it took a relatively long amount of time to find these guys? >> Yeah, well I think there's a lot of reasons for why that happened and I guess the first one is because the stages of development where DNA demethylation happens are quite hard to study, so we'd mentioned the one cell embryo and the other place where it happens naturally is in the primordial germ cells. And so each of the cell types are rare and don't provide lot of material to study technically in the lab so- >> So biochemistry is a bit impossible then? >> Exactly. >> Because you've only got a few cells >>Biochemistry is very hard. >> Yep >> And then the other problem was well, we've had recent advances in the techniques so with advent of next gen sequencing now, we're able to look more closely for more subtle differences and so this has allowed us to see enzymes that are perhaps acting more subtly. And then the major reason for why it took so long to find it is we haven't yet a DNA demethylase. The methyl group is attached to the cytosine through a carbon bond, covalently carbon bond, which is very, very hard to cleave. >> So, you've then got a carbon-carbon bond, because the methyl group is a CH3, so you have carbon-carbon linkage which is incredibly stable. >> Exactly, exactly and we still have not found an enzyme that is capable of cleaving that bond and perhaps we never will because it doesn't exist. Instead we found enzymes that either degrade the methyl group from the carbon or degrade the base, which then feeds into the base excision repair pathway and which will flip out the methylated cytosine and replace it with an unmethylated cytosine. >> Right, so the TET enzymes, they end up performing an oxidation reaction. >> Exactly. >> So what are the intermediates in that reaction? >> So first of all, the TET enzymes will oxidize the methyl group to a hydroxymethyl group and from the hydroxymethyl to a formal methyl cytosine, and from that to a carboxymethyl-cytosine, and a carboxymethyl-cytosine base is able to feed into the base excision repair pathway then, and can be replaced with a completely unmethylated base. But potentially it's possible a de-carboxilation reactions are technically possible, feasible, but we haven't found an enzyme that will directly do that yet. >> Okay. So the other pathway that leads into base excision repair is, as you said, by degradation. So, you have a deamination for AID, the other enzyme that can perform some, or lead into base excision repair so that, in the end, you have a demethylation event. So it was found a little bit earlier. Do you think that was just by chance, or it was- >> [COUGH] Well, people had the theory that the base excision repair pathway would be involved for quite a long time. Whereas they went looking for oxidation reactions, so people weren't actively looking for genes that could modify the base so that it would feed into the base excision repair pathway and deaminases were prime candidates for it. So people were specifically looking for AID in the places where they assumed it would work. >> Well, that makes sense. So, I guess the hydroxymethylation, as you said there are been advances in our ability to look for, with using next generation technology. So we actually cannot only perform bisulphite sequencing which we discussed in other lectures. But also you can also look to find hydroxymethylation in somewhat more indirect measures I guess in a way, chemically. >> Sure. >> We weren't going to the details of that but I guess now what seems to be happening is that people are now able to perform this more widely. And they're looking for hydroxymethylation throughout the genome at different stages of development and using this as an indication of where active demethylation occurs. And it's quite surprising to me that it's not only during these stages we've already mentioned so preimplantation development or primordial germ cell development, but it also seems to be quite common in the brain and most recently, also, shown in various immune subsets, so B cells and T cell development. >> Sure. Sure, yes, so that's a really interesting question. It kind of raises the possibility that, perhaps, hydroxymethylcytosine is an epigenetic mark in its own right, with its own function and well, we already know that there are proteins that specifically bind hydroxymethylcytosine in preference to methylcytosine or unmethalated cytosine, so this really suggests that it may really operate in its own pathways. >> So, it's not perhaps well, that we're using it at this stage to say that it's active demethylation, and of course it is part of the active demethylation process by the TET enzymes maybe it's meaning something else too. So it's interesting that it's been found in the brain and this was found a couple of years before the finding of hydroxylation, or active demethylation in B cells and T cell subsets and that kind of fits, I guess. Why would you need active demethylation rather than passive? Well most of the brain, the neurons are postmitotic, so they're not dividing. So you can't dilute out DNA methylation I guess. >> Sure. Yep, that's right, and yeah, I guess you need a rapid sort of, ways to evolve, or differentiate in the brain. And it might be that we have a DNA demethylation assisting with that in the brain, in more somatic cells. >> Yep and the other thing when we think about active demethylation, is that it in some ways it goes against what we've thought especially, actually the fact that it occurs, it would appear, more commonly than we originally thought. Would be, it somewhat challenges the idea of having DNA methylation as a very stable epigenetic mark. I mean, it's the one that was first described to be how it was mitotically heritable through the action of DNMT1 and most commonly the canonical view of DNA methylation is that it's a very stable epigenetic mark, that can be maintained for the lifetime of an organism. And while I still think that's true, we know of many cases where DNA methylation is established and then maintained, for example, on the inactive X chromosome. Maybe, now, with active demethylation, it means that in some instances, DNA methylation shouldn't be thought of as being an extremely stable epigenetic mark. >> Yeah, well, I think that's an interesting question, too, and it's entirely possible. We see the TET proteins come on in us promiscuously in somatic cells. And if they're expressed in somatic cells, I mean that really challenges what we think of DNA methylation doing, because presumably if they're on, they're demethylating to somatic cells. >> So it would be really interesting to see in the future which regions they are demethylating at those times. So one can imagine that there are going to be regions of the genome that are prone to demethylation, or that are being actively demethylated, and regions that maybe are protected. So maybe there are regions that are protected from the demethylases, and they are retained in a stable fashion. Perhaps like CPG islands on the inactive X chromosome. And then there are regions where they are actively being demethylated and the reasons as to why this occurs, we could postulate on lots of them. I mean, it could be that you need to maintain demethylation of most CPG islands. We certainly find that most CPG islands aren't methylated, so maybe there's some process that keeps them unmethylated by removing any methyl marks that are added there. >> Sure, well there certainly are processes that control the DNA methylation state of particular regions of the genome. We already spoke about the one cell embryo having the male pronucleus behaving slightly different to the female pronucleus and we know that this is because of an oocyte specific factor called STELLA which comes from the maternal oocyte and selectively binds the DNA of the maternal pronucleus and then offers protection to the maternal genome from demethylation. >> From that active demethylation. >> From active DNA methylation, right? >> So we also know the DMR's or the imprint control regions that are found, they are protected from any demethylation, active or passive, during that pre-implantation period, so there are proteins, I believe that also protect those regions at that time. >> Yeah, that's right, too. So, STELLA is believed to do that actually. To bind two DMR's in the paternal genome, whereas it leaves the rest of the paternal genome unbound, and to protect from DNA demethylation. There's also a protein called zinc finger proteins 57 which will bind the methylated DMR of an imprinted region and then it recruits other factors, including the denovo DNA methyltransferases to make sure these imprinted regions stay methylated. >> Stay methylated. >> Through rounds of global DNA demethylation. >> So perhaps, just like we know about these ones for these really critical regions for the imprinted genes, maybe there are also proteins that bind to other regions in the genome that you really need to keep faithfully methylated and that's how we get the stability. >> Sure, definitely, and there are regions such as repeat regions that we know have to remain silent through DNA methylation and indeed they maintain their DNA methylation so there's still a lot to learn there. >> Yeah, I guess the interesting thing as well would be to think how does this happen? Why does it happen? And so there's going to be a really active field in years to come. It's already an active field and I imagine over the next few years lots of the mechanisms that we're discussing will hopefully be revealed. >> Sure that's what makes it still an exciting field I guess. >> [LAUGH] Exactly, that's why we both work in it. So I guess one of the interesting things that demethylation has also been studied in, other than in the brain and in primordial germ cell development and in early embryonic development, has also been in reprogramming. So this is where we've discussed in the other lectures in week four that we have, and week five I think, where we take a somatic cell and it gets reprogrammed back to an induced pluripotent state. And this is really important for therapy I guess. People are really interested in doing this so that you can create a pluripotent cell line from a particular patient and then create differentiated cells, for example blood cells or new epithelial cells of different kinds so that they can have them for transplantation. So to be self transplantation with their very own cells, rather than having the problems that you have with graft versus host disease, normally. But, reprogramming hasn't just existed or been known about since this discovery was made about ten years ago, in about 2006. >> So, that's right. The first reprogramming was first done in the late 1950s, by a young scientist called John Gurdon who had a fairly simple question by today's standards, but really wanted to know whether a somatic cell contained all the genetic information to produce a whole new organism. >> So, by that you mean that he thought that there was a potential differentiation as we know it might actually be a result of throwing genes out to feel that. >> Exactly, regressing, loss of- >> So you're not having him any more. >> Loss of genes, exactly. And so his question was if the somatic nucleus contained all the genes required, then it should form a new organism. So what he did was to take a somatic nucleus from a frog and put it into the oocyte of a frog, and see if it produced an embryo, and sure enough it did, and that really nicely answered his question I guess. That there really are all the genes required as we know now. But yeah and I guess suggested for one of the first times that it was, rather than a loss of the genes, it's a silencing of the genes. So it was one of the first times we noticed epigenetic silencing in action. But I guess what John Gurdon didn't know when he did these experiments was that there had to be complete reprogramming of that somatic nucleus to completely remodel the epigenome in the way that we understand reprogramming now. >> So then a long time later, 50 years later, then Yamanaka's group came along, and they used four transcription factors. In fact, they screened for all sorts of different factors that might, the combinations of different factors that might enable reprogramming of a somatic cell back to something that looked like an embryonic stem cell, which they called an induced pluripotent cell. And they happened upon four particular transcription factors, that are now called the Yamanaka factors. And they got this reprogramming to work, but it's incredibly inefficient. Maybe, less than 1% of cells will do this even if they all have these four transcription factors. So extremely inefficient. >> That's true. >> When you just add these four factors. >> That's true, but we know it can be a lot better or made a lot more efficient because the reprogramming by somatic cell nucleotransfer happens with almost 100% efficiency, compared to 1% efficiency. It also happens much faster, and the reprogramming that you get appears to remodel the epigenome more faithfully than by the [INAUDIBLE] method. So if you compare a somatic cell nuclear transfer reprogrammed cell, it very closely resembles an ES cell and in a more homogeneous way than what will a Yamanaka factor reprogrammed cells. >> Yeah. So that, normal biology does it better than we do with. [LAUGH]. >> Yeah. >> This does not recapitulate no more situations. >> Exactly. Which doesn't surprise me at all. >> Right. >> But it means that there are more factors to find and we can improve the, improve this process. And there's a lot to learn from whatever is contained in that oocyte. >> Absolutely. As an interesting note, because in the early 2000s, there were groups and probably still are groups that wanted to, for livestock purposes, they wanted to be able to reproduce their bull, if you like, that had just the right genetics or a particular deer that had just the right genetics for their livestock purposes. And so before the Yamanaka factors and actually still probably the best way to do it. They used to use an oocyte and of course in livestock, it's okay to get more oocytes, that's not really the case for humans, of course. There are ethical issues and problems of getting enough of them. >> That's right. >> But that's not an issue with livestock so they, or not such an issue, I guess. And what they would do to try and increase the efficiency because while it can be close to 100% efficient I think this is organism specific so I think some organisms are more efficient than others. And the frog being more efficient than most because they have maybe because they have a massive oocyte. But what they used to do was sometimes fuse together two oocytes and take out their nuclei and put in the nucleus and figure well twice as much is going to help, and actually it probably did so yeah, biology happens normally and if we could do this relatively well without knowing anything about the factors that are actually involved, but that doesn't help us for the human therapeutics angle because we still really want to identify what's going on, because we're never going to be able to use human oocytes in the same way. >> No, no, that's right. >> Nor should we of course. >> Exactly. >> So, what's interesting to me, was that these Yamanaka factors, the transcription factors, when it first came out in 2006, I remember having what we call a journal club and we would have one of the scientists discuss this particular new and exciting article and what struck me, was that they weren't the factors that I thought would be involved. They were transcription factors rather than epigenetic regulators and to me I thought well, but it's all about epigenetic reprogramming, why aren't there epigenetic factors in there? But it's interesting that now the factors that increase the efficiency of reprogramming seem to be a sweep of epigenetic factors. >> Yeah, that's right. So it's true that reprogramming is an initiated by the transcription factors, but what happens after that is largely epigenetic, epigenetic changes. So the reprogramming happens in a sort of three phases that we term the early phase, and the intermediate phase and the late phase and the early phase is initiated by the introduction of these exogenous transcription factors, which we call OSKM for short, but they're genes. Oct4, Sox2, KLF4 and C-Myc >> Mm-hm. >> And the early phase is this sort of stochastic phase where some cells will apoptose and some cells will syness and other cells will undergo mesenchyme to epithelial transition. >> Um-hm. >> And it's these which we call MET. >> [LAUGH]. Yeah. >> So these these cells that undergo MET are then the cells that are able to enter into the intermediate phase and they have a range of different transcriptional profiles, these epithelial cells and the intermediate phase is sort of characterised by adjustments to the transcriptome via the epigenome of these epithelial cells and this is really where it's an inefficient process at this stage because only some of these cells will pass through the intermediate stage and it's the cells that end up expressing what we call early pluripotency factors that will make it through the intermediate stage and these early pluripotency factors tend to be factors that were originally marked with H3K27 methylation so that's the first histone mark to be reprogrammed in the process. So the cells then that are expressing these early pluripotency factors then go into what is the late phase, which is a deterministic, and very highly ordered process where the later stage pluripotency factors become activated, so that includes Oct4, Sox2. So, once they're expressed, the cell is no longer dependent on the exogenous transcription factors and then the process is kind of unstoppable at this stage, and so this is when we get most of our epigenetic remodeling so, the genes that were marked by H3K9 trimethylation and dimethylation are then reprogrammed and activated we get the remodeling of DNA methylation at this stage, and the resetting of the demethylation for the pluripotent state. And one of the final things we get is the reactivation of the silent X chromosome, which is really indicative of the process having worked at a lot of levels. >> And I guess the inactive x has H3K9 methylation and DNA methylation as striking features. >> That's right. >> And so those are some of it is one of the large bodies that would remove those and so it's representative in many ways. >> Sure exactly. >> That's just it. >> Exactly. >> Yeah. >> So that you would expect this to be one of the last things to happen in reprogramming. >> One of the interesting things is that the induced pluripotent cells or the IPS cells that you take and compare between are a really quite variable, more variable than what the underlined genetic difference would explain. >> Mm-hm. >> And so this is telling us that well we can get back to that point, all those inefficiencies along the way, even when you do get back to the final point, perhaps we still don't do it entirely correctly and perhaps we can still improve the situation. Now only in the efficiency of getting there but also in how well they represent ground-state pluripotency maybe. >> Yeah, sure so it's not perfect and there's a lot of, the epigenome isn't remodeled in exactly the way that we would hope, and this is one of the reasons why adding extra epigenetic remodellers into the process often aids the process. >> Yeah, so that, yeah, we can increase the efficiency, perhaps of stage two and stage three, but certainly stage three. The ones that have been described so far seem to in Increase the efficiency of this final stage. >> Yes. >> And really getting it back maybe reprogram your last final steps. >> Yeah, sure. And we get this idea of epigenetic memory so, this comes from the fact that if you take the parent's cell, reprogram the parent's cell and that parent's cell is more able to differentiate back to- >> What it came from. >> The IPS cell produced from that parent cell, more able to differentiate back to what it came from. >> So, what you mean is if you started out with a B-lymphocyte, and you'd de-differentiate back to an IPS cell, you're more likely going to be able to make another B-lymphocyte than you are to make skin or muscle or other cell types. >> That's right, that's right, and it seems to be because of some kind of epigenetic memory. >> Yep. >> Often- >> Or inefficient clearing I guess is another way of saying. >> Inefficient clearing. Exactly. So you're left with remnants of the DNA methylation profile of the parent cell. >> Yep. Which could be helpful potentially if you actual want to take a B cell to create more B cells. But is not very helpful if you want to take maybe something that's easily accessible like a swab from the cheek of the patient and end up producing red blood cells. That's not going to be helpful to have a memory that you came from a cheek cell. What we want to do really, is remove any memory that there is. >> That's exactly right. Although for workarounds, it would be possible to take, to make sure you were using cell types that were related. >> Yeah. Potentially. Maybe sometimes genetically not possible. Because one of the ideas now is that, there have been some recent developments with CRISPR Cas9 genome editing, which you can read about, and we usually have discussions on this in the discussion forum. We might be able to, people that have a genetic mutation which means that they're unable to create a particular type of cell, you could create an induced pluripotent cell and then from any cell type, repair its DNA and then reproduce the cell type that that patient was otherwise not able to produce. >> Sure. >> Which would be wonderful in theory. One of the dangers of this at the moment is that if you have any remnant induced pluripotent cells around, these can really cause havoc if you transplant them into a patient. So and that's because a pluripotent cell can create all sorts of different cell types, for example, they can create hair, eye, tooth, liver, skin, all sorts of different cell types and so if you transplant these pluripotent cells what can happen is that you have what's called a teratoma, and that's a tumor that actually can create all sorts of different types. So normally in a person, these teratomas would occur maybe based on an oocyte for example, and so there can be a tumor that occurs in the ovary and when they section and they do indeed find hair growing inside the ovary [LAUGH] and some tooth and some other bits of tissue, so they're quite distressingly horrible tumor types, but we don't want to have that sort of thing occur in patients so this tells us that of course we want to get it right in terms of the differentiation again, so we're talking about dedifferentiation but other people work on trying to get this reprogramming or differentiation down to a particular path perfect and get rid of these pluripotent cells. But the other way to manage this is to never make them to an induced potent state in the first place. And so there's a new field, a relatively new field again, although it's existed for a very long time as well, where they performed transdifferentiation. So they might take for example, a cell from your cheek and try to directly make it into a red blood cell and so if you don't, if you avoid going back to this pluripotent state then you avoid that problem. >> Sure. >> And there's some progress happening in terms of being able to get this to work, it seems like you still go through some sort of a progenitor like state. But you're not forcing it back to that stage then differentiate again, you're instead trying to get it to morph straight into that other cell type. >> Yeah, it's getting that cell to do the minimum possible amount of reprogramming and then send it back out. >> Yep, so there is still going to be reprogramming and it's still going to be an epigenetic event. And so it will be interesting to see in the future how epigenetic modifications and epigenetic regulators contribute to this process just like they do for reprogramming back to the induced pluripotent state. >> It will be. >> Okay, thanks, Andrew and so hopefully you've enjoyed this chat that we've had on these two topics and you'll find also reviews that we've given you to read on this area and hopefully you'll find it an interesting area to discuss. Thanks. >> Thanks.
