Hello everyone. Welcome to our talk about how we make modified histones. This is going to be an important aspect of our module on the ruler over time and space specifically the first series of lessons, because we're going to study the interaction with a specific chromatin binding protein, PRC2 with DNA or indeed chromatin. As you've learned, histone modifications are particularly important in terms of how transcription is regulated. We've learned that acetylation of histones is generally an up regulation of transcriptional processes. However, methylation is a lot more nuanced. Indeed, methylation can signal up or down regulation depending on where the specific methylation event has happened and how many methylation events that has potentially been. We need ways of creating specifically chemically modified histones with a specific number of methylations at a specific lysine. This is a very pinpoint chemical procedure, which actually in a test tube is very complicated. Biology can do this because it uses enzymes, but in general, those enzymes may not be as effective or indeed work particularly well in a test tube. So how indeed are we're going to make these methylated histones. What we need to consider that there are indeed multiple different types of histones, as I've talked about. You can have monomethylation, dimethylation and indeed trimethylation. That methylation is possible at any specific lysine in the specific histone. There's many different possibilities that we need to think about. If we wanted to try to recreate that methylation directly chemically of a specific lysine on a specific histone at a specific position, it would really be very difficult to do chemically. How are we going to make a specific monomethylation of a specific lysine on a histone? Actually what we're going to do is we're going to cheat. We're going to create a mutant where a specific lysine on that histone is changed to a cysteine. Now this is a nucleophilic residue as you all know, and so we can label it with a specific alkylating agent. Because certain histones like histone H4 do not contain any cysteines, mutation of one lysine to a specific cysteine engenders a chemical reaction with an electrophile is shown here. This particular amino chloride reagent can form this as aziridinium ion, which is then trapped out by that specific cysteine we've installed to make a modification that looks an awful lot like a specific histone methylation event at the specific lysine because we had encoded that specific cysteine. Indeed we can perform this experiment using a dimethylating analog and also a trimethylating analog. The dimethylated analog proceeds to a similar, as aziridinium intermediate, whereas the trimethylated analog cannot form that aziridinium intermediate and proceeds via direct displacement of the chloride by the nucleophilic cysteine. These alkylated species are quite similar to the methylated lysines that we want to make. The only difference is that the methylene that I've shown you on the bottom series of structures has been replaced by the S atom that came from cysteine. Obviously this is slightly larger, the sulfur atom is slightly bigger than the methylene group. However, in general, this is a reasonably good representation. We can look here at the structure of histone H4. I'm showing the specifically mutated in cysteine at the particular residue where it was engendered, and we can show the alkylation event and then we get the structure. This is mimicking the trimethylated version of histone H4. We can overlap this with the non-methylated version and we see that realistically there's not a huge amount of change between the two. There is a slight change in the way the side groups are orientated, but it's difficult to see precisely how this is going to transpire to overall changing how the histone packs and interacts with DNA. Of course, Histones are present in quite large structures, the nucleosomes, so it is possible that this could regulate interaction in some way. We can see similar behaviors looking at different histones, we see here. There's also a slight change in the orientation of side groups and we can also see a dimethylation event of histone H3. But if we were to look at a protein that had more than one cysteine. This method is not particularly effective because we can have alkylation at multiple different residues, and again we run into this chemoselectivity regioselectivity issue that we talked about in terms of direct methylation of lysine. Let's talk a little bit about a different method that can actually get us to the answer and use a more chemo diverse way of introducing functionalization. We use a strategy that's actually used in nature. This is Intein chemistry or splicing peptides. What we're going to do is we're going to take Extein 1 and Extein 2 and splice out that green Intein portion and link the two together. That sounds like a complicated process. Let's see how it works. The first thing to notice is that there is a cysteine residue which is directly appended close to Extein 1. This can undergo thioester to a amide into conversion as I'm showing you here. Having made this thioester shown by the red bond, you can actually do a trans thioester esterification across the Intein. That makes this unusual bridged species shown by this red bond that we've made, which is a thioester linking Extein 1 and Extein 2 and Intein is sticking off the other end. Now there is a nucleophilic amino residue, although this is part of a primary amide and that can trap out and essentially spit out the Intein, liberating the primary amino acid of the original cysteine residue. Because we now have a thioester that can undergo thioester to amide into conversion to give us a canonical peptides structure. Hence, we've spat out the Intein, which forms a cyclic structure which can then hydrolyze, and it gives us the products we're really interested in. We can see evidence of this from crystal structures. There's actually several crystal structures of these in the literature. What I'm showing is this Extein 1, Extein 2 branched intermediate linked through an ester bond. The ester bond is more stable and hence it is easier to crystallize and the portions of the two Extein as shown in red in the crystal structure on the right-hand side. How do we harness this to perform modification chemistry? If we don't have the Extein 2, then essentially all that will happen here is the Intein will undergo this amide to thioester conversion, as I'm showing here. Now, the thioester, is a relatively small product at equilibrium and this will go back to the amide, which is not particularly exciting. We've essentially cutoff the chemistry by removing the second portion of the Extein. However, if we add a nucleophile, particularly a thiol nucleophile, we can displace the thiol group from the thioester and do a thioester interconversion. But now it's not an intramolecular reaction like it was for the Extein, Intein, Extein. This is an intermolecular reaction which we drive by adding a large amount of the thiol. The Intein is liberated. Typically this has a tag like a chromatin by a chiton binding domain and we can just precipitate that using chitin beads. Now what we can do is we can add a nucleophile and trap out our purified thioester intermediate. We do that by adding an armed nucleophilic C-terminal protein fragment, which is shown in blue. This is able to undergo nucleophilic attack by the cysteine atom that can displace the thiol group from the thioester, forming a new thioester. This also has an amino function and it can undergo thioester to amide exchange to give the product. This is very interesting because that blue protein can be almost anything and it doesn't have to come from biological system, it can actually come from external system such as synthesis. We can synthesize peptides of 50 to maybe a 100 amino acids relatively simply, and we can functionalize them with dyes, we can functionalize them with specific methyl groups, we can functionalize them with acetyl groups. This is actually a very diverse way of making Extein group, which is synthesized with a specific C-terminal tail. We'll see how these can be used in general and it's very useful chemistry for making diverse, specifically modified proteins semi synthetically. Let's move on and see how we can use TIRF microscopy to study interactions between DNA, chromatin and PRC2. Thank you.