BIOL21101 Lecture Notes - Oct 9, 2024 PDF

BIOL21101 - 09-Oct, automated transcript Oct 9, 2024 --- Thank you very much. Okay, good morning everyone, can you hear me okay at the back? Is the microphone on? I think it is. Okay, all right, so for today we're going to be looking at nucleosomes, which are basically proteins that bind to and pac...

BIOL21101 - 09-Oct, automated transcript Oct 9, 2024 --- Thank you very much. Okay, good morning everyone, can you hear me okay at the back? Is the microphone on? I think it is. Okay, all right, so for today we're going to be looking at nucleosomes, which are basically proteins that bind to and package DNA. This is the attendance code for today, let me move that out of the way, and the PIN code is 909643. Okay, so for today's lecture the learning objectives for the first part are on completing this module, you should be able to explain to your colleagues about the different histone subunits, how histones combine to form the nucleosome, how nucleosomes are packaged to form chromatin, and also about the role of chromatin in regulating gene expression. And there's just a little bit of guided reading here at the bottom. So to start with I've got just an example of a transcription factor, which is a basic helix helix heterodimer, brain muscle aren't like one or beamal one and clock, and these are two transcription factors that bind together and when they're bound together they're able to bind to DNA at enhancer regions, so these are obviously the specific transcription factors. And they bind to this sequence CACGTG in genes that are regulated by these proteins. And the reason I've chosen these factors is because they basically do everything. So clock has a transactivation domain which is the poly-Q glutamine rich sequence, and so when DNA bends over, the poly-Q sequence from clock can then interact with the general factors, transcription factor two, and the RNA polymerase two, and stimulate, it acts as that trigger, touches it, and then you get the RNA polymerase activated to chug along the DNA and make a messenger RNA transcript. And this at the bottom is just to point out it could be any other specific transcription factors or even a transcription factor complex, helix tone helix, transcription factors, zinc fingers, or basic Zipper transcription factors binding here to their canonical enhancer sequence, regulating the general factors to drive the expression of a gene. Problem is, how do they do that? Because the DNA is actually wound up in these proteins called nucleosomes. And so the whole point of today's lecture is to talk about what these nucleosomes are and how they can be relaxed to allow the polymerase two to move along and transcribe the DNA. This is just showing a schematic of DNA, and the DNA basically gets wrapped around these proteins that form this nucleosome. Okay, and you have two turns of DNA wrapped around each nucleosome, and then stabilizing this structure, you then have a H1 histone nucleosome attaching to the outside. These nucleosomes then get packaged up into this three nucleosome dense or wide structure. Each nucleosome is 10 nanometers, and so when you package them into this structure is 30 nanometer width. And the whole point of this packaging is so that you can get all of the DNA basically packaged into the nucleus. Okay, problem is when you want to transcribe genes you have to somehow overcome the fact that it's bound to these histone proteins. Okay, now this shows the basic structure of the nucleosome. Each nucleosome consists of two coils of DNA and eight histone proteins. You have two histone 2A, two histone 2B, two histone 3, and two histone 4 proteins. These will bind together, forming the nucleosome, and this is where the DNA then gets wrapped twice around, and then you have the histone 1 just stabilizing it. So the core subunit of the nucleosome is eight histones, two of each with two turns of DNA, and then this one linker histone. Now all of these histone proteins have a similar structure. They have a three-alpha helical structure, so you have amino acids that form an alpha helix, then there's a little linker, and then another alpha helix, and then a linker, and then a third alpha helix. And they all have this structure, and the important thing is that this structure, the alpha helix in the middle, is the histone fold domain, and this where histones combine to one another. So for example here we can see a histone H2A, and below histone H2A is in green, and histone H2B is in yellow, and they bind together to form this dimer or heterodimer. And on the right it's just showing the histones actually within the core with two strands of DNA wrapped around forming the nucleosome. You have the central H3H4 tetramer, so this is two H3 and two H4 histones shown in white, and then you have two H2A H2B dimers, one that attaches on this side, and one that attaches on this side forming the nucleosome. And this is just showing it in more detail. Here we have the core tetramer of H3 in green, and H4 in purple, and then we can see from this side angle you have the two separate H2A which are yellow, and H2AB which are blue. There's one forming here, and one forming here, or if you look down straight on it you can just see one of them because the other one's underneath. Okay, and this is the DNA wrapped around the outside of the proteins. And so this basically forms initially due to the H3H4 tetramer. This complex of proteins will bind to DNA through the phosphate backbone and wrap the DNA around them. Once you've actually got this initial forming of the nucleosome, the H2B dimers then come in and then finish off and bind within this structure. And what you'll notice is that the histone folds obviously are these structures here, allowing the histones to bind to one another, but on the tails of the histones you have these amino acids which stick out from the nucleosome. And these tails are crucial because they have certain amino acids on them which can be modified. Okay, and so they're essential for chromatin remodeling. And I've just got an animation, oh it's not going to do it with this. In this animation we'll see the remarkable way our DNA is tightly packed up to fit into the nucleus of every cell. The process starts with assembly of the nucleosome which is formed when eight separate histone protein subunits attach to the DNA molecule. The combined tight loop of DNA and protein is the nucleosome. Multiple nucleosomes are coiled together and these then stack on top of each other. The end result is a fiber of packed nucleosomes known as chromatin. This fiber, which at this point is condensed to a thickness of 30 nanometers, is then looped and further packaged using other proteins which are not shown here. This remarkable multiple folding allows six feet of DNA to fit into the nucleus of each cell in our body. An object so small that 10 000 nuclei could fit on the tip of a needle. The end result is that the DNA is tightly packed into the familiar structures we can see through a microscope. Chromosomes. It is important to realize that chromosomes are not always present. They form only when cells are dividing. At other times as we can see here at the end of cell division our DNA becomes less highly organized. Okay so that just shows you how the nucleosomes get packaged up ultimately to form the chromosome. All right so I've got a quick test. How many histone units form a nucleosome? Eight. Yep. So the actual nucleosome consists of two copies each of H2A, H2B, H3 and H4. Once they wrap the DNA around the H1 histone then comes in and just attaches to the outside. But the histone itself is formed from eight, the nucleosome is formed from eight histones. Which histones form the tetramer core of a nucleosome? H3 and H4, correct. They're the ones that come in first and they actually bind as a tetramer. So you have two H3 and two H4. They will then bind the DNA around them and then the H2A, H2B come in. How many loops of DNA are present in the nucleosome? Two, correct. Which histone links the nucleosomes together? H1. And what is the main tertiary structure of a histone protein? Three alpha helices forming the histone fold, correct. And then sticking out from those alpha helices you have the tails which are the things that get modified to determine how the DNA is going to be transcribed. Okay so now we're going to look at the modifications that actually happen to the tails of the histones because that will determine whether a gene is able to be transcribed or not. And that's just for those of you who turned up a bit late. That's the attendance code 909643. Okay so for this second part you should be able to explain to your colleagues about the different types of modification that occur on the histone tails, how lysine acetylation and lysine and arginine methylation affect transcription, whether it causes activation or repression, about the role of chromatin remodeling enzymes. So these are enzymes that can come in and actually shift the histones within the DNA to either open up binding sites to make them more accessible or even hide them. And how the facilitates activation of chromatin transcription. FACT shuttles histones to allow the RNA polymerase II to transcribe the DNA. So FACT is a protein that in front of the RNA polymerase actually takes the histones from the DNA, allows the RNA polymerase to transcribe the DNA and move past and then reassembles the histones after they've moved past. And I'm also going to talk about the role of locus control regions insulators and barriers and their role in gene expression. So insulators and barriers are basically regions that prevent adjacent DNA from interacting. And if I get time hopefully I'll talk about the higher order domains and the role of transcription factories. Okay, so histones can undergo various post-translational modifications. And this includes acetylation and lysine amino acids get acetylated. You can also have methylation of lysine and arginine amino acids. So this is just the single letter code for these different amino acids. You can also get phosphorylation of serine, threonine and tyrosine. I mentioned the RNA polymerase. The protein actually gets phosphorylated on serines as it's actually undergoing transcription. The histones can also be phosphorylated on serine, threonine or tyrosine residues. You can also get citrullination of arginines, ubiquitination of lysines and sumylation of lysines. So there's all these different modifications that can happen to amino acids. So there's all these different types of post-translational modification that can be added to amino acids in those histone tails. And it's still being determined which modifications, and often in combination, are important for various processes that are within cells, including mitosis, meiosis, DNA replication, DNA repair, apoptosis when the cell actually terminates itself, and also transcription. And today we're going to be focusing on the modifications that are involved in regulation of transcription, and in particular acetylation and methylation of the histone tails. So this is just showing a schematic of the four histones, H3, H4, H2A and H2B. And the major modifications that occur on these histone tails, which are these bits sticking out here, are acetylation of the lysine or K amino acids. And you can see that happens in histones 2A, 2B, 3 and 4. And this is just showing which amino acid in particular it is. So amino acid 1 on H4 is acerein, which can be phosphorylated. And then as we go on we can see here that lysine 5 or K5 can be acetylated. And you can also get, so acetylation here is shown in pink, methylation is shown as residues in red, and this is occurring on lysines or arginines, and mainly in H3 and H4. So the methylation is really occurring in that core tetramer of histones and their tails. So for example here we can see arginine at position 2 in histone 3, which would be H3R2, has got a methyl residue added to it, so it would be H3R2Me1. And here you can see that the lysine at position 9 can be methylated as well as acetylated. Okay, so if you're reading the literature and you want to know which particular amino acid is being referred to, it's always the histone that it's involved with first, so H4, then this would be K20Me1. Histone 4 at position lysine 20 or K20 has a single methyl residue added. Okay, so that's how to work your way around the literature, knowing which histone tails they're talking about. The complexity of the post-translational histone modifications, these modifications occur in situ whilst the histones are bound to the DNA, and they correlate with the functional status of the relevant genomic locus. The changes define an epigenetic histone code and corresponding functional landscape, and this will then determine whether that region of the gene is going to be transcribed or open and accessible for transcription or closed down and hidden so that you can't actually transcribe from the gene that's associated with these histones. So this code defines the binding potential of chromatin-associated factors, so it acts as markers for other proteins to come in bind to the tails of the histones, and those proteins are then going to determine whether you're going to activate the gene or actually inhibit transcription of the gene. Okay, so this is just showing histone 3 and histone 4, and for example in this region here we can see that histone 3 lysine 18 can be trimethylated, it means it has three methyl groups added to it. I'll show you how that happens in a minute. And you can also have an acetyl group added to this lysine. Okay, so it can be quite complex, this landscape within the histone tails that determines then what factors combine to the tails and regulate transcription. So one of the first modifications is lysine acetylation. This shows the lysine amino acid, and you can see here that we have the NH3 end, and what happens here is that the protons get removed and a acetyl group COCH3 gets added to the end of this amino acid. This process of adding on the acetyl groups is done by histone acetyltransferases or HATs, and you can also reverse this process and remove the acetyl group, bringing this back to the lysine end using histone deacetylases or HDACs. Okay, okay. And acetylation influences the structure of chromatin and transcription factor access, so this will change whether the general and specific transcription factors can actually get in and bind to the DNA in the region that's associated with these histones. And acetylation is associated with transcriptional activation. So in areas where a gene is going to transcribed, you often find that the histones are heavily acetylated. And one of the reasons for this is that the NH3 end is charged, and so it allows these tails to bind and cross-link with one another, condensing the DNA. When acetylation occurs, it causes less charge on the end of these amino acids, so you get less cross-binding of the histone protein tails, and it leads to a relaxed state of the chromatin. So basically, non-acetylated, the histones are keeping the DNA all wrapped up. When it's acetylated, they're more relaxed, and it allows other proteins to come in and bind. And this just shows a few examples of histone acetyltransferases and histone deacetylases. And the main one in human is HDAC1. Okay. Okay, so one of the other reasons I used BMAL and CLOCK as my initial example is that in addition to having, well, in addition to being able to bind the DNA in enhancer regions, bending the DNA and activating the general factors to allow transcription, CLOCK also has histone acetyltransferase activity. So these factors, just these two proteins alone, can basically do everything. So they come in, bind, activate the RNA polymerase too, so it can transcribe the gene, but it also acetylates the nucleosomes within the gene, opening up the DNA to then allow the RNA polymerase to chug along and get access to transcribe the gene. Okay, so CLOCK contains HAT activity, and it can acetylate the H3 and H4 histones. It opens up the core tetra and making the DNA accessible for transcription, therefore allowing the RNA polymerase to move along. So not only are these activating this to allow RNA polymerase to be released to actually transcribe, they're also opening up the DNA in front of the RNA polymerase, allowing access to transcribe the gene. Okay, now that's a relatively simple example because it's two factors that can do everything to transcribe a gene. In most situations, it's made up of lots of different sub-proteins, and so this is an example of a very common setup. We have the enhancer element here, which is cyclic AMP response element. This is a particular sequence in the DNA that is bound by this activator or transcription factor, CREB, cyclic AMP response element binding protein. CREB binds, and it then binds to protein 300 and CREB binding protein. Okay, once these are bound to this, they can then bind to the protein CAF. Okay, this is the cyclic or CREB binding protein associated factor, and so you have this complex of proteins that bind, and once they're bound, they can then come in, the DNA bends over, and they will then acetylate the histones within the gene, opening up and allowing it to be transcribed. Now interestingly, Tata binding protein is also bound to the Tata binding protein associated factor 2, 250, and it too can acetylate the DNA. So we have here the specific factors, and one of these factors that's associated can acetylate the DNA, but also within the general factors, we have one of these factors that can also acetylate histones bound to the DNA, opening up for transcription. Okay, and this is actually something that happens at a lot of genes. Obviously, if the general transcription factor here has acetyl histone, acetyltransferase activity, you can say that a lot of genes, if TAF2, 250 is present, it can actually open up the DNA for transcription. The second major type of modification is lysine methylation. Okay, so here again, we have the lysine, but instead of having it as NH3+, we've got it as NH and then HH. It's basically the same thing, it's three hydrogens, and this is just how they're actually attached to one another. Methylation of lysine can occur due to lysine methyltransferases, which add a methyl group to the lysine. Okay, so they can take the lysine, use a substrate to then remove one of the protons, one of the H plus residues, and add on this methyl group. However, this process can continue, and the second hydrogen proton here can be removed, and a second methyl group can be added. And we've also got this third hydrogen here in this tail of the lysine, where a third methyl group can be added. So we can basically generate monomethyl lysine, which is known as hypomethylated, dimethyl lysine, or which is known as hypermethylated. Okay, interestingly, when lysine is hypomethylated, this is often involved with a state for activation of the gene, so for transcription. But when lysines are hypermethylated, either at the two or the three stage, that actually sends to transcription. The reason is because of the different factors that are recognizing the monomethylated lysine, or the dientrimethylated lysine, that then come in to regulate transcription. Okay, so methylation can add one, two, or even three methyl groups to the NH3 plus residue of lysine, and it does this by replacing the proton with a CH3 each time. Lysine hypermethylation, this form and this form, is generally associated with transcriptional repression. It acts as a tag for other proteins to then come in and bind and actually block transcription. Okay, and this just shows a few examples of lysine methyl transferases. And just to point out that you can reverse this process with lysine demethylases. So you can take ME3, lysine demethylase can then convert it back to ME2, etc. Okay, and methylation can also occur on the arginine residues. So in this case arginine at this tail has H2N and NH2, so it's basically a nitrogen with two hydrogens, and using a catalytic reaction, one of the hydrogens can be removed and replaced with the methyl group. And this is performed by protein arginine methyl transferases, and they catalyze the transfer of the methyl group from S-adenosylmethionine, which is this here, the substrate, and then adds that Now what's interesting with arginine is it can be methylated again on the same, or the hydrogen on the same side that's already been methylated. And this generates asymmetric dimethyl arginine, or it can be methylated on the other NH2, forming symmetric dimethyl arginine. You can see how it gets quite complicated. Just with these little modifications can make a completely different landscape for these amino acids in the side tail of the histones. So protein arginine methyl transferase 1 methylates histone 4 at arginine 3, and that's generally involved in transcriptional activation. Okay, so unlike lysine methylation, which is usually involved in repression, arginine methylation is often involved in activation of the gene. Okay, and this is just to point out that protein arginine methyl transferase 4, also called CARM1, methylates histone 3, arginine 17, but it also methylates P300CBP, which is this protein here. Okay, so what this protein arginine methyl transferase does is it actually also activates this protein that itself is going to come in and help acetylate the histones within the DNA. Okay, so in addition to opening the histone proteins by methylating H3R17, this protein also activates this acetylase complex, okay, which will then come in and acetylate lysines opening up the histones allowing for transcription. And so if we actually look at promoter region under conditions of activation and inactivation, you can see that the histone modifications are very different. Okay, so within the actual promoter where the general factors bind, you find that histone 3 lysine 9 is acetylated. In contrast, when histone 3 lysine 9 is hypermethylated, this actually is the condition that the locus will be in when that gene is being repressed. Okay, so these different modifications of the same histone molecules, histone proteins, at that particular point in the DNA, when they're acetylated, it's going to allow for transcriptional activation. When they're hypermethylated, it's going to actually switch off the gene. And what you also notice in, so that's in the promoter region, in the coding region, which is the exons and introns, you find that in the active state, histone 3 lysine 9 is hypomethylated. Okay, so in the same gene, you've got different modifications in the region where the promoter is, which is acetylated, and the region actually within the coding region, which is hypomethylated. And so the RNA polymerase is going to bind here start transcribing and go through here until it gets to the end, which will be over here. The AATAAA sequence for termination of mRNA transcription. Okay, and you can actually use that information to do a screen of the genome to find at any one time which genes are active and which are repressed, because we know that the ones that are active, the histones associated with them, are going to be acetylated, whereas genes that are being repressed, in particular H3K9, is going to be hypermethylated. Okay, and so the modified histone tails alter chromatin function by driving the association with specific bound regulatory proteins. For example, histone 3 lysine 4 when it's hypermethylated, and also histone 3 lysine 9 when it's hypermethylated, bind to the PhD finger domain proteins such as ING2 and BPTF, and because I've got them shown in red, that tells us that they're repressing and preventing transcription. You'll notice I've coded things, red means stop, so it stops transcription, green means activate, so you can drive transcription. H3K9 when it's methylated, hypermethylated, and H3K27 when it's hypermethylated, bind to chromo domains such as HP1, and again that will inhibit the transcription of the gene. General acetylation of lysines, and in particular at H4 lysine 16, binds to broma domains such as PCAF, and that will drive acetylation, obviously opening up the chromatin, allowing for transcription. And general acetylation of lysines, and in particular at H4 lysine 5 and lysine 12, these bind to double broma domains such as HTAF1, and again that will help to drive transcription. So what these tails are doing is they're actually acting as markers within the tail of the histone to allow other proteins to associate with them to either then trigger transcription or inhibit transcription. And this is obviously an ongoing area of research, people trying to work out what combinations of these tags on the tails of histones are doing in terms of what proteins that get combined, and whether that then leads to activation or repression of transcription. Now in addition, there are chromatin remodelling complexes. Okay, so what are chromatin remodelling complexes? Well, if within the DNA you have an important, for example, enhancer element that's actually wrapped up in the histone, if you want to transcribe that gene, it's not very handy having it wrapped up in the histones, because it's basically being hidden. So what you'd prefer to do is actually make sure if you've got this gene that you want to be transcribed, you know, quite a lot, you want to try and get it so that that enhancer region is actually in one of these linkers between adjacent histones, so that it's more accessible for factors to bind. And there are various remodelling complexes that can come in and actually shift the histones along the DNA to open up or actually hide important DNA binding sequences. You have the ATP-dependent chromatin remodelling histone sliding molecules, SWI-SNF and SNRF, SWI-SNF and NRF, and you also have the histone acetylase-deacetylase-containing complexes such as SAGA and PCAF. Okay, and just to show you what the remodelling complexes do is here we have an important region of the DNA, it might be an enhancer, it might be a TATA box for example, and it's actually hidden in the histones in a nuclear zone. Now slowly and very slowly over time the histones can actually shift along the DNA making this accessible. What the remodelling complexes do is they come in and go and open up so that you now release this important DNA binding sequence into the linker part of DNA so it's more accessible. Okay, so it would then fix the DNA in this position so that this can be bound by various proteins and even fix it in an activated state so now this gene is there in a situation where it can be bound again and again and again to drive transcription. Or they could shift this important sequence into a histone actually to make it less accessible. Okay, so there are remodelling enzymes that can shift the position of the histones locally within the DNA to make certain sites more accessible or less accessible in a cell dependent manner depending on which genes are going to be expressed within that particular cell. So in a, let's say in a neuron we want to activate this gene so it shifts it into this configuration. In a muscle cell this gene isn't actually activated so you switch it into this configuration for the same gene, different cells. And you also have remodelling during transcriptional elongation. So obviously I told you when the RNA polymerase II moves along the DNA, the DNA is bound to the histones. So how does it actually get access to the DNA when it's bound to these proteins? So there's actually a protein complex of FACT, Facilitator of Active Chromatin Transcription. It's a heterodimeric factor and what it does is it removes the H2A, H2B dimers ahead of the polymerase so that the histones can disassemble, therefore allowing the polymerase access to transcribe the DNA. And once the polymerase has gone past that region, FACT then will lead to the re-association of the histones to repackage the DNA after the polymerase has moved along. So basically what FACT does is it's taking off the histones, allowing the polymerase to go past and then reassembling them behind it. Okay, so I now just want to talk about gene structure and transcriptional activation. We've looked at enhancer elements and we've looked at the core promoter, Tata binding sequence, RNA polymerase II. I've explained to you how the enhancer elements in cases can contain histone acetyltransferase activity to actually open up the nucleosomes, allowing the RNA polymerase to get access. They also have the activation domain that triggers the polymerase to actually start transcribing. Okay, so transcriptional activation domains contact other proteins which facilitate recruitment of a functional transcription pre-initiation complex. There are three common types of activation domain, which are on these enhancer elements. I really skipped over that at the end of the last lecture, but there's basically proline-rich, glutamine-rich or acidic-rich. I mentioned today that clock and BMAL have this glutamine-rich activation domain which basically touches the general factors and that is a trigger to then allow the RNA polymerase to start moving along the DNA and transcribing the gene. The activators work at a distance by bringing proteins associated with key regulatory elements into a single protein super complex at the promoter. So these activators or transcription factors bend the DNA and then they bring everything together in this complex, activate the general machinery to allow the transcription to occur. Locus control regions, which is what's shown here on the left, are similar to enhancers, but they work over much longer distances. So if we think of an enhancer being maybe 5 kb kilobases upstream of the promoter region, a locus control region could be 30 to 100 kilobases away from the gene that it's going to be driving the transcription of. So locus control regions work over much longer distances and what they also have is multiple binding sites allowing complexes of transcription factors to bind and the reason they're interesting is because rather than then activating just one gene, they can activate several genes. Now there's I think about a dozen LCRs been identified in humans so far. So there are certain genes in the genome which are involved in a particular metabolic process and they will actually get activated in tandem due to a locus control region. Okay, so there are some regions in the genome that have these locus control regions. Now also within the DNA you have insulators and these provide boundaries for domains within the chromatin. Okay, so what does that mean? All right, so let's say we've got this chunk of 10 kilobases of DNA. All right, we have an enhancer up here or even one in the intron and the transcriptional start site tata box is here. Okay, we then have this section of DNA next to it and there's an enhancer element here for its gene. What these insulators will do is prevent that enhancer from bending over this way to activate this gene. So any area which has an insulator on either side is actually unable to be influenced by DNA that's the other side of the insulator from it. So it's basically partitioning off a gene for transcription with its enhancers and its promoter. Okay, does that make sense? So it's to prevent unwanted enhancers from bending the DNA over and regulating the incorrect gene. Now there's another thing that's similar to that called barriers and barriers are like insulators but they actually are found at the boundaries between the euchromatin and the heterochromatin. So the euchromatin is the area where the genes are that you transcribed. The heterochromatin is the area near the centrosomes and also in telomeres which doesn't contain genes. It actually has slightly modified histones which bind it up tighter. But if possible the heterochromatin could spread into the euchromatin and that's bad because that might then prevent you from activating some really important genes. So what these barriers do is they act as regions where proteins combined and make sure that the heterochromatin doesn't actually spread along into the euchromatin of the DNA. Okay, so those are the various structural regions that you find on or within the genome that are regulating transcription. I've got four minutes to skip through. So the nucleosomes are the fundamental structural units of chromatin but that's very local regulation. How is higher order folding organized? So this is just showing the DNA being bound into nucleosomes then into the solenoid which is this 30 nanometer three nucleosome deep structure. These then get folded over and this is just showing the scaffold that they fold onto. These then get looped ultimately to form the chromosomes. Okay and if you actually look at regions of the genome within a cell you find that you have stretches of the genome that are gene rich and other stretches that are gene poor. So in regions where you have ridges this is showing the genes that are transcribed and you can see you have a mass of genes being transcribed in this region of the DNA. And then in anti-ridge regions you have very few genes being transcribed and what you can see is if you actually label these genes with a fluorescent colour they're all within a particular domain at the three-dimensional level within the cell. Whereas these two regions where you get very poor transcription anti-ridge one and two they form two distinct domains within the nucleus. Okay so basically you have regions of DNA that are gene rich end up within a particular domain within the nucleus of the cell. And you can see this if you actually look at sites of RNA synthesis it's not spread throughout the whole cell you get these regions that are very dense in RNA polymerase activity and then other regions where you have basically no transcription going on. And what actually is occurring is that you get these transcription factories where in a particular region of the cell you have multiple genes which are coming together and they're being transcribed by the RNA polymerase too. And this is obviously occurring in the euchromatin not in the heterochromatin. Okay and this just shows one example I've got one minute where you have three different genes which when you actually look to see where these genes are being transcribed within the cell you find that all of them so this is a fluorescent in situ hybridization it's a probe different colors for each of the genes that recognize the mRNA and you can see that they're all actually being transcribed in the same region in the cell. This dot white shows that all of these are co-localizing in what we know as a transcription factory. And interestingly these genes actually are coming from different chromosomes so this gene here comes from chromosome 11 and this gene here comes from chromosome 14 but they actually come into this same region within the nucleus where they get transcribed in concert with one another. Okay so there are some genes within the cell that they'll actually be brought together into a transcription factory and be transcribed together. And again these will be genes that are in the same pathway for some functional aspect of the cell and so they all get transcribed together. Okay and that's my time up.