The Wnt / Beta-Catenin Pathway Part 3

okay so welcome back to this next video in which we are discussing the winds be taken in pathway which is also known as the canonical wind pathway okay so now this is just the off state of the wind B ticket enum pathway which is basically where there’s no winds in the extracellular fluid there is therefore no activation of the frizz or lrp 5/6 receptors and therefore the beta-catenin distribution complex is functional and it binds beta-catenin molecules phosphorylates than a full separate sites and then releases those beta catenin molecules which then get bound to by the enzyme beta t RCP which then ubiquitin lanes the beta catenin protein which is then destroyed by the proteasome so it’s a short life for me to get in and when these going to be to getting in perf ways in the off state so now let’s discuss the wind beta catenin halfway in the on the state which is where there is wind in the extracellular fluid okay and it is going to be activating frizzled and the lrp 5/6 receptors okay so let’s now discuss this on stains then so once again that’s in fact actually let’s go back to our other picture wherever it’s gone okay this picture here remember here is our Frizzle receptor with our lrp receptor with the wint bound between them okay and let’s now talk about how this is going to inactivate the beta catenin destruction complex so coming back to the story there now the Frizzle receptor once it has the wind bound to it like this is going to binds now to a protein called dishevelled okay so I’ll draw this here so a protein is going to come and bind to the Frizzle Center once it has went down threat and this protein is within the family of proteins known as disheveled proteins so there is not just one disheveled protein there are many to shove all proteins okay but they all effectively do the same things to will just abbreviate more downs being represented by this green rectangle called disheveled okay now disheveled proteins are usually abbreviated to the SH proteins are short or you can also see disheveled proteins abbreviated to DVL proteins whichever one you want to use whether I’ll probably switch between them in fact I’ll probably stick to the SH I preferred the SH to DVL okay right so a disheveled protein comes and binds to the intracellular aspect of our frizzled receptor here once the frizzled receptor has went down to it okay now what does the dishevel protein now do well basically it is going to break down the pizza Catina destruction complexes okay so remember what the beta-catenin destruction complex is consisted of okay so in the off state of the wind’ beta-catenin pathway a beta catenin destruction complex consisted of an accent protein here okay with an a’ denim aters polyposis coli protein here okay then we have the glycogen synthase kinase free beta enzyme here and then we have our casein kinase one alpha here so let’s just label these up so here’s CK one alpha here is glycogen synthase kinase free beta so it’s color all of these so I have glycogen synthase kinase free beta in in green here okay we’ll have casein kinase 1 alpha in blue here or have axon in red here okay and then we’ll have a denim ATIS polyposis coli in orange here okay so this is our wint arts IRB ticketing and destruction complex in the off state now what’s going to happen is once you’ve got this disheveled protein here now it’s going to bind to certain portions of this ticketing in destruction complex and separate them off from being able to participate in a beta catenin destruction complex like this it’s going to break the beta catenin destruction complex as a part basically so dishevelled binds to XM

okay now the axon which is going to find a dishevelled can bring with it casein kinase 1 alpha which I’ll draw here and also glycogen synthase kinase free beta but it’s not going to bring along with it its animators polyposis coli okay so that’s color these wants in again in okay so here’s our glycogen synthase kinase free beater in green here here’s our casein kinase 1 alpha in blue here and then we’ve got our accent protein here in red okay and the axon has bounced to the dishevelled and then the axon has bound to it the casein kinase 1 alpha and the glycogen synthase kinase fee beta but no longer does it have a denim ators potica cisco live band to it okay now what then is going to happen is this is going to bring our glycogen synthase kinase free beta very close to the NRP 5/6 receptor here okay and then the glycogen synthase kinase freebie tree is actually going to phosphorylate residues within the NRP 5/6 so LRV 5/6 the cytoplasmic tail of this is going to become phosphorylated now before I discuss what the purpose of that is let’s just take a step back and talk about the fact that we have now isolated three components of the beta-catenin destruction complex ok that means that the number of beta catenin destruction complexes are now going to start going down because we’re going to have these receptors becoming activated all over the cell ok and they’re all going to be sequestering components of the beta catenin destruction complex and therefore the number of functional beads Catina in destruction complexes is going to go down now that’s just from these binding to the dishevelled the phosphorylation of the lrp 5/6 is going to act as a positive feedback basically because now once you have phosphorylated the cytoplasmic tail of lrp 5/6 what can happen is this phosphorylated tale of lrp 5/6 can actually also bind to actin molecules which have glycogen synthase kinase repeats attached to them but this time not casein kinase 1 alpha ok so what’s going to happen now is um and I’ve covered it in the exact wrong but never mind this should be in green I do apologize for that there we go overridden ok so here is our accent bound to our glycogen synthase kinase free pizza okay so this is axon here this is GST free beater ok so these two components can now bind to the phosphorylated cytoplasmic tail of the lrp 5/6 so basically this is further sequest stream components of the beta catenin destruction complex so overall what is happening when you activate the frizzled receptor in the NRP 5/6 receptors and dime rise them together like this is you are assembling these protein complexes which just sequester the loads of the components of the beta-catenin destruction complexes so that the number of beads are getting in destruction complexes in the cytoplasm is just going to go down and down and down now then what is the result of reducing number of beta catenin destruction complexes within the cell well you’re no longer going to be phosphorylated in beta catenin as were out today okay so B tickets helium molecules are not going to be phosphorylated which means that they’re not going to end up being bound to by the beta ercp protein okay which means that they’re not then going to be ubiquitinated and they’re not going to be destroyed so the effect of removing them the while reducing the number of beta catenin destruction complexes is that the amount of beta catenin within the cytoplasm goes up okay so be 16 in levels within the cytoplasm is now going to go up basically okay right so we now want to study what is the effect of Butyrka t new proteins going up well basically when the beta catenin builds up within a cytoplasm what’s going to happen is the beta catenin proteins are going to start going into the nucleus okay so all of what we’ve been discussing so far has been occurring in

the cytoplasm okay but now once the beta catenin builds up here it’s going to start going into the nucleus okay and there it’s going to act as a transcriptional activator okay right now before we actually discuss what the bitter gets human is going to do we the new crest it’s best to go back and discuss the off state again so remember before we discussed the honor state within the cytoplasm it was best first to discuss the off state again is now best to go back to the off state and discuss what was happening in the nucleus when the wind beta catenin pathway was in the off state before we then look at what happens in the nucleus when the wind beta catenin pathway is in the on state okay so let’s firstly start with what is happening in the off state okay so in the off state you have very little bita Catina okay so we now need to discuss something known as the TCF LEF family of transcription factors okay so we need to discuss TCF LEF family of transcription factors okay so before we actually discuss this specific family of transcription factors i firstly want to discuss broadly what a transcription factor actually is firstly and then we’ll look at this specific family which is the T cell factors and then lymphoid enhancer binding factors okay so firstly then what is a transcription factor so let me draw these two parallel lines here and now these two parallel lines this time do not represent the inner leaflet only after they fit at the phospholipid bilayer instead this now represents a double stranded piece of DNA a DNA into plaques okay so the two complimentary strands of DNA now and this portion that I’m boxing here this is going to be achieved within the double stranded DNA so outline this mmm here okay so this is a gene now one of the strands of the gene will be what stands the coding strands so let’s say that this strands are highlighting here in purple this is the coding strand okay and this is the Strand that the RNA polymerase 2 enzyme is going to work its way along and synthesize a piece of mRNA this is complementary to it okay so I’ll show this here so this strand here that’s now highlighted in blue this is a piece of mRNA this is complementary to that coding strand which can then go off and be translated into a sequence of amino acids okay into a polypeptide now upstream of all genes in the eukaryotic genome you have a region known as the promoter region okay so I’ll call this portion here the promoter region now the promoter region is not involved directly in actually being translated into a sequence of amino acids so it’s not actually going to be a part that codes for a portion of protein however it’s important in controlling the expression level of the downstream gene is important for controlling how much the protein associated with this gene you’re actually going to produce okay now how does it exert that control epigenetic control basically well it exerts that control by controlling how often you’re going to actually transcribe the genes are the fancy word for transferring the sequence of organic bases of a coding strand into a sequence of organic bases within mRNA is that we are transcribing the genetic code okay and this process is known as transcription okay so the promoter region is going to control the rate at which we transcribe the strands so how much transcription is actually going to occur now how does it clear all this well basically it is the promoter region where the RNA polymerase 2 enzyme is going to bind to the DNA okay now for short RNA polymerase 2 is often abbreviated to our nap – like so are they p4 polymerase and then – so RNA polymerase 2 bytes the promoter region and then will work its way along the promoter region to produce this piece of

mRNA that mRNA will then go and only translated into protein so if the promoter region a is a very good at getting RNA polymerases to binds to it and work its way than the long the coding of strands of the DNA and synthesize a piece of em are they then you’ll get loads of em are they being produced you’ll get therefore loads of protein being produced for this gene whereas if this promoter region is not very good at getting the RNA polymerase to bind here then you’ll get RNA primase to working along its way along the coding strand very rarely and you’ll get very little m are they being produced and therefore fairly little protein being produced okay so that is how the promoter region controls the expression that the downstream gene okay by controlling how likely the RNA polymerase 2 is actually to binds to the promoter region and work its way along the coding strands at the downstream gene now then the concept of a transcription factor then ok a transcription factor is a molecule which is capable binding to the DNA within the promoter region and it will bind to a specific sequence of organic bases within the promoter region okay and what the transcription factor will do is it will change how good the promoter region is binding to the RNA polymerase to okay so transcription factors generally binds to a huge number of different promoter regions okay generally they have sequences of around six to ten organic bases which they recognize and bind to and the promoter regions of many different genes will have these sequences of six to ten organic bases in so the same transcription factor will bind to the promoter region of a plethora of different genes okay and when it binds to those promoter regions as I say changes the likelihood that that promoter region is going to bind to RNA polymerase 2 and therefore the likelihood that the downstream gene is actually going to be transcribed now there are loads of different mechanisms by which transcription factors binding to the promoter regions can alter the likelihood that the gene is to be transcribed and we’ll discuss examples of mechanisms by which they achieve this later on okay but that’s the general principle but they change the likelihood that the downstream gene is going to be transcribed now they don’t always necessarily increase the probability that it’s going to be transcribed sometimes transcription factors will decrease the probability that the downstream gene is actually going to be transcribed so you have enhancer transcription factors which are going to enhance the expression of the downstream gene by increasing the probability but the downstream gene is going to be transcribed and you always still have repressor transcription factors which are going to decrease the probability that the downstream gene is actually going to be transcribed okay right so now let’s turn our attention specifically to the TCF le s family of transcription factors okay so firstly what does TCF and le s stand for well GC f stands of t-cell actors okay so the tears fatigue or the sea is cell and then the F is a factor okay and lef so that’s what TC f stands for okay and the F stands for lymphoid and hunts a binding factor okay I’ll just bring this up a little bit okay so the Alma is therefore for lymphoid the e is for enhancer okay and then the binding is connected to enhancer by – so therefore doesn’t get a letter and then the F is form factor okay right so the T C F and the F family of transcription factors often is just referred now to the TCF family of transcription factors so if you just see this referred to as the TCF family without beyond EFM it’s referring to the same family of transcription factors so within this family there are four separate genes for transcription factors okay so I put these over here so first either is the t-cell factor 1 which is the first member of this family the second member is then the LEF for lymphoid enhancer binding factor 1 okay and that’s effectively functions as t CF 2 then there is t CF 3 okay so t cell factor

free and then finally there is t cell factor for t CF 4 so those then are the four members of this t CF an EF family of transcription factors okay and as I say it’s often now just referred to as the TCF family of transcription factors and you just then acknowledge that one of the members of this family is not called t CF but lef okay so basically these t CF transcription factors they are going to bind to a specific sequence of organic bases within DNA so many promoter regions have the specific sequence of organic bases which these TCF family transcription factors bind to and recognize okay so let me now give you this sequence of organic bases which you need to have in order for a TCF family transcription factor to bind to you okay so it’s an an eight membered sequence organic basis so it’s cytosine cytosine then we’ve got three Phi means it’s by mean by mean Phi mean then followed by guanine and then followed by something which I’m going to put W here okay so what does W stand for well thankfully it’s not some organic base that you’ve never heard of before W just means either a T or an A so it’s fine mean or adenine so if we put the complementary sequence here it’s going to be G G and the confidential kind of based if I mean is adenine the complementary organic base to guanine is cytosine and of course the complements your comic base to a fireman or another name is going to be another thymine over an adenine so it’s going to be another W and then another W here okay but obviously these two are going to be the opposite so if this one’s off I mean this one will be an adenine if this is an adenine this one will be a thymine now came right so this is the sequence of organic bases which you need to have in order for a TCF transcription factor to bind to you so now a TCF family transcription factor which remember is the other name for TCF area family transcription factor is going to point to this sequence of eight organic bases here okay so I’ll color this thing in purple here okay in this sequence of eight organic bases like so this has a special name and it’s only many different promoter regions for many different genes okay it’s known as the wind response element or also the wind responsive element whichever you prefer I’ll put winter response element okay and for short the winter response element is often abbreviated to the WR e so you find this amenity from promoter regions so promoter regions are much much longer than just a tour panic basis but you know portion of it could be this sequence of eight organic bases and we would therefore say that the promoter region contains a winch response element and therefore is capable of binding a TCF any f-family transcription factor so either t CF 1l e f1 t CF 3 or t CF 4 okay right now we are currently in the off state so we have no beta catenin present at the moment in the on state later when we do have beta catenin present be tickity mean is going to bind on top of this T’s the fnaf transcription factor however we’re currently in the off state and even in the off state when we have nobita Catina the TCF an EF transcription factor will still be bound to the winch response elements in all of the promoter regions that contain which response elements okay now this is the thing now on top of this instead of Abita Catina molecule binding because we have no beta catenin molecules but present at the moment you are going to get a molecule within the family of TL e protein is binding there okay so let’s talk about this family of protein so has another name as well it’s also known as the groucho family of proteins as known as the Groucho tle family of proteins so one of these proteins within this family of proteins is going to bind on top of this TCF lef

transcription factor now first B what does TL e stand for well this stands for transducing Mike and hunt serve split proteins okay so the t is for transducing okay the L is then for life the ears for enhancer and then it’s the transducer mic enhancer of split proteins okay so that’s what T le e stands for okay so this is not just one protein this is a whole family of proteins would you pretty much do the same thing okay so one of these Groucho DNA family proteins is going to bind on top of our t CF an es transcription factor even alum when there is no beta catenin present when there’s bits of continuum present eticket in Ian’s games buying there instead now then on top of the TL e what you’re then going to get binding is an enzyme there’s a histone deacetylase okay so I’ll put this here so here is this enzyme binding to the groucho / t le e family protein and I’ll color in this enzyme in turquoise here okay so this enzyme is what’s known as a histone deacetylation for short histone deacetylases are abbreviated to H Dax okay so the anxious for his stone and then the dank is for the Isetta days okay so D is for D and the AC is for a settle A’s okay so this is an enzyme which is capable of removing acetal groups or a famous specifically for a mu removing as tile groups from histones okay so in order to take this discussion further we now need to make sure that everyone is on the same grounds with regards to understanding his okay so let’s just have a little discussion of histones then okay so there are five families of histone proteins okay so the five families of histone proteins are the h1 histone proteins then there is the h2 a histone proteins the h2 b is stone proteins the h3 histone proteins and then also the h4 histone proteins and I want to stress that each of these family of proteins in the human you will have a loads of h1 histones which will all be slightly different there will have slightly different sequences of amino acids but they all perform the same function okay so each of these as a family it contains multiple different proteins that all perform the same function okay so we’re not going to discuss the different individual proteins that are within each of these families we’re just going to say you’ll have a histone to a family member histone here okay we’re going to say things like that basically okay so what do the histone proteins do well basically they assemble into structures known as histone core complexes okay we shall optimist of histone proteins which DNA molecules are going to wrap around so firstly let’s discuss the histone core complex on its own and then we’ll discuss the DNA wrapping around the histone core complexes okay so I’m going to draw my akhter of histone proteins here then again I drew it as a cube so this is the octamer of histone so I’ll separate it up into eight parts then okay like so so here are the eight different parts we’ve got 1 2 3 4 and then we’ll have the same underneath 1 2 3 and we can’t see the 4th one because it’ll be right at the back there ok so this is our octamer of histones okay right now in this Optima you’re going to have two histone to a family histones two histone to be family histones two histone free family histones and two histone four family histones okay so let me show you the arrangement here so we’ll start with the histone four and the histone free histones okay so this one right at the front here which are now coloring in blue here this is going to be a histone or protein okay so it’s one of the histone four family histones I don’t know specifically which one it is because remember that’s an entire family of proteins but it’s one of them

okay it doesn’t matter which one it is they all perform the same function okay here you also have another histone for protein okay so two histone fours are there in there then you’re going to have a histone free protein down here in red okay and you have another histone free protein back here again in red so both of those are histone free proteins this is the histone free protein and this is also a histone free protein now that combination of four histone proteins that we’ve got there – histone fries and two histone fours that actually exists in its own right outside of the Optima so when in fact you’re assembling one of these opportunist you don’t just bring eight proteins together and hope that they somehow assemble into an Optima instead what you do is interesting stages obviously okay so one of the things that you’ll bring along when you’re building one of these Optimates is it’ll bring something you’ve made earlier effectively which is one of these tetras of histone freeze and histone force okay so it contains these two histone four histones and these two histone free histones okay so if you imagine these four that I’ve now colored in separated from those ones I haven’t color then that is the Tetra for histone proteins so here are the histone fries in red and then here are the histone fours in blue okay right so this is what’s known as an h3 h4 tetra mer over case it’s made up of histone freeze and histone force okay and this is one of the components that you’re going to use to make the Optima okay right so let’s now talk about the other four histones okay so this one right at the front here this is going to be histone two a family history so I’ll cover that in in turquoise here okay like so so that’s stone to a and then you’ll have another histones way but we won’t be able to see it it’s the one that we can’t see right on the back there okay we can’t see a single bit of it okay then the other two that are visible this one here which I’m now coloring in Mike green and this one down here which is also in light green these are histone Julie’s okay so that’s a histone to be right at the back there and this is a histone to be in the front here okay right now again coming to this discussion of how we construct a histone call complex Optima here when we’re constructing a histone core complex we bring in a histone free histone for tetra here and we also bring in two diamonds of histone to a with histone to be okay so here is his stone to a in turquoise here and there is his don’t you be in green here okay and this dimer of histone to a with his don’t you his name as a histone – a histone to be dimer so if you want to construct a full Optima like so what you do is you bring in a histone free histone for tetramer you then bring in two of these histone two A’s don’t you be dinars and then they can assemble into the for histone core complex which is an Optima here okay right now what you’re going to do is you’re going to wrap DNA around this histone core complex okay so let me show the DNA molecule here so this in purple is going to represent the do planks of the DNA strands so it’s a double stranded piece of DNA it’s going to come round in front of the histone core complex here it’s then gonna wrap around the back like so coming back round to the front here it’s then going to wrap around again and it’s going to come out here okay right so it wraps around the histone core complex twice and then it comes back out now we’ve discussed all the things stone families except histone warm family histones okay the histone warm family histones are not involved in the formation of the histone core complexes however histone one family proteins bind to the two pieces of DNA that are coming in and then coming out of the histone octamer effectively okay so here this portion in yellow which probably won’t show up in this light but that portion there which holds the two incoming and outcoming strands of DNA together that’s where the

histone warm family histone is going to bind okay so this is the function of histones the DNA within the nucleus of a eukaryotic cell is not just free okay it wraps around these histone core complexes okay to create a structure known as chromatin ok so just two more pieces of terminology I want you to be familiar with chromatin and also the concept of nuclear zone okay so let me draw your picture chromatin them so that’s a this is a histone core complex here and this line here is the DNA coming in and what’s going to happen is the DNA is going to wrap around the histone core complex like so then it’s going to wrap around again so it’s right drawn out twice then it’s going to come out of this histone core complex and that’s going to go on to another histone core complex down here okay once again it’s going to wrap around the histone core complex there’s once then it’s going to wrap around again twice and it’ll go along to the next histone core complex this is what chromatin is this strands that you’ve got which consists not just at the DNA now but the protein as well okay the histone core complexes it’s often compared to beads on a string basically and the string is the DNA which links that the two histone core complexes with DNA wrapped around it that are neighbouring to one another together basically okay and this is known as the linker DNA that’s between the two histone core complexes so that’s what chromatin is it’s not just DNA its DNA wrapped around histone core complexes and as I say the good way of remembering is just beads on a string now the final word that I want you to know before we discuss what a histone deacetylase is going to do is the concept of a nuclear zone okay so a nuclear zone is the repeating unit of chromatin so chromatin basically consists of this repetition where you’ve just got DNA wrapped around the histone call complex followed by the linker piece of DNA and onto another histone core complex now it’s a common misconception nucleosomes just refers to the histone core contacts with DNA wrapped around it’s close but not quite correct because if we just had his stone core complexes with DNA wrapped around that isn’t the rotating structure of chromatin we need the linker DNA as well so a nuclear zone refers to a histone core complex with DNA wrapped around it along with the linker strand which takes you to the next histone core complex which has got DNA wrapped around it this is the repeating unit of chromatin and we join loads and loads of nucleosomes together to make a piece of chromatin basically so that’s just some terminology now okay so this is the fundamental idea with regards to histone deacetylases do you think it’s going to be particularly easy to transcribe DNA which is wrapped around a histone core complex like so do you think RNA polymerase is going to have an easy time getting in when you’re wrapped tightly around this histone core complex or the answer is no okay so in order to actually get genes which are in this portion of DNA wrapped around this histone core complex which is very likely okay because most of the DNA is wrapped around histone core complexes very rich with the DNA small fraction is actually present in the links between the portions that are wrapped around the histone core complexes so most of the genes are going to be in sections that are wrapped around histone core complexes so if you want to actually transcribe those genes you need to loosen up how tightly they are wrapped around the histone core complexes and a way that you can loosen up how tightly the DNA is wrapped around the histone core complex is by a set elating histone proteins within the histone core complex specifically on lysing residues okay now histone acetyltransferases acetylenes my scenes on the histone proteins and this loosens the DNA around the histones whereas histone

deacetylases remove those asset R groups okay and therefore they are going to tighten the DNA back up and reduce transcription of those genes so when beta catenin is not present these TCF nef transcription factors are actually going to be recruiting these histone deacetylases which are going to be reducing the transcription of the downstream gene by tightening up how tightly the DNA is wrapped around the histones and we’ll discuss that in more detail in the next video