so we've already talked about how gene regulation is accomplished in prokaryotic cells at the level of transcription by repressing or activating expression of for example the loc lac operon using transcription factors like cap or the repressor protein to either activate or repress expression of the operon under different environmental conditions so that's a really typical mode of gene regulation that we see happening in prokaryotic cells what we want to do now is turn our attention to eukaryotic cells which as we've talked about many times before are much more complex in terms of their genetic composition they're more complex in terms of their cellular structure structure and likewise what we're gonna see in terms of gene regulation and the regulation of expression of genes in eukaryotes is that that's a lot more complex as well and there are many mechanisms for regulating gene expression that are available in eukaryotes that are not in prokaryotes and we've talked a little bit about this before for example when we talked about transcription and translation and discussed alternative splicing that's definitely one way that we can regulate the product or expression of a gene at the level of the mRNA and that happens in eukaryotes and not prokaryotes alright so what we're talking about here just to refresh our memory and and get on the same page here as we're talking about genes in the genome genetic instructions to the cell for how to synthesize either a protein product that's going to be encoded by an mRNA intermediate as we've talked about before or a functional RNA molecule we know that all of those types of gene products are encoded by genes specific sequences in the genome so when we talk about regulation of gene expression what we're really talking about is regulation of the production of that product now usually genes do encode protein so we're going to focus on that but you know when we say regulated gene expression what we're really saying is is the protein there or not is it functional or not and in eukaryotes that regulation can happen at multiple steps along this progression that we see occurring between transcription of the gene into mRNA and translation of the protein at the ribosome and even beyond that regulation of you know how long the protein sticks around and what its structure is so we can sort of describe multiple steps at which eukaryotic genes are regulated all the way from transcription of the gene down to degradation of the protein to get rid of it from the cell now if we think about regulated gene expression from a larger perspective from the perspective of the cell and ask ourselves for example in a human cell we have 20,000 genes would we expect all of those genes to be regulated in their expression or would we expect that there would be some genes that are not regulated in other words constitutively expressed so can you think of any any proteins or gene products that we've discussed in the course so far that might be important enough for the life of any cell that it would have to be expressed all the time and essentially not regulated and if you think about it for a little while you'll see that some of the basic cellular processes that we've discussed for example in our metabolism unit you can see that it sells pretty much always going to have to be expressing those proteins right so for example the glycolytic enzymes proteins that are in the electron transport chain things like that so those are referred to as housekeeping genes or housekeeping proteins because they're carrying out basic biological functions and so those would be what we would call constitutively expressed in other words not regulated in their expression their basic biological functions that they're carrying out have to be carried out all the time and also so things like you know components of the cytoskeleton do we always need to make tubulin protein so that we can create microtubules yes absolutely so those types of things how many genes out of the 20,000 would fall into that category of housekeeping genes about 4,000 estimates vary but aside from those the other you know three quarters or so are regulated in their expression in other words in some cell types they may be expressed in other cell types they wouldn't be expressed perhaps they're expressed in a given cell type under some environmental conditions or situations and not in others but regulated in some way right so the vast majority of the genes in our genome are regulated in their expression now of those genes that are regulated about 70 percent of that regulation comes in here at step one transcribing the gene right do we transcribe the gene to make the mRNA or not that's approximately 70 percent of the regulation that goes on in regulated gene expression now in addition to that though we have all these other mechanisms by which we can regulate the expression of a gene and whether a gene product is present in the cell or not we know that alternative splicing can lead to different proteins from the same gene by splicing together those exons and different patterns removing introns in different patterns but we haven't talked much about the fact that exporting this mRNA from the from the nucleus is an energy-requiring process it's actually regulated and so some M up mRNAs will be transcribed from their gene but then they'll be held in the in the nucleus until a signal is received at which time they'll be exported so we can regulate nuclear transport we have talked about the fact that mRNAs can be regulated in their stability within the cell some M mRNAs can be rapidly degraded or targeted for degradation whereas others will have a very long half-life and will essentially stick around for hours and hours and hours all right so we can regulate at that stage as we're going to see in in our next discussion looking at development in Drosophila many mRNAs are localized in other words once they're transcribed they're actually physically transported to a specific location within the cytoplasm and tethered there and then finally at the level of the mRNA we can we can regulate the interaction between that mRNA and the ribosome such that we're regulating the initiation of translation and therefore we're regulating how often this protein gets synthesized by the translation of this mRNA at the ribosome now all of these mechanisms of regulation as we talked about very briefly briefly are going to be mediated by those regulatory sequences that we have in the mRNA which are going to bind RNA binding proteins that are going to be responsible for regulating these types of events right how long the mRNA sticks around whether an exonuclease gets recruited to degrade it or not etc so so that regulatory function of the of the untranslated region comes into play when we're thinking about these modes of regulation and then finally once we've synthesized the protein we have additional mechanisms by which we can control the structure of that protein and how long it sticks around in the cell very often proteins will be processed in which specific amino acids are cleaved off this is called processing and so we end up with a shorter polypeptide sequence that gives us our functional product so we could synthesize a protein but it might be inactive in its initial precursor form and only when it's enzymatically cleaved does it become an active protein that can carry out its function so that's another mechanism by which protein or gene products are expressed and then we did talk about protein degradation as a means of regulating gene expression and remember in our mitosis unit we talked specifically about the fact that cyclin protein for example gets degraded at specific time points in the cell cycle and you'll recall that that was mediated the enzymatic activity of the anaphase promoting complex which added ubiquitin to that cycling protein and that targeted it to the proteasome for degradation so we're not gonna spend much time talking proteasome yeah practice spend much time talking about these other mechanisms mostly what we're going to talk about in this video is regulation at the level of transcription initiation and then in the next video we'll talk about mrna localization and how that can lead to transcriptional regulation such that we get specification of different cell types and an overall body plan during development but in this video let's talk about transcription initiation and how it's regulated in eukaryotic cells because we see some similarities with what we saw in prokaryotes but we also see some distinctions that are unique to eukaryotic organisms as well all right so if we think about a simple question like the fact that we see different functions associated with different cells in the body right we think about that how does that how does that arise is it because these different cell types in the body are actually genetically unique in other words they have different genes and different genetic instructions if we look at a pancreatic cell for example specific pancreatic cells within the pancreas are responsible for synthesizing the protein hormone insulin so there's an insulin gene that gets expressed in the pancreatic cell if we look at red blood cells in their immature state we wouldn't see that insulin expression is that because the insulin gene is lacking in those cells or is it some other mechanism well it turns out that it's not that the insulin gene is lacking in those in those cells it's that the insulin gene is present but not expressed right likewise in this red blood cell in its immature state we would expect expect to see expression of the hemoglobin gene such that we'd see hemoglobin proteins alpha and beta sub it's expressed and assembling into that hemoglobin complex that we talked about before the reason that hemoglobin protein is not expressed in the pancreatic cell is not because the hemoglobin gene is not there but because it's being specifically regulated in a way such that it's not being expressed in the pancreatic cell but it is being expressed in the immature red blood cell just a note why immature well it turns out during the maturation process red blood cells actually get rid of their nucleus they extrude extrude their nucleus out of the cytoplasm so the red blood cells in their mature state are one of the only cell types in the in the body that actually don't have a nucleus they don't have a genetic program still being in play what they are essentially is big bags of hemoglobin enclosed in a membrane okay so what's responsible for this differential gene expression well as we talked about briefly before it's gonna be the activity of these regulatory transcription factors regulatory transcription factors influence the expression of specific genes in the genome either activating their expression or repressing their expression now remember we talked about general transcription factors which were required for any gene to be expressed because they assembled at the promoter and they recruited our RNA polymerase enzyme to the genes so that that gene could be expressed now those are used for all genes any gene whether it's regulated or not right here what we're talking about are specific regulatory transcription factors that interact with regulatory sequences in specific genes in the genome and either activate or repress their expression typically what we're talking about is activation in eukaryotic cells because it turns out that in general the default state is off until switched on and so most of these transcription factors are going to be activating in their function rather than repressing in their function and so we could imagine that in order for this insulin gene to be expressed there are going to be specific sequences in the gene which are sort of illustrated by these different colored boxes these would just be specific sequences of nucleotides to which different transcription factors would come along and bind in a sequence-specific fashion and when those transcription factors bind to those sequences we see an interaction between those transcription factors and the complex of proteins assembled at the promoter that leads to activation of transcription and lots and lots of mRNA being transcribed from this insulin gene and so the reason the insulin gene is expressed in the pancreatic cell is because the correct transcription factor population is being expressed that can bind to those sequence and activate the gene and its expression why is the hemoglobin gene not being expressed well we may see expression of one or more transcription factors but not all of them that are required to activate expression of the hemoglobin gene right so two additional let's say transcription factors are required but the pancreatic cell doesn't express those and so therefore the hemoglobin gene is not switched on in its expression we'd see the reverse situation in the immature red blood cell the correct transcription factor population to express the hemoglobin gene would be present but we'd be missing essential transcription factors that are required to activate the insulin gene right so our proteins that bind here at yellow and red that were expressed in the pancreatic cell are not expressed in the red blood cell and so we don't see the insulin gene being switched on right so regulatory transcription factors interact with RNA polymerase and the general transcription factors at the promoter and can activate expression of specific genes in the genome aside from all the others right and regulate their expression now one thing that it's important to note here is that we're typically not talking about one gene being regulated by one transcription factor and if you think about it that makes sense it wouldn't really work to have Jeane regulated by a specific transcription factor if we've got 15,000 genes we have to regulate we'd have to make fifteen thousand unique transcription factors pretty much our entire genome would be made up of regulatory transcription factor genes right that wouldn't work so what we see in set instead is that it's a specific combination or set of transcription factors that have to bind to their regulatory sequences in order to activate expression alright so and this is referred to as a combinatorial code of transcription factors rather than unique transcription factors binding unique genes to regulate their expression so as we saw here this gray transcription factor whatever it is was actually in common right between the two genes that were being regulated the the insulin gene and the hemoglobin gene both had binding sites for that specific transcription factor but for the insulin gene it was combined with sequences that bound red and yellow transcription factor and for the hemoglobin gene it's interacting with pink and orange right these colors just representing different specific regulatory transcription factors that are involved in this process so it's the specific combination of transcription factors that confers the specificity of regulation alright and that's our combinatorial code so this is sort of simplifying the situation absolutely seen before where we're just scratching the surface of how this regulation works if we actually look at the real promoter region of the human insulin gene this is much more representative of what we would see here's our +1 nucleotide which we know is our first nucleotide that gets transcribed here's our tata box and the promoter we know that's at about minus 30 ish from our plus one nucleotide what we see in the insulin promoter and this is fairly typical is a large region with many many many binding sites for transcription regulators and they all pretty much have to be expressed in the recked timeframe in order to activate this from promoter now they might not all bind at the same time but specific sets of them would have to bind all at once in order for us to see RNA polymerase efficiently recruited and transcription of this gene taking place now this highlights one feature of eukaryotic transcription already at snot typical in prokaryotes and that's the fact that these regulatory sequences what are often called enhancers because again typically we're talking about activation of gene expression rather than repression these sequences in the gene are in the gene region that these transcription factors are binding to often are quite some distance away from the promoter and so whereas in prokaryotes we saw that the regulatory sequences for example the cap binding site and the repressor binding site the operator were right next to the promoter and we saw direct interaction between those transcriptional regulators and the RNA polymerase complex in eukaryotes very frequently what we see is what's called promoter distal enhancers so not sitting right at its promoter distal not sitting right at the promoter and interacting with RNA polymerase which would be bound somewhere right here right along with the general transcription factor complex but some distance away I mean this regulatory sequence here the vntr is over 300 nucleotides away from the promoter sometimes we can see regulatory sequences that can be thousands or tens of thousands of nucleotides away well how does this transcriptional regulator that so many hundreds or thousands of nucleotides away interact with this transcription factor or sorry this RNA polymerase general transcription factor complex at the promoter typically what we see is sort of looping effect so if here's our plus one what we would see so here's our gene what we would see is that that distal enhancer site would be brought close to the promoter so this would the n:t are over here that's being bound by this pure one transcription factor and so it would be brought by a looping of the DNA into contact with our RNA polymerase enzyme that's bound here at the promoter so it doesn't matter that it's hundreds or thousands of nucleotides away because it just gets looped around the other consequence of that is that it can be actually displaced you can experimentally take an enhancer sequence and move it further away move it closer flip it around so it's backwards it doesn't really matter the looping will bring that sequence into close approximation to the promoter site it doesn't really matter what orientation it's in so promoter distal binding of transcription factors to their regulatory sequences in the gene is far more common in eukaryotes and rare and sometimes not even observed at all in prokaryotes to spend depending on the specific species here okay so this really nicely illustrates that combinatorial code that we were that we were talking about and so we do see these kinds of direct interaction effects between these transcription factors and the RNA polymerase complex at the promoter that does occur but one thing that we also see and that's actually more common in transcriptional regulation in eukaryotes is that it's not a direct interaction of a regulatory protein with RNA polymerase and the general transcription factor complex instead what we see is regulation of chromatin structure instead right chromatin structure that in the region that the gene is located in and so if we remember back to our discussions of what DNA really looks like in a eukaryotic cell you'll recall that we don't see naked DNA what we see is DNA that has been coiled around complexes of histone proteins which are illustrated in this little cartoon diagram as the oval blobs here with the DNA looped around twice nice form those beads on a stream structures that we talked about before the nucleus zone as we discussed before this first level of compaction or chromatin structure then gets folded or coiled into higher levels of chromatin compaction so first we see the coiling into the cylinder and then the folding and looping out into the higher order compaction of chromatin that we see all the way up to that extreme level of condensation or compaction that we see during cell division so for example during mitosis when we look at a metaphase chromosome so chromatin structure can change right by changing the coiling and looping and compaction level of the DNA so if we think about it there are multiple states that chromatin can be in and they kind of range from you know a state where the chromatin is in a pretty open and uncondensed form to a form where the chromatin is more highly compacted and closed down and so these two different versions of chromatin are referred to as euchromatin for the open chromatin state and heterochromatin for the closed or condensed chromatin state typically you chromatin is associated with regions of the genome that have lots of genes in them so these are gene rich regions with high levels of expression of those genes whereas heterochromatic regions are typically gene poor not a lot of genes present and very little transcription going on there typically this is the state of chromatin that we see for example at centromeres and telomeres which as we've talked about before are really important structural regions of the chromosome that in the case of the telomere confer protection from degradation we saw that capping by the telomere protein complex centromere that site where the kinetochore assembled during cell division these are typically associated with heterochromatic regions of the chromosome and not many genes present right and a very compacted chromatin state even when the cell is not dividing if we have a region of the genome where we've got genes and they have to be expressed it makes sense that the chromatin would be in a more open state where RNA polymerase could then access it right but what we see in eukaryotic cells is that this open chromatin - closed chromatin state of the DNA is actually dynamic and it can change over the life of an individual cell depending on conditions that are going on and it can also change during the development of a multicellular organism such that different chromatin regions of the genome are being compacted or condensed down and other regions are not right and so this is a mechanism by which we can control access of RNA polymerase to specific genes or gene regions and it's very commonly used as a means of regulating gene expression in eukaryotic cells now obviously prokaryotic cells don't have chromatin remember they super coil their DNA as a mechanism for compacting it whereas we associate our DNA with these histone proteins and other chromatin proteins to coil it and condense it so that it's regulated and organized and compacted within the nucleus so we're going to talk about two different ways that chromatin structure can be changed and is typically changed in eukaryotic cells and the first way involves actual modification of the DNA itself and we refer to this way back at the beginning of the course when we talked about methyl groups as functional groups we talked about the fact that we'd come back to this at the end of the course and see that covalent addition of methyl groups on to DNA nucleotides themselves would be associated with gene regulation and so here we are the other main mechanism by which chromatin structure can be modified involves modification of the histone proteins that the DNA is wrapped around so these are fundamentally two different types of modification in the case of DNA methylation we're actually talking about covalently modifying the DNA itself whereas here we're just modifying the proteins that the DNA is wrapped around let's talk about DNA methylation first so one thing we need to know is that that methylation is typically taking place on cytosine nucleotides within the genome it can occur on other bases as well but typically we're talking about cytosine methylation and most often what happens when a gene region has undergone DNA methylation is that we see long term or even permanent silencing of that gene in other words that gene is taken into a heterochromatic State closed chromatin state and really made unavailable for transcription because of that now why would addition of a methyl group onto a cytosine base and the DNA caused the structure of chromatin to change well it doesn't do it directly so here we see sort of a diagrammatic representation of a methyl group that's been added on to a cytosine nucleotide in the DNA and there's nothing about that methyl group that changes the interaction of the DNA with the protein instead what happens is that it serves as a binding site for recruiting proteins that can come in and remodel the chromatin structure itself so it will recruit other proteins that will then alter chromatin structure so not a direct effect but an indirect effect but pretty much this is generally irreversible all right so once we methylate this DNA region we don't unmethylated it stays methylated and it stays silenced by this permanent alteration of chromatin structure that we see going on so when would we want to permanently silenced a gene what type of a gene or genes might we see a cell wanting to shut down permanently and never hear from again essentially never Express again well there a couple of scenarios that we could imagine if we go back to our example of our different cell types remember we talked about pancreatic cell versus an immature red blood cell is this cell ever gonna make hemoglobin no I said we're gonna make hemoglobin is this cell ever gonna be a lung cell is it ever gonna be a neuron is there ever gonna be a muscle cell no it has a specific cell fate that it has taken on which is an endocrine and possibly exocrine depending on what pancreatic cell we're talking about but it has a specific function that's associated with it which requires a specific set of genes in the genome to be expressed all those other cell types specific genes that are not required for this pancreatic cell to function they can be shut down right so one category or class of genes that we can see DNA methylation permanently silencing is cell type specific genes in other cell types other than the cells that need to express that gene right so muscle versus long versus bone versus neuron whatever that's one class of gene that we thought we might want to permanently silence the other class of gene that very frequently DNA methylation is associated with permanently silencing is a class of genes that's functioning early in embryonic development so if you think about the massive waves of cell division that have to happen during early embryonic division it's really incredible in scope we start with one fertilized egg and we have to expand that one cell out into millions and millions and millions of cells that will then differentiate and take on specific cell fates well there's a whole set of genes that drive those rapid rounds of cell division early in embryonic development once we get past to that early stage of zygotic and an early embryonic development we shut those genes down right we shut them down down down because never again in the life of the organism are we gonna have to such rapid rounds of cell cell division taking place so much mitosis going on as we see early in embryonic development and in fact reactivation of those genes so if methylation goes awry and isn't maintained in those genes is a genetic change that has been associated with some types of cancer so we can see reactivation of those zygotic genes and rapid cell division being initiated as a result of that but typically those classes of genes would be permanently silenced following that specific stage of development all right so that's DNA methylation permanent silencing what's the other form of modification involved with our histone modification this is a more dynamic form of rebel regulation this is reversible so we have enzymes that work in both directions here and confer this short-term dynamic very very responsive to changing conditions form of regulation and what we're talking about here again is modification of the histone proteins that DNA is wrapped around so if you remember earlier in this semester we looked at this more structurally accurate representation of a nucleus ohm here's our DNA wrapped around twice around the exterior and here's our complex of histone proteins in the middle and remember that I pointed out to you these little tails that project out so these are n-terminal domains that sort of protrude out from the nucleosome complex right and these are the parts of the histone proteins that get modified get they get modified by covalent modification so we see specific types of functional groups being added on and then removed from these little and terminal tails right and so what types of modifications do we see well we can see phosphorylation which we talked about before in terms of enzyme function and enzyme regulation we talked about reversible phosphorylation of as a mechanism to do that same thing here nothing new we can also see methylation coming into play right so addition of methyl groups on to these n-terminal domains of the histone proteins what we're going to talk about is actually the most common modification that happens which is a set elation addition and removal of acetate groups onto these little N terminal tails that project out so typically when we have enzymatic medicine some enzymatic modification of histone proteins such that an acetate group is added so that would be called a settle a ssin that is associated with less condensed chromatin and as we've talked about before less condensed chromatin a more open chromatin state is going to be more accessible to the transcription machinery and we're gonna see transcription activation as a result of that enzymatic modification so adding an acetate group on is going to tend to result in activation of transcription of that gene in the nearby region so the proteins that carry out that a set elation reaction are called histone acetyl transferases or hats so hats modify chromatin such that chromatin structure becomes more open the transcriptional machinery RNA polymerase general transcription factors have more access combine the promoter more easily and therefore transcribe the gene on the other hand histone deacetylases or h dax catalyze the reverse reaction removing this acetyl group from the histone tail and as addition of the acetyl group was associated with a less condensed chromatin state removing it is going to be associated with the opposite right a more highly compacted or condensed chromatin state which is going to lead to transcription repression right and so we go back and forth we have these enzymes sort of competing with one another right hats adding the acetyl groups on htx removing them obviously in a regulated fashion and in so doing changing the structure of chromatin in a gene region which is going to change the activation state of that gene and whether it's being transcribed or not now what would cause a hat or an H stack to interact with this specific gene region as compared to some other well very often these regulatory transcription factors that we talked about before binding to enhancers these could actually be recruiting these hats and HTX to this gene region so some of these transcription factors could actually not be directly interacting with RNA polymerase but actually binding to a hat enzyme and recruiting it to a promoter region to change chromatin structure and so these are chromatin modifying enzymes that are modifying chromatin by covalently modifying the histone proteins that DNA is wrapped around right now again what specifically does a settle a Schnoor phosphorylation or methylation of these N terminal tails do to alter the structure of chromatin well we think nothing directly once again what we see happening is that when we see these modifications being added we recruit these chromatin remodelling proteins to come and sit down and cause the chromatin structure to be organized in a different way to go from a more highly compacted state to a less highly compacted state changing the spacing between these nucleosomes changing the level of coiling and condensation etc and so this is very much an indirect effect not necessarily a direct interaction of proteins with RNA polymerase to influence the initiation of transcription but to more globally change the structure of DNA such that it either favors the ability of RNA polymerase to come and bind and express that gene or not very different from what we saw in prokaryotic cells