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Fundamentals I 9/18/08 Dr. Ryan 10:00-11:00 These slides are numbered based on the NEW powerpoint Dr. Ryan used in class** There’s a lot to cover and get right to it. Slide 2: Eukaryotic RNA polymerase • I mentioned yesterday that in eukaryotic there are three RNA polymerases. They are called RNA polymerases. There are three DNA dependent RNA polymerases: Labeled RNA Pol I, II, and III Each of these their own sets of promoters and genes that they transcribe. All 3 are big, multimeric proteins (500-700 kD). All have 2 large subunits which are very similar to prokaryotic. RNA polymerase subunits and ' which we talked about yesterday. And the catalytic site may be conserved between prokaryotes and eukaryotes. All of these polymerases interact with general transcription factors-GTFs. WE will go into detail on what they are. How do you discriminates between these three? How do you know if they gene they transfer by Pol I II or III? • There is drug called -amanitin and Pol II shows the most sensitivity. This is an inhibitor of pol II. -amanitin is a bicyclic octapeptide and blocks the elongations. Slide 3: Lecture Part 3 RNA polymerase II inhibitor -amanitin The sensitivity is shown there. Pol II is more sensitive than Pol III and Pol I is hardly sensitivity at all. This drug -amanitin comes from the mushroom called the “destroying angle.” So if you’re picking mushrooms for your morning omelet, stay away from these. Slide 4: Yeast RNA polymerase II subunits These are the subunits of RNA Pol II from yeast. There is a total 12 subunits. The nomenclatures is RNA. 1 o This is RPB1, that’s for RNA Polymerase. B stands for the Pol II. RNA Polymerase I would be RPA 1-12. For Pol II subunits, they are all RPB and then the subunit 1-12. The first subunit shares homology with the beta prime subunit of prokaryotes. One of its important features is the C-terminal domain, its repeating heptapeptide, has lots of serines and theorinines in it that get phosphorlylated from the process of going through transcription initiation to the actual start of transcription. We’ll talk about that later. The second subunit and these are labeled 1-12 according to their size, they discovered or purified these later. The second largest subunit RPB2 shares homology with the Beta subunit of E.coli. It has the NTP binding site and forms half of the catalytic site with the RPB1. There are two subunits that share similarities (Dr. Ryan corrects himself by saying one shared similarity) with alpha that’s RPB3. The promoter recognition has homology to the sigma factor is RPB4. You can see, number of these factors are shared between the three polymerases: RNA Pol I, II, and III. Slide 5: Transcription Factors In eukaryotes, the transcription factors are going to very important. All three polymerases interact with transcription factors. A lot of these interactions are protein-protein interaction or protein-DNA interactions. We’ll talk a lot about types of transcription factors and these general transcription factors used by all these polymerases. These transcription factors (TFs) recognize and initiate transcription at specific promoter sequences. So when 6 billion base pairs of DNA, these transcription factors are going to help land the polymerase on the promoter to initiation transcription. Some transcription factors bind to specific recognition sequences within the coding region. So they are not upstream where the promoter is seen in prokaryotes. Sometimes they bind internally to the genes. Slide 6: Helix-Turn-Helix Motif 2 I would like to talk about three classes of transcription factors. These are figures from your book. They talk about these three classes. First is the helix-turn-helix motif. These transcription factors all bind DNA and what’s special about these is the helix-turnhelix motif is shown in orange in these figures. And these are two alph-helices that are separated by beta turn. The first helix sits down in the major groove in DNA, the second one locks in place. You get very specific type of binding. Most of these helix-turn-helix motif bind has homodimers or heterodimers. Yesterday, we talked about DNA having dyad symmetry, having inverted repeats. An inaudible question is asked about not having the exact slides. Answer: These are in the start of Day 4. This is the lecture of combo of 3rd and 4th lectures. All lectures are in one of these two lectures. They are mixed. So just listen carefully. You will have a copy of these available to you. Another question: paraphrase: Is there a PowerPoint with your notes that we can get? A: It’s written in your book. You see when you get a test questions and you see helix-turn-helix or alpha helix beta turn alpha helix you will know what it refers to. Another question: paraphrase: What is important ? A: I won’t ask you anything that I don’t have on this slide or is not in your book in the reading material. Back to lecture: Alright, by forming a homodimer or heterodimer, you can increase the specificity of DNA binding with these factors. One dimer binds one sequence or one protein in the homodimer can bind its cognate sequence on the other side and they have an interaction domain so you can increase the specificity of binding of these factors. Now we talked about these factors yesterday, some are of these are prokaryotes and eukaryotes that have these helix-turn-helix. Down here, is the cap protein that we talked about. The activator protein of the lac operon. It has the helix-turnhelix motif. 3 Here is the trp represson and lac repressor that we talked about yesterday. Eurkarotic helix-turn-helix motif that is called the antrion (sp?). o Helix-turn-helix motif is located in the homodomain. o This is important for DNA binding. o The specific sequences upstream are very important developmental genes. Slide 7: Zinc-Finger Motif : C2H2 Class The second class of transcription factors is these Zinc-Finger transcription factors. There are 1000s of these in our genome. The typical structure is a pair of 15 amino acids apart separated by about 12 AA in the finger, and 2 histidine, 2 or 3 amino acids apart. o There is a zinc atom that coordinates these 4 amino acids. o Now the actual DNA binding part of the zinc-finger is located in the 12 AA space (between the cystiene and the histidine). Here’s the actual structures. So it’s got an alpha helical structure. o Each of these fingers can contact 5 base pairs of DNA, and these fingers always come in 2 or more fingers together. o So you can have up to 17 fingers in some of these transcription factors. So if you have multiple fingers they can bind/interact longer sequences of DNA. o Now they each interact with 5 base pairs, but they have a tight interaction with three and interact with a neighboring one. o So each finger, if you have 3 fingers are together they actually interact with specific 9 base pair sequence in the DNA. o If you have 4 fingers, you would have 12. o The middle finger is has interaction with the neighboring. Each contacts with three specific bases. S o If you mix and match these fingers- there’s a company do this- they can build you a transcription factor that will bind any DNA sequence you want to make- so they say. So they again they use these fingers, there multiple finger they can bind to longer and longer stretches of DNA specifically. Slide 8: Region Leucine Zipper Motif bZip 4 The third class I would like to talk about is the basic region leucine zipper motif (bZIP). These transcription factors are mostly alpha helical in structure. They have a basic region that makes contact with DNA. And the similar to the helix-turn-helix motif and the zinc finger factors, these bZIP factors also bind in the major groove of DNA. o All three of these major classes all bind to major groove of DNA. Now what’s special about these is that the single factor will dimerize with another bZIP factor. o They do this via this alpha helical domain called the leucine zipper. o If you turn this alpha helical regions and look at it, look at the amino acid that are located- every 7th amino acid is a leucine. o Now the Leucine all lineup on one face of alpha helix. It’s called an amiphatic helix. It’s very polar on one surface. On the surface with leucine, it’s very non-polar. Non-polar on the other surface. **NOTE: this is what I got out of what he was trying to say. He had broken thoughts. *** o See the two leucine surface come together-zip together. You can picture the zipping action. That’s why they get the name bZIP. o So these can bind has homodimer or heterodimers. You can get two different bZIP coming together. o Their DNA recognition sequences may be different. So when they contact the DNA, you can be specific in your DNA binding. Slide 9: bZIP transcription factor The next figure is from your book showing a heterodimer coming together and interacting in the major groove of DNA. Here is the basic region sitting in the major groove. Here is one of the proteins, and there’s the second one behind the helix sitting in the major groove. This is would be the leucine region. They cut of the rest of the protein which would have activation regions that will activate transcription. Again, there’s description in your book for all three of those. Slide 10: General Transcription Factors 5 Now the general transcription factors help position the RNA Polymerases on transcription initiation sites. (The next few slides are from the end of Lecture 3) These general transcription factors help the polymerase come in and land on the initiation site and form along with the polymerase a transcription- initiation complex. The nomenclature is determined by the polymerase that they are associated with. o So, the TF- transcription factor, the II means it associates/ or its general transcription factor for RNA polymerase II. o And then various complex of the proteins that have been isolated, and as they were purified and given letter designation of TFIIA TFIIB etc. o We will in more detail in a moment. So again these help the polymerase associate with the promoter find the initiation site. Slide 11: General Transcription Factors • • Of the general transcription factors, TFIID is the largest and consists of a TATA box binding protein, that is abbreviated TBP. It has 8-10 transcription TATA box binding protein associated factors which are abbreviated TAF. Again the II there for TAFs II are because they are isolated from TFIID of RNA pol II. The TFIID is composed of TBP and the associated TAFs. Those together make up the TFIID. TBP is a “universal transcription factor.” • It associates with promoters of all three RNAPs (pol I, II, III) • It is also used even though it’s called the TATA box of binding proteins, it’s also used with promoters that don’t have a TATA box. These promoters that don’t have a TATA box, have to rely on other general transcription factors that help the polymerase find the initiation site. TFIID has two roles: • Foundation for the transcriptional Pre-initiation complex. • Prevents nucleosome stabilization in the promoter region. • If you have to transcribe a gene, you don’t want nucleosomes to bind there and wrap it all up. So if it’s gene that’s expressed all the time, these so called “housekeeping genes,” you want to have this factor around 6 keeping the nucleosome from closing up the promoter gene. Slide 12: TBP is used by all 3 RNA polymerases TBP- TATA binding protein is used by all three polymerases. It has different names depending on the other proteins its associates with. When it’s in a Pol I gene it’s called : SL1 factor. When it’s interacting with Pol III, its called Transcription factor III B. We have already talked about Pol II, it’s TFIID. It does not always to bind to TATA boxes. Pol I and Pol III, and sometimes Pol III promoters don’t have TATA boxes. But, TBP is still used as a general transcription factors. Slide 13: Yeast TATA binding protein TBP. This is diagram of the yeast TATA binding protein. What’s unusual is that TBP binds in the minor groove. Previous transcription factors we talked about were all major groove binders. This is actually bound in the minor groove, while in binds it bends the DNA by 120 degrees. It also melts this region. If this region is usually A-T rich area, it helps melt this region upon binding. As it binds, it’s got amino acid chains that go in the minor groove and that helps pry the DNA apart at that region. I will talk briefly talk about the promoters for Pol I and Pol III and get back to Pol II. Slide 14: Promoters of RNAP I So the promoters of RNAP I… Pol I is basically there to transcribe the large ribosomal gene. Our genome is 100s of similar copies of the rRNA genes. Remember from yesterday talk, rRNA is the most abundant RNA in the cells- it’s over 80% of the RNA is rRNA. We have one polymerase in Eukaryotes that does nothing but synthesize/ transcribe through these genes. There are 100s of them. The RNAP I promoters are called class I promoters because they are transcribed by the RNA polymerase I. There are two elements: upstream control element and a core element. The two different transcription factor binding sequences: SL1 and UBF. 7 We already know that TATA binding protein is a component of the SL1. Slide 15: Transcription of rRNA genes by RNA polymerase I So this is example of what the rRNA gene looks like. It’s an array of gene after gene with a spacer in between. The spacer is red. The transcripts are initially one long precursor transcript about 45s. Out of the transcript, it’s heavily processed to get the 18S and 5.8S and 28S rRNA. You will get this more when they talk about translational of ribosomes. He then asks the class: Have you had a lecture on ribosomes? It’s coming… These ribosome are ribonucleoprotiens- they are mixture of many proteins and RNA, There are the three RNA components18 S associates with 30 proteins to form a 40 subunit, 5.8 S and 28 S . The 5S, which comes from Pol III transcription, joins with about 50 proteins to make the 60S subunit- a large subunit. Now third polymerase, Pol III. This polymerase transcribes various small RNA’s. The : 5S rRNA, tRNAs (15 % of total RNA in the cell), vary abundant messages. There are various small nuclear U6 snRNA that are also transcribed by pol III. Eukaryotes have specific polymerase that will transcribe tRNA because of the larges messages. Slide 16: Promoters for RNAP III Genes like the 5S rRNA and tRNA are called class III promoters because they use RNA polymerase III. Some of the genes transcribed by Pol III are similar to pol II promoters- two these are used U6 small nuclear RNA and 7SL RNA genes. But they are used pol III. They determine this by used alpha amitain to see if these are sensitive or not. These genes were not. The 5S rRNA promoters are entirely within the coding region of the gene. I will show that on the following slide. The tRNA promoters contain two elements. Slide 17: PIC Assembly for RNA Pol III Genes 8 Here’s the tRNA gene and the 5S rRNA gene. There are series of these general transcription factors. Notice there are called TFIII, see here- for transcription factor for pol III, complex c. They load on these box a and b in a particular order forming this preinitiation complex. The TFIIIB has the TATA binding protein in it. And you will notice that for the tRNA gene, both of these boxes are located downstream at the promoter in the sequence of the trNA genes. Similar for the 5S ribosomal, RNA gene, there’s a series of factors that bind TBP is part of TFIIIB again. It helps position these general transcription factors that help polymerase over the right startt site for transcription of this chain. Question: What is a good thing to take from this slide? A: TBP is used for all three of polymerases. These binding sites for these general transcription factors don’t always occur upstream at the transcription site in these genes. Some of these TF RNA some sRNA don’t use pol II to trassncibe their genes; they use pol III. Look for the broad picture. GF’s help orient the polymerase in the right spot to intiated transcription. Question again: Can’t here it. A: The only time I would ask you memorize specific will be for pol II, that we will talk about. You have the general concept, if I have what TFIIIB to bind Pol II gene, that’s not correct If I have TFIID is used as general transcription factor for tRNA gene transcription is incorrect. For tRNA transcription, is TATA binding protein utilized? Yes. Slide 18: RNA polymerase II General Transcription FACTORS (GTF) Let’s switch to pol II. These are the GTF’s for pol II. The first one of these is listed sort on in the order that they from the initiation complex. The first factor, TFIID –again it’s composed of TBP and all the TATA binding protein associated factors (TAFs). Together, TBP and TAFs make TFIID. I have the function of these in the next series of slides. We are going talk about the function as we go. This is in your note, so you can get basic idea of what they do. We are going to make 9 the pre-initiation complex and start transcription in the next series of slides. Slide 19: TBP-associated factors (TAFS or TAFII) The TAFs or TAFII use the TBP associated factors- these are important to bind TBP to promoters that lack TATA boxes. Originally it was thought all genes had TATA boxes, but we are finding out that majority of genes don’t have TATA boxes once you analyze them. There are different TAFs in different cells. So these TAFs complexes were isolated from cell type. If you look at a different cell type, the complexes that you isolated from the GTFs could be slightly different or very different. In vivo these factors are associated with additional proteins forming a larger complex of about 50 polypeptides. So again for every cell in a given particular gene, there are different times of expression or development, things can be different. It is hypothesized that a single large complex come together first, assemble, and get loaded on to the promoters. Question: If it lacks TATA box, how does it work? Answer: It’s still associated; it brings all these TAF’s. SO there are associated through the TBP. Some other TAFS can also interact with DNA. So those TATA-less promoters, those other TAFs are more important. Or finding the start site of transcription. Ok lets make a pre-initiation factor here for pol II transcription. Slide 20: Eukaryotic RNA Pol II Transcription Formation of PIC If this is a gene that has a TATA box, will bind TATA via the TBP subunit. The TBP, part of the TFIID, will bring this complex of proteins, so TBP and TAFs down the TATA box and bind. It will bend the DNA and binds to the minor groove. It bends the DNA and melts the two strands apart. Next the TFIIA stabilizes interaction on the TATA box. Slide 21: Eukaryotic RNA Pol II Transcription Formation of PIC 10 Next, TFIIB which is a monomeric protein comes in and binds. This kind of helps place the TFIIB, it kind makes contact with the initiation are the start side of transcription. It helps ground things- getting ready to for the polymerase to come in the right location. TFIIB also interacts with a lot of other transcription factors that may be binding distal promoters that we will talk about in a moment – like enhancer like sequences. So these promoters not only have this massive transcription factor sitting at the initiation site, but there are also other transcription factors that are upstream elements and downstream elements. All these interact to bring in the polymerase and get efficient transcription. TFIIB plays a role interacting in some of these other things – accessory transcription factors. Slide 22: Eukaryotic RNA Pol II Transcription: Formation of the PIC TFIIF binds to RNAPII, and they make a preformed complex. This (the TFIIF) helps direct, the RNAPII to the TFII D, A, and B already setting at the promoter. Next the TFIIE binds to the TFIIF/RNAPII complex, and this further helps to cement that initiation site. TFIIE is a DNA-dependent ATPase. This is probably what generates the energy for the helicase that’s the next step, which comes in with TFIIH. Now I might mention, the Carboxyterminal domain (CTD), so that’s in the Beta-prime subunit of the RNA polymerase- that’s the largest subunit. It helps form the catalytic site. It has this long CTD with all of these amino acids: Ser and Thr that can be phosphorylated. It has to be in an unphosphorylated state to come in and bind at this step. Later we’re going to see that it gets phosphorylated, and that enables transcription elongation. Slide 23: Eukaryotic RNA Pol II Transcription: Formation of the PIC Once you have Pol II and TFIIF and TFIIE down at the promoterthat activates TFIIH. TFIIH now contains nine subunits. 11 It has helicase activity, and it’s going to help unwind the transcription bubble. It needs ATP to do this, and it’s probably coming from the TFIIE. This TFIIH also has It has protein kinase activity, and it will phosphorylate the CTD of the Beta-Prime subunit of the polymerase. Once the CTD tail of FNAPII is phosphorylated, it detaches RNAPII from TFIID, and you get the beginning of transcription and elongation of the gene. So this is just a pre-initiation complex. It’s sort of like this is the basal level of expression. All of these general transcription factors bringing in polymerase just helps you initiate transcription. We haven’t yet talked about all the other factors and enhancerlike proteins and other transcription factors that help stimulate or repress this process. Slide 24: Carboxyl-Terminal Domain (CTD Tail) The carboxy-terminal domain is a heptomer, so it’s a stretch of 7 amino acids that is repeated many times: Tyr-Ser-Pro-Thr-SerPro-Ser In the mouse there’s 52 of these. (Referring to the repeating sequence above, I think) on the CTD. It’s very important for transcription that you delete this. In the mouse you can make knockouts, you delete that tail- the animal’s dead. Critical for viability CTD tail becomes phosphorylated on some of these Ser and Thr residues, not Tyr- that’s a typo there. Well it can be hydroxylated on a Tyr too. They have a hydroxyl group. This is what allows the polymerase to transcribe away from the promoter. Slide 25: RNA Pol II Promoters There are other elements in the promoter that also affect the level of transcription. We’ve talked about the TATA box bringing down TFIID and the initiator that’s located right at the +1 transcription start site. There’s also upstream elements downstream elements. We can consider these proximal promoter elements. They can be upstream within the gene or downstream. 12 Slide 26: Eukaryotic TATA Box This is a schematic figure that is from your book. It’s showing the eukaryotic TATA Box. Again, if you line up a bunch of genes that have TATA boxes, there’s a consensus sequence that can be derived, showing the frequency of A’s and T’s at these various positions. On average there’s about 25 spaces upstream of the +1 transcription start site. Again you’ll notice at transcription start sites there’s normally a purine. Slide 27: Eukaryotic Promoter Regions Further upstream of the TATA box you’ll find some of these proximal promoter elements. They can be arranged… you know the sky’s the limit. Different genes will have different combinations of factors. Sometimes there’s a repeating of the same element over and over again. These are just some of the…. For these three genes, some of the factors that have been shown to be in the promoters and be necessary for the basal layer of transcription for these promoters. Slide 28: RNA Polymerase II Promoter Consensus Sequences of Transcription Factor Binding Sites Some of the factors that bind these promoter proximal elements are shown in this table. The CAAT box binds a factor called CTF or NF1. The GC box binds a factor called SP1. The octomer box binds Oct-1, Oct-2. There’s a B box that binds NFB and so on. For any particular gene, the factors that bind there depend on the sequence upstream of the promoter. In vitro transcription experiments people have determined which of these transcription factors are important and which ones aren’t. Slide 20 (on old ppt): Synergistic Activation Just to nail this point down: these proximal promoter elements can be upstream- they can be downstream. They’re usually multiple elements in these important sequences. They can be considered enhancer-like sequences when they’re downstream 13 or far away from the promoter. We’ll talk more about enhancers in a minute. Slide 29: Proximal Control Elements of Genes: Modular Factor Binding Sites You can see the same factor can be repeated over and over again. In this case, GATA-1, they’re showing a globin gene here, and the GATA-1 factor binds at sequence GATA. There’s multiple GATA-1 binding sites, upstream and downstream of globin genes. Slide 30: Coordinate Regulation Via Response Elements Just like in prokaryotes, sometimes we want to coordinate expression. This is done in eukaryotes with response elements. These are transcription factor binding sites again. They are specific sequences where you can control a whole set of genes by upregulating a specific transcription factor. Similarly responsive genes will have a DNA sequence located in cis to the gene called a response element. It’s just a DNA sequence that this particular factor will bind. This factor can bind multiple genes to coordinately regulate increase expression or decrease expression of genes that have these response elements in their promoters. Slide 31: Response Elements: Coordinate Regulation We’ll show that here. This is a picture from your book showing the metallothionein gene promoter. Here’s the transcription start site, at 0. This is -20, -40, and so on upstream. Each of these colored boxes are a specific DNA sequence that binds transcription factors.Here’s the TATA box. (@ -25) Each of these (BLE, GRE, MRE, ect…) are response elements. Some of these elements are shown here (in the box) with their name. Here is the glucocorticoid response element or GRE. You can seen in this promoter there’s a GRE upstream from -240 to -270. This metallothionein gene would be responsible to the hormone cortisol. If you have cortisol on cells the glucocorticoid receptor, 14 upon binding cortisol will come and set on this GRE element on the promoter and cause the up-regulation of the metallothionein gene expression. To clarify how response elements work, what they’re for: you can coordinately regulate numerous genes. Again, you can look at the specifics of these different elements in your book. I’m not going to hold you responsible for knowing the sequence that each of these bind or anything, but you should know that a GRE would bind a glucocorticoid receptor. Question from the audience: Do we need to know what the other ones do? Answer from Dr. Ryan: Heat shock elements are, and some of them are… I will spell them out. I just won’t put GRE. If I say heat shock element, you know it’s a heat shock protein, so it’s probably binding there, huh? Slide 32: Coordinate Regulation by Hormones/Steroid Receptors Question from Dr. Ryan: Have you had a lecture on steroid hormones? Answer from class: Mutterings of no… No? Well you can get coordinate regulation by hormones with their steroid receptors. They have a structure. If you line them all up they have a hormone-binding domain. Usually they have a DNA binding domain. Then they have a variable region which can be the activation or repressing domain. Slide 33: Steroid Receptors Let’s stick with the glucocorticoid receptor here, GR. Here again it has hormone binding that’s going to bind cortisol. It’s got a DNA binding, and it’s got this variable region. Slide 34: Activation of Transacting Factors This is an example where the glucocorticoid receptor is usually masked inside the cell, waiting for its hormone signal to activate transcription. 15 It’s masked by an inhibitory protein that sits and interacts with the hormone binding domain and the DNA binding domain. It keeps the DNA binding domain from being accessible for binding to its glucocorticoid response element. This masked receptor and inhibitor is sitting it the cytoplasm. If you dump cortisol on these particular cells, cortisol will go through the membrane. It’ll bind to the hormone-binding site here, cause a conformational change that results in the removal of this inhibitory protein complex. So this steroid cortisol comes in, the inhibitory protein goes out, and now you’ve exposed the DNA binding site. Upon binding hormone this glucocorticoid receptor complex moves from the cytoplasm to the nucleus, and now the exposed DNA binding domain can find the gluococorticoid responsive element. Now whether it activates or represses transcription depends on the context of all the other factors binding and sitting on that promoter of that particular gene. Ok… there’s…. we’re discovering new ways transription factors can be activated all the time. This is just a schematic showing a few ways. Just by making a transcription factor. You synthesize, you translate, it’s active, it goes to work. You can also have ligand-binding which activates. In the case of the glucocorticoid receptor, this would be an example of ligandbinding. You can have a protein that gets phosphorylated to become active. When it’s not phosphorylated, it sets there inactive. You can add a second unit so you might need a subunit to bind to it to become active. You can have mixtures of these things. Here we talked about again the glucocorticoid receptor where you have an inhibitor. When that inhibitor comes off it becomes unactive. In the glucocorticoid receptor, when the inhibitor comes off it helps it move from the cytoplasm to the nucleus, and it uncovers the DNA binding domain. 16 There’s also where you get release from a membrane. You have a proteolytic event that activates a factor, making an active transcription factor. Slide 35: Control of Cellular Differentiation By TFs Transcription factors can also control cellular differentiation. This is just a schematic showing in a simple form how this could work. If you have an embryonic cell that divides, and in this daughter cell it induces regulatory protein number 1 here, that makes this daughter cell a little different from this daughter cell here. This cell here may start to express 2 and 3. You can see depending on which of these factors are expressed in particular daughter cells, they could have a different developmental fate. Again, this is all done by TF’s. Different factors binding different sequences can activate the transcription of different sets of genes in different daughter cells. So, you can go from an embryonic undifferentiated cell to multiple different cell types. This was a hematopoietic stem cell, and by expressing different TF’s at different times, you can wind up with RBC’s, T-cells, Bcells, NK cells, macrophages and whatnot- all the lineages in the blood. Slide 36: Enhancers These are Control elements that stimulate transcription. They usually bind multiple different transcription factors. These transcription factors usually have activation domains will interact with general transcription factors to boost polymerase loading or just increase the amount of transcription at a promoter. You can also have enhancers that work in a negative way, and those are called silencers. Slide 37: Enhancers Enhancers can stimulate expression of genes over great disances. 17 In the human Beta-globin locus, all the human Beta-like globin genes are controlled by a master enhancer that is 50kb upstream of the locus. It controls the high-level expression of all these downstream genes. How it does this is most probably by looping, and it comes and interacts so that all the factors binding this enhancer sequence, the DNA between the enhancer and the structural genes are looped out, and there’s a proteinprotein interaction. These enhancers can be upstream, downstream, in introns or in exons They normally work in an orientation independent manner. So if you cloned a piece of DNA that has an enhancer upstream, if you clip it out, turn it around, and put it back in, it usually works. These enhancers can be cell-type specific. So, the enhancer upstream of the globin genes only works in erythroid cells. You put that same enhancer upstream of some other genes or if you cut out the whole globin locus and put it in a lymphoid cell, it doesn’t work. Ok, only in erythroid cells, and that’s because in erythroid cells you have erythroid-specific TF’s already bound at the promoter and these proximal elements to increase the erythroid-specific transcription. Slide ?: Proximal Regulatory Regions of Genes/ Slide 38: Enhancers: Action at a Distance This is just a schematic to show an enhancer working at a distance. Here again, if this is 50kb away from this transcription start site, there’s DNA in between that’s looped out, this enhancer binding protein here can interact with the general TF’s that are going to sit on this promoter and can bring in these general factors and other receptor proteins such as a mediator and increase polymerase loading on the promoter and increase transcription. Slide 39: Insulators / Boundary Elements There’s also special elements called insulators or boundary elements. Insulator boundary elements have two functions. If this is a gene B- let’s call this a globin gene again, and this is that powerful enhancer upstream. As part of the upstream of this enhancer there’s an insulator element. What this does is that it 18 prevents the enhancement of genes on the other side of the insulator. So this, (gene A) is not a globin gene. In the case of the globin locus, these are ordinate receptor genes, and it keeps these ordinate receptor genes off in erythroid cells. These insulator-like elements can also stop the encroachment of closed chromatin, the heterochromatin form neighboring loci. In an erythroid cell, this insulator element keeps heterochromatic regions next to the globin locus from coming in and shutting down the genes. This enhancer sequence keeps all this region open, and keeps these genes expressed at a high level. These insulators block heterochromatin from coming in, and it also blocks the enhancement of genes outside of the locus. There was a question about the insulator and RNA Pol going back to transcribe DNA which I could not understand. This is what Dr. Ryan said: Polymerase is going to come in and bind right at this promoter, and what direction do all polymerases transcribe? 5’3’ Once that polymerase is done transcribing, and the transcript’s terminated, it will come off, and it can be recycled right back on the promoter. So, as long as the general transcription factors are binding here, they’ll bring in more and more polymerase, and it will keep transcribing. This gene in an erythroid cell….let’s say this is a globin gene. You get a lot of Pol II transcripts of the Beta-globin gene, but thos ordinate receptor gene would not be open and accessible. The erythroid-specific TF’s don’t have binding sequences. They don’t have the binding elements upstream of the ordinate receptor genes, so they’re not going to be bound there. They’re not going to bring in the TATA binding protein or TFIID. They’re not going to bring in all those general TF’s because they weren’t meant to be expressed in this cell. The right milu (sp?) or TF’s aren’t present there. The right combination of apples, oranges, and bananas aren’t there for that one, but they’re in there for gene B. Another question which I couldn’t understand on the audio. 19 Dr. Ryan’s Response: If there were general TF’s sitting here, but they needed this enhancer to get a high level of expression, this insulator would block that. Another indiscernible question or comment. Dr. Ryan’s Response: Now, if you put an insulator sequence right here (downstream of the enhancer), then you’re going to block the enhancement of gene B also. People have used these insulator sequences to flank genes of interest that they want expressed in certain areas but not have the flanking sequences influence their expression. So they’ll flank their gene of interest with these insulators. Another question, but I couldn’t make it out on the audio. Dr. Ryan’s response: Again, these insulator elements work by binding factors- transcription factors. So there’s a whole bunch of binding sequences or binding elements that are specific types of insulator elements. Question: Response: Only in erythroid cells is this whole locus open. Questions: Response: These insulators aren’t all that common. They’re typically found in… in the globin locus there’s all of these globin genes. They’re all regulated the same way- only in erythroid cells, and the whole locus is flanked by these insulator cells. Not every individual gene is flanked by these things. Now the whole region of this chromosome may be flanked. If you have a whole bunch of housekeeping genes, they may be flanked by these insulators. Now, this erythroid locus in a non-erythroid cell, none of this stuff is happening. All of this heterochromatin is closed up. The proteins that bind this enhancer aren’t around. The erythroid specific TF’s which opened up this whole locus by binding to the enhancer and these promoters aren’t there in non-erythroid cells. Again the type and number of TF’s that are expressed in any specific cell control all this. We’re going to finish the lecture today talking about a very specific example of…oh boy… I believe he skips the next few slides due to running out of time at this point. Slide 41: Silencing: Histone Deacetylation HDAC & Slide 36: Histone Acetylation and Deacetylation There’s ways to open and close chromatin. We talked yesterday about histones and chromatin. There are enzyme complexes called HATs (Histone Acetyl Transferases) and HDACs (Histone 20 Deacetylases) that add acetyl groups or remove acetyl groups with the deacetylase. This will help open and close the chromatin. If you remove this positive charge (from the NH3+ group of Lys) by putting an acetyl group there, it will help repel neighboring nucleosomes and help open up the chromatin. I believe he skipped this next slide(#42). Slide 43: Histone Code This is a very hot topic of research today: the histone code. On these amino-terminal arms on the various histones, they can be modified. They can be acetylated, methylated, phosphorylated, and ubiquitinated.. These modifications have a profound effect on gene activity. They can increase gene transcription, or they can silence gene transcription. There’s a whole code that’s just now being understood that the histone is not a static thing. It’s very active in setting up which genes are transcribed in a cell. Slide 44: Chromatin Remodeling Now, the chromatin can be remodeled. If you’re closed up tight, how do you open it up and express the Beta-globin gene? If you have a hemopoetic stem cell that’s making a RBC progenitor, you have to be able to open up this chromatin and get these erythroid-specific TF’s in there to bind their promoter elements and enhancer elements. So there are complexes of proteins that come in and can help remodel the DNA and histone complex. This is an ATP-dependent process. Slides 45: Gene Activation By Chromatin Remodeling This is just a schematic showing a remodeling complex coming in that can help pry the DNA off the histone, and these TF’s can come bind, and that will set the stage to bring in more and more factors and open up the chromatin for gene expression. Slide 46: HAT’s open chromatin ( Image slide) & 47: Assembly of preinitiation complex on open chromatin Let’s say you have a hemopoietic stem cell again, where the Beta-globin locus is totally closed. It’s not expressed, and we 21 want to turn this into an erythroid cell and express the Betaglobin gene. So if you’ve got closed chromatin, you need to make an erythroid-specific TF, it’s going to come in and associate and make things a little more accessible. Other HAT’s can come in and recognize this TF and start acetylating the histone tails, opening up the region even more. You’re going to have the nucleosome remodelers come in, and that will make it more accessible for more TF’s that will come bind these transcription sequences and open things up even more. Once you get to a certain point you can start bringing in the general TF’s and the polymerase and sit at the promoter and start transcription of the gene. Slide 48: Chromatin remodeling (Cow–cloning slide) The big picture: what happens in development. We have a single-cell fertilized egg- a zygote that’s going to make every cell in the body. I just want to impress upon you the power of these TF’s with this experiment. You can think about it when you go home tonight. Cloning: we can clone cows today… right? In this cloning experiment we take an unfertilized cow egg. We pull out that nucleus- that haploid nucleus. In this case they’re using an epithelial cell from the oviduct. This is a cumulus cell from the oviduct. They take a diploid nucleus from this cell and put it along side this enucleated egg. They electrofuse these together, so this diploid nucleus goes into this egg without a nucleus. All those proteins: TF’s, chromatin remodelers, all those protein complexes in there go to work on this incoming epithelial cell nucleus. Within hours that epithelial cell nucleus is reprogrammed into a zygote. All the genes that were on these epithelial cells are shut down, and all the genes necessary to be a zygote and begin all of development to produce a calf have to be turned on. 22 This can all happen in hours time. Mouse cloning is 3-6 hours. Cow cloning is about the same time frame. So all these proteins are setting in this egg. So, all the things we talked about: chromatin remodeling, HAT’s, HDAC’s, ect… All this stuff is going on to remodel this epithelial cell nucleus into thinking that it’s a zygote. Things like this reprogramming are going to be in the news more and more as we talk about it. We can now take skin cells and reprogram them into something that looks like an embryonic stem cell by expressing four factors. It’s amazing the plasticity there, and it’s all done by these protein factors. 23