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Lecture 15

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University of Toronto St. George
Jennifer Harris

Lecture 3: Transcriptional Regulation 1 - In the last lecture, we took a look at 2 chimps & we talked about the role that the transcriptome plays in giving rise to the differences b/w those 2 chimps. - Considered a problem with regards to the generation of that transcriptome, the diversity of RNA transcripts that one finds in an organism that defines the organism itself, different tissue types, different cell types or different cells in different points in time or under different conditions. - Problem that we had to deal with is this: the challenge that the genome has to deal with – not such a big challenge with procaryotes but big challenge with eucaryotes. - Last lecture: considered a model that would allow one to accommodate all of those factors so that one could have a tightly compressed genome within the nucleus but access it at appropriate points in time or in appropriate cells to give rise to differences in the transcriptome. - In circumstances where one does not need to access the info contained in the genome, it’s tightly condensed within the chromosome – heterochromatin. But then when genes need to be accessed for the process of transcription, they’re de-condensed, made into euchromatin. Slide 1 - Take a look at the interplay again b/w genome organization & the regulation of transcription. - The idea is that we have to access the 4 letter code in the DNA and the idea that the chromatin has to be remodeled and exposed to allow for these RNA polymerase to give them access to the chromosomes. - Last lecture about transcription of RNA how they were made and now a little more about regulation. Slide 2 Slide 3 - How is this orchestrated? How is it controlled? How are specific regions of the genome regulated in such a way that only those genes are expressed? - Movie: organization of nucleosomes (octameric core comprising histone proteins & their interactions with DNA). Wavy bits are histone tails. DNA is going to interact with the histone proteins. Can see the tails whipping off the end & waving out – that’s the nucleosome with the histone tails wagging off around. Important point with regards to those histone tails: the 8 histones, pairs of 4 different histones, present in the nucleosome core (2A, 2B, 3 & 4) have tails that are accessible & can be post-translationally modified. - The chromatin is remodeled for the purposes of gene expression, how is this orchestrated? - Missing slide showing DNA that is going to be wrapped around histone proteins. The histones have tails whipping around and what you see is nucleosome with DNA wrapped around and then the nucleosomes are assembling into the 30 nm chromatin fibre. Slide 4 N terminal Covalent (post-translational modification) - There are 4 types of nucleosomes, the octamer all have tails. - The histone tails can be chemically modified and can create their own code. Slide 5 Amino acids - The AAs that are susceptible to this modification are Lys and Ser residues. Specific AAs in the histone tails are These specific AAs are substrates for post-translational modification where they substrates for post-translational have particular groups added to them by a covalent bond. modification - Can have acetyl groups added to the end of Lys. 1, 2 (di), 3 (tri) methyl groups on the Lys as well. The acetylation & the methylation are mutually exclusive events that occur at this Lys – that is, you have one or the other, or neither. - To the OH group of Ser, a phosphate can be added to give phosphoserine. - By these modifications & their specific location within the tail, you establish the code of sort, a way of flagging things to say ‘here, this is a region that you want to make a specific chromatin remodeling event’. - Histones that you remember, they are high in two amino acids which are serine and lysine. - The lysine can be methylated either mono di or tri or acetylated - The charge once acetylated becomes neutral and in terms of DNA histone interaction, it becomes weaker because - This is a somewhat new idea where before you just thought histones packed DNA Slide 6 - Histone modifications can occur in either H2A, H2B, H3 or H4 – can see that they may be comprised of either methylation, phosphorylation, acetylation or ubiquitylation (generally occurs at carboxyl terminal end). Methylation, phosphorylation & acetylation activities all occur at the amino terminal tail & we can see the different Lys & Ser groups are susceptible to that post- translational modification in the different histone proteins. These together comprise what is known as the ‘Histone Code’ – a way of tagging histones to indicate that specific sort of chromatin remodeling event should occur there (like a flag – ‘make heterochromatin or euchromatin here’) – this is an important concept in eucaryotic cells as a transcriptional regulatory mechanism. - There are these specific types of modifications you see there are 4 types of histones - Looking at the N terminus of the amino acid for H2A then H2B and you look at all the modifications that occur on all these amino acids - You can get methylation, acetylation and phosphorylation or adding a molecule called ubiquitin - Just know that all these modifications occur but is there some meaning in regulation of transcription or structure? Slide 7 - Example with Histone 3 – can see # of modifications that can occur on the amino terminal tail. Note that dependent on the modification is taking place so for example, methylation at K-9 (Lys) – indicates that heterochromatin should be formed there (condensation of the chromatin) – as a consequence you get gene silencing. - By contrast acetylation at Lys-9 (K-9) & methylation at Lys-4 results in gene expression, similarity phosphorylation of Ser-10 & acetylation of Lys-14 gives rise to gene expression – formation of euchromatin (decondensed DNA that can be accessed by the transcriptional apparatus). Yet another example: methylation of Lys-27 silencing a particular set of genes – X chromosome inactivation. - Different modification will signal different types of things to happen, here are a few examples and these are the ones known to happen on histone 3 - Below that are specific modification for whom the meaning have been worked out - Its known that on lysine 9, if it gets methylated its known that it makes the chromatin to become heterochromatin so that it silences or reduces transcription, heterochromatin formation and gene silencing - In other things like acetylation on the lysine and phosphorylation makes the DNA associated with those histones get transcribed whereas methylated K 27, are silencing hox genes - They determine your body segment that allow you to have arms coming out of your torso instead of arms coming out of your head if you play with them then you get flies with tails coming out of their head - Gene expression for the phosphorylated S 10 and adenylation of K 14 is just increased gene expression Slide 8 - What is it that is reading the histone code? – protein modules that detect specific modifications on the nucleosome – this is a so-called histone code reading complex – it detects the covalent modifications that have occurred at the amino terminal tail & as a consequence, recruit particular protein complexes, catalytic activities & additional binding sites that are going to end up modifying the chromatin structure – things like the chromatin remodeling complex, ATP utilizing catalytic activities that converts one kind of chromatin to another. - Got proteins that are able to read the code, recruit other proteins & together they function like a machine to either scrunch proteins up here or de-compress so the gene expression can occur. So depending on the proteins that are binding to the DNA, these are non-histone proteins, you get the conversion of one chromatin type to another. - That’s great you’ve got these modifications but how does this information get used and also how do you get these modifications in the first place you have two parts of the problem - One part is who writes that code and who reads that code - Notice how important it is that the tails are sticking out of the nucleosome and there are specific proteins that bind to the histone tails and you see them all assembled on a scaffold protein and they recognize a very specific grouping of histone modifications - Those come in and they are what reads the code and what they do is attract other factors, maybe the general transcription factors that allow for transcription to happen or chromatin remodeling factors allowing for less interaction with histone - They lead to gene silencing or transcription or other biological function - The histone code has important information that helps to regulate gene expression Slide 9 - In this specific example, can see the spread of the histone code down the length of the DNA so that now, the length of the DNA scrunches up to become heterochromatin or become spread out to become euchromatin. So what happens is a specific protein binds, it in turn recruits a histone modifying enzyme (so-called “writer enzyme”) – these function then to recruit reader proteins that then read the code and then recruit other writer proteins and so on and so forth so that these reader-writer complexes read the code and hit the next nucleosome & transmit the next code to it – have spreading of one chromatin type vs. another down the length of the chromosome structure (chromatin). - What often will happen is that there is a special gene regulatory protein that will recognize a very specific sequence in the DNA - The protein will bind to that sequence and what that will do is set up a whole cascade of events and that is how we get the histone modifications - We have regulatory protein attracting enzymes that will write information to the histones - As well there will be a code reader protein that will bind to the code that is written and that may be linked to another enzyme that will write the code there - What you have initially is that the reader gets recognized by regulatory proteins and histone modifying enzyme brought in, then a code reader protein comes in that modifies the next histone - You get spreading of the code so that the histones around that area will have the same information - You see the reader writer complex so you get regions where you get these information altering the transcription silencing or promoting transcription Slide 10 - A protein has been recruited or has found a stretch of DNA – it in turn recruits a writer protein that then modifies the histone that then you have a read-writer complex & as you can see, as we go along with the chromatin remodeling complex, we’re scrunching up the DNA – it started out nice and spread out euchromatin then on the basis of these histone modifications, reading, subsequent writing step then at the next nucleotide the signal gets passed along so eventually the whole region is made into heterochromatin. - You can spread heterochromatin just as you saw with the fruit fly eye, the white gene was sometimes expressed or not and what happens in those cells, the heterochromatin has spread to those genes shutting them down while in other instances they are still being transcribed - The reader writer complex and chromatin remodeling complex brought in and with nucleosomes over to cause chromatin to be very highly condensed - Conceivable this can keep going over and over and shut down the entire genome and what regulates that? Slide 11 DNA Barrier - Of course we don’t want this to proceed all the time – don’t want all of the chromatin to be made into heterochromatin b/c we need to gain access to some regions of it. Recall in the last lecture, looked at the white locus from Drosophilus – the white locus was necessary to make red eye colour in Drosophilus & recall there was a heterochromatin region right next to the white locus (the white locus being the white gene that codes the white protein that makes red pigment) – in b/w that heterochromatin region & the gene was a barrier that stopped under normal circumstances the spread of heterochromatin into the gene so the gene could be expressed. If there was a mutation such that the barrier was now no longer blocking the spread of the heterochromatin, the gene was made into heterochromatin & expressed, you got a white eye. - The barrier functions to stop the reading of the histone code & make sure that there isn’t spread of heterochromatin into regions that you want to retain as euchromatin. - Here is an example of barrier DNA & an associated barrier protein that recognizes that barrier DNA, stopping the spread of heterochromatin there on the left by virtue of its association with the nuclear pore. Recall all of this is taking place in the nucleus of the eucaryotic cell – the nucleus has pores that allows transmission of info from the inside to the outside – that is transit of RNA from the nucleus out into the cytoplasm & conversely, transit of proteins from the cytoplasm into the nucleus but association of particular proteins with that nuclear pore where the transit (of RNA from nucleus out into cytoplasm & proteins from cytoplasm into nucleus) occurs function to stop the spreading of the heterochromatin into the euchromatin region. - Other proteins simply recognize a specific sequence & function themselves as a barrier for this spread of the heterochromatin into euchromatin area – they function rather passively & here we’ve got a protein that is functioning actively. - There are actually DNA sequences called barrier DNA that prevent the spreading from going too far - There are different mechanisms postulated on how that barrier DNA words - One example is that a barrier protein binds to the DNA sequence called the barrier DNA sequence lie the nuclear pore complex (that allows stuff to go in and out of the nucleus) but it would now act as a physical hindrance of the spread - Or a large protein may just protect subsequence DNA - OR it may acetylate the protein and prevent methylation on lysine 9 Slide 12 Sequence specific DNA binding proteins - Who/what writes the histone code? – sequence specific transcription factors that play an absolute crucial role in this chromatin remodeling – sometimes these are referred to as gene specific regulatory proteins. The proteins bind to specific sequences & then recruit the chromatin remodeling machinery. - What are sequence motifs? A motif, when talking about a DNA motif, is a series of nucleotides, the combination of A, T, C & G (whatever order they are in). This is what specific sequence transcription factors bind to. - Many people in the outside world call them transcription factors, they are different from the general transcription factors that bring RNA polymerase in -
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