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Lecture 7

BIO240 Lecture 7

11 Pages

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Jennifer Harris

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Lecture 7: Eukaryotic Transcriptional Regulation 2 **Lecture 7 begins** - Today we’re going to start to move forward, going from where we were, showing the interplay b/w chromatin regulation, the regulation of transcription, and now finally getting around to making proteins and the regulation of translation – turning it on and turning it off. - Gene expression actually just starts with chromatin & actually ends ultimately with the proteins that interact together. - Need to consider the multitude of different factors that function to regulate transcription itself & modify chromatin structure. - Movie: Modifications can occur at different steps: capping enzyme, putting 7-methyl-guanine one, we’ve got splicing that can occurs (introns being removed), poly-adenylation occurs & eventually have migration out of the nucleus out into the cytoplasm & now the events involved in translation & the control thereof. Transcriptional regulation includes insulators/barrier sequences and gene regulation (initiation of transcription involves) regulatory proteins? Enhancers Heterochromatin - Insulators & barrier sequences are important for defining zones of gene regulation, making sure that where gene expression occurs is going to be euchromatin & where gene repression occurs is heterochromatin. The 2 zones are effectively, to a good extent, isolated from each other. - Insulators function to make sure that genes function as discrete units, that is, they block the activity of DNA motifs known as enhancers – this is where transcriptional activator proteins would bind to these enhancer sites. Insulators prevent the info from the enhancer to spread to the next gene. - Diagram: we’ve got enhancer that’s going to function to turn on gene B once the gene regulatory protein is bound – and we don’t want that binding necessary to influence the expression of gene A. Consequently, an insulator element, a segment of DNA, occurs b/w the enhancer and gene A so only gene B is regulated by the enhancer – ensures that each gene is a discrete regulatory element in and of itself. - Diagram: we’ve got a heterochromatin domain on the DNA near the end and the barrier sequence is functioning to prevent it from spreading into gene B, silencing gene B & making it inactive. Saw this with the example of the white locus – saw barrier sequence preventing spread of heterochromatin normally except in white mutants where it gets flipped around. - Insulators function to ensure (and barrier sequences do the same actually) that you’ve got regions that are structural (heterochromatin vs. euchromatin) and functional (active and inactive). - Diagram: the fact that insulators function to create such domains is very nicely shown in the diagram. Here we’ve got a chromosome that has been stained so we can effectively see the compression of the DNA & those regions of the DNA that are highly condensed are in a nice bright red. Below what you’re looking at is an anti-body that has been made that recognizes proteins that bind to the insulator regions and makes them function as insulators. That anti-body has been tagged with a fluorescent green dye so wherever the proteins are that bind to the insulator, the insulator binding proteins are green showing that they function to divide the genome up into these different domains of condensation and de-condensation or heterochromatin or euchromatin or active and inactive. Epigenetic inheritance* - It turns out that such info with regards to what regions of the DNA are condensed or de-condensed can be transmitted from one cell to its daughter cells. So when mitosis occurs, that info is carried forward to the daughter cells. How does that actually take place? There are 3 mechanisms (there is a 4 one that will described in a later lecture): 1) Histone modification: here we have active chromatin and now we have inactive or heterochromatin that’s been made and in the daughter cells, this heterochromatic state is going to be maintained, that is, that region of heterochromatic DNA in the daughter cells is also going to be heterochromatic. 2) Positive feedback: a protein is not made in this particular cell but now in the next cell the protein is made, and b/c the protein is made and will be present in that cell when it divides, the protein will function in the daughter cells as well & if that protein itself is active, to activate its own gene, it will perpetuate the activation of that gene in daughter cell after daughter cell. 3) DNA methylation: here we have unmethylated DNA regions that become methylated as shown by these little red dots & this new methylated state is now effectively transmitted to the daughter cells. - Note that he’s saying daughter cells, not mother and daughter cells – talking about mitosis, not talking about the products of sexual reproduction where something different occurs. This mode of transmission that is mitotic where daughter cells inherit the gene expression state that their mother’s have is known as epigenetic inheritance. Note that it does not involve a change in any of the nucleotide bases in the DNA but what has been inherited from the mother cell to the daughter cells is the activity of that DNA region, either active or inactive. - Inheritance through the somatic cell line: cells that make up everything but the reproductive tissues in our body – the inheritance of the info from mother cell to daughter cell. X chromosome inactivation - Recall we inherit one set of chromosomes from mom and one set of chromosomes from dad. Women differ from men in that women rd have two X chromosomes and males, their 23 pair of chromosomes, is an X and Y. - There’s 2 ways of looking at that: how is it that men only get by with only one X chromosome? Or how do women contend with having 2 X chromosomes? From a biological perspective, having 2 X chromosomes is the more difficult thing to deal with so as to equalize effectively the dosage of X b/w males and females, what happens in females is one of the X chromosomes, either the paternal copy or maternal copy is silenced – and it’s silenced by condensing it into a highly heterochromatic chromosome. - Dosage compensation, that is, to make sure that women effectively only have one dose of X is manifest by shutting down one of the X chromosomes. - At random, either the paternal or the maternal copy is shut down & depending on whether the paternal or maternal copy that is shut down, what ends up happening as a consequence is that all the cells that are derived from that cell, for example where the paternal copy is shut down inherit that state of the Barr body residing in the paternal copy of the X chromosome. Whereas the other lineage, where the maternal copy has been shut down, it’s now highly condensed, that is transmitted from one cell to the other. - So the net result of that is that as opposed to men, which are with their single X chromosome, they are, in terms of their X chromosome, they are just one great blandness – it’s only the one X chromosome that they inherited by their mom that is going to be expressed. Women, by contrast, are a mosaic – they have the X chromosome from mom and the X chromosome from dad – some tissues, some organs will only be using the X chromosome from mom, others the X chromosome from dad – a mosaic. - How does this shutting down of the X chromosome actually occur? - Non-translated RNA: This is an RNA that begins and ends its life as an RNA – never is translated into protein. - Hypoacetylation – decrease in acetylation, resulting in heterochromatin formation (condensation of chromatin). C-G (cytosine-guanine) - Diagram: we have an unmethylated cytosine (see C-G pair in diagram) and there is a methylated cytosine at another pair. Upon DNA replication (recall it’s semiconservative), we’ll end up with one of the methylated strands up here and a non-methylated strand & the other methylated strand and a newly synthesized, non- methylated but these are recognized by the maintenance methyltransferase so that what is transmitted to the daughter cell is something that is not hemi-methylated (half-methylated) but totally methylated at that C-G dinucleotide & that’s passed on from cell to cell (that info). Epigenetic – not transmitted through genetic mechanisms, it’s “in addition to” or “over top of” genetic inheritance. - Diagram: What we have occurring is the transmission of methylation through reader-writer complex as we’ve seen before – methylation being transmitted down the length of DNA, such that by the time that we get to the bottom, we’ve got a purely heterochromatized region that has been locked in this silent state. Again, this does not involve any changes at all to A, T, C or G themselves outside of the fact that methylation occur – no actual change in the DNA code. - When gametes are made, these signals are all wiped clean. - The epigenetic changes can create differences in highly related individuals – even monozygotic twins (identical twins with the same genetic material), by the time that they are older, despite the fact that they may still look identical, have diverged dramatically in terms of their epigenetic state so that things are very different in the future. - Monozygotic twins, that is identical twins, twins that were derived from the same zygote, from the same fertilization event that when that zygote was cleaved & those genetically identical individuals began their individual trajectories in life, that despite the fact that they are genetically identical, there is something different about them in the molecular sense. **Finishes here at the end of the lecture 7** **Lecture 8 starts here…** - Last lecture: we’ve been covering an elaboration of the central dogma of molecular biology, working from DNA to RNA to protein. We’ve been talking about the organization of genomes, the interplay b/w that & the transcriptome & finally we’re moving on eventually to proteins, just starting to touch on translation. Recall: we were talking about the complexities of gene expression & the regulation thereof in eucaryotes – saw how there are multiple steps at which gene expression can be modified, taking gene expression in the broadest sense to mean the conversion of DNA info to corresponding protein info – so a # of steps that can modified along the way, each of them regulated. - Then we started to work through a list of different types of regulation along this pathway. Finished up with transcriptional regulation by insulators & barrier sequences & we finally finished on DNA methylation and this concept of epigenetic inheritance – the idea that what can happen in a mother cell, mitotically speaking can be transmitted to daughter cells – this does not involve change in DNA sequence but rather the way in which DNA is used, be it in the euchromatin vs. heterochromatin state and the relationship of that to DNA methylation. - Implications: Epigenetic changes can create differences in highly related individuals. - Recall 5-methylcytosine occurs at those C-G sites where methylation is going to occur. - Acetylation of H3 & H4 is going to bring about differences in the chromatin state where those histones are localized. - 2 different experimental approaches that allow them to take a look st nd at 5-methylcytosine content in the 1 instance and in the 2 instance, the acetylation states of H3 & H4. - In the 1 case, there is very little difference b/w 3 year-old twins in the global 5-methylcytosine levels. By contrast, when we look at 50 year old twins, there is significant difference b/w one twin and the other. - Similarly when we take a look at the acetylation of H3 – no difference effectively b/w young twins, significant difference b/w old twins. Same case with acetylated histone 4 –
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