 Hey, ScissorG. How's everyone doing today? Q is the placement of the board and everything okay? Safe and sane. That's pretty good. Yeah, we're gonna hope my Internet holds out. There are times it's been bad. I can. I think right here should do it. True, ScissorG, it's not a done deal as of yet though. Yeah, there's a lot to it. And you'll see by the end what I really imply by the word should. I had a conversation with someone who substituted the word deserve for should, which was certainly not my intent. Well, true ScissorG, but I think there was also a little bit of an uptick when Chantal was advertising to other education groups in local chat. That helped. I have 10 o'clock. Should any objections to starting? All right, so let me welcome you all to another edition of the Science Circle weekend presentations. I am Stephen Gager, known here locally in Second Life as Stephen Zootfly. And the topic for my talk today, genomic parasites nibbling away at us, why I should win the Nobel Prize, is going to talk a little bit about some work I did at a postdoc, which was at the Tulane Cancer Center. And that one thing I want to point out early on is that my involvement in this field and the title is a narrative structure that I wanted to present this as. And you'll see kind of by the end what I think the importance is of the research of this field and that within 10 or 20 years, I would say it's a virtual guarantee that the science keeps going the way it is that somewhere somehow people in this field will be rewarded with a Nobel Prize. So I'm going to try and structure the talk to give you this basic background narrative of kind of context and then give a little bit of a sideways introduction to another field, which is typical of many important discoveries is two things converging and I'll talk about my work and I'll talk about the the impact on the work after that. Now one thing I have to apologize for, for anybody who's a real aficionado with the field or science, I have to bias a lot of the data that I show and I'm going to show a lot of data today or it's things that are a little more accessible and visually and accessible to me and not behind a paywall and today there will be a lot of data don't get intimidated by the slides I will be walking you through the experiments and I have a little extra arrow here to help me point things out and on top of that I also like I have another presentations I have some green text which is the takeaway message for many of the more complicated slides so I have also tried to make this relatively short so there's time for people to stop me with questions if there's something really confusing and to pause a few places to to reflect on the first segment. So again like a good science talk I want to try and go back as far as possible in the science and what I want to talk about is this to lead off with is this kind of general phenomenon these syndromes that are called progeria syndromes and these are progeria is a way of saying aging getting old and these are called segmental progeria syndromes because they're not necessarily specifically in advanced aging but they have elements of aging that then manifest themselves in these syndromes and one of the oldest and most classic one of these is Werner syndrome which was first named and characterized in 1904 and again you can see from the picture that the normal looking young woman by the time she reaches 45 looks more like she's a relatively unhealthy 85-90 years old and then there are other syndromes that have been described in the literature in the medical literature of that have more characteristics of developmental neurological delays although they do span other symptoms like predisposition to cancer they actually show DNA sensitivity in cases this one on the top right is Nimogen breakage syndrome and again you'll see this odd morphology of the skull they are smaller brain sizes and have developmental delays and actually usually they do die relatively early this one is also particularly characterized by when people look at the cells from people with the syndrome they have a lot of breaks in them you can actually observe these breaks when you break apart cells and take a look at them and then the bottom right or so the bottom one is sechal syndrome again another syndrome characterized similarly by these abnormal brains case sizes and shapes and then other sensitivities early death susceptibility to cancer so i have some further reading links that talk a lot about these syndromes but i just want this to stick in your head is that there are these common features of these syndromes but they're not always exactly the same presentation they seem similar and related but they're not always the same now i'm going to step back and step very small and talk about a lot of the interesting molecular genetics that happened in the early or the mid 1900s and what the power here was that with single celled organisms like with bacteria and yeast you could mutagenize their genomes you could take a whole culture of them so that they all have different mutations in different places and then screen for phenotypes and the phenotype that i'm showing here is on this top plate you have this mutagenized group of yeast and you just put them out and then you'll notice that the arrangement of colonies is the same as on the bottom plate but there are two there are two things that are happening here is that you expose the bottom plate not the top plate just the bottom plate to either x-rays as represented by the lightning bolt or say UV light again and what do we know about x-rays and UV light and this is a question for the audience what do you know about those types of things happening in cells vixas mutations definitely a part of it breaking bonds yes they're attacking DNA and chemically modifying it in ways that's bad and if you have enough of a dose you die um but in fact many cells can survive certain doses of of damage and but if you have a mutation in something that helps you repair that DNA damage then you'll see these two arrows those colonies did not grow and so now yes mic shaw points out exactly what i was saying is that a lot of depending on the dose a lot of cells are fine but the ones that are mutated and less than more sensitive to damage they die so you can go back and what jenesis did which went back and identified what these were use genetics and name them and what i'm showing here are some complicated pathways you only need to know a couple key lessons from these pictures is that there are different types of damage so UV damage that links up DNA together is repaired by nucleotide excision repair again it has to be recognized as that type of damage and then a variety of different proteins come in to accomplish different steps of the repair process and you'll notice that some of these genes are called rad genes if you look really closely at the picture on the right we have a double strand breaks the whole DNA is broken on on both sides the ladder is broken and so different repair proteins have to come in this is a repair pathway called homologous recombination or hr and again you'll see different proteins involved on this side than on the other pathway and again very stepwise there's four proteins coming in and doing things and so we see the names you know rad something or xrs x-ray sensitive those are indications that some of these were found in these screens although they have other names too because they're sometimes like the gene uh replication protein a that's a protein that's involved in repair but it was actually first found as being involved in DNA replication now what I want to do is take one example of these proteins and talk very specifically about how science understands how they work and so one of the most well-known rad proteins is rad 51 that I'm showing here on the top is both an assay and a demonstration of its activity and so the idea here is that that little squiggle you see on the left hand side is single strand DNA that's a circle and what rad 51 protein will do is it'll coat that it'll coat that DNA and form is called a nucleophilament so a complex of all these different rad 51 proteins winding around that DNA and vixas point out yeah the bottom gel we're gonna have about gel electrophoresis in a minute and then you present this nuclear filament with linear double-stranded DNA where you see that little five prime that is double-stranded DNA that's just linear not circular and if you put these things together what'll happen is the rad 51 nuclear filament will displace one of the strands of the double-stranded DNA resulting in the end over here in circular double-stranded DNA versus single-stranded linear DNA what Vic is pointing out is that this is an assay that's been known for a long time where just run this on a very simple gel where anything that's faster migrates farther and that's just one the simple lessons of gel electrophoresis with DNA but that these different molecules either the nicked circle DNA or the double-stranded linear DNA have different migration patterns and the fact that over time this nicked circle the one that is now the new product increases over time with the with rad 51 present in the media and so what this is saying is that the specific role of this protein is to bind up get around single-stranded DNA and then use that to go find double-stranded DNA to accomplish repair to make sure you're finding the same sequence to repair that double-strand break and I think it's important I have one citation here of Patrick Song in the Roberson uh lab that first demonstrated that the rad 51 protein from yeast was able to accomplish this but there was actually a whole host of papers Steve West Steve Kolsky who showed that these rad 51 from either yeast or human can accomplish this and this is something that if you actually go back even farther to bacteria rad 51 is known as a wreck a homolog wreck is a protein that's been known to accomplish this type of strain exchange uh for a very long time now like I mentioned already we have these diagrams a lot of the proteins that helped us out and this is where I'm gonna come back to again making this a little bit about me and I'm gonna talk about my gradual work and so the question was is we know these this rad 51 protein accomplishes this but both from genetics and what we can kind of predict from E. coli that there are other proteins that must be helping to do what it does and so the technique that you see here in the top left hand side this is another way of getting about do proteins interact and this is actually in exploded yeast cells we basically take the nucleus explode it and try and uh detect protein complexes using the technique known as immunofluorescence this is antibodies that recognize the protein so these glowing balls what you see are these glowing balls that basically represent that particular protein and what we argued was that this other protein known as rad 52 associates with rad 51 it also associates with rpa inside cells that are doing double strand break repair arguing that they they may be working together but then also from a molecular genetics standpoint the idea that if you have a yeast cell that's completely missing rad 52 and that's why I have the lowercase rad 52 name there that rad 51 doesn't form foci and so that's saying rad 51 depends upon rad 52 to form its complexes and to do its job and so that's what we argued here with the molecular genetics and really the basic results of this is the diagram you see on the right arguing that there's a stepwise formation of repair complexes they recognize the break they form complexes then they accomplish the goal and again I think this technique of doing yeast immunofluorescence was very powerful it was um coming out at the same time that people were doing very simple or the same type of biochemical experiments that you saw before show that rad 52 also helps mediate rad 51 and this is a citation I have your colleague of our of that lab Akira Shinohara Tamoko Ogawa who demonstrated the same thing so again this is the way we get at how genes work and whether um and how we understand what's what genes and proteins do in a cell and now I'm gonna just pose a question everyone do you guys know of any genes involved in cancer what what are some of the first genes that come to mind that have to do with cancer that you may have heard about yeah so sumo it's Hano specifically mentions BRCA BRCA 1 as well as BRCA 2 are the um probably most well known I mean 503 is the one that's known I think most well among scientists but BRCA 1 BRCA 2 is a breast cancer susceptibility genes and this was coming out and it's cloning what it was doing in cells came out around this time in the late 1990s and then was really being characterized as a part of a DNA repair protein and so again what I have here in the diagram is a very again a summary of a huge amount of work in literature saying that BRCA 1 as we understand it and what's mutated has a very bad effect on cells that because it's places crossroads role in coordinating repair processes then when it's missing then of course cells start to go bad they have error prone repair processes that leads mutations and now we know when the basic underlying concepts is that um uh you know the mutations that arrive from poorly done repair is a big problem so Vic actually mentions a quick thing I'm gonna I'm gonna correct your numbers on this real quick so Vic says I had to look it up but it appears a p53 has a role in 50% of cancers and p53 is something that helps coordinate damage response more from the cell cycle not the damage repair part but the cell cycle and that 50% number is actually a little bit misleading that p53 mutations are found in about 50% of cancers but about 90% of cancers have a mutation in some gene that affect the p53 pathway so there's another protein called MDM2 which controls how much p53 protein there is or how much phosphorylation there is and so these are things where p53 really is kind of considered a central tumor suppressor so and and something that had been known for a long so I'm going to go back to talk about the right hand figure here which is what's been known about cancer for a long time and a lot of this was pioneering work by cytogeneticists with lymphocyte lymphatic cancers leukemias but then has been characterized as a general phenomenon of cancer cells because if you look at the picture you see lots of colorful blobs and what those colorful blobs represent are ways of painting human chromosomes now in theory the way these are designed any given chromosome should just be one color the whole length of it should just be one color so these little examples here where you see a combination of blue and red on one chromosome is an indication that things have got rearranged and so again I'm citing the paper here from Janet Rowley a very nice visual one she's also someone that helped identify the so-called Philadelphia chromosome she won the Lasker Prize and she actually was a nice old lady who was faculty of Chicago when I was there and was always riding around on a bicycle that's a little part of it but the key lesson here and this is the crossroads what we are understanding about cancer is that there are genes involved in DNA repair that play an important role in how we basically try to not get cancer and then when those can't then when those genes do something wrong they're not working correctly that leads to cancer and what I want to talk about now is just let's bring it back to my first picture we talk about Werner syndrome, NBS and Seckle syndrome we know the genes that cause those it's relatively easy to map and understand the genes now but even at the time people did a lot of work to understand and name these genes and what we and when they start sequencing the genes people discovered that they are basically radiation based repair proteins that were found in yeast again that the gene involved in Werner syndrome is an SGS1 homolog NBS1 has a functional ortholog known as X-ray sensitive 2 and Seckle syndrome is caused by defects in the ATR gene which is basically an ortholog of mech 1 and also known as rad 3 and a different type of yeast and I think this is a critically important part is that we really do understand a lot about how human biology works by studying very simple organisms and the other thing is that the power of a lot of like your genetics for most of the 1900s really came from trying to understand what a protein does by what happens when it's absent and that is an important way to approach science from a genetics point of view is that we can infer what something does when we design assays that tells very exquisitely what might it be involved in and so anyway that's the first package and story talking about DNA repair cancer aging syndromes and I'm going to go to a little bit of a sideways story called talk about repetitive DNA and this is another story where again if you go far back enough in the 1900s I think 1930s 1940s people did very simple experiments to demonstrate that there was something unusual about eukaryotic chromosomes or genomes and that is in this top left hand corner if you were to basically heat and boil up all the DNA that you extracted from some organism cells and then let that reneal and measure how fast it goes from being single stranded DNA to double stranded DNA this would be a straight line this top chart but the fact that human mouse other complicated organisms dna had these weird bumps in it meant that things were coming together faster than you would expect from normal single copy kinetics and I'm going to give a quick analogy here that if you're say at a catillion and you have a dance card and your dance card is one specific other person that you have to go find you can just imagine you let the whole crowd go and it's going to take a while for everybody to find that one specific partner but let's say it's a 4th of july catillion and all the men are wearing red white or blue and all the women are wearing red white and blue that if your sound card is or sorry if your dance card is just saying dance with someone who's wearing the same color as you are then people come together much more rapidly that's because they don't have to search for something specific they can search for something more general more matching and that's the way these things work and it indicates that large proportions of genomes basically are the same little copy of something over and over and what's for the human genome one of the first clonings and demonstrations of what that was was on the top right hand side again prescott dinier in the lab of carl schmidt carl schmidt a big pioneer in the field what he showed was that they could clone out this specific element and sequence it and they ended up calling it alu because it has an alu restriction site in it but it is a small element that exists in like about a million copies in the human genome and what it is it's just a small little sequence that as far as they could tell expresses RNA doesn't make a protein it's not a gene it's just some small element that expresses itself and so people have used that type of power to just basically make nice pretty pictures and what we have here from the wikipedia talk about alu is this nice picture where they again they stained all the human chromosomes and wherever you see a lot of green that tells you there's a lot of alu elements and so that's just a demonstration of the prevalence of these within the human genome but as far as we know they didn't do anything right they're just there so let's talk about where i think this field really got it's one of those interesting start and that is in the lab of hay kazazian and what we have here in the top is this diagram of the human uh clotting factor seven uh factor just sorry eight sorry factor eight where these numbers and these lines are basically saying here are the exons for this this is how this gene gets made this is the structure of the gene and what they discovered through cloning and looking at an individual patient was this big chunk of DNA sitting in the middle of it for someone who had hemophilia and then another patient they found a similar large chunk of DNA that was a different chunk of DNA in them now when they went and looked at the parents and sequenced the parents dna these chunks weren't there so something must have happened either very early in embryogenesis of the individuals or in the germ cells of the parents and so this is a unique active event and what they've sequenced it and said this is what's known as a line one element a known repetitive sequence in human genomes and they even went through the work to find the source elements and this is the key thing is that these things don't jump in general unless you have an intact element to help it accomplish all the things it takes to jump around in the genome and so what I have in this diagram is showing uh there's a promoter the five prime utr there's an open reading frame that encodes a protein that helps bind the RNA helps bind to dna there's orph 2 which has an endonuclease activity that's the en it has a reverse transcriptase activity and that's where you take RNA and replicate it into dna and so this life cycle allows an intact element like we saw in these patients to accomplish something that's now called transcription prime primed reverse sorry transcription primed reverse trans transposition and so again there's a target site it cuts the dna knows being it's this black flap is coming off so that one strand is cut then it has to come in and cut the other strand in order to then synthesize dna from RNA and then make a second copy and so now you have this insertion of an element just like we saw in the factor 8 sheet now one thing that i'll point out here and this was was very intriguing to me as a in terms of thinking about doing going into this lab was this looks like a double strand break and so i'll come back to that idea i want to pop that idea in your head real quick before i talk a little bit more about why these become relevant this is not just an exercise and these things being in genomes but we now know from this work that these are active that these are jumping around at least in our germ and so again the capstone of the the story of repetitive elements in in certain ways was the human genome sequencing and this was a again international consortium that published in nature there's also the kreg vetner company that published in science and the important thing and the take home message from the first part to the slide is that probably over 50 percent of our genome is based on repetitive elements there's more of them than us when we think about in this top diagram is showing a kind of quantitative comparison of repeated elements versus exons again exons are things that make the genes the proteins in ourselves and they're a lot more red than there are blue and then the other interesting story here this is a diagram i always hesitate to show but i think there's an important lesson to come from it is that this chart is showing the different types of repetitive elements that are historically been in our genome and how active they are and so the x-axis is basically showing how much things have mutated over time and so the x-axis represents n-frame and that what we have now if you look very closely at just these small little peaks here this is the last you know uh 500 000 years and it's not not very active compared to the past so again many many ways we're lucky to have a genome that's more stable with these than it was before anybody want to hazard a guess as to what the total time frame of this chart actually represents this 34 percent substitution in terms of time i'll give you a hint the light blue is an element that started in primates yeah what sort of evolutionary timescale are we looking at here certain primates yeah we're actually looking at 200 million years this just graph actually represents about 200 million years of eukaryotic genomes and our and and human ancestry okay so let me summarize this first part and that is in our genomes there are these intact elements that carry proteins that allow it to jump around and these are known as line one long interspersed nuclear elements i prefer the old time name and it encodes proteins that allow to jump around and move around and their activities dependent upon those and then they accomplish this by a process that means they have to break dna to order to get in there right it's like having to break open a door to get inside a house and so to me when it came time to think about what i want to do for my postdoc and had an offer on the on the table is that in the context of what's going on in the cancer field in the context of what was going on um that to me there was this intriguing idea that these are double-strand breaks that are being recognized by cellular repair factors that may be important for their biology and i'm connecting this back to the picture with the cancer i didn't necessarily know which ones it would be i had a couple good guests i come uh wanted to test a few ideas in the first place but then of course the idea that happened as i was doing the work was how well does this relate to aging how well does this relate to actually a real true biological effect in terms of pathologies or just our normal biology and so that was really the big question was can we really say these are making double-strand breaks is that an intermediate in the pathway does it work that way and so this is uh my main paper from 2006 where again we did one these assays where we use immune fluorescence we have these prot we have this protinos h2ax that have been discovered by other people as being something that is at double-strand breaks very early and we did this assay where we took both human cancer cell lines we took mouse cell lines we basically expressed l1 elements in them and what you can see from this first diagram again the first one's a control so you don't see any spots this next one here lots and lots of spots again if this if we had to try and make this many spots with radiation with an x-ray machine it would have been a highly lethal dose and so this was that kind of wow moment of wow there's a lot more there's a lot of double-strand breaks here than we otherwise would have expected and what we also just double-checked was that this high level of double-strand breaks required the endonuclease function of the orph 2 again there's still a little bit here and that's there's an interesting story there but there's still a little bit of double-strand break activity now the reverse transcriptase is missing in this particular diagram but there's still a little bit of double-strand breaks so it's missing the rt but you don't need the rt to make breaks maybe you need them to stabilize them and then there was also another interesting aspect of this was you don't need that first protein in order to make double-strand breaks you do need that to typically accomplish retro transposition but in terms of at least making some degree of breaks you don't need that and so that was one way of characterizing the double-strand break activity the other one here is this thing anybody want to hazard a guess of what this assay is called anybody know this one this is where double-strand breaks migrate in gels but there's actually an acronym for it so i understand there may be a problem with the chat extender so i'm sorry if i've missed any answers that were from outside of 20 meters away from me uh anyway what do these look like these look like little little comets so this is known as a comet assay and these tails the tails of the comet represent broken dna from double-strand breaks and so again demonstrating that these are creating these in cells when you express them and that again having high levels of double-strand breaks requires having that end of nucleus function this was published in 2006 there actually were other people in the field working on similar work uh the howdy lab looked at um again expressing an element that wasn't you wouldn't expect to express as well and be as active but again they saw the same thing here where in different passages and generations of uh human cancer cells they saw the h2o x foci indicating some level double-strand breaks that persist and go on this other group again i'll talk about this a little more later they basically allowed expression of of elements in meiosis and what they're showing here is the expression of the orphan protein once you allow derepression you get rid of a gene that helps repress retro elements and then over here um these little red spots here this whole black and white the white spots here and then what they colorize red here this is rad 51 our old friend rad 51 that i used to work with in grad school as an indicator that when these l ones get expressed these also cause double-strand breaks in uh myotic cells so one thing that was occurring before i started the postdoc was this uh researcher named john moran who was in hey kazazian's lab developed an assay and the basic idea of this assay is that you express the l one element again by introducing in the cells on expression vectors but you also include a reverse gene and a reverse gene that's gene that is split by an intron and so this reverse gene once it goes through retro transposition gets rid of that intron again notice the yellow bar that you have here actually gets removed here and now that blue gene is intact and what that blue gene represents is a resistance marker that allows the cells to survive in the antibiotic and so what that actually represents these assays as shown here that any sort of colony that's growing in this petri plate represents a retro transposition event that individual colony came from one cell that had a retro transposition event and so what's really useful here is you have a quantitative assay for retro transposition in cells and again this is the type of thing that molecular geneticists and scientists love is a nice quantitative assay that you can work with and then start making lots of charts so and that's they're just demonstrating here that these elements work and heal the cells and make lots of colonies if they're missing the ability to be expressed or if they're missing say the retro the reverse transcriptase part of the l1 they don't work they're in a very nice assay and this is something that they applied try to understand how these things interact with DNA and we'll just cover briefly is this work by Tammy Morish that's now separately in John Rand's own independent lab of looking at cell lines and trying to get colonies from the endonuclease deficient mutants and so the horizontal arrows the horizontal green arrows represent what normally happens with endonuclease mutants in a normal cell but what happens in the vertical arrows those are cell lines that are missing DNA repair proteins so suddenly there's an elevation of how much retro transition you're getting when you're lacking repair proteins and so again that was it's not talking much about how l1 retro transposition works normally but does start to show this interface between repair proteins and how these things jump around and that was this assay and this idea was also the basis of the work I did and what I'm gonna show here is work from my first paper as well as a follow-up paper with Nick Wallace other people in the lab where we looked at inhibitors of DNA repair proteins including caffeine wort man in vanilla these are things that you might have ingested not the wort man in hopefully but the caffeine and the vanilla and so what's represented here these are just colony counts these are just quantifying the number of colonies you see in the bars compared to untreated so the very left hand untreated that's set at 100% and then the black bars represent how many how much l1 retro transposition is going on so the diagrams representing that when you add caffeine to the media retro transposition goes the way so you're inhibiting something that's important for retro transposition and then this is also true of the wort man in that the wort man in also seem to decrease retro transposition uh additionally the one gene of you've might have seen a little bit of atm we co over express something that should impact atm activity something known as a dominant negative kinase deficient and what we saw there is that by impacting atm function you decrease the amount of retro transposition so um we also characterize another protein another repair complex known as ercc1xpf and again similar here we had a cell line that's missing the ercc1 gene and so we looked at the baseline amount of retro transposition that dark gray bar and when we added ercc1 back to the cells we saw a decrease in retro transposition so again this is this argument that this protein limits the ability for retro transposition to occur and we did the converse experiment where ercc1's partner in DNA repair xpf1 if we decrease the amount of xpf1 in cells then these big gray bars go up in terms of how much retro transposition is occurring so again a bottom line is that the work i was doing in in the lab was demonstrating these interactions of how repair proteins play a role are involved in either promoting or limiting l1 retro transposition so any questions about the types of work i was doing to demonstrate the relationship between l1 retro transposition making double strand breaks and how cells reply to that and i'm a step a little bit closer now i have about 20 more about 10 more people in my chat range this is a good point to pause because i don't talk about the impact of these data i don't know if i'll say much about caffeine caffeine inhibits a lot of things and sunbathing is not a good thing uh i'm not necessarily sure they're bad together but all right so let me talk i'm gonna get through the impact of this of this work and this is something that again this was published in 2006 some publications 2008 but i wanted to take again one more little side point here of talking about methylation and what methylation really represents is the chemical modification to genes that help turn them off and cells can turn them back on by getting rid of methylation and this review uh covers this idea that uh in young healthy cells repeat elements again most specifically are methylated to be quiet and that a lot of genes that are being expressed are unmethalated but then there's different status of methylation for different parts of the genome for different genes and the um the thing that he's also trying to represent he's trying to he this is again a review talking about a wide amount of data that's gone into this is that people have noticed that as cells get older methylation repression doesn't work as well it starts to go away you can chemically measure this by measuring the methylation and then if you think about cancer cancer is the same way in that uh when people look at cancer cells and say what's the methylation status of these things then the methylation is is gone from a lot of repetitive elements and so um well again so i think there are some technical points that i want to make from the diagrams but i hope that some of the summary um slides and summary statements i'm making are helping you get the the bottom line the impression of what the the data means and what it's about so this is you know this is one of the key points of what we're working on in the again this was i did my work in Prescott diner's lab one of the key kind of take home lessons from this was well this ability to look at these molecularly doesn't mean a whole lot unless there's actually expression of these is actually happening themselves and so um victoria baloncio who's in the lab was doing some very intriguing work of trying to detect these constructs the expression of l1 elements inside both cancer cells as well as non-cancer cells and what she found was that these can be expressed at a very low level they can be found but then also elements of it that are only expressing the damaging parts the endonuclease and the reverse transcriptase from orph 2 can also be damaging the cells and so this was something where i think a lot of the field had been very focused on the expression of full length as being important and look at methylation of full elements as important but in fact the ability of these parts of it to be expressed could also be damaging and be something related to cancer as well as aging and so i'm going to talk through again again a variety of different talks different papers are published subsequent to this looking at the relationship between l1 and different biology by their mice or human cells and the first one here is looking at how line one seems to play an important role or it's very important to suppress these elements in the cells that make sperm and egg and so this work from the timothy bester lab on the left is showing that when you get rid of a gene that methylates repetitive elements again so now we have un-methylated line one elements are showing that it's being expressed that these green lines you have on the left this is how normal meiosis looks but these green squiggly lines that are basically all messed up and not correct and not going to give you a correct sperm is what you see when you allow l1 expression to run rampant and consequently those mice are infertile and then this was work also from other groups showing that again these little diagrams on the left hand side we have normal a normal mouse on the right hand side they're missing a gene that allows l1 expression to basically go all over the place in gametes and those development cells and you'll see that this cloud of green that you have on the left hand side and these are sperm cells going through myiosis and trying to basically make functional gametes that those populations are missing and once those populations are missing you can't have sperm they're not going to develop and thus you have infertility and they developed this as a model saying that these factors that help with methylation help regulate the RNA basically if you don't have if you have weak repression then you have no progeny of infertility and you need to have strong repression in order to have functional sperm and again they connected this to the expression of these l1 elements and this is a similar study looking at female mice where they're talking about the development of oocytes and the overall impression I want to give here is that they look at a gene that again maelstrom that the red represents maelstrom missing so a mutant and so what's happening in the red cells is that you have more expression of l1 and what's represented here on the left hand side are different phenotypes of oocytes and so this very first diagram is the number of oocytes and you'll notice the first thing you notice right away is that you have a lot less functional oocytes when you're missing this gene the red one and then this is looking at phenotypes of things that have gone wrong and they're lacking crossovers the metaphase is not working this the chromosomes are messed up that when you are missing the maelstrom gene boom you have a lot more defects in cells and then finally this is just looking at aneuploid embryos that the actual you know fertilized eggs develop with chromosome abnormalities when you don't have this repressive gene now this is one of the key things I think is happening that we had suggested and proposed way back when is that the lack of this gene that causes all these problems in oocytes can be partially alleviated by reverse transcriptase inhibitor right now reverse transcription doesn't do anything in terms of human biology right does anybody happen to know a therapy that we use reverse transcriptase inhibitors for is there a well-known popular yeah hiv right so hiv is a retrovirus and you can inhibit its life cycle by adding reverse transcriptase inhibitors and that's what they're doing this example two is they're trying to use reverse transcriptase inhibitors to alleviate the symptoms and that's where you see the light pink the fact that the light pink bars are showing higher functional oocytes than the dark red means that you're alleviating the phenotype which is arguing that again the best example of probably what the cause of this is is l1 being inhibited so that the damage it's causing or its ability to do reverse transcription is what's causing the death of cells in the first now move on to a different topic that talking about the brain and again in summary the several labs primarily fred gauge was very interested in how neurons develop and how brain development occurs and they had this connection that l1 expression can actually get alleviated during developmental cycles and what can happen here is and what these diagrams all represent is bars that represent how much l1 jumping they can detect in these different types of cells so when they look in the hippocampus you see this higher black bar indicating that l1 has been jumping around in those neurons as compared to heart or liver or cardiac tissue and then in this diagram in a different paper they um tried to decrease the amount of a denier of hair protein and in fact in this case they're also decreasing atm the gene that i said earlier seems to play a role in helping promote l1 in this case they saw an increase the amount of retro transposition that was occurring using a modified construct that allowed it to uh jump around more so maybe and their hypothesis was if these are jumping around more in atm deficient cells maybe that explains the phenotype in individuals who are deficient in atm and then these are some nice green cells so there's another assay you can do so i showed a colony assay where every time a retro transposition event occurs a colony is allowed to grow on a plate well this is another way of doing the assay where if the retro transposition occurs things get green so this is a green fluorescent protein and they were showing that in normal cells you have low levels of retro transposition but in a again another a gene that's not able to methylate retro trans retro transposons you get a lot more jumping around inside neurons and then these bars here all these dark black bars the fact that they're bigger is demonstrating that when you've knocked out this gene that you're getting retro transposition at a much higher rate than you otherwise would expect in neurons and so again the summary idea there is that these retro transposons may be contributing to neuroplasticity neuronic variation but importantly when we think about all these syndromes that show brain or developmental delays or other phenotypes in this progeroid syndrome progeroid syndromes that may be one of the causative agents of that is l1 being active in them and causing again DNA damage or retro transposition or other things happening in those neurons getting back to cancer that what these bars represent is expression of retro transposons in different cancer cell lines so this has been shown by a lot of different groups where they've looked at the amount of expression the amount of demethylation other things but they're actually functionally able to see these proteins as a common feature of a lot of different cancers that are actually coming in through you know patient samples and what we have here on the right is again another example again there are multiple in the literature where if you're missing the ability to or if you sorry if you overexpress a gene that demethylates retro transposons again this is a gene that's commonly found in many cancers to be overexpressed that now or two from l1 is being expressed again when you're overexpressing this de-repressor and that in combination you're also getting these h2a x foci that are marking double strand breaks and so again this is connecting l1 expression in actual a molecular phenotype representing cancer that may say hey these things being expressed is causing double strand breaks as mutagenic is a part of cancer and from the patient perspective what this diagram here represents is looking at again cancer samples and just asking what is the prognosis of cancer patients when we try and connect their outcome versus how much methylation of l1 there is in their tissues right this is like a basic question like some sort of marker of survivability and so the green bars represents l1 that's well methylated in those patient samples and the blue bar which is survival so more people are dying in the blue bars when l1 is unmethylated and being expressed in their tissues again similarly and a connecting story back this is something that just came out this year from the jeff bokeh lab is that we can talk again about brocco one and so they did a screen and measurement of denier repair genes involved in l1 and what we're seeing here is when you get rid of brocco one from tissue from from cell lines you actually get an increase in retro transposition and that's what you see in the second and third bars or columns here of the different tissue plates and then when you also decrease the ability of phanconis anemia you get an increase in retro transposition and this is a quantification of this over here on the right that again showing that again compared to a negative control which is always you have to find some sort of baseline in the experiment that by getting rid of brocco one or getting rid of an associated protein called ctp ctlp that you get more retro transposition so then again their argument in their paper was you know in the absence of brocco one or ctlp you get more retro transposition that's occurring all right and then the final example of an impact i want to talk about is l1 and a model of aging so has anybody heard of like this great compound in red wine that might help you live longer there's a researcher named david sinclair who's been studying this resveratol yes so synergy has it and resveratol is something that helps activate a class of genes known as certuans so certuans again actually something discovered originally i think in yeast that had to do with aging phenotypes and so what they've what people have found is that if you knock out the gene serr t6 that mice have this accelerated aging phenotype and that's been well known and their argument and the experiment they did here is really really interesting is that they basically took serr t6 mice who are prematurely aging and basically gave them reverse transcriptase inhibitors right so if we look at this diagram this is comparing survival over time just again overall organismal survival and that the green bars represent normal the black bars represent the serr t6 mice and the red and blue represent serr t6 mice that have been fed reverse transcriptase inhibitor and so it's showing this intermediate phenotype there's some again alleviation of the phenotype when you get rid of reverse transcription in these cells this one is showing body weight so again red and blue is intermediate and then this is this cognitive test where again the black bar represents a long time to get out of a circle get out of a maze type of thing and that adding reverse transcriptase inhibitors to these mice's diets helps them do better and what we also see here is looking at you know various tissue phenotypes how badly damaged are replicative tissues and that the fact that the reverse transcriptase inhibitors are larger and taller black bars means they're doing better off than the mutant mice and then finally connecting this all again together to DNA damage they're measuring how many h2a x foci which as we've discussed represents double strand breaks where again you have wild type normal mice that you're comparing them to the serr t6 mice that have these very large bars and then when you have reverse transcriptase inhibitors you see a lower level of double strand breaks in those and so again their model is that maybe line one activity is very important for contributing to the aging phenotype of these model mice and again maybe something that is important for normal aging as a process so again thanks for bearing with me on some complicated biology i want to make sure i showed the breadth of the biology that's been done in this area try and connect it to the basic biology that was occurring and again as a narrative structure connected to the work that i did all of which i do recognize understand involves some technical details but again hopefully as you're looking at some summaries as i'm about to do and going through this as i talk about it that the basic idea that's coming across makes sense and so this is the summary slide i want to say is that you know l1 these elements that express themselves are considered you know repetitive elements they're selfish dna they may be sources double strand breaks and rearrangements and cdna that's happening themselves and that uh the basic living circumstance we have with them is we try and have genes that keep them from doing this stuff but there are times when this repression uh goes away and so um i think i've been trying to make the case and there have been lots of literature saying hey maybe the activity of these when unrepress leads to cancer or aging or again some defects in neuronal activity and neuronal development and that in particular we think this may be the case because there are times where people are missing these genes in terms of syndromes or mouse models where there are very dramatic effects of these happening so again now going back to the narrative structure i said yeah what about noble prizes in this area and i think if you look at the history of noble prizes in medicine you know there have been technical achievements with a huge impact so the structure dna pcr um embryonic stem cell knockouts uh the protein gfp that i showed is this great molecular tool and fire mellow doing RNA interference so again amazing but they didn't necessarily teach us anything per se conceptually new but important information now there have been discoveries that weren't necessarily incredibly technical or brilliant but had a major impact on human health so penicillin is i think the classic example there but then also the discovery of nitric oxide as a vasodilator right to some degree that was new in terms of thinking about it as a messenger but then also the idea that you know this is now the source of viagra um and then there are these noble prizes where the awarded for something that turned science on its head people have gotten very comfortable with what they thought science was but the noble prize said hey that's actually wrong so mclintok with transposons is a classic example i think my favorite one and he even wrote a book about it that stanley prusner talking about prions again infectious agents that are only proteins and then marshall and warren talking about helicobacter pylori causing ulcers and then fire mellow i think that also changed the way we think about how gene expression is done as well and so when we think about these types of criteria for noble prizes i think one i think if the biology of l1 elements keeps going on the track it does then i think the idea of cancer aging fertility neuronal diseases maybe some others as a source of dna damage or as a source of as a causative agent then i think that case will be relatively easily made and i think the question is how do you award or what is the story the noble prize wants to say and i think if you want to take the people who've done the most for this field you think about hey kazazian who again trained john moran as well as goodier a prescott dininger who again this was the work done in his lab but also victoria's work there are two people haven't mentioned astrid engel and mark batzer who've done a lot of allu biology and then of course is jeff bokeh who i think is probably like the most da Vinci of all molecular biologists that i really know of right now he's done so many things and he actually um just an amazing scientist in many different fields and then maybe methylation so it depends what story people would want to tell like i think if people say that the double-strand break story is really an important thing then recognition for that work would of course make sense uh if you really want to focus on the genetics the fact that these are active then the people who've been doing work and showing these are active in genetic diseases is really important uh so again it depends there's lots of biology yet to be done but i think in terms of understanding where the noble prizes may or may not go then again at a minimum i think this work would have contributed to the idea of how these things cause pathologies and so i've had an hour i do want to make one last dedication so there was a person luminary in the field known as yours yurka uh he is someone who's very involved in the computational understanding of repetitive elements and uh you know i didn't know he died i had actually worked with him helping a student do a research project um and he was just super kind and generous with his time and very helpful so um again i didn't work with him very much but people in the field i think really liked him and he did a lot of really amazing work so a dedication to yours yurka who passed away in 2014 and then there's some background reading in this in the slide so i will end my talk there and i have time for questions happy to stick around for half an hour or whatever to clarify anything to give some more summaries whatever would help with um the talk so i emailed the pdf to shantel this morning yeah i mean there's this let me just also say i know that there were some complaints in the text which i think i missed the most of that beginning but you know some science is complicated and i wanted to make sure that there was both the breadth of the importance of this was to make the case for the nebel prize as well as trying to get into some of the details so vick is asking what kind of medium do you use for the plates and he only worked with plant dna uh the only human cells are doing again one thing that's actually kind of funny about a lot of human biology that we understand through tissue culture is that we want to use adherent cells things that actually adhere to plates and so um um people you know plastic makers make these plates that have relatively good substrates for things like hella cells or human epithelial cells to attach to and so those are the types of plates we're using there again i don't remember the brand okay great violet thanks for enjoying the talk nice seeing you again oh wait i think i saw i think i might have that one pretty fast okay there's a question from radra and ral could this research result in medicines repressing the aging process so i think yeah that's the big idea right um and let me say let me give the positive and let me give the pessimistic so the positive is if these elements are something that are really responsible for aging for causing dna damage and that's something that's just normally happening happening in normal individuals then finding either a that reverse transcriptase inhibitors are effective would mean more people should take those all the time or again maybe more importantly find a very exquisite way of stopping the endonuclease activity there's no drug that specifically targets that i know of the endonuclease activity of these elements again that's possible to make maybe somebody will at some point or maybe there would be a genetic engineering solution to this i'd find a way to repress the expression or to use crisper to inactivate them these are all things that would make sense and so to me i think the the argument of the biology is that that's what we're seeing in these models and then that's also my pessimistic point is that one of the things um uh and i'll get to your point sissuji in a second you know is that when you start from from a science point of view you have to do stuff that you can study and understand and interpret experiments from and so a lot of times working with mutants is a very powerful way to understand stuff but could also be slightly misleading in terms of what the science really really means how you interpret it and what it's useful for so i think that's kind of did i answer your question rater on that would be the way we'll see i think to me the case has has been made is that if you can inhibit this we should definitely find out what happens we should do those studies uh so i have a question here well sorry so a point from um sissuji talking about according to david sinclair DNA damage is not central to aging it's epigenetic change clones are not older than the original organism and so i think you know um there definitely are different models that david sinclair talks about in terms of what aging really represents and i think there's also the hormonal change model uh again a part of aging could be the depletion of stem cells which has to do with the fact that all cells that were born with have a limited lifespan because of telomeres and we don't replenish telomeres um so there's always a cap on aging and that's certainly going to be effect on aging but is that necessarily the contributor to a lot of damage that makes us age faster or makes aging a bad process um so i think you know there definitely are other models for how aging occurs how DNA damage occurs the sources of DNA damage um you know DNA replication is something that is that can lead to double strand breaks and maybe that's the main source we always need to replicate our DNA so maybe that's the issue there okay so uh max asked an interesting question repeated by shantel steven i came in a little late but is there a relationship between l1 retro transposition and the increase in miotic errors that you see in older females so this is the part of the complicated biology i didn't want to get into but my basic answer would be no that the um what's called the um advanced maternal age where the segregation of chromosomes doesn't work very well largely has to do with the fact that female oocytes are arrested before they divide their chromosomes they're they're held there for a really long period of time and that that stringency of holding them that way just doesn't work as well after the age of say 35 and so that's really um that's really the main thing that's happening in that biology and in fact what this this last paper i talked about before you get the oocytes you want to use for fertilization there's this restriction process where the ovaries are recognizing bad eggs and they get rid of them and so that's a part of where the l1 biology may be playing a role it's not so much in um the reason l1 biology may not play as much a role in female fertility as male fertility is because there is this checkpoint where they say are these good eggs or bad eggs and if l1 has made something to get bad egg it never becomes an ovum is i think the question is the way i would answer that question so suit well sorry um let me go back chronologically there's a couple questions about what is the dna damage maybe we're talking about here oh yeah so so actually i answered was suma's question so suma's question was when a noble prizes awarded there's always a one-line phrase that describes what the award is for what is my one line phrase now again i didn't necessarily want to propose one but let's propose it where they do want to award me for the primary observation that i'm talking about for my and there are other ways to phrase it but let's bias it towards me i hope you'll forgive me for that one but it would be you is awarded for the discovery that retro transposons make double strand breaks that contributes to aging cancer and disease that would be the one word phrase and i think that's again that's the key insight that came from from my work was the first one to attempt to really look at this double strand break activating activity of l1 that as it's trying to integrate itself into the game i'm with a life cycle of l1 is is trying to jump into dna is trying to get in there so it's got to break it to get in like a thief which is asking me to repeat the one line phrase and it is the noble prizes awarded for the discovery that double strand breaks caused by retro transposons or retro transposition causes or is a causative agent for aging cancer and other diseases so mushrooms actually ask a really interesting question which is why does it do this right and i think um this gets into some very deep questions because there's not an answer per se but very deep questions about what is the replication of dna what is evolution what is biology that from their perspective like i kind of wrote it in the title these are just parasites these are things that are interested in their own replication they have the chemistry for their own replication they jump around that's what they do and so they're just parasites that do this and so that's what it is i mean it's just something it's a phenomic parasite that's trying to get jump around and get get in there and of course we build defenses for it right biology says hey this is not good for us let's build defenses to try and stop it and then when those defenses break down they go wild no i think okay so yeah that's a very deep question so mushroom asks you know where does this element come from and the answer is people don't per se know they're ancient right these types of activities are ancient the reverse transcription i'm sorry the reverse transcriptase seems to be related to retroviral reverse transcriptases but maybe they're also related to the reverse transcriptase you find in telomerase um so maybe some sort of mad scientist mother nature point happened where you combine the native reverse transcriptase with an n new place so it's just got combined together somehow and then they became active so i think so i think the only thing i'll say about this talk was it was a way for me to not think about COVID-19 uh erin that this is an opportunity to think and do stuff that's not thinking about the current conditions then there's no real relationship between the two well so again so well erin you're not asking about COVID-19 you're asking about a solution to aging okay so mushroom asks again this question which um you know reverse transcriptases so the central dogma of molecular biology as proposed by James Watson is that DNA is made into RNA which is a similar related molecule and then RNA makes proteins and so that's a stepwise that's a forward path of how we make make genes how we make proteins from genes and the reverse is basically taking RNA and turning it back into DNA and so that step we've heard of retroviruses hiv that's the reverse of that process so that's why it's what we call reverse transcriptase so i think so there's a question i think i again because of the chat range extender i've missed some questions uh there was one that was just what's a brief summary of what l1 is or does i think i've kind of answered that question that it's a small parasite it expresses itself expresses proteins to move around um again just to summarize other aspects of the talk to say that this activity of trying to jump around genomes can impact DNA and cells negatively and specifically it looks like it's x-ray damage like making double-strand breaks that's breaking DNA rather than ask an interesting question will this be used to insert genes or for gene therapy people have um developed constructs that do that the problem is that this reverse transcription and the insertions elements is variable you don't always get the full length thing back in fact most of the time this reverse transcription maybe because of DNA repair proteins maybe because it's just not very good you don't get the same size the the thing the size of the thing you're starting with you never get that same size back and so when it comes to like gene integration gene delivery vectors so there's so many other ones that are much better at it than that so you don't want to use this process for that yeah tagline mentions again a classic nibble prize david baltimore showing again he was awarding the little prize for this again this this um lateral thinking type of thing that viruses particularly um reverse transcriptase viruses can be causative agents in cancer and that was something that people had been kind of not thinking as as a cancer source so david baltimore developed the baltimore classification of viruses based on their mechanism of replication well so again the source of the reverse l1 you're asking is it a non-human virus and i think you know that's a reasonable way to connect and think about it that when we think about hiv or reverse you know um retroviruses it's similar it's trying to do something similar but it's never trying to leave the cell so a lot of retroviruses of course make coat proteins and then try and infect other cells whereas these are just genome parasites because they never leave the cell on their own so len brings up the point that they say Alzheimer's is cancer of the brain you know brain pathologies have a lot of different causes and thoughts for where they're coming from you know some of that has to do with inflammation some that has to do with maybe pre odds misfolder proteins some that may have to do with just the lack of sleep you know there are these things that happen in brain biology that's a little bit different these may be related to certain types of pathologies where the DNA damage the fragility of the genome may be relatively maybe important for how brain development occurs oh welcome david so shiloh asked about leukemia is this a condition of perpetual DNA damage and so a lot of the classic leukemias like um the jant rally covers this in some ways and the diagrams in there that a lot of leukemias especially the childhood ones are an example where translocation occurs and makes a novel fusion protein so maybe you take the ability to express and be a stable protein from say gene a and then gene b part of it that tells the cells to grow rapidly without the fact without the ability to suppress that activity and so these gene fusions these chimeric genes and proteins are considered a primary driver of leukemia now there are other leukemias that seem to be related to dna damage uh i don't know that anybody i haven't delved into it particularly about how much dna damage is related to lots of classifications of leukemia yeah no i think the entire line mentions the right thing that cancer requires a disruption in the control of cell replication again there are four main things considered in neoplasticity and that is one unregulated cell cycle unregulated and driven proliferation the ability to metastasize moved to other parts of the cell and then also immune evasion and so this model we have for how cancer occurs means that lots of things have to be going wrong and that these are changes in again in many cases in mutations but maybe as mentioned before epigenetics were expressions different again the genes are mutated but the expression patterns are different you know it's very complicated cancer is like a super complicated thing but mutations that help create these steps the gene dysregulation that leads to a cancer phenotype mutations are one of the underlying i would say features of it especially when we consider that when you compare the chromosomes of cancer cells that have all these translocations disruptions wrong numbers of chromosome copies that seems to be an underlined feature of a wide variety of cancers yeah yeah scissors is right this depends on how you define whether cancer is a disease or a multitude of different ones i think it's important to classify them as a multitude of different ones because the way you treat them is very different so breast cancer one of the classic ways of treating breast cancer is that there's a proliferation of cancer cells you treat them with estrogen inhibitors and then in fact if you're like two-thirds of breast cancers if you treat them with estrogen inhibitors this proliferative signal to grow goes away and there are examples of older women who go through menopause and then their cancer goes into remission and that's a kind of one of those interesting aspects of thinking about how the specific tissue you're dealing with is important to treating the cancer well and that's sometimes part of the design which it is that you um you try and get the main message across your audience get them thinking about it even maybe they don't understand all it but they get thinking about it you can answer those types of questions delve into it well okay so shall I ask an interesting question what about men who face prostate cancer they are not undergoing hormonal changes what kind of cell damage is occurring yeah prostate cancer is not something i'm an expert on uh one of the things that is an indicator of prostate cancer is this although this is also controversial is prostate specific antigen which i think is something related to prostate cell growth and then release into the bloodstream you know um i don't other than the general idea that DNA damage is important for maybe being a causative or beginning agent for prostate cancer that that's a problem and the fact is you know prostate cancer oh i'll come back to your point the middle of tagline that you know prostate cancer again is not one of these cancers of aging you get older and maybe you um have some alleles that give you a predisposition to it but again it's an you know cancer as an as an aging related pathology is kind of still out there so tagline mentions that you know the way to treat prostate cancer is chemical castration that again testosterone is something that still drives the growth signal for prostate cells and so if you get rid of that driving signal that doesn't mean you've cured the cancer you just mean you've cured a part of the issue that's driving the cancer to do with no no i know i mean cancer i mean surgery i think it's usually a main option for prostate cancer i don't know no no again i'm i'm a microbiologist i'm not a medical doctor so i can't necessarily answer those types of medical questions well thank you ariana we'll see um you know the one thing that of course is important to recognize in Nobel prizes is that they usually weren't the people who have a long history of of contributing to the field so i think um i'm not in that field anymore i don't do retro transposition biology anymore well any other questions about l1 biology DNA damage thanks erin by a mushroom you're welcome everyone thanks for coming yeah i guess this will be a long video although the talk itself did hit an hour all right with that i'll close it up thanks everyone for coming um i'll probably be online for a little bit longer if you have any questions just i am me otherwise i will um see you all next week or next time