Scientist Stories: C. David Allis, Epigenetics Inheriting More Than Genes

Well, good morning, ladies and gentlemen. My name is Agata Smogorzevska, and I'm an assistant professor here at the Rockefeller University. I'm the head of the Laboratory of Genome Maintenance, and it's my pleasure to welcome you here today. The Rockefeller University is one of the world's leading biomedical research centers. Founded just over 110 years ago, we have had our home on this site for over a century. In that time, the university has created a culture resulting in an unparalleled record of achievement. Scientists associated with Rockefeller have won more Nobel Prizes in medicine and chemistry than any other institution in the world, 24 to be exact. If Rockefeller University were a country, it would rank 4th in the world in Nobel Prizes after the United States, the United Kingdom, and Germany. Scientists in our 73 laboratories conduct innovative studies aimed at improving the understanding of life for the benefit of humanity. To give you some more background on what makes Rockefeller University special, we now have a brief, brief video for you.

Rockefeller University was formed in 1901 by John D. Rockefeller Sr., and the reason it was formed was that we had reached this the point in the state of medicine where you could diagnose what was wrong with people, but we had no ability to stop the disease that they had from killing them. Rockefeller had an advisor named Gates, and so Gates came up with the idea of bringing together the best and the brightest in one location to basically study how do we interact with disease and how do we stop disease. My own laboratory studies a rare group of patients who, amazingly enough, have an immune response against their own cancers. We're able to discover something that people haven't seen before, which is human beings have a natural ability to get rid of cancer. Not only do the best scientists want to come here, but the best students, the best fellows, the best clinicians want to come work here. This is the number one place in the country, possibly the world, to do this kind of research. So I'm privileged to have an absolutely spectacular group of people working with me in the laboratory now and hopefully in the future.

Rockefeller is important to what we're doing because it's one of the only places in the world that actually prizes risk-taking and has allowed us to do completely improbable scientific experiments that we just wouldn't be allowed to do at any other university. Mosquito disease transmission is an enormous problem in the developing world. Malaria kills a million people a year. Several hundred million people are sick and infected with the malaria parasite. To try to find out what what that substance is that's either attracting or repelling mosquitoes, and that substance, if bottled, could be an incredibly important tool in preventing mosquito disease transmission.

Rockefeller is just an incredibly exciting place to be because of the strength of the basic sciences on the one hand, and the commitment that's always existed here to taking that knowledge and translating it into medicines that can help patients. We need to identify those with passion and with the intellectual capabilities to back up that passion to go into medicine and science. Biomedical research is our only focus when we're here, as opposed to being at a large medical center where often your research competes with the time that's spent in clinical care. Rockefeller is second to none in providing that protected time. Obviously, I'm very thankful for the investment that Rockefeller has made in me. You know, as part of the Clinical Scholars Program, I had 3 years of protected time. That gave me enough time to focus on my research, generate data. Now I'm an NIH-funded investigator, and that was not a small accomplishment, and I could do it thanks to the investment that Rockefeller made. Rockefeller University is very unusual in that it is not divided up into departments reflecting different disciplines, because departments can form barriers between different sorts of research workers. What we have here, no barriers between the basic researchers and the clinical researchers. Having no departments is critical. Every head of lab, tenured or not tenured, reports to the president of the university, period. This is really the golden age of disease research, translational medicine, and drug discovery, and I think that medicine is going to be transformed profoundly in all of those different areas.

So before I talk about the tradition of this holiday lecture, let me tell you how I started in science and became a professor here. I was born and raised in Warsaw, Poland, in Europe. When I was 14, I spent half a year in junior high school in Los Angeles. My English was very rudimentary, but I hope it quickly improved, as hopefully you can see. My major achievement was playing first cello in our orchestra. And when I came back to Poland, I joined a high school class which emphasized math and physics. And we had a lot of math and physics. But we also had some biology and some chemistry, and I quickly fell in love with biology. I remember distinctly, uh, when I realized that experimental science might be for me. We were discussing cellular division, and I imagined being the first person describing mitosis, the process where chromosomes are being divided into new daughter cells, and what a thrilling experience that must have been. The promise of discovering something that nobody else knew propelled me into doing science in 3 different laboratories while I was an undergraduate student at the University of Southern California. During the first summer, I studied how pieces of DNA were being moved between bacterial cells. Then I studied how Drosophila, the fruit fly, aged. And finally, I studied how human cells aged. These hands-on experiences were the most important in preparing me for a career in science. To continue my education, I joined the MD-PhD program here in New York. My medical school was just next door at Cornell University Medical School, and my doctoral work was here at the Rockefeller University at the— in the laboratory of Dr. Titia de Lange. For my doctoral thesis, I studied how chromosome ends known as telomeres function to protect the information that is stored in our genome, in our cells. Rockefeller is a terrific place to do science, and it's so terrific that after my further medical training and postdoctoral work in Boston, I was thrilled to become a professor here. I started my lab 4 years ago, and we study how the DNA, which is constantly being damaged, is being repaired, and how those repair processes are essential in preventing cancer formation. My lab has trainees of all levels, including graduate students, medical students, and postdocs. During summers, we have undergraduate students as well as high school students. I will be joining you for lunch, so if you have questions about my decisions to become a scientist, do ask me. And before lunch, I will also introduce you to some other students and postdocs who are here with us in the audience. So they can also share their journey with you. So let me now tell you about the holiday lecture. Part of the Rockefeller University's rich history is its long tradition of giving back to the community. We continue the tradition today through our many science outreach programs, which offer education initiatives to New York area students and teachers. Today's Talking Science event is presented as the 54th Annual Holiday Lecture on Science for high school students. The lecture series was established in 1959 by Alfred Mirsky, a biochemist and Rockefeller University librarian. Dr. Mirsky modeled the lectures on a popular series of science talks for children established in London in the early 1800s by Michael Faraday, who is considered to be one of the greatest experimentalists in the history of science. It had been my hope to mention all of the schools represented here today by name, but I'm pleased to say that there are way too many to do that. I can tell you that there are approximately 90 schools represented, the most we have ever had at this event. And you have traveled from Manhattan, Brooklyn, Queens, Staten Island, the Bronx, Westchester, Long Island, New Jersey, and Connecticut. Thank you all for being here. I also— I also want to give a special welcome to those of you who attended this event as a student and now are here as teachers. This is an inspiration to us, and we like to believe our— your experience at the Rockefeller University helped cement your decision to pursue career in science. Now on to today's event, Epigenetics: Inheriting More Than Genes. For today's lecture, it's my pleasure and honor to introduce to you Professor David Allis. He has been at the Rockefeller since 2003, and he's a Joy and Jack Fishman Professor and head of the Laboratory of Chromatin Biology and Epigenetics. He obtained his Bachelor of Science degree from the University of Cincinnati and his PhD from Indiana University. For his thesis, he worked in Dr. Anthony Mahowald's laboratory, where he was studying the development of Drosophila melanogaster, the fruit fly that I already mentioned, a very useful model organism that a number of laboratories here also study. Next, he did postdoctoral work in Dr. Martin Gorovski's laboratory at the University of Rochester, where he started to study a component of a cell that is continuing to study today, that is the chromatin. In his postdoctoral work, he used a unicellular organism, Tetrahymena thermophila, another model organism that brought us amazing information about fundamental processes, processes that occur in cells. Dr. Allis has published over 300 articles in the field of epigenetics, and you will get a glimpse of this field and his discoveries in today's lecture. He has so many awards that I'm not going to mention all of them, but I'll just like to highlight a few of them. In 2003, he received the Masry Prize, and I mention it only because it's given by the University of Southern California professor who I personally know. In 2004, he received John Wiley Prize for distinguished research in biomedical sciences. In 2007, a Gardner Foundation International Award given by Canada. In 2005, he was elected to the National Academy of Sciences, and I know that there are many awards in his future. So please welcome Dr. Allis, who will present What is Epigenetics and Why Isn't Your DNA Enough?

Thanks. Certainly, let me thank Agata for that overly, overly kind introduction, and then let me echo her comments welcoming all of you, whether you're students— I'm so impressed you're here— your teachers. This is really an overwhelming privilege for me to give this year's talk on what my lab is doing. Thank you so much for coming, and here we go. So my first real slide other than the title slide gives you a glimpse of either different colored fly eyes. We've heard about melanogaster already. Obviously different mice with different coat colors, different sizes. Some of you might recognize the calico cat as a footnote. That is our calico cat. So that's for my wife who's here. And two women that might look identical to you. Oh yes, they are identical twins. But in fact one of them is autistic. And this actually made the COVID of a, a symposium at Cold Spring Harbor dedicated to epigenetics. The point I'd like to make is no matter which one of these biology examples you like, if you think you see a different phenotype, a different coat color, a different eye color, obviously one woman phenotypically normal, the other perhaps sister not So, the question would be for you, and one of the main points I'd like to make is their DNA is all identical, giving rise to these different phenotypes. And this really, that identical aspect of these different examples giving rise to different phenotypes is really at the heart of sort of epigenetic problems that we want to discuss. Now, I looked for cartoons. You'll get the feeling through my talks I'm a big cartoon fan. I don't draw any cartoons, but I think this is the oldest cartoon I found. Believe it or not, I wasn't even born when this cartoon appeared. But you can see the problem here. This poor soul is now turning his report card over to his dad and literally saying to his father, "Hey, Dad, what's the problem here? Do you think it's heredity? Ouch." Or environment. And I don't know what the father said after the cartoon happened. Of course, how exciting was it when now over a decade ago the human genome was sequenced? And these two covers of these two prestigious journals, be it Nature or Science, really displayed on their covers the excitement that we anticipated as scientists. Because, after all, now we knew literally, at least in this example, the sequenced genome of a human being. And a lot of you, I know, stay up with this. You're going to think about the beauty now of what we've been able to tap into with regard to personalized medicine. It's really quite staggering. And so a lot of money at the time was spent on this endeavor, and we were excited about it as scientists. I mean the community at large. Because now we were gonna— so the logic of the Human Genome Project— now we were gonna understand that genes will determine disease states. This is important for me. The older I get, genes determine aging. And ultimately genetic analyses, whatever form they take, will give us diagnostic tools and therapies. That would be the best-case example where we could correct human pathologies, giving rise to personalized medicine treatments. And I'm happy to report this is happening, and so this is a very good thing. I don't want to leave you with that misconception. Now, if you go and spend not a lot of money, and it's just a cheek swab, you can go to companies— I just lifted this from their commercial homepage— 23andMe, and you can actually find out quite a bit about yourself if you want to spend $50. And so let's just sort of walk through some of this. I won't ask you to really— and I apologize if you're in the back, you may not be able to see this very well, but, you know, carrier status. So let's highlight one. Well, sickle cell anemia, absolutely a red blood cell oxygen hemoglobin carrying disorder. It's more rampant in the Afro-American population. You can find out if you're a carrier for that disease. But wow, drug responses. I thought the one that you might be able to relate to as a group is Caffeine metabolism. Ouch. 6-hour, 5-hour energy drinks, popular in our household. Traits. Eye color, a no-brainer. This is what we think about when we think about Mendel. But now, maybe more to the heart and soul of what we might really care about, if you're willing to take this test, is breast cancer susceptibility for women. Very popular in the news media. I have to worry about this longevity. I don't know if I want to know the answer, but here we are nonetheless. And for the men in the audience, just to be fair and balanced, prostate cancer risks absolutely exploding with real genetic insights into what genes are potentially altered in underlining these traits, these diseases, and various phenotypes. So, this company will tell you the more you know about your DNA, the more you know about yourself, and they hope to make some money from you. This cartoon was sent to me, which I think really sort of ramps up exactly what we might be thinking about. Maybe there's a gene for everything, and perhaps the right scientist can, you know, break through the laboratory door in a eureka moment and say, I found the gene that makes us think that everything is determined by genes. But what I want to have you think about is, is that the whole story? What doesn't add up here? So these were some of the puzzles for what sort of the Human Genome Project left us with that were sort of head-scratching. Just the dots didn't connect. First of all, even in us there was a reasonably small number of protein encoding genes. Truthfully, not that many more than flies and worms and fish. And I think that's a bothersome idea for scientists. How do we wrap our heads around that? We ought to be the kingpin species. And so why was that? Of course, like the example I showed of the two identical sisters, where does individual variation come from? And how can you have one autistic monozygotic twin where the other sister is normal? Past experiences, do they really influence us? Or wait a minute, is it possible there's something even in the past from our parents? That, that's absolutely mind-boggling. And again, diseases. Can every disease be explained if you knew the genetic makeup underlying that person's entire genome? So, what is epigenetics? That's one of the things that the title was supposed to sort of advertise. And you might think of genes as being the words in a written English language. And epigenes is how you might sort of alter how those words are read. So, do you boldface the words? Do you italicize the words? Do you underline the words? You're not changing the words at all, but you're certainly changing how those words are read. And for more of you, I think the analogy of if you thought of genes as being the computer hardware, maybe just the software is the epigenome, epigenes or epigenomes. And of course, the excitement now in epigenetics is really, I think, illustrated nicely by this puzzle piece. I'm not saying epigenetics is the center of the universe, so this artist thought that it might be, but if you look at what might now be explained by epigenetic links that are not explained by your genes alone, it's a pretty staggering amount of biology. You could pick your own favorite puzzle piece, but I would make a strong case standing here today that it's the fact that human diseases and human biology are now, and I hope I can develop that in my two lectures, the fact that those are now being explained by epigenetics has carried this into another level. As you, or some of you, go into science, perhaps, you know, you'll have to sort of think at what suddenly carries your, the community's interest, and I think certainly human disease is way up there. So epigenetics is not new, even though I might be advertising it as somewhat new. It actually traces back to Conrad Waddington, in the '40s. He was a developmental biologist and he was trying to explain how in development, and this is his famous highly reproduced sort of contour epigenetic landscape map, that a single genome early in development might take different paths. So this is his original drawing. Here's a newer version. But should that genotype, think of the ball as a genotype, should that fixed genotype roll to the left or roll to the right would indicate at this level different troughs that gave different phenotypes. So now think of the fly eye colors, the different size mice, the calico cat fur patches. And the term epigenetics is just a fusion of the Greek prefix epi, or above or in addition to, to genetics. And genetics again is I think, fully accepted. So you might— this might be insulting for some of you, but I was shown— or I liked this slide. It's very simple. So if you took a cake and you baked the cake, how might you change that sort of to give you different flavors? Just add different frostings. And I think that really works well because, again, epi, I'll remind you, is above, on top of the genetics. And here we're just adding frosting. We certainly get many different tastes, but you might be saying, get real, why is he up here? And I think now relevant to real biology, and especially for those of you that might be interested in development, if you took the early cells of an early embryo, we're gonna ultimately have to try to understand how these can give rise to very different cell types and cell identities. A process called cell specification. Now, the important part here is that the DNA almost in general is fixed. It's not able to change. So it's always been a puzzle how you could get rise to different blood, nerve, muscle, skin. But you're not really changing the genome. You're not altering the DNA. So of course you might just cop out and say, well, but there's factors then that bind to the DNA and change these programs. Fair enough. But I hope to convince you there may be even more. And sadly, once these landscapes are sort of locked into their fates, there can be mistakes made. These cells will dedifferentiate. They will go backwards. They will take on a proliferative potential, giving rise to now what is clearly a devastating human disorder, various human cancers. Now, the idea that really genetics and a gene was what we should really be thinking about is a magnificent Rockefeller discovery. This is the very hospital that you saw in the movie that we just saw, and it was these three Rockefeller physicians— Avery, MacLeod, and McCarty— who literally proved unequivocally that the transforming property of cells was DNA itself, was the Watson and Crick molecule that we've all grown up with. But it was these Rochester physicians in this hospital who literally laid the groundwork for it is DNA that is a gene. Now, here's my first sort of audiovisual sort of question. We understand DNA. The question for you all is how much DNA, if you were to take every chromosome and line them up, not a fair thing to do, but line them up, how long would it be? So, just for a second, think about your answer. And I hope that a lot of you might say, let's just see a show of hands, a meter plus, You know, the 3 feet plus? A yardstick length? How about a meter's worth of DNA? I didn't see that many hands. 2 meters? Ah. 5 meters? Well, the right answer would be 2 meters worth of DNA. So that's a problem for cells. And so our cells, have had to struggle with how to pack that DNA in the confines of what we call a true nucleus. Every true eukaryote, meaning true nucleus, has to package those 2 meters worth of DNA into what turns out to be a distance in diameter of, on average, 2 micrometers. A eukaryotic nucleus might be roughly that size. Now, how you're going to accomplish this seems to be an elegant packaging routine that ultimately takes you through this sort of remarkable bead-on-a-string chromatin fiber, and then a lot of you will recognize metaphase chromosomes. And let's just focus on the simplest unit of this packaging, and that is a famous now chromatin particle that my laboratory and many in the world work on called the nucleosome. It's a fixed amount of DNA wrapping around 4 conserved, almost invariant histone proteins. And some of you— so they're sort of like the inside aspect of this, and the DNA wraps around the outside. And some of you may see these remarkable protein sort of extensions that are hovering out that are often known as histone tails, and, and they are turning out to carry many of the signals that I'll talk about in my lecture that literally dictate epigenetic regulation. A better look at the nucleosome is shown here. This is actually the published crystal structure. What does that mean? This is an atomic structure done by Tim Richmond and colleagues where we got the first look at atomic resolution of how these four histone proteins that make this core, hence core histone proteins. This is their conventional now color code, come together to make this internal scaffold upon which roughly 147 base pairs of DNA wrap.. And because this was a crystal, they weren't able to sort of really see these tail protrusions well because they're too disordered. Now, I'm not sure how many of you think this is a beautiful structure, but this is in every organism from yeast to man. There was no change on how nature decided to package our genome. That inspired Lindsay Baker when she entered my lab from Harvard University. She's now a PhD and she's moved on to do her postdoc. She made me this incredible— I wouldn't know really how much work went into this cross-stitch. But it's like— this was actually a Secret Santa present. And this caused my family to take me out of the Secret Santa pool. Too much pressure. So Lindsay put the bar that high, but you've got to admire her hard work. Nucleosome, sweet nucleosome, LB. So what we have less clear understanding is how this bead-on-a-string fiber literally packs, but we have seen literally higher-order structures. And this makes me want to introduce to you Dr. Aaron Goldberg. Aaron was an MD-PhD student in my lab. And because I can't juggle a lot of things, but I can't drip— so this turns out to be another gift. Yeah, this is Vanna White. So, but Aaron seriously was in the same MD-PhD program that Agata was in. He's now doing his residency. But what I want to have you notice, this was a gift to me, but the inside sort of white discs would be these histone proteins. And then you can see the DNA. It's actually two turns, two superhelical turns of Watson-Crick DNA wraps around each of these nucleosomes. And thanks to Aaron, if you get the impression there may be more extended open regions and more compacted regions, then we would be asking, you know, what would be the molecular switches? Thanks, Aaron. No problem. He's going to be a terrific surgeon. I've had very good people. And as a footnote, to get you back here, Aaron's going to be a star of my lecture 2. His work is going to be highly featured in my next talk. So what would you think about for making a switch? And I don't think it takes a rocket scientist to think that if you had it open, that might be more conducive to genes being on. If in fact it was highly compacted, that might make it more sort of, you know, resistant to gene expression, more silent chromatin. And so this leads us to sort of these cartoon versions, but what I was just trying to show with Aaron's help on the model, and the most important part of this cartoon— and I'm sorry it's a little small— But on these histone tails, I'm depicting little tiny sort of green balls as a chemical on mark or red balls as sort of a chemical off mark. And those chemical marks were also discovered— I kid you not, I'm not making this up— they were discovered later in time at Rockefeller University again by Vince Alfre and his colleagues, who was under Mersky, who you heard about starting this holiday lecture. And it was Vince Alfre who said, I can figure out who that green and red mark is. And his landmark studies, now in the roughly the mid-'60s, called attention to two chemical modifications of proteins, one referred to as acetylation and one referred to as methylation. And I don't need Aaron for this now. I think I'll just say very simply, you know, this is far too simple, but if some of you can see these little flags, acetyl, although I apologize it's not green, and methyl, although it is a stop sign in red. Now again, we can help Aaron, but it was Alfrey's idea And you can see this nice GIF. They have little holes. Pretend this is the histone tail. If you could add these chemical modifications selectively to one region, Alfre's idea was that this might cause the chromatin to open. And believe it or not, think how much he got right in the mid-'60s. If you had another chemical mark, and his data suggested the methyl was more like a tag or a flag for this compacted chromatin, this would be repressive. And I just have to say, he got it right. So, thank you again, Aaron. So, okay, thanks. So, let me show you, for those of you that are more chemically inclined, just how simple these groups are in real chemistry. The acetyl groups are shown here. And it's a CH3-C double bond O, and it will always attach to a lysine residue in histone proteins, or for that matter, other proteins as well. Here, for some of you, is a lysine residue if you know amino acid structures. Amino acids, of course, are the building blocks of proteins. There's roughly 20 of them that are scrambled together, encoded by genes. But they have a very reactive amino terminal group. It's positively charged, and when you add an acetyl group, you change that charge. And that was Alfred's idea. That was the kernel of his idea. Negating that charge on the histones would weaken the interactions with DNA. Why? Because DNA is negatively charged. Plus likes minus, but if you lost that plus, It would open the chromatin up. Methyl groups— think about this. How similar is a methyl group? It's just CH3. It also is added to the amino terminal ends of lysine residues, but it doesn't change the charge. And how remarkable that that turned out to be, in fact, an off signal. So where did my lab enter the picture? Now we're literally years later. And so about mid-1990s, and a couple of brave people in my lab decided, well, if we really want to understand the switch that might interconvert off to on, we better understand the enzymes responsible. Enzymes, as you know, are proteins. They do the chemistry of adding these chemical flags, and the one we went after first was called a histone acetyltransferase. It was transferring the acetyl groups onto histone tails, and it was nicknamed a HAT. In converse, there had to be opposing enzymes that stripped these groups off—histone deacetylases. And that seemed to be a popular switch, and I'm not going to change the chemical flags,. But if you get this, if this was a red decoration, there would be enzymes that add methyl groups, not to be confused with acetyl. They would be histone methyltransferases. There would be enzymes that oppose that activity. They are demethylases. And the question was, for you, how would you identify these enzymes? You're me. I'm a nobody, assistant professor. I'm trying to get students and postdocs interested in You know, you have to make judgments in your career, and I decided, as Agata mentioned, to do what we were gonna do, for good or for bad, we were gonna use this ciliated protozoan Tetrahymena. Now you gotta be kidding me. This isn't attractive. This is a pond water organism. And what attracted me to it as a model, I wanna stress as a model, I mean, how far removed is this from a human disease? It has two nuclei. That's a unique feature of ciliated protozoan. They have a little micronucleus and a larger macronucleus. Here's a real stain of the two. And what I thought was just too fascinating from a biology-alone perspective is the micronucleus makes no genes expressed. Zero. It's dead. It absolutely, for those of you that know transcription, it transcribes nothing. Nada. The macronucleus, on the other hand, is probably the eukaryotic most active nucleus known to man. It transcribes its genome like no other nucleus does. And the lab that I joined in Rochester, New York, had learned ways to grow Tetrahymena separate the two nuclei, literally pots of Mike and Mac. And I thought, let's look at the histone proteins. And I— this is too much detail, but I wanted to show you one experiment of my own just to let you see how bad my handwriting is. And this was like from the Civil War era. But when I did my best attempt to run the histones on a particular gel system, unimportant, from macronuclei, that's the active, or the micronucleus, you could criticize unloading, but I could tell there were these ladders. I could see these rungs of a ladder. I think you can see them very nicely here. There was a band, a band, a band, a band, and when I looked at the micronuclear compartment, I saw the first rung of the ladder but not any of the others. And I just thought, holy cow, I tracked this down. It was all, all this laddering was due to acetylation. I made the emotional commitment to then purify this enzyme system from the tetrahymena macronucleus. And most importantly, I recruited Jim Bernal into my lab to do it. So this is the Jim Bernal who literally loved the cold room. He always wore this hat— get it? Hat?— going into the cold room. This was his passion in life. And now here's another audience sort of test question. You're Jim Bernal. You're growing Tetrahymena. You're going to make a decision to try to purify this appropriate nucleus and then extract the enzyme, the mystery enzyme. Here's the question for you. Are you gonna do it from a flat— this is just water. If I drop this, it's okay. Ah, it's probably not okay with Rockefeller, but 200 milliliters, option 1, there's a choice. So you got this on a flask, you got it on a shaker, or 2 liters, an order of magnitude different, 2,000 mL. So 200 milliliters, 2,000. I'm just gonna leave it for one of our test questions in the afternoon, what you think Jim did to start the purification of this enzyme. Importantly, Jim succeeded in doing it. And Jim won the 19— I can't— these are real pictures. Jim won a year later. This is important. He was in Stockholm, Sweden, rubbing shoulders with the real Nobel laureates. He won the prize for the best PhD thesis for his discovery of the tetrahymena hat. A first. And my question is, where is the real Jim Brunell? Who looks happier? There's— I've never forgotten this. I've even gone— Jim's now a successful person in a big pharmaceutical company. He still looks miserable in a tie. I think of Jim as the left-hand person. And when Jim was in Sweden being interviewed, not necessarily by all the press corps that the Nobel laureates were, but this was printed. I just thought it's a quote that I hope you remember from my lectures. Jim said literally, "Science is so funny. You're famous with about a dozen people." And I thought that is Absolutely right. Nobody recognizes us when we get off a plane. There is no paparazzi. So you do this because you love it. And about 11 other people think it's cool too. So little did we know, and we're going to fast forward 10 years, these enzymes were identified, both the on and the off. They were crystallized, meaning their structures were obtained. They were drugged and they went into clinical trials. And here's a patient that was suffering— it's the same patient. It's a CAT scan from the upper chest cavity and the lower chest cavity. Importantly, on the left, this is before they received drugs that are now FDA-approved. You can see the large solid tumor masses. This patient was treated across the street at Memorial Sloan-Kettering. Here's the patient after 8 weeks of continuous drug treatment. Here's this tumor. You know, this is before, after, before, after. I'm going to go through this quickly, but now these drugs have been treated multiple types. This, this was actually a metastatic lung tumor, as you can tell. This was a woman across the street that was being treated for skin cancer. Here's the after. It's more important to look at the after than the before. But if you caught the face before, not a pretty picture for this woman, but now a much happier outcome. So, Jim now is, you know, his work is, I think, much more regarded than the 12 people that he talked about in his quote in '96. Now here's a fly example. See, I'm confusing you. We're now kind of going way out of human biology. But I want to make an important point because it's like, why do science? So Drosophila, it's easy to spot eye color. And I apologize, you'd think red eyes would be encoded for a gene by red. But nope, fly people call the gene that makes red eyes white. That's pretty scary because they name their genes based on the mutant phenotype. But nonetheless, there is a gene here on the tip of this chromosome that when it moves close to heterochromatin, that's that silent chromatin, it turns off. And so what are wild-type active genes that give red eyes here, now when they're maximally silent, give white eyes. But the only thing that's changing— here's the point— this white gene— I apologize, I should cheat and call it the red gene— It has only translocated because of an X-ray break that's now moved it closer to heterochromatin. But the DNA— here's the point— the DNA has not changed. It's identical, and even this gene has been sequenced. There's not one base pair change, but it's on here, it's off here. And so it's called position effect. You're changing the position of the gene. It's running into a bad neighborhood. I moved to New York and I told somebody, "Oh, you know, it's amazing. Heterochromatin is a bad area." And somebody told me, "Well, in New York, silent neighborhoods are good." So I've never forgotten that. But gene people, Drosophila people, they screen for mutants. And I want to tell you what is the key gene. Now we're talking gene. That determines this position effect. And get this, you gotta, you gotta be a believer if you decide to do science. The gene was picked up in a screen for suppressors of this position effect. The 3 stands for it was found on chromosome 3, and it was mutation number 9. That's all you need to remember. Chromosome 3, a mutation I should say, a mutation on chromosome 3, mutant number 9. We decided this was so remarkable, we should find out what SUVAR39 encodes. We did the biochemistry with the very person who sent me this gift. I'll show you his picture. He was from Austria, Vienna, Austria. Thomas Genuine, and this is a picture of a board in my office to this day. I like these colorful histone, but I just want you to note, believe it or not, you won't think I'm telling you the truth, SUVAR39 was an H3, histone H3 methyltransferase, that's a writer, that adds methyl groups selectively to lysine 9. I mean, go figure. Did you get that? Do I have to say that again? The mutation SUVAR39 in reality was the first ever— now we're not talking HAT, we're talking methyltransferase that literally takes the histone 3 tail and adds a methyl group at lysine 9. I say, whoa, you got to be kidding me. And to fast forward a little bit, now we'll go back to humans. Because I figured there'd be roughly a 50-50 split, I like this cartoon. I only changed the cartoon by adding the epi. Clearly these hardworking parents are a little bit troubled by their kids. The sister is clearly annoying her brother. I assume it's her brother. And she's— he's actually got it right. He says, you know, she says that her epigenome is more complicated than mine. Well, that's because every woman in the audience has 2 X chromosomes. You all know that. Males are XY. And the cells will not allow for that imbalance. So early in development, in all the women in the audience, you inactivated one of your X chromosomes. You have to. That's, that's a key. It doesn't matter whether you inactivate the one from your mother or potentially the one you inherited from your father, but you will always inactivate one X chromosome. The men in the audience will keep their single X chromosome active. And believe it or not, these are literally two nuclei traced from female human cells, human cells, where when you develop a methyl antibody, they stain the inactive X chromosome chromosome beautifully. And believe it or not, this is again our cat. Sorry, it was a blurry picture. But this is our cat where literally the patches of different coat color are literally due to this random inactivation that's all being driven literally by methylation of the histone tails. And that's really quite remarkable. —finding. Back to our picture that we had earlier of the two identical sister twins. This was the COVID of a Cold Spring Harbor symposium dedicated to epigenetics. And I just want to say that autism spectral disorder has a strong genetic component, so I want to be fair, but some of the autism disorder spectral aspects, most importantly Rett syndrome, are now accepted to be a strong example of a true epigenetic disease. Why? Because this woman, Huda Zoghbi, who is my partner in crime at my first faculty position, we shared a lab. So Huda and I shared gel equipment. She's a pediatric neurologist. Literally, she was just here at Rockefeller, we're talking December 5th, winning the Pearl Meister Greengard Prize for top women in science. This is a prize that Paul Greengardt started to pay special credit to outstanding female-only scientists. Huda was this year's recipient for her discovery that the protein that this gene encodes that gives rise to Rett syndrome is responsible for binding methylated DNA, which in turn recruits the enzymes that we were talking about that Jim Bernal and others now have identified that change these landscapes in Rett syndrome in neurons in the brain. So we can relax a little bit. We all know we are what we eat, but would you have thought from a new book on epigenetics we're also what our parents ate? Or maybe even their parents? That's pretty spooky. And this leads to that other example I gave you. These mice were all bred genetically identical. The only change in these mice were that they were— the mother in utero was fed a different methyl diet. And these methyl donors in the diet, in the chow, in the mice chow, caused these mice— to really all genetically identical to have these dramatically different sizes and coat colors. And this is now proven to be another example of you're not changing the DNA, you're changing what they're eating. Not only what they're eating, what their mother ate. And that's pretty remarkable. And this has been now chased out to be due to DNA methylation and histone methylation. That simple chemical group. That I talked about. And these authors wrote, "Wow, this may be a stunning example of changes in the environment affecting changes in gene regulation that might lead to disease." I'm almost done. Time magazine, now we're a Newsweek family, but nonetheless, Time magazine decided to put epigenetics on their cover in 2010. With the comment, "Why your DNA isn't your destiny." It's actually a fascinating read. And they awarded epigenetics or the human epigenome as the number 2 scientific discovery in 2010, second only to some sort of ancestral prehistoric findings of who gave rise to what. And if you— I'm sorry. That's just not— that's a different area. But if you read this Time magazine article, "Why DNA Is Not Your Destiny," you will see really a remarkable study that was led by this physician in the very northern part of Sweden. Sorry if you can't read this, but this is above the Arctic Circle. And as a pediatrician, he carefully kept track of feast or famine properties that were all dictated by the harsh winters or, conversely, the not harsh winters that affected patients under his care that led to changes, profound changes in their weight, their lifespan, their disease susceptibility, and even their mental health. And I think this was a nice cover of the family. This was again the physician, Dr. Lars, I can't By Green, and his son, and his grandson. So, sorry, son, father, grandfather. And so, my nearly last slide for this lecture is focused on just the left half. I wish this was animated, but I think when I was a student, this is what we heard a lot about. The DNA was often depicted as naked DNA. We were told that the genes, sort of this is the blue box, The genes are critically important, no contest, again discovered here at Rockefeller, and that we had transcription factors that bound the DNA, a very important group of proteins, nuclear proteins, that literally bind the DNA sequence and make genes on or off. I want you to remember that this is true genetics, and yet it's fairly fixed. You can't really do a lot to change the DNA. You can regulate the synthesis or the degradation of the transcription factors, but you're stuck with really this sort of genetic blueprint. Can't change it much. Yes, some of you will say you can mutate it, you can alter it some. On the other hand, the epigenetic landscape on the right is very, very fluid, very dynamic, and literally signals can be put on these chemical tags of DNA methyl marks histone tail methyl marks, histone acetyl marks, and these enzyme systems are just feeding in and off of this sort of balance, this yin-yang going on in, in ways that are often now turning out to be regulated by things that are really outside of us, things that we can control. Of course, some of you will say, but yeah, sunlight and cigarette smoking can cause mutations for sure, but it's looking more and more like there's a whole other a really big part of this that is really being regulated on this side of the coin. And I'm making equal credit to whether the modifications are affecting DNA or histone proteins. And lastly, I said I'm not a cartoonist, but I've had the privilege of having very talented people in my lab, including Sean Taverna. I'll show you his picture in a minute. This is my last slide for this lecture. Sean is an assistant professor now at Johns Hopkins Medical School in Baltimore. He's going to do very well, but he has this passion for cartoons that are scientific. Some of you may know Gary Larson. I think Sean's the next Gary Larson of sort of another generation. And so, I really like this cartoon for this lecture. You know, clearly the senior mechanic is saying, "You don't change the DNA," to this assistant. You change, you know, it's an epigenetic transmission. And you can see sort of this nice nucleosome, histone 3, histone 4. So that's what really Sean was trying to depict. And here's Sean Taverner, because he'll play another important role in another cartoon in my next lecture. So here's Sean when he was in the lab. So I think that's the end of my first talk.

We'll try to do diseases next. So we have, we have time for questions, and we have microphones in front, so just raise your hand and Dr. Alice will answer questions.

And if I can't answer the questions, Aaron will. No, just kidding.

I'm going to ask the first question. Can you get the microphone there? And can you switch between the micronucleus and macronucleus? Is that tetrahymena?

So did everybody hear the question? So the tetrahymena, you know, are those two nuclei that all this sort of biochemistry came from, are they sort of interconvertible? Sort of, let's throw this out to the audience. The macronucleus, I said, was the active one. It's where these hats came from. What do you think the role of the mic is? And let me even be more bizarre. Let me tell you that you can suck the micronucleus out of the cell with a little micropipette. Pet. The Tetrahymena is fine. So why would you keep the mic? What's the mic good for? Who knows that? I'm just deflecting. I don't know the answer, so I'm deflecting it. No. The micronucleus is the germline. When Tetrahymena has sex, there's a male and a female strain. They will pair and have sex, and only then does the micronucleus nucleus come into play. So Agata said, "Can the mic and the mac interconvert?" That was the real question. They do in the sexual phase. If they're just growing in food, there's no connection. Again, you can suck the mic out. It's totally silent. We didn't need it. It's like that inactive X. It's just off. But in the sexual phase, you'll think I'm kidding, the macronucleus that was keeping the cell alive is trashed. And the micronucleus divides to give rise to a new mac and one stays a mic. It's just stem cell biology all over again. I got to differentiate and I got to self-renewal, but Tetrahymena does it as a single cell. How cool is that? But the question is, would you have ever picked it to do any work?

Okay, go ahead.

Does being a product of your environment have anything anything to do with epigenetics? Being a product of your environment? Like if it's sunny outside or— Oh, that's a great question. Yeah, does living in our world, does living in our world, whatever that means, our environment in total, does it— I think absolutely. And now more and more people are trying to sort of— that's a hard experiment to control. But people are now trying to sort of like recapitulate environmental changes sort of in like, say, mice. And you— I mean, believe it or not, this is sort of a Rockefeller thing, but there's a researcher here at Rockefeller who studies behavior in mice. And I think you're going to think I'm making this up, but they have models now where you can have good mice What does a good— I'm sorry, I mean good mice moms. They lick their pups, they take care of them, they play with them, they nurture them. And then, believe it or not, there are bad mice moms. And literally, you could say, "Oh, that's awful." But they're terrible, they don't do anything. And then they've bred those mice and their next generation, and this good mom or bad mom is now being inherited through their epigenome. So like, that's an amazing story, and you can think of the social ramifications in our society of good moms and bad moms. So it's good, it's way past sunlight, you know. Hi, I have a question about the position effect. I wasn't really sure. You talked about the gene that controls the eye color. Yes, the fruit fly. When that gene that controls the, say, the white eye color goes to the heterochromatin. What specifically on that heterochromatin determines whether it's homo? Great. I won't go back to the slide. So here— yeah, I can do it with a ruler. No, I can do it with my chromatin model. Even better. But I'm not going to ask Aaron to come up. I'll try to handle this myself. So let's say the white gene, which really, I'm sorry, codes red eye color, is out here. And the experiment was to break this chromosome with X-ray and move that close to off-chromatin, heterochromatin. And you're asking a great question. I said the DNA doesn't change, so what spreads? What moves? And believe it or not, it's this Suvar39 methyltransferase, that unbelievable methyl discovery is literally able to literally move from like this bad region, we'll call it the bad neighborhood, and it seems to now move. And believe it or not, if you now make more of the SUVAR39 expressed, it moves further and silences this. Express more of it, it silences this. And these genes are not changing. So there's a remarkable spreading effect And now that makes people in my field wonder if there's boundaries, like is there a fence post? Is there something that keeps this from moving? And think, if that's going on in a fruit fly, can you imagine what the human chromosome potential is here? And translocations, and we just got a good question about sunlight. So, you know, if you can have that kind of spreading and that kind of movement of these enzymes in Drosophila, Drosophila, it's unbelievable to think about the ramifications in us. Thank you.

Sure. What happens to the epigenetic tags during replication?

That is an absolutely spectacular question. So if it was a DNA, if it was, this will be, you're going to laugh at how badly this is handled. So if the methyl mark is on the DNA, Then we understand how it's replicated, which is what you asked, because Watson pairs with Crick. Everybody knows what I mean, the double helix? We know that there's methyl trans— so you have to pretend that this is a Watson and a Crick, but it will replicate. So pretend I had a mechanism to spread this apart, and the methyl group here will be copied over onto the methyl group here. And then once that's kind of in place, you know, it's faithfully propagated to daughter cells. What we don't understand is in the histone tails where you had sort of a methyl mark on the histone. Again, to replicate through chromatin, I have to somehow unwind this, pull this apart, pretend like I had a fork. A lot of you know— question, yeah? Yes, it's just— oh, that's a great question. So in general, it does not interfere with the base pairing. It is sort of on the cysteine base. It's on a nitrogen in that base, and it's to the outside. But because it's to the outside and not really embedded in the double helix, inner part, you know, this is where the famous Huda Zoghbi Rett syndrome protein bound and was really— we like to call these readers. So that's not a problem. But what we really don't understand is how you copy histone marks through replication. That's a great— that's a very active area of investigation.

So when you have replication and you have a free nucleotide that is going to base pair, are those free nucleotides already pre-methylated? No, no, they're added—

So the complementary strand is not methylated? The complementary strand is not methylated for a moment and then it's copied.

Do tags open and close the histone or are they just there to let you know that they're open or closed?

Okay, another excellent question. So the question was when you add one of those chemical Let's make it acetyl because that'll more illustrate my point. When the acetyl was added, now you have to pretend this is a histone tail. Because that's negating a charge, you know, it was thought that that might just change the chromatin structure right there and there, you know. But now we also know, I think the second part of your thought was so charge would affect the chromatin structure.. But if this was planted in my arm as a tail, then again, we now know there's acetyl readers. So that acetyl group also can be read in a very elegant way by proteins that are— don't confuse it with the Rett syndrome, but there's proteins that read acetyl marks on histone. There's proteins that read methyl marks on histones. And frankly, those are amazing drug targets now too. We now know how to drug those readers. And they're going into every cancer, they're going into every disease. And I should have made the point, and I didn't make the point, but this is a good time to make the point. How do I say that? Is that you didn't change the DNA. So when I was like an assistant professor at Baylor and I overlapped with Huda, I mean, the rage was to do gene replacement therapy. If you had a disease DNA in an important disease, The idea was replace it with a good gene. But now in epigenetic therapy, you haven't changed the DNA. You've changed this on-top layer. So now people are excited. They can drug these molecules. And so, you know, I think there's a whole other excitement that you don't have to replace the DNA here. The DNA may be mis-silenced or mis-expressed. Because it's, you know, not doing the right thing with the molecules I talked about today. So therefore, if we drug them, we may be able to reverse these outcomes. And so, but back to your real question, you can change the chromatin structure or you can change the reading potential.

Okay, I have a few remarks, so stay seated. But first, thank you so much for the lecture.

I'm super impressed some of you are back. Looks like a lot of you are back. I hope you had a great lunch, a little downtime. And again, let me say, should I forget to say how appreciative I am on behalf of all of Rockefeller that I know some of you are up at the crack of dawn. This is for some of you, and many of you, holiday time, and I have to say, wow. I think I said that before, but I need to re-say that. Thank you so much for coming. So the part 2 of this, we're going to try to take what we talked about in the first lecture and build on it. And I promised you Aaron Goldberg, so I want to deliver on Aaron Goldberg. But this was one of the things that we talked a lot about, the fundamental repeating unit of chromatin, the nucleosome. This sort of tail domain where a lot of these modifications lie, methyl, acetyl. But now my question for you would be, what does this asterisk represent? And in the spirit of epigenetics, genetics, what's he talking about? Let me say that what I really mean this to represent is a mutation. Now we're talking genetics in the histone protein that encodes histone 3. So again, say that one more time. This asterisk means a mutation A real mutation in histone 3 at, believe it or not, one of those lysine amino acids, and in fact one of those special lysines that gets methylated. And behind it in the backdrop are patients that sadly died of a brain tumor. Make it worse, these are pediatric children that passed away, and this is an autopsied stain of their Here are cells, many cells staining with a methyl antibody. But with no exception, the children that passed away, and again these were pediatric brain tumors, and died knowingly carrying, not they didn't know, but their genotype revealed they have this histone mutation, they're stained in the background. So these are human pediatric brain tumors carrying the histone mutation on the right or not carrying the mutation on the left. So stay tuned. So this is the front of Rockefeller. When you came in the front, this is the Founders Hall. I believe that's the first campus building. And here you only need to recognize the FedEx delivery trucks that will be a theme of my talk because we haven't talked, although there was a great question about replicating chromatin. How do you get these histones copied? And so that'll make you think about delivering, delivering histones to where they need to go in the genome. That's okay if you're the regular histone and you go everywhere, but a lot of you asked after the lecture, how do you get special marks to special places? So that might require some delivery machinery. This I just want to show you quickly is Rockefeller in the spring. It's a different view, but talk about— somebody's described this to me when I was thinking about coming here as the greatest place on earth to do science. But boy, when you catch Rockefeller in the spring and these azaleas are in full bloom, the same Founders Hall but a different vantage point, it's a pretty amazing place. Just walk the grounds, and besides the history, you say, wow, this is just like a beautiful Central Park. So now to the science. Last lecture, whatever the modification was, we talked about enzyme systems that are dedicated to putting the marks on—writers—enzyme systems that oppose that activity, take it off—erasers. This could have been on the acetyl side, a HAT, Jim Bernal. This could have been an eraser, an HDAC, histone deacetylase. But I've never said that you could vary the histones, and hence my talk title, Varying Epigenetic Landscapes. So now I don't mean to change the modification. I don't mean you change the chemical mark. I don't even mean that you add different enzymes into the equation. I mean you vary the histone. You vary the histones. And I, I kinda lied. I said they're conserved. This is how everybody packages their genome. But I didn't tell you there are minor histone variants of each of the major histone types. And so that will get to a question of, could it be that every amino acid— and again, those are the building blocks of protein, but in this case histone proteins— might they matter? And so if nature's changed a histone just a tad, does that matter? And so this gets us to the heart of what textbooks tell you chromatin is all about. Because they're talking about dividing. Here's that great question we got about replicating. You can see the replicating fork. You're copying Watson and Crick. You've got replication machinery. These are the old nucleosomes, but you do have to copy and put in new histones. That makes sense. And you do that with bulk histone proteins. You do this during one stage of the cell cycle. Some of you know it as S-phase, and you do this with regular H3, and it comes in with its own machinery. The name isn't important. And yet, roughly in the early 2000s, focus became paid to a small, very minor histone 3 variant. Now we're back to varying a few amino acids. Now some of you are going to be bothered, those of you that are more mathematically inclined. Why is there a 3.3 3. It turns out there's really a 3.1 and a 3.2, but I'm grouping them together, so don't be bothered. They only differ by one amino acid, so I feel like that's a fair lumping. But what made this 3.3 so special is two properties. One, it was deposited into active chromatin selectively. Think about that. It went into genes that are being transcribed. Transcribed into RNA, expressed genes, and this is the most remarkable part. If that wasn't good enough, it also was deposited by separate machinery— the name doesn't matter— into non-replicating. I mean, that's just your bulk nucleosomes that somehow are literally capturing 3.3 selectively. And neurons would be a great example. And that I think is part of what— not only is Aaron Goldberg a very smart person, he's got extra neurons, but he decided what a great place to study a variant that is deposited into non-replicating chromatin. So I've kind of already sort of alluded to the conservation, but here's an audience question. How many amino acids distinguish distinguish 3.3 from the other, you know, again, in reality, 3.1, 3.2? How many of you want to say less than 5? A lot of you. And how many want to say 10 or above? A lot of you. And the histone 3, I have to tell you, is 150 amino acids. How many want to say 100? Just to be extreme, not many takers. Well, the answer is 3, so a very small number. And so that gets to the problem of it can't be important that you just varied a few amino acids. Come on, 3 can't matter, no way. So Aaron Goldberg, who you've seen demonstrate many things as a former Rockefeller MD-PhD student, decided that he would turn to embryonic development and particularly focus on now a very hot group of cells because they give rise ultimately to many different cell types. I'm sure a lot of you have heard about embryonic stem cells. Because embryonic stem cells are pluripotent, they can give rise to many different cell types when you trick them and you push them down different lineages. Aaron wanted to specifically ask what might 3.3 do in sort of neuronal differentiation. And he decided to ask two big questions. So this is very current research. This is about 2010 when Aaron was in the lab. One is ask, where is 3.3 in the genome? Is it everywhere? Is it select? And who does it run around with? Who's its partner in life? And so very quickly, Aaron's work took us to a whole new system, system 3. Now that's remarkable for a graduate student. You enter Rockefeller knowing that there's bulk chromatin everywhere copied by one delivery system. Then there's the literature of about 2002, 2003 that says no, no, no, no, 3.3 goes into active genes, expressed genes, genes that are transcribing RNA.. And it uses a different delivery system. You get the play on delivery. And finally, Aaron's work said in a nutshell, this active histone, that's too simple. And that's the great thing about science. It's just not always the way it is. Also goes into silent chromatin with a separate delivery system, DAX. And it's going in selectively to either the ends of chromosomes, into telomeres that Agata and others work on even here at Rockefeller, T.C. DeLong and colleagues. It also goes into silent chromatin that's around the centromere where the microtubules attach and you pull the sister chromatids apart during mitosis. These are silent neighborhoods. Back to that sort of position effect variegation from the first lecture, flies. You put a gene near telomeres or this chromatin in orange, You shut it off. You silence it. You don't change the gene. This is epigenetics. So Aaron's work said, whoa, we got to pay attention to a whole other system. And this I want to show you. Aaron's work was published in this most prestigious or highly prestigious journal, Cell. What blows me apart is when you get a paper published in Cell, they invite you to submit a cover submission. How cool is that? Now you don't normally get a cover, but they invite you to submit something. And one of Aaron's MD-PhD friends painted— painted— that's why, I mean, give me a break. And I want to show you, she's here. She may still be here. Hey, she's in the back waving. So Yifan— I agree. She actually, as a friend of Aaron, as a, you know, a friendship thing, drew this remarkable cover. I've only modified it slightly because I put on some of the labels. But I've never asked her, but I'll just sort of say now, put her on the spot, I always thought this had to be Aaron. I've never asked the question, but the white coat might give it away. So both of them, congrats on their accomplishments. But if Aaron was gonna now pursue not only science but medicine, then clearly the next thing Aaron should have been talking about, and I mean this in a cartoon way, but I also mean it seriously, we weren't driven, and by we I mean Aaron and colleagues, weren't driven to understand a disease. We were driven to understand a process that we thought was interesting. What would this 3.3 be doing in neuronal differentiation? And could Aaron learn anything about it? And this whole other delivery system to silent chromatin came about from Aaron's work. But then this paper appeared a year later. This is just the title. Most of the researchers are from Johns Hopkins Medical School, very famous cancer biologists if you know some of the names. And right in their Science paper title, the machinery that Aaron discovered as being responsible for depositing 3.3, that's that delivery system that Aaron discovered, was frequently, here's the key words, frequently mutated in a version of pancreatic neuroendocrine tumors. I might slip up and call it PANNETS. But to put a celebrity face on this disease, this is the disease that Steve Jobs passed away from, sadly, in 2011. And so how remarkable— put yourself in our shoes. We're doing this work as a chromatin lab, and suddenly this paper from other individuals pops up. We didn't get it to review, and it says that this pathway is mutated in a high frequency in this form of pancreatic cancer. But in looking at the author list, we had— and their affiliations— we had the good fortune of recognizing that one of the buried middle authors is Laura Tang. And what's important about Laura Tang is she's a physician, MD-PhD herself, who's employed at Memorial Sloan-Kettering. And it turns out she's the world authority on pancreatic neuroendocrine tumors. Although she wasn't the senior author, we learned later that she is at least the New York City New York City, as opposed to Baltimore, expert on this disease. And so we immediately hooked up with Laura Tang and her lab. And one motivation was Simon Elsesser in my lab, a graduate student, especially motivated by the disease connection, solved the crystal structure. That's not important per se, except I just want to show you that the protein that Aaron discovered as being the the delivery system protein called DAX is shown here in sort of yellow-orange. The star of the show is 3.3 in blue and its partner in life is the other histone H4. This crystal structure was solved. It was really one of, first of a kind, and the 3 amino acids that literally distinguish 3.3 from the regular H3 are right here. And literally armed with the information, here's the important point, of what human mutations in the pancreatic neuroendocrine disease are carried by these people, we're able to now understand what's going wrong with those people. And if you now engineer DAX to carry the human mutations, you literally abolish this delivery system. How remarkable is that? And it looks like what's gone wrong is now those patients are not delivering the proper dose of 3.3 to silent and activating regions of chromatin. Go figure. So with Laura, this is a humongous data set. So let me walk you through it. This is unpublished. So if you walk through all the samples on the left half. These are from patients. Each row down is literally a different patient. It's— he or she has been genotyped. This is either primary tumor in the pancreas, or importantly, perhaps, where the tumor has metastasized primarily to the liver. So Laura is, you know, being right across the street, she's able to get us primary and metastatic tumor samples from these patients. And these are all patients on the left that literally have either expressed genes in red or low-expressed genes in blue that literally don't have, don't have a mutation in DAX. They're tumors, they may even be lethal, but they're not a DAX tumor. But every patient on the right is someone who's carrying a genotyped DAX mutation. And, and that's just remarkable. I don't think you have to be a biologist to say these are different from these. The genes that are now highly expressed here in red are not the ones that are expressed over here, and the ones that are sort of expressed here are not expressed here. And these, you can't read them, it doesn't matter. They're just genes. They're genes because we've sequenced the human genome. I told you that. This was— and these that are highly expressed are largely pancreas proteins, and these that are largely expressed here are liver proteins. And this is unpublished because we're now excited that these DAX mutations on the right may be, again, take-home, mis-depositing 3p3 into genes that are now misexpressing liver proteins, which, how ironically, is where this tumor likes to go and settle in, which probably ultimately is part of the disease problem. So I should stop now. I mean, that's pretty fulfilling. We're talking about unpublished work. Aaron's work was 2010. The structure was published in 2011 or '12. This is, so we're really current. But this is the drop dead, I mean, if you said, why is he in science? This is it. In 2000, late '12 and '13, so we're talking this calendar year, albeit it's almost over. The histone that I've been telling you so much about, histone 3, and more specifically, histone 3.3 was found to carry mutations itself in young children that are carrying brain tumors in the cortex of the brain, albeit deadly but not as deadly as what is truly a death sentence is children that are diagnosed with brain tumors near the brain stem. You don't have to be able to tell how lethal this is There is no sort of good therapy or treatment for children that are diagnosed with this. There's no operation that you can get this tumor out. And so, and so really, these are perhaps some of the most devastating— there's going to be no cancer that's good, but these are really death sentences. And believe it or not, the sequencing from two groups, one in St. Jude's in Memphis, Tennessee, Another from— these are all pediatric powerhouses in McGill, Canada— published back-to-back papers that found mutations in 3p33 in these hotspots of pediatric brain tumors. And more remarkable to me is the mutation is always the same. And you're just going to think I'm making this up. It's a lysine. It's a lysine in the tail. I'm connecting my talks now. And it's always mutated to methionine. Don't let this M confuse you and don't think of it as methyl. Now methionine is another type of amino acid. It's encoded by the one letter M. I should have said the lysine is encoded as a shorthand in the one letter K. So you'll hear me talk about K to M. K to M, K to M, and that's just unbelievable. So this prompted Peter Lewis, a former postdoc in my lab— now he just started his faculty position at Wisconsin— to ask, what does the K to M mutation do? And this was just published, but it takes me back to my setup slide. We were able to connect, because this is such a devastating disease, even in Memorial, and this this red-hot medical neighborhood, there just aren't many people that are willing to give up their child's, you know, sadly deceased brain material for these kinds of analyses. But Oren Beecher, who trained at Memorial as an MD, who's now starting up his lab at Duke University, literally was able to send us autopsied brain tumor samples from either— this will sound familiar to you— patients that were genotyped wild-type. And by wild-type, I only mean that they don't have mutations in the histone. Is that clear? And every, every sample that came from patients that had died, and Oren was able to send us some tumor samples that carried genotyped K to M bona fide mutations in histone 3, this was the staining we got with some methyl antibodies. And should you be bothered by this little patch of tissue here that looks positive, it's actually a nice internal control. This is probably non-tumor vascular tissue. So when you look at the tumor part, there's no staining here. And I didn't want to bore you with too many details, but Peter Lewis did a remarkable series of experiments And the K to M mutation, believe it or not, in the tail of this histone is acting as a poison and it is totally poisoning or killing the methyltransferase, the HMT, the writer that's responsible for methylating this lysine. It's unbelievable to us. And so, you know, this is about, for me, as good as it comes. This has turned the brain, the pediatric brain tumor field upside down. This is a close-knit group. Again, you know, it's so devastating for the parents and they are looking for a breakthrough. And now I think they think this is off scale for the field. Now, somewhat with apology, I want to tell you about one of Peter's real experiments.— but again, it was published in March of 2013, and here it is. All you need to know about this is this is— we do this in the lab, we run these gels, this is what we do. We buy these antibodies, we and others have developed them. The complication is that they're against different lysines, so what have I done a bad job? I've specifically not told you what lysine it is, but the original mutations happened to be K27M mutations. The point is that it is against 27. There is a lysine, K, at 27. And Peter literally ran a gel and looked at these samples, and I hope you just can spot there's a hole. There's like no staining here, but that matches what we saw by the tumors. No staining in the tumors, no staining in this gel thing. The unexpected part is Peter decided to look at human cells that carried other lysine mutations. So I'll just take you through it. K9 happens to be another lysine. I'm not making this up. And I hope some of you might see the blue box looks like it's a little understained. Everything else looks okay, but maybe the signal here a little down. Let's, let's skip over 9, 27, Let's get deeper into the tail. There's a lysine at 36. Maybe some of you will agree there's— I mean, talk about a hole. There is a total hole. And Peter wrote at this point in time, maybe, just maybe, this is written in his paper, there will be other mutations found at these other lysines in other human tumors. But we didn't know that they'd all be pediatric. Pediatric. And now, fast forward to now. This was the original one, brain cancer, K27M, March 2013. This was published just a short while ago from a group in Cambridge University in the UK. They found bone cancers that are specifically carrying K36M mutations, all pediatric. And lastly, unpublished from a group at McGill, I now know that pediatric leukemia has now been sequenced and identified to have a K9M population. Why pediatric? Why specific cell types? Why K to M is now a hot area of investigation for all of us. And so I would have thought, you know, brain tumors are enough for me. Saving a kid's life, that's enough for me. But now we're getting more and more examples of histone mutations in cancer. Unbelievable. So to walk you through this a little bit, here's a chronology, you know, kind of a timeline of people that went after these enzymes and marks and readers in a systematic way because they thought they could drug the epigenome. I mean, it started in 1996 with HATS and HDACs. Then we have to jump over to the methyltransferases and the demethylases. 2000, 2004, readers. I talked in my question and answer period, you know, a lot of these marks are read by— they've been drugged in 2010, in part by people at Rockefeller.. And all of this was done intentionally. People recognized since you didn't change the DNA but you were changing this additional layer that was not mutated really, you could maybe reverse things. And so, you know, I might be confusing you. There were mutations in histones, real mutations, but here we were trying to target the epigenetic layer. And the idea was this might change balances and sort of either prevent or take care of cancers primarily as a disease. Now, I mean, this was published— I haven't really even read this article— but 28 days ago, a group published, forget the genes and the enzymes and the drugs that were meant to be targeted, these are other drugs and substances I won't begin to show you their chemical structures that literally were being added for these disease indications. And, you know, I've marked the ones that fall into cancer, but all the other ones— let's just go through some of these: AIDS, multiple sclerosis, sleeping sickness, inflammation, addiction, major depression, major depression, hypertension, pain, epilepsy, bipolar disorder. These These are cases where people were administering these substances to improve this life, but they now know they're regulating— these substances are regulating epigenetic molecules. That's unbelievable. It's not just a cancer problem anymore that's being targeted. It's that this is going so far, so deep into various human diseases And we're talking about published December 2nd, you know, this month. So here you can download this graph. I didn't do it. I would never do this kind of thing. But if you PubMed epigenetics— this was done by Reuters— and just look at the citations in the literature, you know, you can see what, what trajectory this is on from '96 to 2000, 2004. You know, the enzyme systems, the readers, the histones mutated are off the chart because this ended in 2011. This was in 2013. Where is this going to go? You tell me. Is it— has it flattened out? Is it going to keep going? Is it going to drop? About 5,000 papers published. About 1,000 proteins are bona fide epigenetic regulators. 196 clinical trials. 18 biotechs. 5 major programs at major pharmaceutical companies. We're talking about the big players, all have programs now in epigenetic space. And let's give a quote back from somebody who can really say this with clout. Jim Watson, one of the Watson-Crick discoverers of the DNA structure, says you can inherit something behind— beyond the DNA sequence. That's where the real excitement is. Now. When he says that, I say, whoa, that's pretty amazing. Now, some of you, you know, want more, especially maybe a high school group, top students like you all are. You want more than these kind of data sets. You know, you want more proof that this is really where it's at. And so remarkably, this was sent to me a year ago by one of my former postdocs who's in Illinois. He's like, Dave, have you seen the Spider-Man movie? And I'm like, no, I don't get out much. I'm in the lab, you know. But here's an audience— we don't have this as a formal test question, but this is the coolest laboratory setting. I wish my office looked like this. So who can spot what Craig— I'm sorry, Craig Mizen was his name— who can spot what Craig saw on this trailer sequence. I just froze it. I didn't see it. I didn't go to the movie. He spotted it in the movie. Who sees it? Some people in the front have an unfair advantage. But check the blackboard out. Now I'm gonna help you all out right here. Now is that not unbelievable? Who knows what you're looking at? This is unbelievable. Ah, see, it's mumbling now. People are waking up from lunch. It's like that Ferris Bueller movie. Okay, so here's a blowup. This is this. It's as big as I can make it without getting too pixelated. It's a nucleosome. I mean, I kid you not. This is not computer graphics. This is from the trailer of the movie. So it's a nucleosome. It happens to be having a modification. It happened— it even happens to be at the lysine that now we know is the famous lysine for the first case of the pediatric brain tumors, lysine 27. He's even got it methylated, and it's a complication, but I didn't tell you that the methyl groups can be monomethyl, dimethyl, or trimethylated. They're most silencing when they're trimethylated, and this is ME3, trimethylated. This is unbelievable. So now, Even my own children, they're all grown up, they don't care about chromatin. They think this is the coolest thing I've ever showed them. Forget the rest of what dad does, it's this that's so cool. So, okay. So I want to sort of conclude with my own private, very personal case study. I feel bad there's some people in the audience that are in my lab, but this is really not good. This is where I went to high school, a rough Cincinnati public high school. And I want to now show you, I really sucked it in here, this is only for you, this is going to be like blacked out real soon. This was my high school, I kid you not, I know some of you are shocked, but this was my high school senior photo. I'm even telling you when it was. And the point I'd like to make is I thought I was a pretty average student. I'm not even saying below average. But I'm not saying above average either. And I thought I was seemingly normal in 1969. I know a lot of you are like, "You're kidding me, how old is that?" So, don't do the math in front of me. But a lot of these epigenetic diseases worsen over time. So if I was normal in high school, what's happened to me since I got into the histone business? Well, one perk of being a scientist, and I really mean this seriously, You get to travel. You get to travel nationally, internationally. I feel so blessed that I've been all over the world. So for some of you that choose this business, it's unbelievable what you get to do and who you get to hang out with. But this is something that spooks me out a bit. When I go to airports, this is not good. This is really not good. I mean, I see things that other people don't see. So, who's to blame for this? What is really going on as I've aged that I see these things? Histone 1 to 4, this is unbelievable. I gotta say, blame it on my epigenetics. No. So I don't honestly know whether it's something I ate, too much sunlight, maybe I wasn't in school, nope, I don't want to say that. But nonetheless, maybe it's something my parents ate. But it's a growing concern. And now another very big serious perk is the fact of the, you know, just terrific people, you know, This was taken, you know, past when Aaron Goldberg was in the lab, but this is my current group at Rockefeller, a terrific group of people. This was taken this past fall. Every one of them are, you know, going in different directions, whether it's PhD, MD-PhD, already have a, you know, a beat on some other career path, and it's just a very big privilege to work with these people. So that's certainly a serious perk for me. And, you know, this is probably boring to some of you, but it makes me make a couple points. You know, there's folks in my lab who have contributed. I think in most cases I've had a picture of them, whether it's past or present, but I want to make a bigger perk for the outside collaborators. So if you just look at where these collaborators are coming from, you know, some are coming from far away in China, but others are coming from Sloan Kettering, Albert Einstein, You know, we can skip to Cornell and so on and so forth. But this group in particular I want to plug because all of these individuals are physicians, either physicians straight out or physician scientists who literally are able to get us remarkable sort of samples, tumor and otherwise. And many of these others are chemists, structural biologists, do mass spec work. Companies that help us make antibodies. It's really remarkable, the outside collaborators. And of course, you know, we don't do this without a lot of help from agencies. So this is where some of our funding sources is. And this program, if I'm not like too badly over time, is, you know, really not, not my show by any means. There's an awful lot of people that have contributed to this, and I just want to especially thank all of them, and especially The student and postdocs who have taken time out of their holiday, and maybe special comment to Kathy Vanchieri who's helped me a lot with the slides, and she's here. Thank you, Kelly. And Marty Imhoff. And really, you know, again, they've done quite a bit. So, Sean, back to Sean. I don't have much more left. But I want to make a couple of things, points very quickly on the theme of why do science. So Sean has one more sort of kind of like cartoon. I told you he's a real scientist at Hopkins. But here's this dense neighborhood we're at. So, you know, I mean, you can just see like here's the Rockefeller campus and then I've kind of got a big oval up here on Weill Cornell. Memorial Sloan-Kettering, of course York Avenue running right here, and you're here. So to orient you, you're in this dome right here, Kaspery Auditorium. So here we are, and you can see you don't have to go far to interact with great people. And, and this I think is really nice because Sean drew this special cartoon. I guess it's on your program, but this was his rendition of the Kaspery Dome, this special sort of— I think it's a hat with various wintry kind of mittens. So, you know, that was really meant to be a special cartoon from Sean. He's very talented. And my last part to this is to say happy holidays, and that's my change.

Thank you very much.

So we have, we have time for questions, and then we have a quiz.

Quiz with gifts. And there's a quiz with gifts, so don't stress out about the quiz, but questions that pertain to this lecture. You mentioned earlier about how epigenetics is connected with mental health, and I was wondering if you could expand a little bit more on how that works.

That's a deep question, how epigenetics might expand or impact mental health. Well, I think, you know, I talked in the first lecture, the Rett syndrome disorder in the autism spectrum is looking like a very bona fide epigenetic disease, with the caveat that it it is stemming from true mutations in the Rett syndrome protein. But back to things that you're really, I think, getting at. There's groups that study here at Rockefeller, there's groups at Mount Sinai that have modeled mouse studies to be looking at things like depression, addiction, other mental disorders, We talked a little bit about abuse, whether it's from the mother, however that works. And they have recognized that in the brain, there are programs of gene sets that are now picking up these cues from the outside that are changing the patterns of DNA methylation, histone methylation, histone acetylation. And in some remarkable studies I mean, it's a nice Rockefeller story. A group at Mount Sinai headed up by Eric Nestler teamed up with Paul Greengard, a neuroscientist Nobel laureate fame here at Rockefeller, and they began to like literally not only study addiction, they had mice coming into cages where they could sort of go left and get a control bottle of water or go right, their choice, and get a cocaine bottle of water. And, you know, it doesn't take mice long to learn they're going for the fix. But then they sacrifice these animals, they looked at their brain, they stained their brain, you know, control and cocaine-induced, and regions of the brain that correlate with reward centers were staining newly with these different antibodies that mark epigenetic Genetic tags. And then remarkably, for some of them, once we know the mark, we know who the writer is. And then there's drugs. And you can get drugs to now— so now let's go back and repeat the experiment where the mice are given the same choice, but they're now on a drug that sort of inhibits the writer. And now these mice aren't making the cocaine choice. Huh. Pretty cool. And so, you know, then you'd say, well, well, well, but, you know, people, people, mice isn't people. Mice aren't people. But, but, so I wasn't good in English. It's one of my— but, you know, I think that's really quite remarkable. Now with Aaron's work on the 3.3, you know, the idea that this is a histone that doesn't require replication, neurons are not replicating, you know, we, we in the lab are working on projects, not with Aaron but with others, that are now finding remarkable changes in the 3.3 pattern in response to— I don't know if this is even cool stuff for me because my lab doesn't really have a history of this, but you can put mice on kind of an enriched environment. They get toys, they get bells, they get whistles, or they get nothing. And then they look at the 3.3. Now forget the marks. Forget the— now look at the variant. That's changing. That's unbelievable. And, you know, Ian Mays, whose work I'm talking about, even has found an access bank in UT Southwestern in Dallas where autopsy brain samples are being obtained from people who have sadly committed suicide from a depression disorder. So they killed themselves sadly, tragically, and their brains have been banked in Southwestern by a physician, Carol Tamigula. Those brains are coming here, those are getting extracted, the histones are being looked at, the histones are different. They're different with marks, they're different with variants, and that's amazing because that's people now. And you'd like to say, well, are they all males? Are they all females? Are they all the same age? Well, they're not because you take what you can get. People are depressed, depressed. You know, that— so, you know, these are some of the studies that I know about that. And then, you know, Bob Darnell is a researcher. He was featured in the video. His work does a lot with autism and some other mental disorders, Fragile X. And, you know, they've now sequenced, sequenced, sequenced, and I mean DNA. This is your genetics, personalized medicine. And they're finding genes that match in those big datasets. And Bob has come to me— it's like why it's great to be at Rockefeller— and he's like, got 12 matches: autism, Fragile X, they're epigenetic regulators, let's team up. So that those become looked at. And frankly, when we go into the cold room and we purify— that's a lot of what my lab does better than anything— is we purify a complex and we get something that's got a mental retardation syndrome tied to it. You know, so there's— I think the brain may be one place where the glory of what I talked about, or I should say all of the glory of what I talked about today, is going to be most manifest. Because the brain's, you know, kind of got to be loaded with inputs, you know, learning, memory, and you're not changing the DNA. So how are you changing? And that'll be a really fascinating part of epigenetics in the future.

Long answer, sorry.

No time for our gifts, no. Another question here. Could you use acetylation and demethylation to help stem cells differentiate? Absolutely. Good question. So stem cell biology is red hot. Some of you know Nobel Prizes were were awarded for being able to go into a somatic differentiated cell and be able to reprogram it back into induced pluripotency, but it looks like it's low efficiency, and people have— right to your question, people have added HDAC and had inhibitors, and they've improved the efficiency of that step, thinking— and it's too much detail, but other researchers in Philadelphia recently published some of these famous methyl marks. They have to be overcome to get efficient reprogramming. So there's like a barrier. You've got to re-erase the blackboard and sort of be able to sort of clear these marks and get them to start anew for efficient differentiation in all the different cell types. Could you talk about how cell signaling comes into play with what you're talking about? Yeah, so cell signaling, I think classically is, you know, most cells, you know, they're taking in environmental cues from the outside, hits the plasma membrane, and then you have transducing signals that will carry signals through the cytoplasm. Many— I was asked at lunch about phosphorylation cascades, but, you know, A will communicate with B, will communicate with C, will communicate with D. Then these get into the nucleus and oftentimes invoke their activation or their repression. And so I think the chromatin layer, what I've talked about, is absolutely down. We've often said in labs, we're signaling to chromatin. But believe it or not, if you work your way back and say, well, I just want to go from a methyl mark backwards or an acetyl mark backwards, it doesn't It doesn't take you too far before you find out you're right in a signaling cascade. So, I think once some of the— and, you know, some people will be mad and say, "Alice should have said, you know, the real business end is getting these into the nucleus and their transcription factors that bind DNA and regulate genes classically." You know, but, you know, some of these signaling cascades unquestionably are feeding their inputs in on histone tails.

Phosphomethylacetyl. Absolutely, absolutely. Okay, one question here. Question? Yes. Um, you mentioned a lot about the, like, the drugs that cancer patients are taking who have cancer that's caused by, like, epigenetic changes, right? Um, like, how does HAT and, like, HMT work in those What does it do in the drugs that changes their cancer?

Okay, well, that's a good question. So how do these drugs sort of really impact and change? I think at least 3 people after my first lecture said, well, how come it's just not willy-nilly? If chromatin's everywhere— my props aren't here— but if chromatin's everywhere, how do you target specific genes for a therapeutically good outcome? And we don't have all those answers. Those are great questions. Others of you said, "I wish I could target a gene, you know, that's like my favorite gene to be on or off." We can't do that yet either, but I think what's happening is when you add a HAT inhibitor or a methyltransferase inhibitor, you shut those enzymes off and there seems to be then, you know, kind of like a change in these marks that will suddenly tip the balance, and obviously some genes are probably more primed into an on state, there are some that are more primed into an off state, and you just throw a little bit of that balance. Let's be more specific for a HAT. You knock out a HAT, most people would say acetylation is going to drop, or you knock out an HDAC, acetylation will come up, and so these genes then open or close the chromatin, and the only mystery was why are they so specific and why are patients getting a good therapeutic outcome? Because if you did that to the whole genome, every gene would activate or repress. But believe it or not, people have now done systematic studies of no drug plus drug, and it's only a small number of genes that change. And, you know, good for the patients. Some of these are or things you want more of, like a tumor suppressor, or you want an oncogenic protein more silent. So, you know, good for some brave folks. The first example that I showed, and, you know, I skipped over the facial melanoma example, but these were all done at Sloan-Kettering by Paul Marks and his colleagues. And, you know, he's told me privately There weren't many people that were going to fund this because they thought it was just too risky, but they stuck it out and then they got good outcomes and then all of a sudden they were bought, they were bought, they were bought, and now it's a big program at Merck. Go figure. So, you know, I think, you know, I don't want to say proof's in the pudding, but, and then you look at the pictures that I did show, whether it was the lung example or the melanoma face this example, just put yourself in those people's shoes. It's very rewarding.

And now we hope that we can work some of this magic on the brain tumors. Yes?

Can epigenetic changes affect the reproductive success of a species? Oh, yeah, absolutely. What's the epigenetic part on the reproductive success? At the end of the day, it's really not me here. It's like kids and passing on, you know, through your, you know, germline— male sperm, female egg, oocyte— and the epigenetic layers and marks have to be erased, cleared, reset. And so that is a red-hot area of research. I would say between the question I got about brain and this question about germ cells, that's going to be where is where I think the chromatin problems are going to maybe be the most profound. But sperm is a highly condensed chromatin package. The oocyte is a very decondensed. And so you've got to work against all that to sort of now have successful embryogenesis development. And so yeah, it's cool. I mean, there's entire—

almost like meetings that are dedicated to germline epigenetics. Hi, thank you very much for your lecture, but my question is, what ethical boundaries are you facing today in epigenetics?

Ethical? None for me. I mean, you know, no. I mean, you know, truthfully, I don't think we cross any dangerous lines. I mean, maybe the most sensitive thing for us have been the pediatric tumor samples that, you know, we published Peter's work, and I probably got 15 emails from parents that were desperate for, you know, what would be the cure for their darling 6-year-old. And, you know, I just had to refer them to the physicians because I can't give them a magic bullet. And so that to me is just heart-wrenching. But under all the appropriate scientific channels, we're able to get these tissues here. These parents are filling out all the appropriate— they're consenting to all of this. I actually think they're heroes. And we're going to try to learn from it. And, you know, I go off in February to a workshop that the National Cancer Institute has asked me and another one of these— the brain tumors at the base of the— are called DIPG for the pons. And, you know, they're taking— the charge for us was you put the best 15 chromatin people in a closed room for a day and a half with the best 15 DIPG And let's see what comes out of it. And so, you know, I can't wait. It'll be a highlight of next year because, you know, these people have no therapeutically good outcome for these children. And here we are paying attention to all this chromatin, and they're hungry to learn about it. And so it's just a true blessing and privilege.

I don't think we have anything too prickly going on in my lab. I heard that the pancreatic neuroendocrine disease that Steve Jobs developed could have been caused by his diet.

So how is it that the H3.3 not being delivered properly could contribute to that? Yeah, wow. Yeah, Steve Jobs did, I think, kind of sadly decide on some pretty radical course corrections that included some maybe non-conventional treatments, but, you know, how his diet may or may not have impacted that particular, you know, SAD, I honestly don't think I've seen anything published where if you eat X, Y, and Z, you know, you're going to be super protected against pancreatic neuroendocrine tumors, or frankly any other. I mean, you know, like now these big sequencing consortiums, you know, whether it's breast cancer, prostate cancer, it's interesting. They're looking at all the mutations in huge numbers of men or women, and when they find a group that are epigenetic, sometimes they contact us and say, you know, can we start researching with you, and we'll get you tumor samples that are coming from men who have volunteered to be part part of a clinical study, you know, you get tumor samples to my lab, extract the histones, look at them carefully, or breast cancer, whatever it is. And so, you know, but how we're going to— and they're taking good records, like are these men what age group, or women what age group, were they— I don't know. I don't know how they can describe their diets, but you know, they ask you all these questions and they're now paying a lot of attention to And hopefully, maybe some common denominators will surface. But Steve Jobs's, I can't speak to that. I really can't. OK, last question. So do you foresee a possible epigenetic project similar to the Human Genome Project where you can map out all the different epigenetic factors? It's been done. It's been done. Yeah, so, well, no, no, no, that's a great question. So the Times 2010 number 2, you know, I think they ranked it as their number 2s. When it said the epigenetics, when I showed the Time magazine cover, so when it said something like discovery number 2 of high importance was the human epigenome, that's what it was. 50 labs came together together worldwide and they took healthy, diseased, this type, that type, and they tried to take this sort of both toxin lines together and literally attack them on the histone side, the DNA methyl side. There's amazing mapping techniques we didn't go into where you can look with high precision for where these marks are, and so this has all been categorized. Analyzed now worldwide, and it's staggering. I mean, it's pretty easy for the mathematical and the computational to look at DNA algorithms because it's A, T, G, or C. Even that's challenging, you know, and is it really a causative mutation in the DNA? But think about what I'm talking about. And I didn't really come close. I'm not trying to insult you. I didn't come close. Just about every amino acid seems to have a flavor of chromatin marking and people have asked about, well, what about signaling pathways? We got that question. Some of these famous lysines are right next to serines and threonines. A lot of you know that those are amino acids that can be phosphorylated. Oh, and by the way, it's awful. If it's phosphorylated, it can't be methylated. If it's methylated, it can't be phosphorylated. That's called crosstalk. Whoa. So, when you go into a particular disease cell extract the chromatin, start mapping it genome-wide, it's unbelievable. It's— I mean, so yeah, there's kind of these rudimentary maps of the epigenome, but I mean, I think it's the tip of the iceberg.

Okay, so I think it's time for our quiz.

Yeah, so this— okay, now let me tell you how these are organized. Organized. There's, I think, 10 of these. This is going to a little bit maybe translate to those of you that aren't, you know, and I'm not going to be the picker or the grader. So in every case, we have a question. Answers are below. You'll get to all participate at least mentally. And then once we get a volunteer or 2 or whoever, you'll be picked on, shout out your answer, And then we've got the right answer circled. I don't know if there's a right answer, but there's an answer circled. And then there's— I'm not involved in the gifts. I don't know what's coming. OK, so question 1. This was meant to be easy.

Calico cats are always— whoa! Whoa! Am I going to be responsible for this? I'll close my eyes. Oh my God, they're in the back. This is way too hard. True. So the answer is true.

Let's see. Let's see. Okay. All right. Okay. Question 2. The props are gone, but 2 meters of DNA in a eukaryotic nucleus must be as small— packaged as small as 2 microns or micrometers, roughly in the width of a nuclear— what is that packaging ratio? 10x, 100x, 1,000x, 10,000x? I would say 1 million. Let's see. Oh, we have a winner! We wanted to make you a millionaire, but it didn't work out. Okay, next one. We're getting through this. Ah, I don't have my flask. What volume of growing— now here there's even some— oh, we still have— Active people in the back. So remember I had 2 flasks to orient you, 200 mLs, and I had 2 liters. So you can see the choices: 10 mL, 200 mL, 1 liter, 2 liters, 200 liters. We need somebody in this side.

Okay, okay, okay, okay. No, I've got it. I don't know, I don't know.

I don't know. I don't know. Oh, we don't have them on both sides? Thank you very much. Oh, okay. I'm gonna go with 2 liters.

Not right. Oh, come on. Let's try it again. Try it again. I need somebody from the middle of the— this looks like a middle.

200 milliliters. Not right. OK.

We'd like to get some teachers involved. 200 liters. No, I'm just kidding. Oh.

Sorry, what was that? 200 liters. So if you think about that, that's 10 of what— no, no, no, that's 100, 100 of the larger flasks. And Jim purified this hat band into like an invisible— I mean, this is a tour de force. Sorry, but good, good, good third guess. I mean, answer. Okay, next question. Yes. Which organism would be your choice for, you know, isolating a molecule and doing biochemistry? Protozoa, the ciliated, like Tetrahymena, flies, mammalian cells, and by humans I mean real humans, not human cells, or all the above because they all have pluses and minuses?

I'm afraid I don't know how you are going to choose the answer here.

Yeah, I would say all of the above. Sorry, what's the answer? Here's it. All the— oh yeah, I think we have a winner. Seriously, you know, this is something that I really think is— you got to pick your organism carefully, and there's perks for all of them. Okay. Oh, this is a little bit of a trick question. In terms of molecules for epigenetic regulation, who should we be caring the most about? DNA, histone protein— so not any protein—

histone protein, RNA, all the above, None. Okay, we have, we have somebody who came from the other room.

Oh good, just to answer this question. All right, all right, I'm gonna say that the answer is, um, answer is, uh, maybe, uh, DNA.

No, uh, next time, next time. Okay, right here. Histone protein? No. She said, she said histone protein. No. No? Oh, it breaks my heart.

I don't know. It breaks my heart. Oh, and I feel bad. The reason The reason why I thought this was a trick question is I don't think I even said RNA. And just now as an educational point, but I feel bad, this was sort of hindsight, but RNA is now playing a big role. And if you got— because I got questions, how do you target? How do you target? Well, you know that RNA is complementary to DNA. And so it's looking more and more that a lot of this epigenetic machinery gets gets to where it needs to go through an RNA molecule. How cool is that? But I'm sorry, I really didn't develop that, so I take the blame on this. No gift from me. Okay. Next one. Oh, yeah, this is important. Histone mutations, we talked about them. You know, what's the right way to call those? Should we call those genetic?

Should we call those epigenetic? Both? Neither?

Okay, you get to decide between each other who is going to answer this. We're almost done, I think. I'm gonna say both. Okay, I really think that's a great— it's really hard because I get challenged. You know, you have a mutation in a regulator, shouldn't that not be called an epigenetic disease? So you get— you're with me a little bit. Okay, next question. You're going to decide to regulate, you're going to do it with an acetyl group, green, methyl group, red. What are you going to set your epigenetic landscape? How are you going to fine-tune it?

I don't want to say Christmas tree, but what would your epigenetic tree look like? Okay, somebody in a hat, would you like to answer?

I'd say both. It's just as important to keep genes silent as it is to turn genes on. Okay, I think we're Ah, one that's dear to my heart. I only showed you one of my experiments. When you do the ladder, although I didn't describe the gel system, are we varying with the acetyl because it's heavy? It's an extra bit of chemistry?

Or it changes the charge?

That's the question. Oh, that's a difficult question.

Okay.

How we doing on gifts? Ready? The Charge. Yes! Okay. We're getting personal now. What's the best part of doing science?

Travel, papers, glory? Surprises people?

Shh. Okay, it will be right here. People. Hey! I think, I think I'll know it when I see the question. This may be the toughest one of all. And there's a little bit of history.

Shh. So now let me—

so I don't know, I don't know, I don't know.

Everybody can read the choices. I don't know. Okay, first row.

Front row.

Um, I'm gonna have to go with chromatin. No? You gotta be kidding me. Good answer. I think she deserves— I think she deserves a— I wish.

Yeah, I think she would. Okay, another one. Charles. In the political climate, I thought you'd all say Camp David, but in any case, it's a true story and then I'll let you go. I got to give a talk this past fall, I think it was September, October, and it was in a big auditorium sort of like this And my wife was there. She's also still here. But she was sitting behind students and they didn't have any clue. And not that I showed this thing, but they had a setup slide and it had my name as C. David Alice. And the students, I think these were undergraduate graduate students, were sitting behind her. And of course, she's like a fly on the wall. You know, she's like just not admitting who she is. And it was so funny. The students behind her said, "What do you think the C stands for?" And one of them said, I kid you not, true story, she's here, she's going to testify, said, you know, I think it has to be Clyde because he doesn't want to use it. So that's why that's up there. And I apologize if anybody's name's Clyde. And I'm done. I'm done.

That's it. I think that's it. OK. Lastly, thank you all for being here. It was a terrific day, and thank you, Dr.

Alice.