to understanding chromatin architecture in all life domains as we learned from yesterday's lecture. She is also recognized with many prestigious honors and awards for her research. Now Dr. Luger is a distinguished professor at the University of Colorado Boulder and HHMI investigator. Finally, we would like to thank to thank the support for the steamboat lecture from our department, Wolf and the summer facial. Now let's welcome Dr. Luger for exciting lecture. I feel like deja vu all over again. I feel like I've done this before. I don't quite know how that happened. Thank you for the kind introduction. Thanks again for hosting me. Thank you, everybody who's here for maybe a second time, which is very hard to imagine. So I'm not going to give my talk backwards like I advertised yesterday but I will talk a little bit about more general aspects of my career with the hope that maybe some of you who are earlier in your career stage might find instructive. Let's just ask like how many how many undergraduate students are here? One to a couple, how many grad students? Many, good. Okay, this is for you.  So, and we'll see how this goes. I don't know lengthwise how we're doing but we'll be done in 50 minutes. I promise I'll just cut it short. When I was like your age, around thereabouts, that was my idea of a typical career path. I thought professors were born like that and then they were of course white and male and they knew everything and that's what happened. And it took me a while to figure out that that's not a typical career path. And in fact, mine really worked out like this. It was a random path, maybe with some attractants and repellents. So I was doing random walk. I had attractants. I knew I was curious about nature. I love planting things and growing seeds and tomatoes and stuff like that. I wanted to know how cells work. I really liked solving puzzles. And I had two older brothers and they were very techie and they always talked over my head and so I wanted to show them up and show them that I could also do science. Repellents were.  So MD was not an option for me. I did not like teaching teenagers. So high school teacher was not an option that really didn't leave a lot of leeway. I didn't really want to be a pharmacist like my aunt and so I really didn't know what to do. I took myself off to the nearest university, which was three hours away by train to the University of Innsbruck, not a great university, entered the major of microbiology, and I was lucky enough to be adopted by the biochemistry department and biochemistry sounded like you know that might get at how cells work and things like that so kind of a random walk into that department. So I searched there. I found it exhausting, because I was working with Bernd, who was a very senior research fellow there, and is almost like running, running, trying to run an ultramarathon with a trained ultramarathon runner, never having run even a 5k. So, you know, this guy kept going I was exhausted. That was my induction into biochemistry. Nothing worked. I did learn to pour a really mean sequencing gel gradients. I brought them home to my mom the films. She was very impressed. I learned how to do mini preps from scratch so that was good. I did an internship in industry, which I really hated, but I met my future husband there so that was good, I guess, and then I took myself off to PhD work in Basel where my then future husband also resided. So again, kind of directed random walk. My PhD in Cosper's lab was amazing because he was a very hands off mentor and a very creative mentor, and he would let us do whatever we wanted to do. And so I did a really crazy project that amazingly worked. And we could publish it very well. And so that really got me hooked into science and I wanted to do more. We were working closely with the crystallographers. They were all a bunch of really unkempt and unwashed guys who spent many hours in the dark room and in the diffractometer to solve the crystal structures. And I was just really intrigued by how you get from these patterns to a structure. So that's what I wanted to learn. And so I decided to interview with Tim Richmond, who was then a new professor at the eth surgery was a superstar, establishing crystallography there, and also postdocs at the eth paid really well. So that was also an attractant, shall we say. And but before I did that, I decided to in the words of my PhD mentor make a really valid attempt at not having a career, because I took a year off with my then husband. And we rented, we bought an RV and we drove it all across every single National Park in the United States and our piece de resistance was to canoe about 2000 miles down the Yukon River in in in this very trustworthy little rubber to me, which was really amazing. And it was a good thing that I took a long vacation because what I did not know at the time is that I was really in for a real, a really hard project. And we were trying to figure out how the human genome is packaged in the nucleus so like that sounds easy right after canoeing down the Yukon River. It seemed like that was manageable. And just to give you all a little bit of a size comparison, if the nucleus were the size of a golf ball, it would contain about 10 miles of DNA, a really, really thin thread. And you'd have to package that in a way that you could replicate it facefully and you could also find the pertinent information in a spatial and temporal accurate way. I give you a little bit of a history on on on the structure biology and what people did before I entered the field which was nowhere near its infancy. So back in 1974, Roger Kornberg had to do is from a lot of careful biochemistry that chromatin is structured based on a repeating unit of eight histones and 200 base pairs of DNA, which we now know is still true. Francis Crick and Aaron Kluge then deduce that in order to package and compact the DNA in this manner it needed to be kinked rather than curve because the curvature would inflict too much of an energetic penalty which turned out to be wrong but that's okay. In 1976 which is way way way before most of you were born. Finch and Kluge developed the solenoidal model of the of the chromatin structure again based on theoretical considerations as well as whatever experimental data were available which wasn't really very much and this is still one valid model of chromatin higher order structure. This was 1976. In 1977. Finch and Daniela Rhodes and Michael Levitt and Aaron Kluge, but these really pretty amazing electron micrographs of chromatin nucleus almost stacks isolated from I think cuffed timers at that time. This is the electron density map projected and based on that as well as some careful DNAs one digestion of chromatin, they came up with this model of the nucleus on. And if you superimpose what is now known as the nucleus on structure onto this model, you see a pretty amazing correspondence. And this is that yes they were really important and really good papers written before you were born. And, and, and we actually already had a lot of information before I or even Tim Richmond entered the field. So this was 1979 in the early 80s. Tim Richmond then managed to get crystals that diffracted to about seven angstrom resolution. And this is the structure. It's really not something that we would throw on the cover of nature, nowadays, and, but we, we could clearly distinguish a DNA from the protein so it was known that the DNA was on the outside that was actually known before then, and it scattered a little differently. But clearly we had no idea how the histones were arranged at the inside and the, the assignment of the histones on the inside turned out to be completely wrong. But a lot of these model was actually facilitated and supported by very careful cross linking experiments by Mirza Beckhoff and colleagues. And that turned out to be those, those cross linking data turned out to be completely consistent with the structure that we know today so again my point is that yes they did very careful very quantitative biochemistry and structurality in the old days and hybrid approaches, even at that time were a thing and really helped to deduce structures that by current knowledge now seem to be correct. So now enter the early 90s and me, when the dinosaurs work walk the earth. This was a dark ages, all the cool kids were working on transcription this was teach and passion and all these guys working on TF2D and T the whole alphabet soup. Nobody cared about chromatin in fact chromatin was thought to be completely unimportant. The post translational modifications were actually known, but they were largely ignored. So I have a textbook from 1984 where all the modifications are described in full detail, but nobody knew about their biological importance. What was niche for proteins, blabology wasn't really applicable to this third generation synchrotrons were just being built so X ray crystallography was really the only game in town, and we needed very hard x rays to solve structures from for very large complexes. So storage and graphic systems were about the iPhone zero level. And if that we actually had whole data sets we couldn't in process whole crystallography data sets because they were like mega basis in size. And so we had hard drives and tapes we had to shuffle them back and forth so I'm that old yes it's true. Chromatin was isolated from chicken blood and cough time was so natural sources. There was also a structure of the histone octamer that was published in 1991. So this would have been amazing, because this would have provided a lot of really good information for facing, but this group refuse to deposit their structure on the PDB, which at that time was not required so we could not really use it for anything. So there are nuclear some crystal still diffracted to only five angstroms and we still had not solved the face problem, which is a problem that's inherent to crystallography because you can't refocus x rays. So unlike in cryo and where you can just look at things, you have to, you have to solve the face problem that's all I will say about this because it brings back really bad memories. So the path towards higher resolution when I started, it was clear that we needed histones that were homogeneous and not heterogeneous because we knew at that time that there were a lot of post translational modifications. Nobody cared what they did but we knew they existed. We knew there would be a problem. The DNA sequence and length of the nucleosomes when you prepare them from these little arrays isolated from cough thymus were not uniform. And so it was clear that we needed to build nucleosomes with defined DNA sequences and with recombinant histone protein so we needed to make recombinant nucleosomes. Okay, so that was my task. And that was four years of my life in the bucket. And what we did is we we duplicated a DNA sequence that we felt would really like to form nucleosomes in a plasmid we then did 12 liter plasma perhaps to isolate enough of that DNA fragment to, to make nucleosomes for crystallography and the diagram amounts of this 147 base pair DNA fragment, a lot of phenol, a lot of ethanol precipitation, I'm still not sure what it did to my brain cells, and I'm sure I don't want to know. Histone expression, we needed to express each histone in E. coli. This was not a trivial task at that time. Because you couldn't really order genes online so it was a different time. That's all I will say about it. We then isolated them. We had to, we had to refold them to form a histone octomer. And then we had to devise means to combine DNA and protein to form mononucleosomes. And while we're at it we also made nucleosomal arrays for higher order chromatin structure so we could do it on duplicates of, of the positioning sequence. So, having done that, this is all great. I did a lot of quality control to ensure what I've made was really the real deal, because it didn't have any enzymatic activity and so how would I know that I was really making something that was not garbage. So we did that, that was all good. Then we set up crystal trays and that was before robots. So there's a lot of greasing and pipetting. And this is what we got. Super pretty to look at. Really bad crystals. They're actually worse than the crystals we got from chicken blood or cough thymus. They were thin windowpane crystals. If I could mount them they would diffract about five angstroms on a good day. And they were really slow and unpredictable in the crystallization behavior so sometimes they wouldn't crystallize for month. Sometimes they would crystallize in a week it was just a nightmare. So this was about year for ish, I think, great for morale, of course, because you see your career chances slowly circling the drain. But I then had an idea that I think, or I did an experiment that I still think was the most important experiment of my career. What we do with crystallographers when we get crystals, it's always a good idea to take them, wash them, dissolve them and analyze them. So that's what I did. So this here is the input. This is a dissolved crystal. Okay. And what you will see is that the input looked fuzzy, and they just always looked fuzzy. This band is very fuzzy, red kind of bloated. The crystal is really not fuzzy nice crisp and so that was weird. So then I decided to take the mother liquor.  And that's where the leftover protein would be. There was no leftover protein there was no leftover nucleosome and everything is gone and incorporated into the crystal is not fuzzy. So by very sharp deduction. I concluded that the fuzzy material is transformed into non fuzzy. Okay, very scientific explanation. And so what we then discovered subsequently shortly after is that indeed the nucleosomes that we make were not uniformly positioned there was heterogeneity in the positioning. It was DNA sticking out this way or this way, or it was centrally positioned. And it turned out that the crystal only like the centrally positioned species. And so, in equilibrium it just pulled everything out of the solution and that explained all of my fuzzy versus non fuzzy and empty mother liquor data. So that's great. What do you do with this. Now, then again, a stroke of genius. It's a very high tech experiment. I stuck my nucleus on up north tube into a heating block 37 degrees, half an hour. Boom, we're not fuzzy. So this is great. You can then crystallize this material, and you obtain resolution that goes from here, which is really not great to hear, which is good. So this extends all the way to about two point angstrom and beyond. If you push it really hard, it goes to 1.9 angstroms. I just want to show this because it is very, very pretty. Okay. So, check, right sample homogeneity diffracting crystals. Awesome. We still have the face problem. And in order to solve the face problem and I will really spare you the gory details, I had to engineer about 2016 substitutions throughout the histones to then hook up heavy metals to then perform multiple isomorphous replacement. Nowadays, we do these structures with Selena Sistine or Selena methionine wasn't really an option here because the structure was too large. And also there was no system to incorporate Selena methionine. And so this had to be done. A lot of these things did not the fact a lot of these things did make gave me very bad crystals.  If you're curious about of a more scientific explanation of what I told you in brief, you can look at this free iBio video that is that that I took with with this with this nonprofit organization. There's actually a lot of really good videos out there by various scientists explaining their key experiments so I really recommend all of the undergrads and grad students to look at this site. Okay, so September 1990 97. Very happy person. This is, this is me at that time already was a big hiker at that time. And so we managed to publish this structure also huge shout out to people who are not on the paper song 10 Thomas rex diner, Yvonne and Andrew my partners in crime so this was a huge team effort. I also encourage you to, to take the call to this this is itoshi Kuro Misaka, and I forced him to write. He's a very talented composer and musician I forced him to write a nuclear zone birthday song and that's what he did. And that was before chat GPT so that's really interesting. So this is a rock song about the nuclear zone. Okay, good. So this is an accomplished we have a structure of the nuclear zone. It's not only beautiful but it also I think explains really well. A lot of the biology and you can you can appreciate how tightly the DNA is wound and bound by the histones and how much of a problem that's going to be for transcription and repair machinery and all that good stuff. I also entered pop culture. It, I'm sure it it inspired the Millennium Falcon this is from Star Wars. I'm sure but that must be that must have been it. And also, I just want to share this because it just gives me so much joy. There's this cancer scientist, Dr Brian Wellman at University of Utah, and he's also an artist and a welder, and he welded giant nuclear zone for me about this big and he gave it to me as a present. I didn't even know the man so this is totally fabulous and and it's in my living room and it gives me joy every day. So, back to back to careers. So eight years one paper. What's next, I should also say that in those eight years. And I don't mean to say this in a mean way, but I received no mentoring whatsoever on the next step. I was there to solve the structure of the nuclear some and I did that. And the rest was my problem. So that was just the way it was at that time. But I did actually around 95 or so Tom check visited our department. And he's very famous, and the last time he showed was the view from his office. So I decided that's where I needed to be. And so I, I moved over, not to Boulder because they didn't want to interview me but to the University of Colorado State University, which was still not well known university but a land grant university with a very strong mission in undergraduate teaching, also a strong vision they wanted to get crystallography going in their department so I thought well I'm, I want to do this the hard way so why go to a department where they already have to set up if I can do it from scratch right it makes perfect sense. So I started in 2000 pretty soon recruited these four rather for Lauren looking graduate students sitting on the side of the road. The lab group pretty rapidly 2005 till 2010 we had a pretty good size already and a lot of fun. A lot of good things happened in 2004. I got a GMI support and also had a baby and at any rate we grew the lab in in many ways. We had a lot of babies, as you can see here. So, so these were really productive times shall we say. At CSU, my first mission was to build an x-ray diffraction facility. And that was good. It was, it was interesting dealing with contractors and such so. And now I was also learning how to teach I was learning how to mentor I was learning how to run a lab I made a lot of rookie mistakes I kind of muddle through it with the help from my friends, and asking a lot of questions. Specifically, we were addressing how epigenetic variations affect chromatin structures so post-insulation modifications histone variants and all that good stuff. And we were really interested in how new plusomes are assembled by histone chaperones. And finally, we started a project that that is also still ongoing and how how the cancer drug target part one senses DNA damage and how it can be inhibited. And how it interacts with chromatin. So these were our project projects in 2015 I felt I needed a little bit of a change of scenery but not too much so I moved down to see a boulder who didn't interview me way back when but now they did hire me which was great. And I pretty soon started hustling and bake sailing and campaigning for a Titan Creos microscope which did not exist at that time and so this is, this is the arrival of Princess Creos on campus in October 2019 about the same time I think your guys is first Creos arrived. This is Shana my administrative assistant who was instrumental in getting her set up and wrestling the contractors and all that good stuff. And we had first slide in 2020 and just to to convey to you how excited we are why we are so excited about this is this is kind of the, the wasteland in Princess Creos sits there's it's the only game in town still and we're very very proud of her she performs really well and we treat her very well. We now have a glass use and and I just don't get over these 2d class images this is not even a particularly good one but now we can just the fact that we can just take a nucleus and put it on the microscope and look at it it's just amazing to me. So, let's talk a little bit about science. So this really allowed us to then study projects that weren't really accessible to x Rikker's biography before and that is that nucleosomes are interaction hubs for nuclear factors so there is about 30 million nucleosomes in the nucleus, a lot. They're very densely packed and they interact with many factors. That's, that's part of their job. And, but the best, the most frequent interact of nucleosomes are themselves because they packed to form higher order structure. So there's primer, you can almost equated with protein structures as primary amino acid sequence. In this case it will be the different flavors of nucleosomes with different ptms and histone variants. These can fold in into secondary structures and that then determines tertiary structure. This is a very simplified way of putting things of course but that's what we were interested in. A really good case point, a really interesting case study would be the centrum or the human centrum. In humans, so the centrum is a specific region on a chromosome to which the mitotic spindle attaches. Okay, it in humans, it's epigenetically and only epigenetically defined by the histone variants and a doesn't really have a special DNA but the only thing that tells a centrum or to be a centrum or is sent a. So we have a nucleosome in which age three is replaced by an age three like protein. Okay, and it's this protein that then recognizes a whole alphabet soup of the inner kinetochore. So a whole slew of proteins converge on this poor sample a nucleosome to then nucleate the outer kinetochore which is again another slew of proteins. And among those proteins there's three different ones that specifically recognize from age three. Now what do they recognize, there is really not a lot to go by. This is the age three nucleosome structure and this is Hitoshi Kouromizaka SEMP-A nucleosome structure, he of the nucleosome song. There's really not that much different they look like nucleosomes. The only difference to write home about is this little dinky loop that sticks out. So we were really interested how does a protein recognize this really minor difference and then nucleate the entire kinetochore on this.  So you have to get this right. So, those were the questions and enter Cody Zhao who said, I will do this by CryoEM. This is when we just started, we didn't have a cryos yet. So Cody said like I will do this with CryoEM. And we were super lucky because CG was at that time in Tom Czech's labs and he was going, he was a little ahead of us with his other project. So Cody and CG teamed up and I don't actually know what you guys did, but you made it work. So huge shout out to CG. This is kind of how our department in Boulder rolls, we really help each other. And what goes around comes around. So thank you for that. And Cody got this to work amazingly. And this is a structure at 2.6 angstroms. And remember, I spent like eight years of my life to get a 2.8 angstrom structure of a nucleosome and now literally undergrads do this on a weekend on a good day. Okay, good. So, so this is our protein kind of stuck and protein stuck to SEMP a nucleosomes and I don't want to go in the detail this is way too much detail here but I just want to say that we now know how SEMP and specifically recognizes this little rinky group that sticks out of SEMP a and that is not present H3. So that's how it discriminates. We've also recognized we've also learned that this protein has a very large protein DNA interaction face interface as you can see here it's almost like it sits with its butt back on the DNA. And the interaction is of course, it can do it with any nucleosome. Okay, so that was interesting and we published this together with Andrea Musakio who helped us with a crystal structure of SEMP a that they had and a lot of other biology. So, what Cody noticed, even in the first days when we weren't really that interested in higher order structure he noticed it form these nucleosomes when he added SEMP and it formed these pesky nucleosome stacks and he tried to avoid them and pick other particles. But then he went back to it and he started to pick dinucleosomes because he figured he could then determine the structure of those dinocleosomes and indeed he did and it turns out that SEMP and bridges SEMP a nucleosomes together so it forms these nucleosome stacks almost like a little bit like a sticky glue or like these. This is one of our collaborators who loves these macarons. We wanted to do a cover for our paper. And so the poor man had to go out and buy macarons to take these pictures. So you know, I was really feeling sorry for him. Okay, so this is a structure of these nucleosomes glued together by SEMP and so very, very cool, and a way to promote chromatin higher order structure, and they do this by employing an additional DNA binding interface that we actually didn't know existed so on the other side of SEMP and that was just like hanging out in the breeze. That also is a very basically charged surface that then is used to interact with a neighboring nucleosome. So this is all good but in real life, nucleosomes do not exist in isolation. They actually exist in nucleosomal arrays. And so remember I told you right at the beginning this approach could also be used to make nucleosomal arrays and so we made 12 more nucleosomes where we just have a very long piece of DNA and then you position 12 nucleosomes on top of that. And you can add SEMP and and you will notice that you find you get very regular, very regular stacks are compacted zigzag arrays of nucleosomes that are promoted by SEMP and you do not get this when you have no SEMP and present and Codi managed to solve the structure of this of this array as well too much lower resolution. To be honest he didn't try that hard to push the resolution because we didn't really need a lot of more additional information and also he was out on job searches and ready to jump to the next adventure. But what we learned from this is that that SEMP and promotes the folding of SEMP A arrays in this manner and these arrays look very different from other arrays that are possibly spawned by the presence of the linker histone H1. So what we think and there's a lot more biology here and we actually teamed up with Aaron straight at Stanford, who is a centrum specialist to test in vivo, whether the second DNA interface was responsible for forming higher order structure and we found that indeed it is and so this second interface really seems to have some critical importance. So we believe that SEMP and is a centrum Eric, kind of a linker histone is like double sticky tape that helps congregate centrum Eric nucleosomes with normal nucleosomes, because that second DNA binding interface does not rely on the presence of SEMP a the only loading function of SEMP and is that little rink it include there's one interaction interface it needs one of those and then it can hook up with a normal nucleosome or any other nucleosome it wishes to do so. Okay. So we think this promotes long range and short range nucleosome interactions and is responsible to form a really specific structure at the center mer. So, so much for that. The point I wanted to make with the stories first of all a huge shout out to city for for helping us. Second, this was our first cryo em structure so we're immensely proud of it. And third, the types of things that you can do with cryo em would literally not be possible with extra crystallography and vice versa. Some of the small things that I showed you yesterday would not have been possible with cryo em so we need them both and we love them both and will continue to use everything, even NMR if we have to. Maybe. I grew up scientifically next to court retreat trick. And so I'm a little shall we say opinionated with respect to anymore. That's all I'll say about this. Okay, so I have a couple more minutes. And I just want to talk about one other big project that really was we've been chewing on for a long time and then really was made possible by cryo em. And that is what happens to paraphrase Roger Kornberg when an irresistible force the RNA polymerase meets an immovable object the nuclear zone something's got to give right. And what gives is that we have to kick out the histones and but we also have to maintain chromatin in the wake of the polymerase. We need histone chaperoning to prevent those histones from aggregating on the DNA on on the RNA so we need his and chaperones. We need these to stabilize these partial nuclear zone so that they don't completely discombobulate and we need to reassemble in the wake. And there's two very diverse classes of protein factors that are responsible for this one is histone chaperones. They are literally like the chaperone at the high school dance. They prevent hanky panky between inappropriate partners. There are also a little bit matchmakers because then they help reassembly which I hope the chaperones at the high school dance do not do. And then we have ATP dependent chromatin remodellers and these guys are big SUVs of machines they use obscene amounts of ATP to shovel nuclear zones out of the way and to remodel chromatin in general. So I'll tell you one story for sure and maybe we'll get to this one because this one is done by John Markert who is a U Wisconsin alum as well I have a great pipeline it seems. So, and the work on fact was done by young Lou, who's now also the who's not the University of Hong Kong. So what do we know about fact fact was originally designed by designed discovered by Danny Reinberg, and also Chris Formosa. It is named very unfortunately because when you search fact in PubMed, you get a lot of hits, obviously. But it's named for facilitates chromatin transcription, presumably by nucleosome disassembly so that's biochemically how it was identified. However, a knockout in yeast leads to nucleosome assembly deficiency so that kind of begs the question, what does it do we now know that is hugely involved in transcription replication DNA repair. And we've renamed it much to Danny Reinberg chagrin to facilitate all chromatin transactions, we've maintained the acronym though so just to keep him happy.  And so it's being explored as a cancer drug target. And we were really interested in how does fact work in these different contexts. And what does it do to new clothes homes without ATP hydrolysis, what's its job. So, our first forays into this was not very exciting, because it turns out that fact actually does not even want to bind to new clothes home so what kind of a factor is that right that's really not very useful. And so when you take fact and nucleosomes or DNA, no binding, but we figured out that if you pre incubate fact with histone H2A H2B. It somehow gets activated, you can then combine it with a partial nucleosome, and it will make a complex that then might be amenable to cry oh yeah, and this is what you see here. So, this is a gel shift. And when you do this experiment, you see some left over his tone at the bottom, but you also see complex one and complex two, there's two bands for crystallography. This would be the kiss of death, like it's a no go for cry and what the heck you just slap it on a grid and see what you can see. In this case, we slept it on a grid. It looked horrible. And we needed a lot of and CT also helped us with this. We needed a lot of help from Bridget Carragher we use spotted on which is now chameleon different freezing techniques so it was really not an easy project. But young and Cody together managed to solve the structure and we fact is a heterodimer, consisting of two multi domain subunits. We, despite the fact that we put the entire fact complex into our complex. We can really see the color bit so there's a large part that is disordered but what we could see was already hugely informative. The resolution is also not amazing but a good starting point. And what we can see is that fact looks, it kind of looks like a, like a unicycle, and it sits astride the nucleosomal diet and each subunit dangles around an either side and there's also this. This is a big terminal domain that seems to hug the histone dimer so just to summarize the findings from this and and the information that we gleaned from this is not so much like where every atom is but really in its mechanism and its regulation. The second point is that. Yeah, we would have liked higher resolution but you should not discard a low ish resolution structure because it can still be very very informative. So fact has an extensive DNA binding interface despite the fact that it does not bind DNA on its own. So that was interesting. And the C terminal acidic domain mimics it acts like it's DNA and tethers the H2H2B dimer to the body of the nucleosome we actually didn't give it enough DNA so usually this dimer would have flown off, but we tether we tether it with a C terminal acidic domain. So this, this allowed us to develop a model for its regulation. So we thought that the C terminal acidic domain is the one that auto inhibits binding when we delete it. We actually can get this thing to bind to DNA. Okay, so this is shown to the gel and the gel on the left it's not great binding, but it is capable of forming a complex with DNA when you delete the tail. What happens is that in the absence of histones when fact is just trundling around and mining its own business. We think this domain is kind of swinging to the inside and binds to this acidic to this basic DNA binding cavity, and it auto inhibits itself. Along comes a free H2H2B dimer because it got kicked off off by a polymerase or something fact fishes it out hugs it to itself, and then can dock it on to a tetrasome and this explains the nucleosome assembly function that we observe in vitro as well as in vivo. Alternatively, and this is also incidentally how we made this complex for cryo EM before knowing anything about this mechanism. Alternatively, fact can also encounter a nucleosome with partially unwrapped DNA and that we know is an intermediate during transcription the DNA has to be peeled off. And this thing is kind of left over fact recognizes that it hugs it grabs a diamond hugs it to itself and this is the complex with structure we actually solve and it prevents the dimer from flying off and this really explains the tethering and stabilization function of fact. So, I just hope you appreciate how much like DNA this the CTD looks like so it's a true DNA mimic. The deletion of the C terminal domain in yeast is lethal and so that really lends credence to the model that we've developed here. I just want to summarize this that fact is a nucleosome maintenance factor it's neither an assembly nor disassembly factor. It's kind of just a repair crew, the cruises around and looks for his stones and bad nuclear zones and just kind of tells them it's okay don't make you hold again and so that's kind of how I see its role. And the regulatory mechanism is such that it only engages partial nuclear zones which is actually a good thing, especially when you have a lot of fact and so fact will be engaging with the DNA that have lost part of their DNA, it will also capture free his stones as they're flying around and reassemble them in the wake of the polymerase. And that model is, and the structure is really highly consistent with all the biochemical data that was in the field at that stage, and was later beautifully illustrated by by some amazing structures by he told she Kormizaka and Patrick Cramer's lab where they actually had fact and nuclear zone and polymerase in a mega complex and they could show it in all in all stages of its life cycle and it's doing exactly what I have actually depicted it's doing here it's doing all of those things. So, all cool explains its central role in all DNA processing pathways because every polymerase whether it's DNA or RNA polymerase needs to peel the DNA off and so we encounter these intermediates. What we do not understand I'm really intrigued in hearing opinions is this dependence of cancer cells and we don't know what the cause or effect is it could be that in cancer cells, you have so much disordered this regulated transcription that you have a lot of disordered nucleosomes and you need a lot of fact to kind of rain them in. It could also be that fact is overexpressed in cancer cells, and too much of a good thing is not good and so factor starts to destroy nucleosomes and that results in destroyed nucleosomes and that then in turn results in in transcription that is this regulated so we do not know what this connection is. So I think I will probably stop here. I just want to give a huge shout out to to john who's been a fabulous graduate student in my lab, who independently worked on a complex that's called smart cat one. I'll show you what what smart cat one does it is involved in transcription replication DNA damage repair. It's a chromatin remodeling factor and john figured out what its structure and its mechanism is and it's really been a tour de force work in the lab we're still working on regulation. So this is an ongoing project and with that I will I will finish and acknowledge the people who did the work so Cody and young who are now in at the University of Hong Kong faculty members, john market who's a postdoc at Harvard but probably will be on the market soon. So if he applies to this place, you should hire him he's amazing. And then, again, a huge shout out to Pamela dire my lab manager of 22 years. So she started with me the year after I started or two years after I started been with me ever since truly indispensable to the success of my lab and with that I'm happy to answer any questions about anything or let you go off and do you think whichever you prefer. Thank you very much. I have a question about the nap one or I'm sorry the fact. Chaperone structure and the extreme sea terminus the acidic. I think you and possibly Rebecca Hill and others have found that other histone chaperones have these poly acidic stretches like nap one that are polyglutamylated and I'm just wondering if you have any evidence from your structures of, of this modification and do you think it might play a role and its chaperoning function. Yeah, that's a blast from the past so so polyglutamylation has been discovered on on jubilant mostly but also on his son chapter on his especially on fact and also on a chaperone called nap one. We did a lot of work on the polyglutamylation in that one. We never published that because there was a key experiment that was missing. And then the student graduated and nobody wanted to pick up the project. What we did find is a polyglutamylation of these of these tales regulates their activity. So, yeah, I actually had forgotten about that thanks for reminding me. Another question about the fact. See terminal domain seem like the knocking that out is obviously lethal in yeast as you described, but is it more the acidic nature or the length of the sea terminal domain that was found that's like really crucial and if so like which residues I guess I'd be curious to know we're found to be really important regarding that. So, it's mostly these and ease with some interspersed other residues. Our density was not good enough that we could have the register we just know it dangles down from there, so we could not build an atomic model of that till. I believe that the team for most of the chopped off the entire sea terminal domain I don't think they did piecemeal deletions, so I can answer that question. If I had to guess, I would say that if you make it much shorter than what we see here. It's probably not going to do. It's probably going to have the lethal lethal effect. Any more questions. Is there something that removes fact from the nucleus home. Or does it just kind of hang out there if it stabilizes the nucleus home. During transcription. He told she Kormizaka like pictured, they were like eight cryo em structures, the whole life cycle so the polymerase eventually shoves it off and we've actually also shown that when you shove it off the DNA it sits on the histones and so it hops over from the DNA to the histone it binds the histones and then it goes off. It's kicked off completely so during transcription the polymerase motor kicks it off when it's just cruising around doing its healing thing if it indeed does that we don't really know we probably. Once it's assembled the nucleus home. I believe what happens is that the DNA competes off the CTD, and that kind of makes it lose its foothold and it's dissociating. And we have in vitro evidence for that but it's not super strong. Do do our key have a version of fact. No they don't know it's a eukaryotic gig. They don't really need it the polymerase doesn't really isn't really that slow down by nuclear something out here. And anymore questions. Like sex doctor look again for the awesome. Thank you.


So John chapter 3, so we went through John chapter 3 last week and I looked at that phrase from John the Baptist in verse number 30 where he said, he must increase, I must decrease. We looked at that but I want to stay in John chapter 3 for one more week and I want to preach just on one verse in particular. I didn't want to leave the last verse of the chapter out of this Bible study because it's hard to have a favorite verse in the Bible but it's definitely my favorite soul winning verse, John 3, 36 where the Bible says, he that believeth on the sun hath everlasting life and he that believeth not the sun shall not see life but the wrath of God abideth on him. So that is definitely my favorite verse for soul winning in the Bible where there is just such a great verse to show people that it's just by believing on Jesus and then when you do believe on Jesus or trust on Jesus that you hath everlasting life. It's in a moment. It's not a process that people think it's not a lifelong, you know, a repent every Sunday or whatever people think it is. It's a very clear verse in the Bible demonstrating the Gospel. So this evening I want to look at this verse. I just want to kind of look at this idea in this verse. I think this verse we're going to look at another unique aspect of the verse outside of the Gospel this evening that makes it very unique. And the question I want to kind of answer tonight is who is God? You know, who is God?  You know, who is God? What's he like? What's his personality like?  And now you want to find out who this God is, what he wants from you, what he's like, what is his disposition towards different things, you know, who is God? All you have to do really is just read the Bible.  And that's what I want to look at this evening.  Is God an angry God?  You know, this one right here is God a loving God.  And I always ask people the question, well, do you think God loves you?  Do you think God loves you? And this is inherently, it's, people know this. People know this right off the top of their head. They know, oh no, God loves me. I've never had one person say that they don't think God loves them. Maybe I have, I don't know. But the vast majority of people know that God loves them. Know that God is a loving God. It seems that everyone knows that. This is really being pushed. It's the only side of God that's being pushed today in Christianity, in this modern culture that we're in. And the problem is, is that if you just teach that God is only a love, what, God is love. The Bible literally says that statement. The Bible says in 1 John 4-8, it says, you know, he that knoweth not love, you know, he that loveeth not knoweth not God, for God is love. In 1 John 4-8. So the Bible literally does say that God is love, that God is love. But if that's all you teach about God and you don't teach who God completely is, what will happen is you will get people that will actually reject the God of the Bible. They will actually say, oh, well that's a, that's a, you know, so by the way, is there an agenda to teaching that God is all loving, all the time, no matter what? You bet there's an agenda there. Because Satan is trying to get people to reject the God of the Bible. So if you have many traits as a person and I teach that, you know, just, you're just all about this one trait.  And all I do is tell people that, you know, you just like to run and you're good at it. I'm misrepresenting you in that case. And people that are just mis, that are just teaching that God is loving of everything all the time are misrepresenting God and what they're doing is they're actually causing people to reject the one true God, the God of the Bible. All right? Because, you know, this is every, you know, every now and then we will watch a Christian movie in our home to try to find a movie for the church for a movie night. It's like 90% failure rate. We usually don't even get through the movie. But every single Christian movie out there is like, it just has these Christians that are just like these super weak, these super, you know, they're just like these super weak men, these super weak people, they're always getting tricked, they're always getting run over by everybody, you know, and they're just like, oh, love, love, love, it's just steamroller. After steamroller it just rolls right over them. It's because of this idea that's being pushed in Christianity today that God is just this one trait. That's why. That, oh, we just have to accept and love everything all the time. So the question is, is God angry or is God loving?  Well the answer in John chapter 3 and verse number 36 is yes. Look at John chapter 3 and look at verse number 36. The Bible says, he that believeth on the Son hath everlasting life. So that seems like a really good statement right there. If you cross reference that statement to John 3.16 the Bible says, for God so loved the world, for God so loved the world that first half of John 3.36, he so loved us, everyone in the world. He so loved whosoever, that whosoever believeth on the Son hath everlasting life. That's how loving God is. That's how loving God is that whosoever would just trust in his Son, he will give them heaven. He will give them eternal life. He will give them something that they do not deserve. That's how loving he is. That's the first half. That's the first side of the coin in John chapter 3 and verse number 36. But let's keep reading because the Bible is the whole verse, not just half the verse. The Bible says in the last half of the verse it says, but he that believeth not the Son shall not see life but the wrath of God abideth on him. So here we see in one single verse. This is why this verse is very unique.  The people that have not trusted in Jesus Christ, they have the wrath of God abiding on them.  And if you can't get that from reading just the first few pages into the Bible, you are not reading the same Bible that we are in this church, which is the King James Bible. God is full of wrath and the Bible, as a matter of fact, the Bible is other than pointing to the Gospel. The Gospel has the most detail about God's wrath. So why is God so angry? Let's explore why God has so much wrath.  Turn to Psalm chapter 5. The first thing God is angry about is quite simply rebellion or sin. So that's why he's wrathful. That's why he's angry. But who is he wrathful towards? Go to Psalm chapter 5 and look at verse number 5. Psalm chapter 5 and verse number 5. Look at what the Bible says here. So let's explore this idea. Is God one-dimensional? God is love, 1 John 4-8. Is that it? That is not it, folks. There are many dimensions to God.  It says, the foolish shall not stand in thy sight. Thou hastest all workers of iniquity. This is the Psalmist saying that God hates these people.  No, God hates these people. God hates these workers of iniquity. And I'm gonna show you what that is in just a minute. Turn to Matthew chapter 7, verse number 23. But these are people in Psalm chapter 5 and verse number 5 that God has wrath towards. God is angry with them. He hates them. Look, I know that that word's a bad word today. But today, hate means, the word hate means to have a stronger version towards or to strongly dislike. That's what it means. Whoo! It's just a word. It's just a word describing a feeling towards something or someone. A negative feeling. Look at Matthew chapter 7. So God hates these people. These workers of iniquity. Who are the workers of iniquity? I tell you what, I'm gonna prove the reprobate doctrine to you right here without even going to Romans chapter 1 because it's everywhere in the Bible. The Bible says that God hates these workers of iniquity. Look at Matthew chapter 7. And look at, actually, let's start at verse number 21. It says, not everyone that saith unto me, Lord, Lord, shall enter into the kingdom of heaven, but he that doeth the will of my father, which is in heaven, the Catholic will say, see, gotcha, aha, you gotta do the will of the father. The will of the father in John 6.40 is that you believe on the Son. All right, not part of the point I'm trying to make, but many will say to me in that day, verse 22, Lord, Lord, have we not prophesied in thy name? In thy name cast out devils, and I named on many wonderful works.  They're telling, they're standing in front of Jesus, telling Jesus why they should be able to go to heaven, why they should have everlasting life because they've done all these great, wonderful works. Look at verse number 23. And then I will profess unto them, I never knew you, depart from me ye that what? Ye that work iniquity. These are the same workers of iniquity that God hates. These people in Matthew chapter seven, so right at Matthew chapter seven in verse number 23, if you write in your Bible, cross reference Psalm 55, because the workers of iniquity are people that are going to hell. So the Bible is saying in Psalm chapter five in verse number five that God hates these people, these are the people that are, these people are going to hell. You say, how am I gonna prove the reprobate doctrine to you? Because in Psalm 55, they're currently working iniquity. They're alive on this earth working iniquity and God hates them now. They're not dead. It doesn't say, I hate us the workers of iniquity that have died.  And then in Matthew chapter seven in verse number 23, we see that that is defined as people, they're damned, they're going to hell. And yes, they thought they were righteous in verse 22 and they thought they had great works, but the point is in Psalm chapter five in verse number five, the workers of iniquity are people that are alive that God hates. God does not hate sinners. God holds out hope for people that would get right and be saved, but these workers of iniquity are something different. These are the people that have turned on God. They're working against the Lord. You know, then refer to Romans chapter one. But the point is you don't even need Romans chapter one to prove that you can reach a point before you're dead on this earth. People can reach a point where God just rejects them.   I mean, I don't even want you to get saved. See the Pharisees. Now go to Psalm chapter 11. In Psalm chapter 59, we won't turn there, but in Psalm chapter 59, David again references the workers of iniquity and he equates them to bloody men. Men that love violence in Psalm chapter 59. Go to Psalm chapter 11, look at verse number five. Psalm chapter 11, look at verse number five. So we're looking at why is God so angry? He's angry with people that have rebelled against him. He's angry with sin. Look at Psalm chapter 11, verse number five. It says, the Lord trieth the righteous. But him, now this matches up perfectly with the bloody men of Psalm chapter 59 that are also equated to the workers of iniquity. It says, him that loveeth violence, his soul hated. So the Bible, this is somebody different here. This isn't somebody that has messed up and has joined a gang and got in a drunk and driving accident and went to prison. That's not who this person is. This person loves violence.  These are the workers of iniquity. But the point is, God hates them. God hates these people. They're alive on the earth at this point. Turn to second Corinthians chapter five. You say, but what about me? You say, but what about me? I still sin.   You were never a worker of iniquity. How do I know that? Because you're saved. Because you were able to get saved. You say, what about me? I still sin. I still sin. And God is angry with sin. God is angry. God's wrath is towards sin. I'm gonna show you that this evening. You're like, I still sin. Turn to second Corinthians chapter number five and look at verse number 21. But here's the difference between you and somebody that is in John chapter three in verse number 36, that is a sinner that has that wrath of God abiding upon them. Look at this in verse number 21 of second Corinthians chapter number five. The Bible says, for he hath made him to be sinned for us. That's how we got out of this mess. But here's the real key, who knew no sin that we might be made the righteousness of God in him.  He's not going to look at you and see how much sin you have or don't have when it comes down to whether or not you get eternal life. What he is going to see, I'm choking. What he's gonna see is the righteousness of Christ. It's the righteousness of Christ that is imputed to you in your life. So it is a matter how much sin you have or don't have as far as that decision on whether or not you get eternal life, because God's righteousness is given to you. He's gonna see the righteousness of Christ. He's gonna see the righteousness of him that knew no sin. And that's how Jesus could take that sin upon himself because he knew no sin. If Jesus sinned one time, he could not be that righteousness imputed upon you because he would have that sin. That's why God had to come and do this himself. Turn to Jeremiah chapter 23. Jeremiah chapter 23, look at verse number five. Jeremiah chapter 23, look at verse number five. So the Bible here is showing us again that when God looks at us, he's not gonna see how much sin you have or don't have when it comes to you getting eternal life or not. Look at Jeremiah 23, verse five. The Bible says, behold, this is a prophecy of Christ here where the Bible says, behold, the days come, sayeth the Lord that I will raise unto David a righteous branch, and a king shall reign and prosper and shall execute judgment and justice in the earth and his days Judah shall be saved and Israel dwells safely. And this is the name whereby he shall be called the Lord, what does that say? Our righteousness. So the Bible here is saying that Jesus, how am I getting to heaven as a sinner? Because Jesus is my righteousness. So it doesn't matter how bad you and I mess up, if we've trusted on Jesus, he is our righteousness. He, I mean, that's what God is gonna see when he looks at us is the righteousness of Christ, him that knew no sin. So that's how we're getting out of it is because of Jesus' righteousness, not how good or not good we are. But look, God is mad because people, nations are in open rebellion to him. When you read the Bible, this is why God is wrathful. All right, look, he can be wrathful towards us and I'm gonna give you a couple points on how to have God's wrath not be on you, not to damn nation, but have God not upset with you, have God not be angry with you, but God is wrathful, back to the main point. God is wrathful, God is love, but he is also wrathful because people and nations have always been in open rebellion towards him. And look, every nation, every nation that has rebelled against God will be judged on this earth by God. We need to keep that in mind. We need to keep that in mind as we walk through this short blip that is our life because every nation in the Bible, look, it wasn't just the nation of Israel that was judged by God. Every single nation was judged by God that every nation that rebels against God is gonna get what they've got coming. It wasn't just Israel that was carried away by Babylon that was judged. Babylon was also judged. And if you look at chapter 46, 47, 48 of Jeremiah, Edom was judged. The Moabites were judged. Egypt was judged. Everybody got what was coming to them because everybody was rebelling against God. And it didn't matter if they were God's people or not, God judged them anyway. So look, I mean, that's why, you know, I like to pay attention to what's going on in the world. You all know that. But I don't wanna get too wrapped up in a lot of this other stuff that's going on with other nations because what you have to understand is this idea that there's good guys and there's bad guys, especially in this world that we live in today, it just doesn't, it's a false narrative. Question the narrative always, especially today. The truth is, I mean, look, I mean the conflicts today that we see, the conflicts today that we see, it doesn't matter.  That's the narrative today. You gotta question that narrative.  They're trying to put one nation or another in the good guy box. But I'm telling you, in the world that we live in today, in the world that we've been in in the last many decades, there's, you know, as a Bible-believing Christian, you're gonna have problems with that.  Neither one of these two fits in the good guy box. God is gonna judge all nations. So it shouldn't really get wrapped up when enemies of God do bad things to enemies of God.  All of them, all of them, all of them. Question the narrative because God is gonna judge all nations that are an open rebellion to him. And you show me the nation today that's following the Lord, you show me that nation. So we shouldn't get too wrapped up in that. But we see that God, look, God is wrathful towards people and nations that are in rebellion to him. God is wrathful towards sin.  That's how it works for the unsaved. So you say, what about me? Me, could I experience God's wrath? You bet you could. You will never experience the eternal judgment of God's wrath, but God can definitely be angry with the saved believer today. And so that's the point of the sermon tonight. I just wanna give you two ways as a saved Christian that you can avoid God's wrath in your life. You will never experience, if you are saved tonight, you will never experience the eternal judgment of God's wrath as John chapter three, verse number 36 is explaining, you can still experience it through the chastisement of God. So the first one is this, turn to Proverbs chapter eight. I'm gonna give you two points tonight. It's a simple message tonight. I'm gonna give you two points to avoid this in your life. The first way to avoid God's wrath in your life as a Bible believing saved believer is to have your own wrath.  Look at Proverbs chapter eight in verse number 13.  The world is trying to rob you of your wrath today. You need to make sure that you listen to what the Bible says and as a Bible believing Christian, we need to get our wrath right.  Look at Proverbs chapter eight in verse number 13. We need to get our wrath right as Christians. The fear of the Lord is to hate evil, pride and arrogance and the evil way, the forward mouth do I hate. That's what's all of us talking right there.  That's what he's saying. You don't have to, look, if you don't hate evil and the workers of iniquity, you will take no action in your life to protect against it. I mean, that's the first thing. It's to follow the Bible to protect yourself and your family.  This is Jehoshaphat. This is Jehoshaphat and the mistake that he made. Turn to second chronicles chapter 19. You say, what if I just want to love everybody all the time no matter what? Well, then you're Jehoshaphat. And the Lord rebuked him for this. Jehoshaphat, of course, made an alliance. He made an alliance with a very wicked man, Ahab. He made this alliance with him. It almost cost him his life and it messed up generations of his family. But look at what the rebuke that the prophet gave to Jehoshaphat after he narrowly escaped with his life. Look at verse number two of second chronicles chapter 19. You say, could God be wrathful towards me? Could God be angry towards me as a Bible-believing Christian? I'm saved, why would God ever be angry with me? And Jehu, the son of Heneini, the seer, went out to meet him and said to King Jehoshaphat, shouldest thou help the ungodly and love them that hate the Lord? Therefore, he's saying you shouldn't love people that hate God. That's what they're telling you today. They're telling you to love people that hate God today. And instead, he says, therefore, he says, because of this, because you did do this, therefore is wrath upon thee from before the Lord. He's like, now God's wrath is on you. What does that mean? That means God has mad at him. God is angry with Jehoshaphat. Chastisement is coming his way. This is the Christian today that loves them that hate the Lord. This is why, if you ask me, if you ask me, like what is the most important thing that the new IFB brought back? What is the most important truth that the new, because look, we haven't invented anything here. It's just Bible doctrine that people stop preaching, stop saying, if you ask me what the most important thing that churches like ours have brought back the most important truth that was not being spoke anymore, it's not the pre-trib rapture. It's not, oh, you know, post-trib pre-rap. That's not it. It's the reprobate doctrine. That's what's going on. I mean, that is super important today. Because why?  They're loving people that they shouldn't be loving. And that's exactly what the narrative is today. I mean, I'm watching, I am watching.  You don't even have to pay much attention to see this. There's this gal that goes around to colleges. And she, I don't know if she's Christian or not, but this, her name's Riley Gaines, I think, and she speaks out against like transgender, you know, like trannies in women's sports or whatever. She's really like fighting against this. She goes to college campuses and like she's been assaulted. She's been like held against her will. The last, you know, video somebody sent, like people went up to her table and just threw everything.  All these people are saying love everything, hate her. There's literally a school board president of a public school, which I don't care about, that refused to fly any flag other than the American flag and his state flag. And you know why he did that. And literally people want to kill him. All these loving people want to kill him.  Look at all these death threats. How many pastors do we know? They've had their families and just threatened.  Yeah, it's pretty loving. It's a lie. The narrative is a complete lie. Turn to James chapter four. So we're not supposed to just love every single person that hates the Lord. We're not supposed to love anyone that hates the Lord, folks. Look at James chapter four in verse number four. The point of trying to get you to understand here is to avoid what happened to Jehoshaphat to avoid God's wrath coming upon us.  God wants you to stand up and choose a side. Look at James four, four. James four, four, the Bible says you adulterers and adulteresses. Know ye not that friendship of the world is enmity with God. You know what enmity means? It means being against, meaning hating, meaning working against him and just being in conflict with him.  You're gonna be against the Lord. That's what the Bible is saying here. Who sort of there will be a friend of the world is the what? The enemy of God. You must choose a side. You can't be in the middle. So for yourself, for yourself, you should have, turn to 2 Corinthians chapter seven. So to avoid God's wrath, you need to choose a side. You need to have indignation against those who hate the Lord, the Bible is saying here. But look, even for yourself, even for yourself, you should have indignation against these things that are trying to get you to fall.  Look at 2 Corinthians chapter seven in verse number 11. This is talking about 2 Corinthians chapter seven and verse number 11. It's talking about somebody that would get right. So 2 Corinthians, the whole concept of 2 Corinthians is kind of like, hey, we kicked this guy out of church in 1 Corinthians. Now he's getting right. Let's get him back in. Let's get him back into church. What does a godly sorrow look like? What does it look like when somebody gets right? And the Bible gives us this great verse here that shows us what godly sorrow should entail. Look at this. For behold, this self same thing. That he sorrowed after a godly sort. What carefulness it wrought in you. Yay, what clearing of yourselves. Yay, what indignation. You know what that means?  What fear, what vehemint desire, what zeal. Yay, look at this. What revenge. So there's two words there. Indignation and revenge. Talking about how angry you should be at the sin in your life. How angry you should be at anything that is drawing you towards sin. Anything that's trying to pull you out. I mean, we're talking about somebody that got thrown out of his church here. It's like, man, it should just make you just just vengeful about that thing, about that person, about whatever. You need to have that wrath against that sin in your life so you can protect yourself against it. I mean, the things, the people that want you to fail, that have caused you to fall, you should have wrath against that to avoid God's wrath. But here's another thing. Here's another thing. Back to go to Matthew chapter 12. I mean, just on this idea of choosing a side. How about this? Could you stand up for God? There's all these people out there trying to destroy the Lord.  There are all these people out there trying to destroy and blaspheme God. And they're trying to teach all this falsehood to all these people that we love in the world about who God is and who God isn't. Could we stand up for God? You say, oh, I just want to love everything and everybody. Could you be jealous for God like he's jealous for you? Think about it this way.  Would you get wrathful then? Of course you would. What if somebody came in and tried to harm someone that you love? Well, do you love the Lord? Do you love the Lord as you consume all the TV that blasphemes him? As you consume all the media, all the movies, all the music that just hates him? You need to get your wrath right. Not only to avoid God's wrath, but hey, do you love God? Are we jealous for God? He's jealous for us. So much that his name is jealous. Look at Matthew 1230. Where is our wrath for those trying to destroy God? Matthew 1230, the Bible says, he that is not with me is against me. And he that gathereth not with me scattereth abroad. We need to get our wrath right as Christians. If you don't, there's wrath on you. That's what the Bible is telling us here. Turn to Genesis chapter 15. That's the first thing.  Get your wrath correct. Not politically correct, folks. What the Bible says. What the Bible says about what you should be wrathful towards. What kind of world would we live in? What would there be any justice if there was no wrath? Think about that. We'll get into that in just a minute. But look at Genesis chapter 15. Here's the second thing that you need to have in your life to avoid God's wrath. The second thing that you need to have in your life to avoid the wrath of God on you in your life as chastisement is mercy. I bet you didn't think I was gonna say mercy there, did ya? You're like, man, I was getting all wrathful. I was getting all wrathful and excited tonight. And then you drop something like mercy on me. Look at Genesis chapter 15. Let's look at what God's mercy looks like. Yeah, God's got a lot of wrath, but let's look at what his mercy looks like. Look at Genesis chapter 15. Look at verse number 16. The Bible says, when the sun was going down, we're talking about Abram here. He's not even Abraham yet. It says, the sun, a deep sleep fell upon Abram and a hole of great darkness fell upon him. And he said unto Abram, no of surety that thy seed shall be a stranger in a land that is not theirs. And they shall serve them and they shall afflict them 400 years. He's talking about the Egyptian captivity here. And also that nation whom they serve, will I what? Will I judge? His wrath came upon Pharaoh, did it not? And afterward shall they come out with great substance. And thou shalt go to thy fathers in peace and thou shalt be buried in a good old age. But in the fourth generation they shall come hither again for the iniquity of the Amorites is not yet full. Don't miss that last statement right there. And here's something that people don't understand because nobody reads the Bible anymore. Nobody reads the Bible. Nobody understands the Bible. Of course, most people aren't saved so they couldn't understand it. But let's just look at the timeline from Abraham to Joshua. Here's a good way to, here's a good way to, this isn't exactly correct, but this is a good way to remember the timeline. Okay. If you look at from Abraham to Moses to David to the return after the Babylonian captivity, just Abraham somewhere around 2000 BC. And then if you just look at those big milestones, it's about 500 years in between, maybe 450 in a couple cases. But it's not really, there's some debate on the actual dates. But if you look at Abraham to Moses, you're looking at almost 500 years from 2000 BC to 1500 BC. Then you go to David, that's about 1000 BC. Then you go to after the captivity, that's about, I don't know, 450 BC or so. And then 450 years from there, the birth of Christ. So it's just kind of like narrow it down 500, 500, 500. And it's a good way to remember those things. But the point I'm trying to get you to understand is this. When God gave Abraham the promised land, it was 500 years before they actually went there. Before he actually went and tapped Moses and said, hey, it's time to get the people to go. It's time to get them to go into the promised land. He already told Abraham here that he was going to give it to him. It was 500 years. Why was it so long, you ask? Well, the Bible just told us in verse number 16, because the iniquity of the Amorites was not yet full. You know what that is? That's God's mercy right there. That's how much God, God, that's how much mercy, Godhead. He said the Amorites, what are you talking about? They're not God's people, but their iniquity was not full. And they're not gotten bad enough to where God decided he was gonna come and judge them. See, this is what people don't miss. This is what people miss. It took God hundreds of years to get to the point where his wrath was full with the Amorites. And then he said, why call Moses at that time? Because his wrath was full. Because his wrath was full and that was the time that he chose to rescue his people. See, the promise fulfilled Abraham. It doubled as a judgment on the people in the land of Canaan. This is what nobody understands today.  No, God spent hundreds of years pouring his mercy out on those people and his iniquity wasn't full for literally over 500 years. Instead, he put up for 500 years, he put up with people hating him. He put up with people turning against him.  He put up with people for hundreds of years.  You know, at some point mercy becomes unjust. Endless mercy becomes unjust. You say, what point is it correct? This point right here. Whatever point God chooses to end his mercy and bring in his wrath, that's what's just. You know, I've thought about that. I've thought about that as a pastor many times. I have reflected on this one thought so many times over the last probably two years I cannot even count the times. But at some point, how much mercy do you give to people without harming other people? See, you just can't tolerate evil things and evil people and people doing bad things for infinity or you end up hurting innocent people. At some point, judgment and wrath equals mercy for the next generations, you see? Tell me God isn't just.  Tell me that that's just. Tell me that that's mercy. Tell me that that's loving. It's not. As long as there is evil people and sin and open rebellion to God on this earth, God must have wrath. Turn to Matthew chapter nine.  At some point, infinite mercy turns into injustice. And that's what these people are calling for. They say we need to love everything and love every evil and love every perversion and love every child and wester. They're calling for injustice and evil is what they're calling for. Look at Matthew chapter nine. Let's get back to mercy. Matthew chapter nine and verse number 13. The Bible says, but go ye. So we see that God's mercy is hundreds and hundreds of years for these nations in the Promised Land. Look at verse 13. It says, but go ye and learn what that meaneth. I will have mercy and not sacrifice for I am come to call the righteous but sinners to repentance. Jesus says this and he was quoting Hosea. Chapter six and verse number six. You can go ahead and turn there if you want. You can write that in Matthew chapter nine and verse number 13. If you write in your Bible what Jesus was quoting this idea in Hosea 6.6 of God wants mercy over sacrifice. And the Hosea 6.6 says it this way. Says for I desired mercy and not sacrifice and the knowledge of God more than burnt offerings. See, Israel on the outside was obeying the laws. Jesus was speaking to the Pharisees. They were on the outside. They were obeying the law and they looked good and everything was great and they had their borders enlarged on their garments and they looked super religious but they did not fear the Lord. They did not love God. God's like keep the sacrifices. He's like I want mercy and not all these sacrifices. Look at 1 Samuel chapter 15. This is a perfect example of this exactly what Jesus is talking about in 1 Samuel chapter 15. 1 Samuel chapter 15 is when Samuel told Saul to destroy the Amorites and he didn't do it. He didn't listen to the Lord. He kept some of the sheep. He kept some of the oxen and he kept the king. He didn't kill him. Look at verse number 18. And the Lord sent him on a journey and said go and utterly destroy the sinners, the amicoites I'm sorry and fight against them until they be consumed. Where for? So Samuel's talking to Saul. He's like why didn't you do it? He says where for? Then didst thou not obey the voice of the Lord? But disfly upon the spoil and disevil in the sight of the Lord. And Saul said unto Samuel, you have obeyed the voice of the Lord and have gone the way which the Lord sent me and have brought a gag the king of Amalek and have utterly destroyed the amicoites. But the people took of the spoil, the sheep and the oxen and the chief of the things which should have been utterly destroyed. Why? Why did he say the people did it for? To give to you God to sacrifice unto the Lord thy God and Gilgall. So here Paul he does two things that are wrong. First of all he doesn't admit it and then he blames somebody else. But he says the reason we did it Samuel was so we could sacrifice to the Lord. We got all this stuff for you God. And what does God say? He says exactly what Jesus told the Pharisees. Look what he said and Samuel said, hath the Lord as great delight in burnt offerings and sacrifices as it obeying the voice of the Lord behold to obey is better than to sacrifice and to harken than the fat of lamps.  He wants you to listen. He wants you to obey. He wants you to be merciful is what Jesus said. He's like put all your sacrifices away.  That's what Jesus came here to do was be merciful to these sinners. Turn to Luke chapter six. Mercy's important. God wants us to have mercy. You say how can mercy, how can me having mercy help me avoid God's wrath? I keep sinning. I'm never gonna be sinless. You're never gonna be sinless. What you should get the sin out of your life. The Bible says if you willingly sin you're gonna be beaten with more stripes. You're gonna be beaten with many stripes. If there's a downside of a church like this it's right there. Is that you're gonna know what's right. You're gonna know what's wrong. Everything from the Bible is gonna be preached. You're gonna know. You're gonna have to willingly go against it. And you know what that fact right there is why a lot of people don't come to a church like this. Or a lot of people end up leaving a church like this because they hit some point where they're like I'm not gonna do that and they don't wanna be beaten with the many stripes over and over again. Look at Luke six, 36. The Bible says be therefore merciful as your father also is merciful. Go to Matthew chapter five. You say but still what does that have to do with God's wrath pastor? Go to Matthew chapter five. We're getting closer. We're circling the target here. Matthew chapter five. Look at verse number seven. Matthew chapter five and verse number seven.  What do you need?  You should confess your sins to the Lord daily. First John one nine is talking about being right with your heavenly father. It's trying to be, it's trying to get you to be in a good relationship with the God that saved you. It's not talking about being saved over and over every day. It's talking about being in a good relationship. Confess your sins. Look at Matthew chapter five and verse number seven. The Bible says bless it or the merciful. It's saying why be merciful though? Bless it or the merciful. Oh, here it is. For they shall obtain mercy. You know what you need in your life? As you try to get the sin out of your life, as you try to change things and get things right, you read the Bible, you hear something. Oh, don't have that right. And you try to get that thing right and you slip up and you fall. You know what you need? You should pray for mercy. How do you get mercy? You give mercy. The Bible is super clear about this. Let's go to James chapter two and verse number 13. I could read you verse after verse after verse about this. He who is merciful will obtain mercy from the Lord. He that gives no mercy?  I'm not talking about the workers of iniquity. I'm talking about 99% of the rest of the world being merciful. Look at James chapter two and verse number 13. It says for he should have judgment without mercy. Who wants God? Who wants God to give them judgment with no mercy? Who wants a heavenly father that will just see them slip up and will just smack them down as hard as he possibly can right away? Who would like that? Well, that's what this guy is gonna get. He shall have judgment without mercy that has showed no mercy. And mercy rejoices against judgment. You will get mercy from God. How you give mercy to others. You're this person, you're just never gonna let anything go.  You're this wife that's gonna bring up these things from 10 years ago. You will receive no mercy from the Lord. That's what the Bible is telling us. You can't let grudges go against something that happened to you three years ago.   You know what's also a very merciful thing to do?  You know what's pretty merciful? Going up and knocking on somebody's door that you've never met before. Taking time out of your week every single week every single chance you can get to go knock on somebody's door and show them how they can get eternal life. It's not like you earned it.  You know what, that's love and that's mercy because you know what? Those people that you're knocking on their door 99% of the people in that neighborhood that wrath of God is abiding on them in an eternal way. You know what they need? They need mercy. They need somebody to come up and show them. And you know what? There's a lot of people that'll listen. There's a lot of people that wanna hear and there's nothing more merciful that you can do in your life. You want God to give you mercy? Go out and preach the gospel to people and then you'll have mercy from God. Because guess what? You're not gonna be in sinless perfection. I don't know about you but I want my mercy tank full with the Lord. When I go and I mess something up, I want God to be like, you know what? I just need to shoe him this way and he needs to be merciful. I don't need God coming in and nuke in my life. And that's exactly what he can do. That's exactly what he can do.  If I just go out and I'll just never forgive a thing, I'll never let anything go. God's gonna go straight to the stick with me and he'll do the same with you. This is a philosophy that is very clear in the Bible. You want to avoid God's wrath in your life? Tonight, folks, on this Wednesday night, you want to, how can I avoid God's wrath in my life? God's chastising wrath?  That's number one. And then be merciful to all others. That's number two. And we will avoid God's wrath in our lives. So John 3.36, God is love, he's full of it.  And we just need to know who God is and how to navigate that in our lives. And of course, the Bible has all the answers. Let's bow our heads and have a word of prayer.