Okay, today what I want to talk about, and I've talked about this before. It was a great post, but I think I need to talk about it again and do a little tutorial on how I deal with this. I've seen, and I had mentioned to me, twice this weekend, somebody told me about a fire situation of, guess what, backstabbing a receptacle. And then I kept seeing it pop up on my Facebook meme, so maybe that's somebody trying to tell me something about, maybe I should talk about this. What I mean by backstabbing a receptacle or device is I've got this here. Here's my device. It's a switch. It's a Decora switch. I've got this guy here, and if you look in here, you've got those little circles. Let me just get a screwdriver to point with. You've got these little, let's see here, these little circles that are in there. Let's go up to there and see that circle. What you can do is strip your wire, stick it in there, and it's called a backstab. Very, very quick, which I guess some people like, but the problem is you don't always make a great connection. And what can happen sometimes is it doesn't make a great connection, and then it starts to arc inside the device itself. That arc can cause this heat, that heat causes fire, and that is a very dangerous thing. So backstabbing your devices, I think is a no-no. I understand you guys who are doing condos and you guys that are doing houses and you're just all about getting it done quick. But I think it is worth it to hook these wires and put them on your receptacles. So I'm going to show you quickly how I do that. Now just to show you quick, I'll show you another problem with backstabbing. So I've got this device here. I've got preloaded here, I've got some Lumex, as you Americans like to call it, or Lumex as we Canadians call it, if you notice.  I'm going to stick this in the back here just to show you what I'm talking about. Stick that in the back. Stick that in the back.  There we go. Grab my number one, screw it down. So I do that. Okay, I'm not sure if any of you guys are timing that, but we're only at three minutes into the video. There we go. I've got this here. Now let's see where I talk about you sticking it in the holes there. Here's the problem I find with these guys, is when I want to change this out, there's a quick release thing in there, but good luck trying to get anything in there, you see there's that little quick release tab, you've got to have a really skinny screwdriver to stick in there, and even then you're going to bust off the back of this.   Yeah you do. Let's face facts. So you cut that off and then I take this off, and now I've pretty well got to use this plug because what I have here is dangerous on the back, right? Because I have access to, if I want to use this plug, that's going to be live right there. So that's, and I don't want to have that. So that's why I'm not a fan of backstabbing. Anybody who's been in my class knows that I tell them that that's an option and that your bosses will tell you about it, but if you can get away from it, do not backstab these devices for two reasons, dangerous, and then when you change it out, it completely like this could have been reused, but now I'm going to have to go through and try to carefully pull that out. It's just don't do it. So quickly hook them. Again, not a big deal guys. Let's just take another piece of Lumex that I've got here, sorry Romex for you Americans. I know I've got more Americans and Canadians watching this. I'm going to strip it. So I strip that. I strip my neutral and I leave a little bit of length there. And I'm not sure if you guys tell me how much length you leave. You don't want to leave too much, but you don't want to go too little.  So now I've got a full hook and I do the same thing on my neutral and then I do the same thing on my ground. Now the habit I get into is I always want to drown first. So I take my device, I ground it. Now here's the trick with these things. You'll see if I can line this up on the video good enough. But when I hook this, you notice that the hook goes on the direction that the screw is going to go this way. So as I tighten this down, it's going to, it could pull this in and it could take this guy and spin it around there and get it tight. If I go on the wrong way, let's go like this. And this I see a lot and I battle with all my students on it until they get in the habit. It's a bit of a mind job. But if I screw this on here and I'm screwing around, you see how this at the same time could come out. So you want to make sure that the hook goes in the direction that you're going to go to the right basically, righty-tidy, lefty-loosey, am I right? Okay, let's go. So I go like that, tighten it down, right, boom. Then I take this, I've got them all already pre-hooked. I just make sure that again, this is an older one so I've got to loosen this off, loosen that off, loosen that off. And make sure that remember when you're doing your hot, your hot goes to the gold screw, your white or your neutral goes to the silver screw. Remember that. You'll be learning that if you're an apprentice.  And then I want to hook this thing on there. It's not loose enough. So I get on there, hook that on, tighten this on. Again I'm talking and stuff like this. I don't think that this is how long it takes. As you get better and better at it, it goes quicker and quicker. Am I right? Again, I know Evan Hussie is going to get on there. He's going to call me on this and say, nope, you've got a backstab because it's just quicker. You want to make sure you get this on there, again, got it on, oops, coming out. Thanks Keith. Going on like that. Urban, it's good to see you buddy. How's Montana? Go like that, tighten it on there. And again, and what I like about this hook, again, it didn't take me too long, is I got a good solid connection there, right? You don't know what kind of connection. I can see physically that this guy is screwed on there. This guy is screwed on there. It's all good. Thanks, Kevin. I got a great connection. Now here's the things you want to watch for on this one. This neutral here, if I was going to talk to my student about it, I'd probably tell him it's a bit too long, just a hair too long. This hot, I think is pretty good. I like to see this guy come under a little bit more, but the fact of the matter is, guys, I can see the connections. And to me, that is key, because then I can feel safe that I've made that good connection. Honestly, I think the more and more you do these things, the quicker and quicker you start hooking faster and faster, and you probably can come pretty darn close to going as fast as those guys who backstab them.  The backstabbing part, I think it's kind of lazy. And I've done it in the past where you just want to get it done.  But you've got to hook that ground anyways. So what are two more hooks and going on there? Just get in the habit of it. It makes you a better, I mean, that's not a better electrician, but it makes you look to me like you take more care of your work. Again, you can see your connections here, that's good, that's good. So you want to make sure you've got those great connections. So that's why I think you should not backstab a receptacle, because you cannot see on the inside. And again, for those of you just joining me now, you see that if I cut that off, basically this device is now useless to me unless I take the time to take it out. And honestly, the amount of time it takes to take those out, I'm going to throw it out anyways. Let's be honest about things for once. Well, not for once. You know what I'm talking about. All right? So that's the way the Electric Academy does their devices. You guys, I've been having so much fun with this stuff. I've been getting actually got a flood of emails in, which I was quite surprising, because I've been running the site now for about three years now, four years. And I haven't been able to get it up and running the way I've kind of wanted it. I've changed the game. I'm talking to you guys all the time, which is so exciting to me. I'm getting into their interactions with you guys, talking on Facebook Messenger, talking through email. So please, if there's anything you want me to discuss, please send me a message. I check them all the time. Send me an email, chat at theelectricacademy.com. And I will make sure I address it. So there's been a few people that I've talked about. Evan, Steve, Irving, I've talked to you guys. I've addressed some of the things you wanted to talk about through Facebook Live. So I do honestly, if you have gotten value from these, please share it. If you want me to keep going on these, please give me likes because it gets more engagement and lets me know that you guys want to see this stuff. And I don't know if you guys want to see more of like little tutorials like this. I've got a pipe bending one coming. It probably will be in probably the end of September. I'm working on that. Just getting that all lined up.  That's sort of stuff I'm into as well. So anything like that. Actually, Kelly gave me, Kelly Smith gave me a great one about grounding and bonding. So for lightning strikes and that, I'm going to get on that as well. So make sure if you have not liked this page, like this page. If you are looking for like a PDF that shows you a ton of online resources for electricians, make sure you go to the top of this page and hit the learn more button. Just give me your email. I'll blast that to you. It's Wednesday. When I send out the newsletter, I'm going to be sending out my top 10 apps that I think all electricians should be using. If you've been following me, you know that Evernote's going to be one of them. But not always. Hey Scott, Scott's another guy that's been, he's been talking and liking stuff. So I appreciate that Scott. Thank you very much. It's awesome. Have you guys engaged on the posts? Let me know of anything else you want to talk about. So again, this Wednesday, the newsletter that comes out top 10 apps that I think all electricians should have on their phone. Cause our phones, let's face facts. I know a lot of older for me hate them, but phones are part of our tools. Are they not? So if you agree with that, like it. And I, that will be coming out on Wednesday. If you're part of the newsletter, if you want to be part of the newsletter, hit that learn more. Not only do you get that PDF of online resources, you get signed up to the newsletter. I'll send that out. I send that out every Wednesday. If you don't like the newsletter, don't worry. It's not a hard sell.  That's all. Okay. I got to go because my class starts next Tuesday. So I've got a crapload of prep to do. I'm getting totally excited because it means a new class. I get them for six months and I love my job as some of you can probably tell by now. All right. Thanks for everything guys. Have a great day. I'm at my 12 minute mark, which always seems to be about right for Chad to sign off. So I'll have a great day. Work safe out there. And hey, Brian, and I will talk to you guys probably tomorrow because I've got a week that I'm still not teaching. And then I hate to say, but when I start teaching, I'll probably slow down to maybe two or three of these a week. Okay. Cool. Again, if you need anything talked about, let me know. Chad at theelectricacademy.com, like this page. Like, hit that learn more button and we will chat with you guys tomorrow. Have a great day guys. See you tomorrow.


University of the Year has been a terrific project for us in Northern Ireland to work with. For many reasons, it's heightened our profile as a research intensive university, it's highlighted our profile of doing really high quality research, and we were working within the theme of arts and humanities, which has a terrific resonance for the city of Belfast and a terrific resonance across Northern Ireland. Ours was a very competitive commission. We had 12 submissions from very high quality artists across Northern Ireland, and we spent a lot of time looking at them. They were all very different. Some of them were live performance, some were sound insulations, and we actually chose Carrie Neely's work because we felt it captured a wider range of activity, but also there was an excitement about using two venues in Belfast City Centre, the iconic Belfast City Hall, but also showcasing our building here in the city centre of Belfast. We felt that we got a significant interplay between the commission and what we want to achieve here in Northern Ireland as a vibrant university. Luminous Curious Journey as a title kind of started with a play on words. I started looking at words that had the OU within the title so that we could really pull that out and have focus on the open university. And then once I had chosen what works were going where, we really went for the title with the OU in it which suited each project. And I think what Carrie achieved in her commission was really getting a strong feel for how the research of the open university interweaved with the very rich tapestry that we have in Belfast in Northern Ireland of the written word. So we've used some very famous poets. We've used people like Michael Longley. We've used Kieran Carson. And we've also involved Belfast's new poet laureate, Sinead Morrissey. So we've got their works being reinterpreted by graphic artists, by digital artists. And also what I've really valued has been the way that Carrie has integrated the work of our students and the work of some of our lecturing staff as well. This piece of work that we've been involved with over the last number of months has also inspired Belfast City Council to run a fringe festival. In addition to the visual displays at Belfast City Hall and at the Open University building, there are live readings at the Ulster Hall. And again, that's a very iconic and very highly regarded arts venue in the city. So we're really delighted that the city has brought in to what we're doing as well. Well, we were delighted here at the Ulster Hall to be part of the University of the Air program and we've designed a program of arts events which are looking at the idea of local literature as well as other art forms. We have a really wide scope of different events covering photography, music, even dance events coinciding with literature. And we're also trying to engage as many different people. So you can see us setting up for a children's event here and all sorts of different things. And I just feel this really buys into the University of the Air as an idea that Open University is for everyone. This is a very eclectic program and we're looking to engage with as many Belfast people as possible this week. I think the exhibition obviously has attracted the attention of the research community throughout Northern Ireland. So, you know, we've had great engagement with the other two universities in Northern Ireland. We've had great engagement with the politicians both at Belfast City Hall and at the Northern Ireland Assembly. So they're really excited by what the Open University is bringing to the city. You know, it's a delight to me to be involved in anything at all associated with Open University. But I also want to say thank you to the artists and the ambition of the artists because if anybody's going to see Belfast from itself it's going to be our artist. I think it's putting the Open University right at the heart of the community that it serves by demonstrating something that people maybe don't realize we do really well and that's research.


Hello, my name is Danny Rogers, and I am privileged to serve as the Chief Judge of the Fort Worth Municipal Court. Here in Fort Worth, our municipal court judges are all appointed by our City Council and Mayor to two-year terms. Currently, we have 10 full-time municipal court judges as well as eight part-time judges. Sometimes, people want to know, how do you become a judge? Well, all of us are attorneys. We're licensed by the State Bar of Texas. And the minimum requirement is that you've worked as an attorney for two years. All of us have met those requirements. And then every two years, our appointment is up for renewal or review by the City Council. And the City Council goes through a process and has reappointed us for some of us for several times. I've been privileged to serve the City of Fort Worth as a judge for 25 years. Many of those years, I worked as a magistrate, primarily as a magistrate, which meant that I physically worked in the jail. Now the judges, jail judges, what I was called, called myself in those days. And what our responsibility is, is to review all of their arrest by the Fort Worth Police Department at that time for probable cause and to make determinations of what bond, if any, should be set and what the conditions should be, that process. And that's a different part of what we do. Typically, all judges are magistrates. And we have judges that serve in that role now. So our judges are all trained to do that work, as well as to work here at the courthouse. At the courthouse, our primary jurisdiction is Class C misdemeanor, traffic tickets, city ordinance violations, that sort of thing. And we do everything from plea dockets, which is an initial appearance by someone, to a walk-in docket where someone comes in and just needs to talk to a judge about a ticket, to trials before a judge and trials before a jury. So our days are very busy. We have a lot of administrative work that goes with that as well. Parts of our operation run 24-7-365. And that would primarily be the magistration I talked about earlier. It is a real privilege to serve. We have a cross-section of folks that serve as judges, both full-time and part-time. There's a total of 18 of us. What I would really want to get across to you is that if you get a ticket in the city of Fort Worth, you know, there are several pages of information attached to that. But I would say the most important part is that you reach out to the court one way or another. You come to the courthouse here at 1000 Throckport and Street or you can come to us through our virtual court. You can get information about that on our website, on the citation itself or addresses and phone numbers of how you can contact the court. If you will come and see us one way or another, then we will help you in every way that we can to move that case forward, hopefully to resolve it in a way that you feel is fair and that you've had an opportunity to be heard. So I would also say that it's very important that you be heard. You have an opportunity to come and speak with a judge or a clerk or whoever it's necessary to help you understand what has happened and to provide you all the rights that the law provides for you. If you will come and see us, we will move it forward and we will get this hopefully taken care of quickly so you don't have to worry about it or carry it. So we hope that you will come see us. We want to help you to walk through this process with you, answer any questions that you have about the case. And remember, it's just a Class C. It's a traffic ticket. It's not about going to jail. It's about coming to court, resolving the case in a way that you get heard and justice is served. So thank you very much for watching.


An optical fiber or optical fiber is a flexible, transparent fiber made by drawing glass silica or plastic to a diameter slightly thicker than that of a human hair. Optical fibers are used most often as a means to transmit light between the two ends of the fiber and find wide usage in fiber optic communications, where they permit transmission over longer distances and at higher bandwidths data rates than electrical cables. Fibers are used instead of metal wires because signals travel along them with less loss. In addition, fibers are immune to electromagnetic interference, the problem from which metal wires suffer excessively. Fibers are also used for illumination and imaging, and are often wrapped in bundles so they may be used to carry lighting or images out of confined spaces, as in the case of a fiber scope. Specially designed fibers are also used for a variety of other applications, some of them being fiber optic sensors and fiber lasers. Optical fibers typically include the cores around by a transparent cladding material with a lower index of refraction. Fibers are kept in the core by the phenomenon of total internal reflection which causes the fiber to act as a waveguide. Fibers that support many propagation paths or transverse modes are called multi-mode fibers, while those that support the single mode are called single-mode fibers SMF. Multi-mode fibers generally have a wider core diameter and are used for short-distance communication links and for applications where high power must be transmitted. Single-mode fibers are used for most communication links longer than 1,000 meters 3,300 feet. Being able to join optical fibers with low loss is important in fiber optic communication. This is more complex than joining the electrical wire or cable and involves careful cleaving of the fibers, precise alignment of the fiber cores, and the coupling of these aligned cores. For applications that demand a permanent connection the fusion splice is common. In this technique, an electric arc is used to melt the ends of the fibers together. Another common technique is a mechanical splice, where the ends of the fibers are held in contact by mechanical force. Temporary or semipermanent connections are made by means of specialized optical fiber connectors. The field of applied science and engineering concerned with the design and application of optical fibers is known as fiber opics. The term was coined by Indian physicist Narendra Singh Kapini, who is widely acknowledged as the father of fiber opics.


In entertainment news now, David Adelike is a man of color and is notably a king on social media. As a stunt has just been pulled to the surprise and of course amusement of fans getting himself a dedicated emoji to celebrate David Doe at ten in the music and entertainment industry. Now the 27-year-old founder of David Doe Music Worldwide celebrate his 10th anniversary of making music in Nigeria and across the borders. With his Dami Duro remix in 2012, David Doe appeared from the onset of controversial singer and that has come in handy for him. Updates


Hello, thank you for joining me. I'm surrounded by flower pots at the Fletcher's Garden Centre near Eccleshaw in Staffordshire. I've come here today to visit the Hillcote Valley miniature railway, which is situated over this side of the Garden Centre. It's a seven and a quarter inch gauge miniature railway. It opened in 1993, so we're going to go and have a ride and see what they have here. So if we come to here and approach the railway, there's the ticket office. We'll just hear them starting up a diesel loco, so that sounds very exciting. We're going to have a ride behind that. So before I get the ticket, I'll just show you there's their ticket office, see the railway track here and there's two trains there waiting, so I think what I better do is go and buy my ticket and we'll go and have a ride. The Valley Miniature Railway. I really like seeing the British class 66 next to the American loco, so we've got American loco's built for Britain and America side by side. I'm just going to show you a couple of our things before we go. So as I've mentioned before, we're here at the Fletcher's Garden Centre, which is near Eccleshaw. Another feature though of this Garden Centre, as well as it's very first-minute railway, is they have a wildlife and bird of prey centre. So if we have a look here, here is the entrance to the wildlife and bird of prey centre. I'm not going to go in there today, but if you do come and visit, it's certainly something you can do to make your visit even more interesting. I'm just coming now into the children's playground. The reason I'm bringing you in here is because from here you get a very good view of the yard and shed. So see some wagons here. There's a new diesel loco, which has only just arrived at the railway. It's their newest loco. There is a steam loco over there on the turntable by the signal box. So I've had a great time here. So if you're out this way in Staffordshire, Eccleshaw is quite near to stone. I would say you probably do need to come here by car, although there might be a bus that passes here, but don't quote me on that one. But you know, why not come and visit, have a nice day out, have a ride in the train, buy something in the garden, centre for your garden, have a look around the zoo, which is all behind this hedge and we did see some of the animals when we were filming the trains and when we were riding the train. So from the Hillcote Valley, miniature railway, thank you very much for watching and goodbye.


So first I added the tens and then I got 80, then I added the ones and I got 21 and then I added those two numbers together and I got 101. First I broke the numbers, then second I added the tens and then there and then 10, 20 plus 20 plus 50 equals 80. Third I did the ones, 7, 8, 6 plus 20 equals 21 and then 80 and I put 20 with the 80 and it equaled 100. Then I added one more and I made 101. Okay, I did a number line, I got 17, jumped 20, I jumped 8 and then with the 20 I got 37 and 8 which equals 45. Then I jumped 50 and then I got 50, I jumped 6 and then I ended up 101.


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Everybody Matt here for late in sports cards rip in 2022 and anyone football hobby 10 box break number 16 pick your team They've Anthony G. Dolphins Texans Bradley R Falcon Steelers Colby C. Raiders Rams Edward P. Jags Edward E. Cardinals Niners Bucks Eric M Commanders Evan Z Packers Harper B. Chargers Henrik J. Patriots Jeff M. Ravens Bears Colts Joseph C. Vikings Josh M. Bengals Justin R. Broncos Lance H. Browns Eagle Saints Matt J. Cowboys Matthew M. Bills Matthew G. Titans Robert H. Seahawks Rory C. Lyons Jets Thomas A. Giants Tony Y. Panthers and T. Tong Chiefs All right, here we go First half of this case is pretty rough. So hopefully Loaded out here. This is Seahawks patch auto 36 and 49 Doug Baldwin I'm not sure I've pulled a Doug Baldwin auto before 36 of 49 Seahawks This is Texans formula one rookie dual patch auto blue Derek Stingley Jr. This is 36 of 75 This is Niners quad relic auto 6 of 49 Ronnie lot a couple patches and couple jerseys. I think nice Ronnie lot for the Niners This is Patriots rookie patch auto 191 of 199 Bailey Zappie nice hit for the Pats Keep it going Move it in the right direction. This is Steelers rookie dual patch auto 123 of 149 George Pickens This is Falcons day one shadow box rookie auto 70 of 75 Desmond Ritter You go nice rookie auto for the Falcons for more boxes, let's see at least two more big hits Falcons formula one rookie dual patch auto Desmond Ritter. That is blue Five of 35 almost got the jersey number five of 35 Congrats Falcons going back to back with the Ritter Falcons was Bradley are That's Bradley Excuse me Patriots rookie dual patch auto six of 25 Bailey Zappie Congrats Pats with two Zappie RPAs Two Zappies two Ritters. This is the wonders auto Rams Marshall Falk awesome hit that is number two of 15 Love these wonders autos pulled Chad Johnson and now Marshall Falk from Panini one so far this year Awesome hit All right last one do a quick recap Done here On the back nice one Packers Lone Star patch auto 18 of 20 Aaron Rogers Congrats Green Bay 18 of 20 Nice way to close it out Excuse me So we had Steelers rookie dual patch auto to 149 George Pickens Niners quad relic auto 49 Ronnie lot Texans rookie dual patch auto Derek Stingley Jr. That was to 75 Seahawks patch auto to 49 Doug Baldwin Rams the wonders auto Marshall Falk Falcons rookie dual patch auto blue Desmond Ritter That was 35 Falcons day one rookie shadowbox auto to 75 Desmond Ritter And we had Patriots with two Bailey Zappie rookie patch autos to 199 and to 25 Rookie patch auto rookie dual patch auto and Packers Lone Star patch auto 18 of 20 Aaron Rogers That'll do it for the break. Thanks, everybody


This is stock number 11018A. We are here at Summit Automotive in Fond du Lac, Wisconsin. Your new and used light-duty truck. Headquarters, today we are checking out this super clean 2014 Chevy Silverado 1500 crew cab, short box, high country. This vehicle has the 5.3 liter V8 motor. It has been fully safe and inspected by our service shop. It has a fresh oil and filter change. All the fluids have been checked and topped off and this truck is 100% ready to go. I believe we did brand new brakes all the way around on it too in our safety inspection. White diamond metallic is the color we shoot all of our videos in 1080p 60 frames per second. So if you have HD capabilities on your computer tablet or smartphone device turn them on right now because it is definitely your best way to check out the quality and condition of the truck before seeing it in person. And for a 2014 this thing is in fantastic condition. It does come with 20 inch Vox painted alloy rims and it does have like new Cooper Discover SRX 275 555 R20 tires. I'd say at least 80 to 90% of the tried left on those tires. They look very new. You can see that those front brakes are brand new as well. Front fender is in nice shape. I didn't see any major dents of dings on that. Front bumper is in nice condition. You do get the factory fog lights. Front bumper parking sensors. No major dents on there. The headlight lenses are nice and clear and you do get the projector lamp head lamps chrome trimmed grille. That's an excellent shape and the hood is in really nice condition as well. Paint is very reflective and shiny. Nice condition. Passengers side front fenders in nice shape and the passengers side rim no major scuffs or scrapes on there. As you go down this side the truck take note of how clean the body is how reflective and mirror like that paint is. We take these HD videos so if you are far away or even if you're close by and you just cannot make the trip down but you're still interested in purchasing the truck you can see the truck hear the truck and have confidence in the vehicle that you're looking at before you even get here so that when you do get here there's no surprises and that is why we offer the Summit Auto Fastpass option in the upper right hand part of your screen a link to our website where you can apply for financing get pre-approved see a payment quote even appraise a trade on this vehicle all from the comfort of your own home so when you get here all you got to do is drive the truck love it you'll be all set to go back brakes are brand new as well and I didn't see any scuffs or scrapes on that room either back tires have just as much tread as the front tires and the frame and underbody other than a little bit of surface rust on the frame the body is in really nice shape and see any corrosion on that and it does have some nice aftermarket chrome step bars on it so framed on it by is pretty nice and as we come around to the back of the vehicle rear bumpers in great shape it does have the backup parking sensors the rear bumper steps and the tailgate is in really nice shape as well with that high country logo there and it does have a tonneau pro hard fold-up tonneau cover on it has the shock down assist tailgate spray and bed liner that's a nice shape the bed itself is a nice shape and it does have the LED bed lighting that's actually right up there turn your cargo lamps on and those will turn on tailgate shuts nice and solidly and as you go down the driver's side just as clean as a passenger side no denser dings on that driver's side box and for full disclosure this back rim really nice condition as well down the rest of the side of the truck looks really good you do get the power mirrors with the built-in directional signals and the driver's side blind spot mirror inside the high country package gives you this really good looking brown leather interior you get the I think it's kind of a gray or white piping on it no rips or tears on the seats both these seats are heated and cooled has a color match weather tech floor mats and then on the seat headrests here you get the high country stitched into the head rest really intricate good-looking power windows power locks power mirrors memory driver's seat you do get the power fold-in mirrors I always like showing both sides so that you know that both sides are working good and it does have the Bose sound system factory brake controller turned out four-wheel drive and auto headlamps you get the tilt and telescopic steering wheel and as we hop inside here turn the ignition on so that you can see the miles and everything see at this one has 59,759 miles instrument cluster is very nice and clean you get the heated leather wrap steering wheel Bluetooth and information center controls on the right cruise controls and forward collision warning controls and heated steering wheel controls on the left you get the six-speed automatic transmission with the optional tap shift close the door here so listen to the dinging you get the factory navigation system on the Chevy my link system and FM and Sirius XM radio capabilities this is also where your backup camera shows up CD player you get dual climate control down here and you got your heated seats air-conditioned seats power pedals stability control cargo lamps that's how you turn those bed lights on parking sensors you can turn those off and on and then you do have lane departure warning as well three USBs to 12 volt power points and then a hundred ten volt hundred fifty watt plug-in passenger side seat is in excellent shape smells very clean inside this truck I don't think it's ever been smoked in and the headliner is in really nice condition as well you do get map lights up here as well as your home link buttons for your garage door security systems and lighting systems and on-star and SOS buttons in that review mirror I'm gonna start it up so I can show you this backup cam works well there and then we'll start it up before we take a look under the hood as well we'll check out the back seats and then we'll check out under the hood back seats are just as clean as the front seats no rips or tears back here does have the latch child safety system same piping back here power sliding rear window with the built-in rear defrost and those seats are in excellent shape you get a weather tech format that goes all the way across which keeps your carpeting looking good underneath and we'll start it up and take a quick look under the hood I would personally like to thank you for checking out the video today and hopefully from this HD video you will have been able to tell just how clean this truck is all the way around inside and out under the hood we have the 5.3 liter v8 motor engine bay is very clean runs very smooth this truck has been fully 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super clean 2014 Chevy Silverado 1500 crew cab short box high country and white diamond tricode thanks again


Good morning. I'm Laura Elnitzke, and I'm a PI at NHGRI. And I'd like to welcome you to this session of current topics entitled the regulatory and epigenetic landscapes of mammalian genomes. Now, due to rules, regulations, and government policies, I want to start by saying I have no affiliations with commercial entities.  Nearly a dozen years ago, as this millennium began, President Clinton called the Human Genome Project the most wondrous map ever produced by humankind. And he was right. The study of the human genome is driving the entire future of medicine. As scientists directly evolved in disease research, we all are likely to use genomic sequence and the extensive amount of data on sequence variants on a daily basis. And yet, how likely are we to see a variant and know that it is the causative mutation we're looking for? To date, the Hunt for Disease Alleles in the genome has focused on only 2% of the whole. Those are the coding regions. Just 2%. This morning, I want to talk about the other 98% of the genome. Now, we typically ignore the half of this 98% that is represented by repetitive elements. But the remaining half likely holds the secrets to human disease. That's because this other half contains the regulatory components, which play a crucial role in delivering gene products in the proper spatiotemporal patterns, meaning through developmental time and within the necessary cells. Yet sequences containing the regulatory elements are so hard to decipher that have been called the dark matter of the genome. But that opinion is changing. Many mutations associated with diseases are being identified. And now we need to think forward to the future about how mutation discovery leads to a mechanistic understanding of disease. This will ultimately enable new therapies. So I really want to emphasize this point. The cell represents an interconnected set of events. Disease is not about a single mutation in a cell. It's about a cascade of events. These happen as a consequence of a mutation, and no event occurs in isolation. So therapies need to target processes that are derailed by mutations. And when the mechanisms of disease are known, optimal therapies will become obvious. We're not there yet, but it's an exciting prospect. For now, there's no map connecting the cascade of processes in disease. In other words, we've got a map of the genes like the celestial stars, but we don't yet know how these genes are connected, like the patterns and constellations. However, these patterns are emerging. And they're partly revealed by a new code, one that coexists with the raw sequence of the genome, known as the epigenetic code. That code regulates processes in the genome and is passed from cell to cell, but it isn't encoded in the DNA. Instead, it is carried along either as methylation marks that are placed directly on the DNA, or as methylation and acetylation modifications on the tails of histones that are wrapped by the DNA. And until we had the genomic sequence, we were absolutely blind to this code. The epigenetic patterns provide a landscape to the genome by identifying a cell type, distinguishing functional elements, and indicating gene expression levels. And ultimately, these patterns may be critical to diagnosing disease. Now, this morning, I'm going to describe some individual parts of the genome, but I'm going to emphasize how the genome is larger than the sum of its individual parts. In other words, just as an alphabet forms words and words form language, the processes in the genome depend upon and react to each other. So the sequencing of the human genome was just the beginning of the story. We have the sequence of 2.9 billion haploid base pairs. But determining what they do and how they contribute to disease is what makes our job so fascinating. Amazingly, we know the genetics behind some 3,000 Mendelian diseases. And many scientists predict that about 4,000 more will be discovered in the next few years. In contrast, the genetics of complex diseases currently evades description. And in the prevailing explanation known as the common disease, common variant hypothesis, many genes are needed to contribute very small individual effects, each slightly elevating the risk of disease. So I ask you, have we really learned nothing but probabilities from the genome, as some of the naysayers say? I think not. New information about the role of DNA in health and disease is discovered every day. The genome sequence has expanded the potential of genetic testing. And it's opened the door for developing new therapies that target specific and diverse causes of the same disease, such as some types of cancer, enabling treatments that can be tailored to each specific patient. Now, if you contrast that with the blanket, non-specific approach of chemotherapy, then the potential payout of the human genome project becomes palpable. So let's look at four important outcomes of the human genome project. They include comparative genomics, mapping of functional elements, the interpretation of disease processes, and reading the epigenetic code. Now, in technological terms, the human genome may not be very impressive. We know that it's three gigabases in size, but as an information storage system, how do you think that equates to other information storage systems, such as Mozilla browsers? Any guesses? The answer? Those three gigabases, when converted to bits and bytes, become the equivalent of 750 megabytes in binary code. In other words, the human genome equates to 2.8 Mozilla browsers, and it's smaller than Microsoft Office, but it's probably almost as buggy. Now, we like to think that the human species is the most advanced species on the planet, and this suggests that genomic complexity has been critical to our success. Yet the origin of that complexity is not obvious from studying the human genome itself. Genomically speaking, we fall somewhere between a chicken and a grape, and if you thought that the number of bases in the genome was significant, well, I'm sorry to tell you that there's not much difference between the person sitting next to you and a turkey or a lungfish. Underlying the genome's complexity is its ability to create diversity, and this diversity comes in a number of forms. For example, multiple non-coding elements are used in combination to define precise, yet adaptable, patterns for gene expression. The alternative processing of genes and the use of alternative promoters also creates extensive diversity in the genome. Furthermore, non-coding RNA, which was largely undetected until a few years ago, now plays a central role in the theory of multicellular complexity, carrying out a massive amount of regulation within the cell. So with this complexity in mind, maybe it's no surprise that the coding regions cover less than 2% of the genome. And the hunt is on to find altered sequences that impact normal regulatory functions to understand how they affect regulation, function, and speciation. And regardless of whether these mutations cause single gene diseases or are involved in complex multigenic diseases, they have subtle and far-reaching impacts that create diverse and unpredictable outcomes. So what do we know about the human genome so far? There are 1,400 elusive disease-associated variants in the genome that have been identified through genetic epidemiological studies known as genome-wide association studies, or GWAS. This is a statistical technique that compares thousands of patient samples to thousands of control samples and has identified sequence variants associated with the risk of Crohn's disease, heart disease, diabetes, and dozens of other conditions. The vast majority of these 1,400 GWAS variants lie in the non-coding regions of the genome. And they're providing us with enormous opportunities to discover what they do. So throughout this talk, we'll take a look at functional categories of the genome to further explain the steps you might consider to ascertain function at these GWAS sites. Now, probably the biggest impact of the human genome project to date is the ability to compare genomes at the nucleotide level. And that's because mutations in functional DNA are less likely to be tolerated and leave a signature of similarity across genomes. Early on, with only human and mouse genomes available, some of the most important insights were gained. For example, while some genes change rapidly, such as immune reproduction and olfaction, other loci containing developmental genes are so highly constrained that no base pair has changed in 75 million years of evolutionary time since the last common ancestor. It's pretty amazing. So how much overlap is there between the human and mouse genomes? Guess this? At the nucleotide level, approximately 40% of the human genome can be aligned to the mouse genome. These sequences seem to represent most of the orthologous sequences that remain in both lineages from the last common ancestor, with the rest likely to have been deleted in one or both genomes. And today, you'll find the genomes of 36 mammals and 10 non-mammalian vertebrates aligned in the genome browsers. You'll recognize that we use genomic diversity to uncover the story of human evolution because it refines the historical account of the early migration of humans out of Africa into the Middle East and beyond. And you've probably read that having the sequence of the human genome available was crucial to the discovery of unmistakable traces of Neanderthal DNA mixed into some modern human populations. So what percent of genomic DNA in people from Eurasia and the Southwestern Pacific was inherited in the Neanderthals? From Neanderthals. Between 1% and 4%. But did you actually know that this inherited material is not concentrated in genes? Instead, it's spread somewhat randomly in the non-coding portions of the modern human genomes. And doesn't it make you wonder what it contributes? Now, the Neanderthal data is available for everyone to view on the genome browsers as well. But let's not forget that getting to this point took integrated and cross-disciplinary approaches in biology, math, statistics, and computer science. And obviously, it wasn't so easy that a caveman could do it. So what is important in what we've learned? Taking a bottom-up approach, let's discuss some sequences in the human genome before talking about how these features fit together. There are millions of sequences in the human genome that perform essential functions. Broadly, they can be defined as protein-coding genes, non-coding RNAs, and regulatory sequences. One way to narrow the search for meaningful pieces of the non-coding genome is to align genomic sequences from species that are very different. For example, 46 species ranging from human to fish spans an evolutionary time of over 450 million years. This computational exercise allows us to identify patches in the sequences where functional words stand out against the background, somewhat like words embedded in a random alphabet. This similarity is caused by negative selection or purifying selection, which is the pressure to remove deleterious changes from a sequence. It is a strong signal of regions whose sequence has been maintained because it is critical for a biological function. Now, in the early days of genome sequencing, the initial calculations of the amount of the human genome under purifying selection were performed when only human and mouse genomes were available. The underlying prediction was that some proportion of the genome was more conserved than expected when compared to the level of conservation found in neutral sequences. So in other words, some proportion of the 40% of the genome that is alignable stands out as being preserved for functional purposes. So the 40% is represented by the blue curve. And when the alignable neutral regions were removed, represented by the red curve, the statistical estimate was that 5% of the genome was under functional constraint. And that 5% represented the biological functions that were common to the human and mouse genomes. Now, keep in mind that this is 2 times larger, 2 and 1 half times larger than the 2% estimate of the amount of coding sequence in the genome. So these perceived regulatory regions outnumber the coding regions. Now, although these statistical techniques could identify the number of bases under constraint, the approach could not say which bases were functional because conservation alone is in proof of function. And the statistics certainly didn't say what functions the regions perform. So scaling up and comparing multiple genomes, the regions under negative selection begin to stand out, known as phylogenetic footprints. And these are often a proxy for transcription factor binding sites. The use of additional genomes adds stronger evidence to find regions under purifying selection. It has also been shown that these functional words can be found using a collection of short evolutionary distances represented only by primate genomes. And this works as long as enough primate species are included. This approach is known as phylogenetic shadowing. And it has the benefit of finding functions that are shared only among primates. So some very extreme examples of negative selection exist in the genome. For example, have you heard of ultra-conserved elements? They are longer than 200 base pairs, and they have 100% identity with no insertions or deletions between human, mouse, and rat genomes. So the funny thing is, they were originally thought to be traces of contamination by human DNA in the mouse or rat samples. It's funny in retrospect. Maybe it wasn't so funny at the time. It was later shown that they actually belong in these genomes. And this extreme level of conservation correctly predicts their functional importance, and that can vary. They act as enhancers. They act as regulators of splicing or domains in protein coding genes. Now, the ones that overlap protein coding genes can be really interesting. They actually tend to serve a dual role. In one instance, they serve as the coding regions for proteins. And in another instance, they represent enhancer elements that regulate the transcription of neighboring genes. So today, the methods to detect constrained elements fall into two broad categories. These are generative, model-based approaches and bottom-up approaches. So an example of a widely used generative approach is available on the UCSC genome browser known as FAST-Cons. Have you seen FAST-Cons? This approach identifies the number of constrained sequences within a genome based on the known patterns of sequence variation among species. In other words, it calculates it all at once. And in contrast to the generative models, an example of a bottom-up approach for constrained element detection is GERB. This approach first estimates all constrained bases in the genome, and then clusters neighboring sites together to calculate a total percentage. So recently, GERB was used to predict the percentage of the human genome under functional constraint and found an additional 2%, bringing the total to 7% of the human genome. Now, it's likely that much more than 7% of the human genome is functional. However, these measurements look at constraint across diverse genomes. And therefore, they're bound to miss some things. Some constrained elements are missed because they are specific to human, or maybe they're conserved only in primate sequences, or some may not have conservation consistent with the way we measure it. In fact, one headline of the 2007 ENCODE paper published in Nature was that roughly 50% of the known non-coding functional elements seemed to be unconstrained across all mammals. The explanations were numerous. Perhaps the functions actually lacked biological assays. That chromatin accessibility might be more important than sequence composition. That the regions were, in fact, lineage specific. That they were functionally conserved, but non-orthologous elements. Or that they did not confer a selective advantage or disadvantage to the organism, and were treated as biochemically neutral features. And now you can understand why it's so hard to find regulatory elements in non-coding regions. Now, on the opposite side of the spectrum, some genes are under selection to increase the number of nucleotide changes in a particular species. And this is known as positive natural selection, the force that drives the increase in advantageous substitutions. So a well-known example, although at first glance, you might not consider this advantageous, is the sickle cell mutation in the beta-globin gene. In its heterozygous form, this mutation actually protects against malaria. So in this case, the blood cells are weakened by the presence of the sickle allele, causing them to be rapidly cleared from the body as the cells become infected. And this connection explains why the mutation has been retained and the incidence of sickle cell corresponds to regions endemic with malaria. Now, another example is a mutation in a regulatory region near the gene for lactase. And this allows lactose tolerance to persist into adulthood. And studies show that this particular variant was apparently selected in parts of Europe as the domestication of cattle was taking place. So I don't know about you, but I'm thankful because I love Dairy Queen. In 2006, a collection of transcripts with accelerated divergence was identified and called Human Accelerated Regions, or HARS, for short. They were conserved across species but had accelerated changes in human relative even to the chimp sequence. Up to 1,000 of these regions have been described. And some HARS are thought to have contributed to the emergence of human neuroanatomy, language, and complex thought. One example contains a gene enhancer that targets expression to the opposable thumb and may be responsible for modifications in the ankle or foot corresponding to bipedalism. Note, though, that hidden processes in the genome have complicated the discovery of accelerated regions. While positive selection is a measure for accelerated evolution, the presence of accelerated evolution is not in itself proof it was caused by positive selection. So for example, there's another process known as bias gene conversion. And this changes regions that are rich in A's and T's to regions that are rich in G's and C's. And this phenomenon actually explains several of the HAR regions rather than positive selection. So now three forces must be considered to interpret the fastest evolving regions of the genome. These include positive selection, bias gene conversion, and the relaxation of negative constraint below the average background level. And all of these would create an excessive number of changes. In relation to the global change, what is that accelerated change? Accelerated changes are typically measured on a local basis. But when I say local, local tends to be fairly large in the genome, a megabase or so. And so it's calculating the background level of changes and then looking in a single species to see if there are an excessive number of changes. Sure, the rates will vary. And really the only ones that can be easily detected are those that are well above the background level. So a second very important contribution of the genome project is the mapping of functional elements. Although comparative genomics got us very far in our understanding of the basic makeup of the genome, it says nothing about the activity of a locus in a given cell type. Promoter and enhancer activities differ by tissue, reflecting dynamic processes that act on the static sequence. And the need to look at dynamic features within cells has fueled the rise of chromatin immunoprecipitation coupled to next generation sequencing. And under the tidal wave of data, the dark matter is rapidly giving way to the encyclopedia of DNA elements that lie within the non-coding regions of the genome. This information is narrowing the search for mutations that cause disease and identifying more functional elements than previous methods that used sequence conservation. Now I know you're all familiar with the static landscape of a cell where there are genes composed of exons separated by introns. Every gene has at least one promoter, which extends some length upstream and is typically divided into the core promoter, the proximal promoter, and even an extended promoter. These are the recruitment and docking sites for the transcription machinery. And moving further away, we find elements such as enhancers or silencers and insulators, which modulate the transcriptional activity. They perform functions through direct contact with the promoter. Most likely through three-dimensional looping. And if we were to look more closely at the gene body, introns sometimes carry enhancer or silencer elements, either for the same gene or for neighboring genes. And as I've mentioned, often alternative promoters are embedded within the body of the gene to provide specialized expression patterns or deliver unique forms of the protein. So let's focus on enhancers for a minute. Looking at the sequence level, we know that enhancers are composed of clusters of transcription factor binding sites, each of which are six to 20 base pairs long, and they bind both activators and repressors. Evolutionary sequence conservation is typically present. If we look at the epigenetic level, these transcription factors are recruiting chromatin remodeling complexes that facilitate open chromatin and leave a signature of DNAs one hypersensitivity. This results from the repositioning of nucleosomes to allow transcription factor access. The chromatin remodellers act by targeting specific histone residues and transferring the acetyl or methyl groups to create the histone code. So in active enhancers, you'll find the marks of H3K4 mono-diamethylation and H3K27 acetylation. You'll also find the protein P300 present. So identifying enhancers is becoming a solvable problem and they are being found just about anywhere. They're adjacent to genes, they're embedded within coding axons of genes, they're in introns of unrelated genes, they're even mega bases away from their targets, and they have been found in regions known as gene deserts. So one important component of enhancer function is physical interaction with the promoter. And DNA looping completely takes us out of the realm of sequence analysis and into the 3D structure of the nucleus. So you've perhaps heard of experiments used to detect looping interactions. They include chiapet, 3C, 4C, 5C, and now high C. And these techniques identify enhancer promoter interactions. And when all of this data is coupled together, the data from long distance interactions, gene expression and chip seek assays, tell us the who, what, when, and where of regulatory function. So the third crucial contribution of the genome project is the ability to interpret disease processes. Given the important role of enhancers in gene expression, it's not surprising that mutations in enhancers cause genetic defects. In the case of the sonic hedgehog gene, a gene involved in the formation of digits in mammals and fins in fish, gain of function mutations in a distal enhancer create extra digits, whereas loss of function mutations cause limbs to be truncated. So gain of function mutations can actually be pretty cool to study. Here's an example of a mouse line that can regenerate lost tissue. While it's currently not known precisely how this is done, the hope is that the regulatory information could eventually be used to regenerate lost limbs. And it just makes me think some amazing regulatory pathways must be involved here. So speaking of a role in disease, we've all heard of the common disease, common variant relationship in which multiple weak acting variants combine to cause complex diseases. But did you know that this idea might apply directly to enhancers? So an emerging hypothesis about enhancers is that their modular construction could allow the accumulation of mutations that carry a modest effect of disease risk. This connection makes enhancers excellent candidates for a role in causing common diseases. And now the challenge is to determine which variants affect function and which do not. So in that same vein, several GWAS variants involved in type two diabetes, colorectal cancer, breast and pancreatic cancers, and even coronary artery disease now appear to reside in enhancer elements. So what about the rest of these GWAS variants? Actually, the majority of the GWAS variants. This is where it gets challenging. Because enhancers aren't validated across the genome, how do we know if a variant disrupts a functional element or is neutral? Does sequence conservation and phylogenetic footprinting provide evidence? Some. Do histone modifications in DNA's hypersensitivity indicate function? Yes. Do P300 and looping interactions show activity? Of course. So primarily to find this evidence, these are a lot of different experiments to have to do. We look to resources like ENCODE. But please keep in mind that these epigenetic patterns are cell type specific. And this is such an important point. Since ENCODE has only looked at a subset of features in a subset of all tissues, most information is still lacking for specific cell types involved in diseases. If that cell type hasn't been studied, the evidence of function might not exist. Now, one big caveat to keep in mind is that the GWAS variant might not be the causal variant in disease, even though the statistics say so. And this is due to the relationship among variants in a region. In other words, any variant in the region of green, the region of linkage disequilibrium, defining the family of variants that travel together could be the culprit. So some fraction of GWAS variants don't actually fall at the functional site, but they are related to one that does. So I'll come back to enhancers in a few minutes as we discuss specific chromatin modifications. For now, I wanna discuss the idea that functional elements in the genome are interconnected through chromatin. So one way to holistically assess a cell is to look at nuclear interactions. And as we all learn in introductory biology classes, eukaryotic chromatin structure can be viewed as a series of superimposed organizational layers. At the root is the DNA sequence, which undergoes higher order packaging being folded into nucleosomes and creating the fundamental units of chromatin. With approximately 147 base pairs of DNA wrapped around an octamer of histone proteins, including two copies each of H2A, H2B, H3, and H4. Now in the nuclei of mammals, the most obvious form of structural genome organization is the compartmentalization of chromatin into eukromatin and heterochromatin. Eukromatin is open chromatin. It is accessible to transcription factors and is represented by the beads on a string model of DNA. Heterochromatin is closed chromatin, packaged into the condensed form of 30 nanometer fibers using histone H1. Now one of the most important recent discoveries in the field of genome biology has been the demonstration that genomes are non-randomly organized in the nucleus. So each of these colors are representing individual chromosomes and the area that they occupy is known as the chromosome territory. So in addition to these chromosome territories, transcriptionally silent heterochromatin, which contains gene poor chromosomes or regions with low overall transcriptional activity are localized to the nuclear periphery. The gene rich chromosomes and the highly transcribed eukromatic DNA is situated in the center of the nucleus. So oops, what makes this really interesting is that some of these localization patterns are cell type specific, not only at the level of the chromosomes, but also at the level of the genes. And in that case, their repositioning reflects the genes activation or repression status. So the holy grail in this field is to elucidate what mechanisms determine where a gene or a chromosome localizes within a cell nucleus. Another goal is to easily detect when a mutation has interfered with nuclear positioning of a gene compared to the positioning of the normal allele. So what remains missing? What we desperately need in descriptions of gene regulation is to integrate information about how nuclear organization contributes to regulatory interactions that occur between chromosomes. So a well-known example is the activation of the interferon beta gene on human chromosome nine. This requires physical interaction with regulatory elements on chromosomes four and 18. How often these interchromosomal events occur throughout the genome is not known, but it might be predicted using these new techniques offered by high C that detect all interactions in the genome. Now in the silenced region at the nuclear periphery, important proteins include the lamin proteins. They form an intranuclear scaffold known as the lamina around the edges of the nucleus. This lamina supports the nuclear architecture and helps to organize nuclear processes such as DNA and RNA synthesis. So how do we confirm that laminar interactions are important? Find mutations that cause disease. Look at the presence of sequence conservation in lamina interacting domains or delete lamina structures. I'd go with the first one. Mutations that cause disease. Sequence conservation is not often present in lamina interacting domains. However, there are tracks on the UCSC browser using chip type assays that are now identifying broad regions of laminar interacting domains. So for example, Hutchinson-Gilford-Pergeria syndrome is a premature aging syndrome with symptoms of early cessation of growth, baldness at the age of two, progressive degeneration of the skin, muscle and bone, and often fatal atherosclerosis in childhood. And in this disease, the architecture of the cell nucleus is abnormal. The mutations associated with this disease occur in the protein lamin A. Without lamina function, nuclear organization is lost. So is transcription and transcription-coupled repair that corrects DNA damage. And it's this reduced ability to mount a response to DNA damage that triggers cell death and senescence, thereby promoting accelerated aging. So Groucho Marx had this great line about aging, saying that aging is not a particularly interesting subject. Anyone can get old. All you have to do is live long enough. And in many ways, the processes of aging are parallel to processes seen in disease. For example, chromatin is altered during aging. There are phenotypic outcomes, such as general heterochromatinization and telomere shortening, which correspond to and promote a decrease in the repair of chromatin aberrations. Other events like the loss of ribosomal DNA repeats through looping and excision, loss of DNA methylation, and the redistribution of histone H1 all create genomic instability. Instability leads to cell death and aging under normal circumstances and to cancer in cases of disease. So let's continue with this idea of interpreting disease processes. Several disease examples come from splicing mutations that reveal hidden regulatory elements. And what I mean by hidden is that they are embedded within the coding sequences. A portion of these mutations happen directly adjacent to the splice junctions and they change the splice boundary information. The rest occur in central positions of exons that contain specialized splicing regulatory elements known as exonic splicing enhancers or ESEs and exonic splicing silencers or ESSs. Also a similar set fall outside of the coding regions. These regulatory elements are known as intronic splicing enhancers, ISEs or intronic splicing silencers. These sequences are the binding sites for RNA binding proteins that regulate splicing. And therefore their nucleotide sequences serve two purposes. In the first, they code for amino acids in the DNA. In the second, they serve as regulatory sites for splicing proteins in RNA. And this explains why synonymous substitutions that don't affect the DNA, that don't affect the protein coding region can disrupt splicing. Now there are well-known examples of coding mutations that disrupt splicing enhancers and cause an exon to be excluded from a transcript. And this happens in cystic fibrosis, in breast cancer, and spinal muscular atrophy. And often the consequence of a skipped exon is a change in the open reading frame of the protein, leading to a loss of the protein function. So think about it. If this happens in a master regulatory factor, the regulation of all downstream targets would also be altered. Now altered splicing happens in the nuclear lamina protein, laminae, causing partial skipping of the 11th exon. And can you guess the outcome? The altered structure of laminae changes its protein function along with nuclear architecture, leading to the impaired physiological functions seen in progeria. This is how a single nucleotide change can affect the course of a lifetime. To find regions, oh, the splicing regulators. Very good question. So the question was, is there a source to find these regulatory elements? Right, that's the question, yeah, okay. So your question addresses the current genomic challenge. And that's that the landscape of these ESCs and ESSs, as well as the ISCs and the ISSs, is not well mapped. And also the consequences of mutations in these elements are not easily predicted. And even worse, the extensive amount of non-coding space to search is actually prohibitive. And so much more work is needed in this area. But this is one of my favorite areas. So my group has developed a tool called Skippy to predict mutations that are likely to cause exonskipping. And it's available on the NHGRI website. And I welcome you to go and look at it. It has a very easy user interface. You simply need a chromosome number, a chromosome coordinate, and the alleles involved in the change. Again, it's only a predictive tool and any predictions need to be experimentally validated. So this tool was built on the knowledge we gained from comparing characteristics of known exonskipping variants to neutral substitutions that don't affect splicing. And using this tool, we've identified new synonymous substitutions that dramatically affect splicing in disease genes. Keep in mind, though, that this tool only addresses mutations in coding regions. Mutations that fall in intergenic or intronic regions are still an open question. That's correct, you won't get an answer. There are other tools out there that map the binding matrices of these proteins. So you could put in your intronic sequence and get a prediction of what proteins recognize that sequence. You could then put in your mutant sequence and see if those combinations of proteins have changed. So there's another connection in the genome, and I don't know if you've ever made this connection. It's that the size of a DNA associated with nucleosomes, about 147 base pairs, is nearly the exact length of an average exon. And the current ideas are that exons may have evolved to this size to facilitate their identification and splicing. And in this case, the nucleosomes serve as speed bumps, slowing the RNA polymerase as it crosses the exon and buying time for the splicing factors to work. So what's the prevailing connection between functional elements in the cell? Is it that aging cells sabotage each other? That non-coding RNA orchestrates many events, or that conserved elements underlie all important features? So let's talk about non-coding RNA. The answer is non-coding RNA. Although the conventional view of gene regulation invokes the central dogma of DNA to RNA to protein, much of the RNA in a cell is never translated. However, this RNA is actually very important. And evidence from genomic studies indicates that organismal complexity is largely due to regulatory functions contributed by non-coding transcripts in the genome. So for example, link RNA are transcribed regions greater than 200 nucleotides in length. They are often poorly conserved, suggesting they might have some lineage specific functions. And they regulate gene expression by diverse mechanisms that are not yet fully understood. It's also possible that they are conserved among species in their functions and they are conserved in their secondary structures but not conserved at the level of the nucleotide sequence. Now tens of thousands of examples are thought to exist in the genome. And recently four roles of link RNA have been proposed. The first, they serve as guides. Link RNAs can recruit chromatin modifying enzymes to target genes either in cis or in trans. In the second, they serve as signals. Link RNA indicate gene regulation as it happens. In the third, they serve as decoys. Link RNA can titrate transcription factors or act as decoy targets for binding proteins. And in the fourth, they serve as scaffolds. So link RNA bring together multiple proteins to form RNA and protein complexes that direct histone modifications. Given these important roles in the cell, it is not surprising that misregulation of link RNA is associated with disease. Studies from the lab of John Rinn are identifying candidate oncogenic link RNA and tumor suppressor link RNA. Now like link RNA, micro RNA are a class of non-coding RNA but with much smaller size. These RNA transcripts are generated as 70 base RNA hairpins from within spliced introns or from non-coding regions of the genome. The hairpin structure is processed through the action of specialized enzymes to create a 22 base message that targets the three prime UTR of messenger RNA and causes either degradation of the primary transcript or interferes with its translation. There are over 1,000 micro RNA identified and their specific expression patterns are now being used as a diagnostic in cancers. So the fourth area in which the Human Genome Project has made significant contributions is our ability to read the epigenetic code. That's a very good question. So the statement was that many genes are targets of the same micro RNA and micro RNA target many genes. So there are an enormous number of interconnecting networks that are regulated by micro RNA. And so the difficulty in this question of how many genes are targeted by micro RNA, probably the vast majority of them, we don't actually know the precise answer to that question. And that's partly because of the way micro RNA act. So they have very specific recognition patterns. Typically in the three prime UTRs, there are some variations on this theme. And also there are some new sites being found within the bodies of genes. So until we know what all the target sites are, it's gonna be pretty difficult to address that question. But if you're thinking in terms of regulatory networks and you're thinking in terms of transcription factor binding sites, you should also be thinking in terms of micro RNA target sites. Quite reasonable thinking. I think that's quite reasonable. And there are tools out there to predict micro RNA binding sites. Some of them use sequence conservation in those target sites. Others use the precise patterns recognized by the micro RNA due to sequence complementarity. Sometimes micro RNA, when they start out in their hairpin structure, they're double-stranded. Sometimes each strand can also specify a different target. So within the last five years, we've all lived through a monumental shift. And I think you've probably recognized it. And this is the fact that we've gone from a gene-centric paradigm to a genome-scale perspective. So in the book, the immortal life of Henrietta Lacks, the author, Rebecca Skloot, wrote, good science is all about following the data as it shows up, letting yourself be proven wrong, and letting everything change while you're working on it. And this is actually very true. In fact, chip experiments are not classified as hypothesis-driven science. They are hypothesis-generating science. And they are changing the face of our biological understanding by enabling new paradigms to be described. While I'm not showing them all here, there are over 150 different epigenetic marks catalogued, and the question remains, how many are necessary to describe the chromatin code? So the most extensive study that I've seen used 51 chromatin states. It subdivided them into combination and based on the statistical occurrences defined regions as being either promoter-associated, enhancer-associated, or repressed. And these results are striking because they indicate the high combinatorial complexity and the information content possible within epigenetic patterns. And of course, they give us the ability to annotate functional elements in the genome. But what's so exciting is that they give a readout of the dynamic state of those elements. So how do you tell a promoter from an enhancer? Active mammalian promoters typically have histone H3, lysine 4, mono-dye, and tri-methylation at the transcription start site. They also have DNA hypersensitivity, the presence of RNA-Pol2, and typically you'll find a CPG island. There are also specialized histones and the presence of histone acetylation marks. As we move away from promoters, we find that P300 is specifically localized to enhancers. A recent CHIP study found over 55,000 candidate enhancers with P300. These enhancers had H3K4 monomethylation and they were depleted for H3K4 tri-methylation. So this is a big difference between enhancers and promoters. H3K4 tri-methylation. In contrast to active promoters, repressed promoters have specific identifying marks as well. And these include H3K27 tri-methylation, which is the prototypical mark of polycomb group repression. They also have H3K9 tri-methylation, which correlates with constitutive heterochromatin and DNA methylation. Now, polycomb proteins are important in negative regulation and their overexpression can cause disease. So for example, polycomb proteins are upregulated in some cancers such as melanoma, lymphoma, breast, and prostate cancer. And in those cases, it is possible that their excessive repression causes the de-differentiation of cancer cells. So in that case, a solution that has been proposed is to interfere with polycomb functions as a therapy. Is polycomb always a sign of repression? Typically, yes. And that's because polycomb proteins are transcriptional repressors, showing dependent co-localization with the H3K27 tri-methylation. But in about 20% of genes in undifferentiated ES cells, a strange phenomenon occurs. In these cells, the activating mark H3K4 tri-methylation occurs with polycomb repression. And therefore, these loci are described as being bivalent. Although the genes are largely inactive in the undifferentiated ES cells, they are marked for possible future activation and they remain flexible, contingent with the needs of the differentiated cell. So taking this back to diseases, some tumors carry these repressive marks at these loci when they shouldn't. In other words, they've never been activated. They've never lost the repressive marks. And the hypothesis is that the repressive marks have been retained abnormally preventing the cell from attaining its differentiated identity. And some have speculated that these cells represent products of stem cells, known as tumor stem cells. So the histone code extends into the body of transcribed genes where H3K36 tri-methylation and H3K79 dye and tri-methylation are elevated. There's also monomethylation at several histone tails, as well as extensive acetylation throughout the gene body. So all of these signals can be used to identify the collection of actively transcribed genes within a cell and to define that cell type. What's really exciting is that the more precisely these signals become understood in the promoter and the gene body, the more predictive power we have regarding the level of gene expression. In other words, we are learning not only to read the code, we are learning to interpret its meaning. One important protein that is central to most regulatory processes is CTCF. It functions at tens of thousands of sites in the genome as an insulator and a boundary element. CTCF is heavily involved in regulation at the CFTR locus. And if you look at the linear view of the CFTR locus, it looks completely different from the three-dimensional view. In the three-dimensional view, CTCF supports and stabilizes long-range DNA interactions that form loops by interacting with additional proteins such as cohesin. These are the types of interactions that are being identified by those looping assays, the three, four, five Cs and the high Cs. So when you see this, then it's easy to understand why CTCF is so important. It's partitioning the genome into distinct regions of open and closed regulatory domains. So although we focus mostly on histone modifications today, another aspect of the epigenetic code is conferred by DNA methylation patterns. Like the histone code, these are specific for a tissue type. They stably alter gene expression patterns frequently by causing gene silencing, but not always. They suppress the expression of integrated viral genes and they prevent genomic rearrangements caused by repetitive elements and they play a crucial role in the development of many types of cancer. So let me finish up by showing you a comparison of tumor and normal samples generated in my lab. Here's a picture of DNA methylation displayed as a heap map. The blue color indicates no methylation and the yellow represents strong methylation. There are about 500 promoter regions on the X-axis and about 50 different samples on the Y-axis. So there are two types of normal samples shown here, uterine and fallopian tube. And you'll see that the tumors of the uterus look very different in their patterns. Oops, sorry, the tumors of the uterus down here look very different in their methylation patterns from the normal uterine tissues. You'll also see that these sites look very similar to ovarian tumors of the same morphological subtype. Those also look very different to their normal comparison which is actually to normal fallopian tube samples. So notice also that in the normal samples, CPG islands are not methylated, that's very typical, and that non-CPG islands carry methylation marks. You'll see that those patterns almost completely shift in these tumors where there is strong methylation in the CPG islands and a loss of methylation in the non-CPG islands. So what we hope is that the genes with heavy methylation should be pointing us to a common pathway or enzyme that is responsible for this targeted methylation. That's correct. It will vary. So the question is, can we derive a common methylation pattern for all ovarian tumors? In the case of ovarian tumors, the answer will be no because there are multiple subtypes and each subtype will have a different methylation pattern. And you can actually distinguish the subtypes because they have morphological differences and from all of the data that I've seen, those morphological differences correspond to differences in methylation patterns just as they correspond to differences in gene expression patterns. So I told you that the naysayers were wrong about the importance of genomic sequencing and I hope I've convinced you. The human genome sequence has advanced our understanding of evolutionary diversity among species. Furthermore, it is essential to figuring out how genomes function and what the important elements are and it drives research of variants that disrupt function in complex disease. Predictions of the future of medicine indicate that we'll all have a sequence genome in the next decade, perhaps on a smart card that we carry around. Knowledge of the variants that create subtle changes in our gene expression patterns could help doctors predict how we respond to drugs, what diseases to vigilantly screen for and just think it could even help us pick our significant others. With that, I'd like to end. Thank you for your attention. Please let me know if you have any questions.