 Hello, my name is Michael Snyder, and I'm here to tell you about some of our recent work to characterize silencers in the human genome. This is a very understudied area in genomics. And the work I'm going to tell you about today is the work of a talented postdoc in the lab, Balshy Pong, who now has his own lab and leads. So, as I'm sure you're all aware by now, most of the human genome is not protein coding. In fact, only about a percent and a half of the genome encodes protein coding genes, and the rest is thought to be non-coding elements, including regulatory sequence. There's been considerable attention paid to trying to characterize the activators in gene expression. Specifically, there's a lot of work to characterize enhancers, such as distal enhancers that might lie far away from genes, enhancers that lie near the promoter regions, and promoter regions themselves. So there's been lots and lots of work to study these. Many actually now millions of elements have been identified that are involved in activating gene expression, or at least thought to be activating gene expression. But if you think about it, there's lots of the genome that's not expressed, and some of it's actively shut off. Some of it might be just through more general mechanisms, heterochromatim, but probably a lot of its regulator regions that are actually characterized by proteins such as silencers. And so we set out to carry out a systematic study of silencers in the human genome. The way we did this is kind of complicated. I'm going to give you a simple explanation. I know it's a complicated slide, but we decided to actually look for open regions of the genome that might encode silencing. So you can actually isolate them by a method called fair. We can pull down the regions, the open chromatin regions of the human genome. It will contain both activators and silencers. And we would take these fragments fairly small. They're only about 200 kb, and we would clone them upstream of a selection system. This is where it gets complicated. The way the selection system set up is that if we turn off a downstream gene task base, we can actually basically stop apoptosis. So we made this giant library, a very large library of short fragments, and actually integrated into the genome. And then what we do is we actually are able to induce induction of gene expression. And if you have a silencer, you actually keep that gene off. And those are the cells that survive. They don't undergo apoptosis. And so you actually kill all the regions that aren't silenced. And what you can do is select for cells that actually have silencer fragments. And then what we do is we actually sequence those silencer fragments using a PCR method, and then we compare it to the starting library. And wherever we've cloned the silencer, there'll be a significant enrichment in those fragments upstream of this region that's turning off the apoptosis gene. So the bottom line is we can select for cells that live because they've had this negative selection with this apoptosis gene shut off. And so we did this, so we actually carried out several times. And what we came up with was that fairly high stringency, roughly about 2,600 silencers. These are all done in a cell line. You've probably heard of a lot by now, K562. It's used a lot in the end code project. And basically we found over 2,000 silencers and they're scattered throughout the human genome. And in fact, it turns out many of them lie upstream of genes. They're in energetic regions. A fair number are also in introns. And interestingly, they're in other places of the genes as well, including genes themselves in some occasions. So the point is we actually find silencers. They're generally throughout the non-coding region of the human genome. We've actually gone and validated whether they're truly silencers. We take them, we clone them upstream of a very active promoter. It's called PGK. It's upstream of a luciferase construct. And this is in a transient transfection. And the silencers for the most part will actually shut off gene expression in this reporter assay. And occasionally there'll be one that fails, but most of them I think over 90% of them succeeded. We've also deleted them from the genome itself. So in a more endogenous assay, you can go in and use CRISPR-Cas9. Actually remove the silencers and basically these are three regions that are really strongly shut off. At least these two, this one's actually expressed some. When you go in and delete them, these are three independent clowns. Basically the genes can activate in some cases well over a hundred fold relative to background. So the point is that these are in fact true silencers who are shutting off the genes in cells. We actually start characterizing them. What's special about these silencers? Well first of all, the ones in K562, if you look over here, they're actually rich for interesting histone modification called H4K20 monomethylation. It's actually not very well studied and its role is thought to be complex. It's not 100% clear what's going on here, but it's been activated, it's been implicated with silencing and activation. But clearly in our assay, the silencers we have are highly enriched for this interesting mark, and there's some other interesting marks as well. We look at the motifs that are enriched by these silencers. We find things you might expect. We actually find AP2, which has been implicated in silencing and other studies. Same with this other one, KLF12 has been implicated in silencing. But then we actually found a new motif. So there's other silencers to be discovered out there, we think as well. And so there's a lot of interest in doing that. We've also looked to see whether what it looks like for silencers and other cell types. We actually took the exact same library even though it was isolated from open chromatin and K562 cells. We transfected it into HEPT2 cells and we in fact pulled out another set of silencers and the overlap is very, very small. So in fact what we think is that the silencers are very tissue specific. If they were the same silencers over and over again, they would get used many times. There's very, very few that are shared. We can show statistically this is highly enriched for being tissue specific. We've looked at the pathways these silencers are involved in and let's just use HEPT2 as an example. It turns out that the silencers are preferentially rich up to neuronal genes and things like that. These are genes that would not be expressed, expected to be expressed on HEPT2 cells. There are so in the case of K562 cells, most of these pathways are not expected to be expressed as well acting inside a skeleton and so on. We'll have to admit this cancer one I might have expected so that was a little bit of a surprise but it must be a very specific set of cancer genes. So the point is these silencers are upstream of regions you don't expect to be expressed. We actually wanted to get a little more insight into the functional role of silencers. So we looked at two of them that were upstream of these drug transporters ABCC2 and ABCG2 or two drug transporters that are actually known to remove anticancer drugs. They're actually involved in cancer resistance. And so we discovered that in fact we had silencers that were upstream of each of these genes. So what we did was we went in and knocked them out. First we tested them in Luciferase assos and sure enough, they were silencers. They do shut off teen expression. We also tested them in knockout experiments so we knocked them out. And what we find is that they will induce gene expression, the expression of neighboring gene. If you knock them out, this is for the ABCC2. This is for the ABCG2 in both cases. When we knock out the silencers are upstream of the genes become highly active. We've also then looked at sensitivity to drugs and sure enough it turns out that in each case you will see increased. Survival, meaning the transporter is not a shut off. And so actually when you knock them out, the genes become active. And so they'll actually crank out the drug. They'll knock it out. This is the oxycin, well, a cancer drug. And here's two others. So the point is in each of these cases, when you knock out the silencer, the gene becomes active and you can transport out the drug and the cells become more resistant. So actually this is really important then for functional assays for cancer resistance, it turns out. It turns out that silencers we think work across domain. So just drilling in on this one a little further. This is the ABCC2 gene. And here's the silencer here. And if we knocked it out, we could show that not only does this gene go on, but then we actually see some other genes. Here's this one CP1. So here's ABCC2 jumps way up in expression when you knock it out. Here's CPN1. It also jumps up in expression. Even this one jumps up a little bit. So all these genes in the vicinity of the silencer, they all go on. So we think it's having a general effect. And even this one next door goes on a little bit as well. So again, we think silencers actually operate over long regions. Not only that, they can work over long regions of the genome as well. Here's a case where for three different silencers that can act at long distances. We showed to show you one example here. There's a silencer here. And it turns out when we knocked it out. It's thought to have a 3D interactions with interactions up here over here, which again, this is a long distance away hundreds of KB. And it turns out this is upstream of this Norexin gene and our XM2. Here's another one, this RAS, GRPB2 gene. And when you knock out the silencer, these genes, this one here and this one here, which lie way upstream, will actually now go on an expression. So silencers we think can operate over long distances. It's true for this locus. We actually showed it for two other loci as well. So the bottom line is we think silencers are pretty cool. We set up a method to identify them. We showed a thousand of them throughout the human genome. And they're in energetic regions, introns and other places. Like enhancers, we think many of them are tissue specific and maybe general repressive ones as well like Heterochromatin that we have not necessarily identified from this. These are open chromatin gene turning off silencers. We validated many of them through surface assays and knockouts. We show that they actually are important for regulating, for example, drug resistance. And that's really relevant for cancer. We can also show they operate over long distances. Once again, I want to impress upon you that this is the work of one postdoc, Balsher Pound, who actually did this beautiful analysis of these silencers, a relatively uncharacterized region of the human genome.