354: How Cohesin Acetylation and ATPase Shape Chromatin Loops and Cohesion

[00:00:20] Speaker A: Welcome to Base by Base, the papercast that brings genomics to you wherever you are. Thanks for listening, and don't forget to follow and rate us in your podcast app. So I want you to imagine your DNA as an impossibly long thread. Like, if you were to unwind all the DNA in just a single human cell, it would literally stretch for miles. [00:00:39] Speaker B: Yeah, it's a massive physical scaling problem, really. [00:00:42] Speaker A: Right. Now, take those miles of microscopic thread and try to pack them into a cellular nucleus that's, you know, just a fraction of the width of a human hair. And you have to do this without [00:00:52] Speaker B: tangling the thread, which is a miracle in itself. [00:00:54] Speaker A: Exactly. Plus, the entire length of it has to remain perfectly readable by the cell's machinery at a moment's notice. So how does the cell manage this monumental, almost paradoxical task? Well, a huge part of the answer lies with a single molecular motor called [00:01:10] Speaker B: cohesin, which is essentially the master architect of your genome. [00:01:13] Speaker A: It is. It organizes DNA into dynamic loops, and it tethers sister chromatids together to make sure cells divide correctly. And importantly, defects in this machinery are directly linked to human birth defects and various cancers. [00:01:25] Speaker B: Yeah, it's incredibly critical for life. [00:01:27] Speaker A: But this sparks an immediate curiosity. How does this single molecular motor know whether it's supposed to tether two strands together or if it should extrude a loop of DNA? And what really happens when its internal control mechanisms are altered? Like, how could this change our fundamental understanding of genome architecture? [00:01:47] Speaker B: Well, answering that requires us to look way past the macroscopic scale and really zoom down into the finest chemical details of the motor itself. [00:01:55] Speaker A: Today we celebrate the work of Lorenzo Costantino, Douglas Koshland, and the research teams from UC Berkeley, UC Riverside, and the Austrian Academy of Sciences who have advanced our understanding of how cohesion's loop, extrusion, and tethering activities are regulated. [00:02:11] Speaker B: And it's such elegant work, because to really appreciate the magnitude of what they've uncovered, you have to understand the baseline biological problem. Cohesin is this highly conserved protein complex. [00:02:21] Speaker A: Right. Meaning it's basically everywhere. [00:02:23] Speaker B: Exactly. In all eukaryotes. So from simple budding yeast all the way up to human cells. And it does two critical things. First, it holds sister chromatids together. [00:02:34] Speaker A: That's called cohesion, which is super important during cell division. Right. [00:02:38] Speaker B: Yeah. You get two identical copies of DNA. [00:02:40] Speaker C: And. [00:02:41] Speaker B: And cohesin acts like a molecular tether, keeping them locked together until they need to pull apart. But the second thing it does seems mechanically, totally different. It extrudes DNA into loops. [00:02:51] Speaker A: And those loops regulate how genes are expressed. Right. And how the chromosome condenses. [00:02:56] Speaker B: You nailed it. It reels the DNA through its ring, bringing faraway pieces of the genome right next to each other. So you have one machine doing two very distinct vital jobs. [00:03:08] Speaker A: Okay, let's unpack this. It's. If cohesin is a motor sipping along a rope, the ATPase is the engine, right? [00:03:13] Speaker B: Right. It burns a molecule called ATP for energy. It's the literal engine. [00:03:17] Speaker A: And then there's acetylation, which might be the steering wheel or the brakes. We know these parts exist, but how do they actually integrate to tell the complex what to do? [00:03:25] Speaker B: Well, the regulation comes from a process called ECO, unmediated acetylation. Specifically, a subunit of cohesin called SMC3 gets acetylated at two key lysine residues in budding yeast. These are known as K112 and K113. [00:03:41] Speaker A: And just for our listeners focused on mammalian biology, those correspond to K105 and K106 in Vamils, right? [00:03:47] Speaker B: Yes, exactly. And what Eco One does is it attaches these bulky charge neutralizing acetyl groups onto those lysines. [00:03:55] Speaker A: So to solve how this all integrates, the researchers basically became molecular mechanics, right? [00:03:59] Speaker B: Yeah. They used budding yeast as a model organism and created a panel of specific mutants. They wanted to systematically alter cohesin's acetylation [00:04:06] Speaker A: and its ATPase activity, selectively breaking parts of the car to see how it drives. [00:04:11] Speaker B: That's a perfect way to put it. They created acetyl null mutants, which are K112R and K113R. That basically means they swapped the lysine for arginine. So those spots physically cannot be acetylated. [00:04:21] Speaker A: They cut the brake lines. [00:04:22] Speaker B: Exactly. And then they did the reverse. They made acetyl mimic mutants K112Q and K113Q. These permanently mimic the acetylated state. So the brakes are permanently engaged. [00:04:31] Speaker A: And they messed with the engine itself too, right? [00:04:32] Speaker B: Yeah. They made ATPase mutants. They had the ti mutant, which is a hyperactive super fast motor, and the DE mutant, which has severely reduced activity, a really slow engine. [00:04:44] Speaker A: So they have all these broken molecular cars. But to see what these mutations actually did, they used a technique called micro CXL on cells arrested in mitosis, which [00:04:54] Speaker B: is just a brilliant imaging approach for this kind of structural question. [00:04:57] Speaker A: But wait, let me push back here for a second. How does Micro C XL actually. Let us see. See these loops? [00:05:04] Speaker B: Ah, right. So it doesn't take a literal photograph it maps out 3D DNA contacts at incredibly high resolution. You chemically glue the DNA together where it's touching in 3D space, chop it up and sequence it. [00:05:18] Speaker A: Oh, so you're finding out which parts of the genome are physically hanging out next to each other. [00:05:22] Speaker B: Exactly. And in the resulting context maps, you get these off diagonal spots. Those spots represent style stable positioned loops that are anchored at specific genomic sites. [00:05:32] Speaker A: And those sites are called cohesin associated regions or cars, right? [00:05:35] Speaker B: Yes. That's where the loops are supposed to stop. [00:05:37] Speaker A: Okay, so to understand the engine, you first have to test the brakes. What happened when they mutated the acetylation sites? Like with those single acetyl null mutants where one brake line is cut? [00:05:47] Speaker B: This was the first big surprise. The single acetyl null mutants, either K12R or K113R, they still formed normal sized, perfectly positioned loops. [00:05:57] Speaker A: Wait, perfectly? Really? The loops were completely normal? [00:06:00] Speaker B: Completely normal. It wasn't until both sites were lost by depleting the Eco1 enzyme entirely that things went crazy. The loops expanded uncontrollably and positioning was just totally lost. [00:06:11] Speaker A: So it's a redundant system. Acetylation of either lysine is sufficient for loop positioning. [00:06:15] Speaker B: Yes. You only need one brake pad to stop the stop sign. [00:06:17] Speaker A: Here's where it gets really interesting, because earlier you mentioned the K113R mutant. [00:06:22] Speaker B: Oh, yeah. This is a crucial piece of the puzzle. [00:06:24] Speaker A: Right? Because we know from other data that this specific K1313 mutant completely fails at sister chromatid cohesion. It can't tether the strands together. [00:06:34] Speaker B: It's totally broken for cohesion. But, and this is the wild part, it forms perfect wild type loops. [00:06:40] Speaker A: Wow. So it can do one job perfectly, but completely fails at the other. [00:06:45] Speaker B: Exactly. And this proves definitively that the activities required for cohesion and loop formation are. Are mechanistically separable. They are not the same physical action. [00:06:54] Speaker A: That is a huge paradigm shift. Okay, so we've talked about the brakes, but what about the motor itself? Does acetylation just slow the ATPase motor down to stop the loop? [00:07:04] Speaker B: Well, they tested that by looking at how a loader protein called CC2 stimulates the motor. And it turns out K1 13 acetylation completely blocks CC2 loader stimulation of the ATPase. The motor just won't rev up. [00:07:18] Speaker A: But what about K112? [00:07:19] Speaker B: K112 acetylation only partially blocks it. The motor slows down, but it still runs. [00:07:24] Speaker A: So is it like driving a slow car if the ATPase motor is slow? Does it just stop at the stop sign easier? [00:07:29] Speaker B: That is exactly what you would intuitively think, but that assumption is completely wrong. [00:07:33] Speaker A: Oh really? Why? [00:07:34] Speaker B: Because of the K112Q mutant. It has a slow motor. Right. And it stops perfectly at boundaries. But then they looked at the DE [00:07:40] Speaker A: mutant, the one with the damaged, sluggish engine core. [00:07:43] Speaker C: Right. [00:07:44] Speaker B: The DE mutant also has a slow motor, but it blows right past the boundaries. It creates these massive random loops and completely loses the tightly positioned loops. [00:07:54] Speaker A: Oh, wow. So simply lowering at pace activity does not dictate loop positioning. [00:07:59] Speaker B: Not at all. And it gets even weirder. They looked at the hyper at pace mutants. Like the TI mutants. [00:08:05] Speaker A: The one with the revved up super fast engine. [00:08:07] Speaker B: Yeah. You'd think a fast motor would crash through the boundaries. [00:08:10] Speaker C: Right? [00:08:10] Speaker A: Right. [00:08:11] Speaker B: But it actually formed more position loops and fewer random loops. [00:08:14] Speaker A: That's crazy. So suggesting a fast motor actually helps stabilize loops at boundaries. [00:08:19] Speaker B: Exactly. It seems like the high speed and tension of the motor reeling in the DNA actually helps slam it securely into the boundary lock. [00:08:27] Speaker A: So what does this all mean? How does this reshape our models? [00:08:30] Speaker B: If we connect this to the bigger picture, these findings heavily argue against the passive loop capture model. [00:08:36] Speaker A: Which was the old idea that cohesin just randomly traps DNA that happens to float by. [00:08:41] Speaker B: Right. Because remember our K113R mutant, the one incapable of tethering if it can't trap or tether DNA. But it can still form perfect loops that strongly supports active loop extrusion. It's actively reeling the thread. [00:08:55] Speaker A: And furthermore, K113 acetylation is specifically essential for engaging the second sister chromatid. It's not just a brake pedal for loops. [00:09:03] Speaker B: Exactly. So we're really looking at cohesin having three distinct systems. CC2 responsive states based on its acetylation. [00:09:10] Speaker A: Okay, lay those out for me. [00:09:11] Speaker B: Sure. First, you have a fully inducible state. When it's unacetylated, the motor runs fast and extrudes loops. Second, a partially inducible state. When K112 is acetylated, the motor slows, but still positions loops. [00:09:23] Speaker A: And the third? [00:09:24] Speaker B: The third is a non inducible cohesion promoting state. When K113 is acetylated, the motor ignores the loader entirely and instead focuses on holding that second sister chromatic. [00:09:33] Speaker A: Wait, the micro CXL was done on cells arrested in my posis. They were paused for a long time. Couldn't even a broken slow motor eventually finish extruding a loop given enough time? [00:09:42] Speaker B: That is a phenomenal question. And you're completely right. This is actually a key limitation identified by the authors. [00:09:49] Speaker A: Because life isn't static, right? The cells in our bodies are dynamic. [00:09:52] Speaker B: Exactly. This prolonged paused state in the experiment might completely mask defects in loop extrusion speed. A sluggish motor might eventually get to the boundary if you give it hours, but in a living dividing cell, it would fail. [00:10:09] Speaker A: So what's the next step for this research? [00:10:11] Speaker B: The suggested next steps are time resolved studies. We need to look at this both in vivo and in vitro to measure cohesin dynamically, to see the movie, not just the photograph. [00:10:21] Speaker A: That makes total sense. So to wrap this all up for you listening at Home Cohesion's abilities to tether DNA for cohesion and extrude chromatin loops are mechanistically separable functions. They're governed by distinct interplay between its at pace motor and specific acetylation marks. [00:10:36] Speaker B: Yeah, and while acetylation helps dictate loop positioning and tethering, it's the ATP hydrolysis rates that stabilize these loops rather than simply determining their size. [00:10:45] Speaker A: What does this mean for our broader understanding of how single molecular machines adapt to perform multiple critical tasks across the genome? This episode was based on an Open Access article under the CCBY 4.0 license. You can find a direct link to the paper and the license in our episode description. If you enjoyed this, follow or subscribe in your podcast app and leave a five star rating. If you'd like to support our work, use the donation link in the description. Now. Stay with us for an original track created especially for this episode and inspired by the article you've just heard about. Thanks for listening and join us next time as we explore more science base by base. [00:11:30] Speaker C: Wow. Midnight on the chromosome line A ring of hands keeps time with mine it pulls a road from folded air Then chew is where to stop and stare One small mark can steer the glow Another holds what must not go Same machine, two different vows A quiet switch inside the house two marks, one map and the engine in between ATP sparks in a loader's restless dream Loots can find their safes in the light but to hold two sisters k 1/3 freeze the tide I turn the dial watch the pattern rearrange Same same binding new design Nothing stays the same. When both marks fade the track runs long wide open arcs where anchors were strong still on the de DNA still in place we're drifting past the checkpoint space Speed it up More loops locked to the rails Fewer wander off in random trails slow it down the skyline stretches then a PDS5 at the door but the order. Can't begin. 2 marks, 1 map and the engine in between. ATP sparks in a loader's restless dream can stand in rows aligned and bright but cohesion needs that single specific tie Separate gears in a single frame pole position hold three. No. 1 Nae Nae. Nae Nae. Sam.