|Type||Kitchen Table Talk|
On January 27, Dewpoint and Condensates.com welcomed Stanford’s newly appointed Chan Zuckerberg Biohub investigator Serena Sanulli for a Kitchen Table Talk. Serena has done all her training in the chromatin field. First she studied chromatin changes in development with Raphaël Margueron in her PhD work at the Université Pierre and Marie Curie. She then went on to UCSF to apply biophysical methods to chromatin structure and organization with Geeta Narlikar and John Gross.
Obviously, Serena loves everything chromatin—the dynamics, structure, and function of nucleosomes to chromosomes. Her talk covers her wide-ranging interests including her work on how HP1 drives chromatin compaction to heterochromatin, studied with a wide range of biophysical and biochemical methods.
Serena must also love the condensates community just as much as she loves chromatin because she delivered this incredibly stimulating talk at 7 am Pacific time! The attendees were fully caffeinated and the discussion brimmed well over our allotted time. And Serena was kind enough to provide written answers to the questions she didn’t have time to answer. You can find her thoughts on those questions and a recording of the full talk and Q&A session below. Enjoy!
Create an Account or Sign In to view the video.
Mark Murcko (00:00:00):
It’s great to see everybody. Good to have you all here. Our speaker today is Serena Sanulli. She is a Chan Zuckerberg Biohub Investigator at Stanford. And it’s been wonderful to watch her career evolve. Serena loves everything about chromatin, dynamics, structure, function. She studies nucleosomes and chromosomes with a wide range of biophysical, biochemical methods, and naturally, no surprise to anybody on this Zoom meeting, that research interest has led her to condensates–quelle surprise!
Mark Murcko (00:00:34):
Today she’ll be talking with us about her work on HP1 and how that drives chromatin compaction into heterochromatin. And Serena, as I mentioned, has worked in chromatin her entire career. She got her PhD in 2013 from the Curie in Paris, studying chromatin changes and development with Raphaël Margueron and then she moved to UCSF and she worked with the Geeta Narlikar and John Gross. And that was to apply biophysical methods to study chromatin structure and organization. And just a few months ago, she took her faculty position at Stanford and she’s just getting her lab up and running, of course, in difficult times, but getting off to a great start.
Mark Murcko (00:01:16):
We’re also very grateful to her for her willingness to give a lecture to all of us so early in the morning, out there in California. So it’s clear that she loves the condensate community just as much as she loves chromatin. Serena the floor is yours.
Serena Sanulli (00:01:30):
Thanks, Mark. Thank you so much for the kind introduction and it’s really a pleasure to be here virtually with you today. It’s also being part of this condensate community and line up with this Kitchen Talk series that you’ve been putting together. It’s great.
Serena Sanulli (00:01:51):
Today I’m going to tell you about chromatin and my long-standing interest. And also going to tell you the journey within the phase separation field as a chromatin biologist. I have to confess that I didn’t really appreciate the implication of phase separation from the beginning as a chromatin person. But what I’m trying to do today is really going to tell you how the perspective of phase separation has really reshaped my view of chromatin organization. I’m going to try to really share with you this vision…
Serena Sanulli (00:02:24):
Okay, so in eukaryotic cells the genome is package into chromatin and is stored in the nucleus. And here in this beautiful image, you just see the nucleus of the eukaryotic cell, and you can appreciate the complexity of this environment and the presence of these sub-compartment or sub-domains inside the nucleus. Some of these are very large, sub-compartments, you see the nucleolus here, some are smaller, such as for example, here at the PcG bodies.
Serena Sanulli (00:02:55):
These different compartments are different for their composition, but also for their function. And recent work has shown that a lot of these compartments are highly dynamic and they form through mechanisms that involve phase separation. [inaudible 00:03:16] trying to understand how the genome and chromatin organize inside this complex environment. If you look at the nucleus of the cell and this, what you see here, is a section of the nucleus, seen by electron microscopy. We can immediately see the chromatin inside the nucleus, and we can appreciate that a true genius structure of chromatin. And we can see that our dark patches shown here, darkly stained, and then light patches of chromatin.
Serena Sanulli (00:03:47):
And based on this cytological appearance, chromatin has been divided into heterochromatin and euchromatin. The heterochromatin are these dark regions, are considered the most dense and packed region of chromatin and they’ve been associated with low transcription. The light regions here, the region that are called euchromatin are considered to be the relaxed and open region of chromatin and associated with active transcription.
Serena Sanulli (00:04:16):
I’m really interested in understanding what’s the structure of chromatin inside these different patches? What’s the difference and how can chromatin structure of the sub-domains be regulated in a dynamic way and during the life of the cell?
Serena Sanulli (00:04:33):
[inaudible 00:04:33] just in terms of chromatin organization, because we realized that the way the genome gets packaged in this chromatin, it really regulates almost every single process of DNA, including transcription and DNA replication. To highlight the different possibility and types of chromatin organization that we can see in the cell, I’m just showing you different cell types by electron microscopy. Up here you have, you can see the hematopoietic stem cell. These are hematopoietic stem cell, and these are different cell type from a hematopoietic lineage.
Serena Sanulli (00:05:12):
If you start to see inside the nucleus, you can see that the dark and light patches are actually very different across different cell types. I tend to think of this as chromatin mesoscale organization of chromatins organized and fold in 3D. The ratio between heterochromatin and euchromatin, dark and light region is actually very different, but then we can also see the respective nuclear positioning of this different type of chromatin is also very different. This is just to highlight how dramatic difference chromatin can have in terms of organization in the cell and also highlight this chromatin regulation is really important to specify cell function.
Serena Sanulli (00:05:56):
So what do we know term of chromatin organization? I always like to show this hierarchical textbook model of chromatin organization, probably most of you have seen it. In the left side of this slide here, you see the cartoon of this model and on the right side, I’m showing the corresponding micrograph from electron microscopy.
Serena Sanulli (00:06:17):
This is considered a hierarchical model because it goes from the naked DNA down here up to the most condensed type of chromatin that we find in the mitotic chromosomes. I’m really interested in looking at the first level of DNA packaging to the chromatin down here. And today I’m going to talk a lot about this. They’re really the first level of packaging of chromatin. When we think of packaging, we think of nucleosomes. Basically it’s when the DNA gets wrapped around a group of protein, which are called histones to form this fundamental basic unit, which are called nucleosomes. And you can see here, if you look in the electron micrograph, if we look at the open stretched out fiber of nucleosome, they have this appearance that has been called as beads on a string, where the nucleosome, the spheres, are beads and the connecting DNA is a string.
Serena Sanulli (00:07:13):
The next level of compaction is in this model. What you see here is this fiber, which is called a 30-nanometer fiber. And this fiber is formed by a chain of nucleosome folded in a very specific way, creating specific nucleosome-nucleosome interactions. And this was thought to be a very static, discreet kind of structure that has a diameter of a 30-nanometer fiber.
Serena Sanulli (00:07:37):
And you can see here how this fiber looks like when you extract chromatin from the cell–you treat the chromatin, and you put that on electron microscopy grid. Of course, things are not so simple. We really have relatively good understanding of the atomic details of the nucleosome itself. So they’re really the fundamental unit of chromatin, the building block of chromatin. Here I’m showing the high resolution, crystal structure that we have available today. A lot of work has been focused in the chromatin field in studying the tail of the histone, which you see here, they’re protruding out of the nucleosome core.
Serena Sanulli (00:08:21):
These tails are considered to be highly flexible. They can be modified by different type of post-translational modifications and a lot of work in the chromatin field has being focusing on these PTMs because they seem to regulate dramatically chromatin. But for some reason, the chromatin people assume that the folded core, the central part of the nucleosome was a rigid unit. And actually, if you look at the initial textbook model of chromatin, they referred to the nucleosome core, the histone core as a rigid lego block coming together to form this very static structure.
Serena Sanulli (00:08:59):
But the most recent work in the last couple of years in the chromatin field, we have started to appreciate that actually there is something going on also in this folded core of the nucleosome. And then we can see some structural flexibility in there. And I’m going to talk a lot about this today, and one interesting aspect for in the future, and that’s what I’m really interested about and we focus on in my lab is understanding what is the extent of the conformation of plasticity in the folded core of the nucleosome and what’s the function of this?
Serena Sanulli (00:09:33):
When I started to look at what’s going on in terms of chromatin mesoscale organization–how chain of nucleosome come together and form a 3D structure in space–I was really surprised to see that what we know is actually much less. And that the in vivo data had failed to basically reproduce and show us what we’ve been able to see when we extract chromatin from the cell and put that on a grid.
Serena Sanulli (00:09:58):
To give you an example, well, we used to think about euchromatin, open chromatin structure, I used to think of nucleosome, like it’s shown here far apart from each other, with the idea that transcriptional machinery could, for example, access the underlying DNA. On the contrally, when we think about dense and packed packages, we have been thinking of a structure that could look like the 30-nanometer fiber, so very compact and dense type of chromatin.
Serena Sanulli (00:10:27):
The problem has been that when we have started to look in vivo into cell, we really failed to observe this 30-nanometer fiber or any kind of discrete defined structure. And if we look at the most recent data based on imaging, and this is an example from my most recent technique based on electron microscopy, but also we think about 3D chromatin conformation studies, all these data are pointing out the fact that in cells, chromatin does not adopt any highly ordered kind of structure. And the chain of nucleosomes seems to adopt a relatively disordered kind of structure. They form clusters that can go from five to 25 nanometers. And you can see up here, you can really adopt many different kinds of conformation.
Serena Sanulli (00:11:20):
An exciting part is that if you compare the dark patches versus the light patches, so heterochromatin versus euchromatin, we don’t see any specific difference in terms of structure. And the only difference is the fact, basically the density of chromatin, how many nucleosome we have in a defined volume. And so really understanding, is there a specific structure for heterochromatin or is there a specific structure for euchromatin, I think has been a quest for the chromatin field.
Serena Sanulli (00:11:52):
If we start to think about chromatin in the cell and chromatin organized through, not a defined structure, but more kind of a disorganized structure, to me is really puzzling to conceive how cell can set up highly regulated processes, starting from a chromatin structure that is disorganized and messy. Is it really that chromatin in the cell is like a spaghetti ball without any kind of organization?
Serena Sanulli (00:12:20):
To understand better the context of chromatin compaction, I used as a model system, the HP1 type of heterochromatin. This is a highly conserved type of chromatin and this is the reason I decided to choose this as a model system, it’s conserved and it’s present from yeast to human. And when I started working on HP1 heterochromatin, we also had a fairly good understanding of the molecular layer involved in this process.
Serena Sanulli (00:12:52):
We know that the key component of this type of compacted chromatin is the HP1 protein shown here in green. These are structural protein. They are known to specifically bind chromatin containing as a post-translational modification, the methylation of lysine 9 of histone H3. HP1s are essential for the formation and the function of heterochromatin. We know that the way in which they work, that the model has been proposed and the way HP1 protein works is that they compact chromatin and, therefore, they induce gene silencing.
Serena Sanulli (00:13:30):
And I always like to share this video that I think very visually show how good HP1 proteins are in compacting DNA. This is a DNA curtains experiment. The DNA stores the label. And then when you add HP1 protein, you can see that the DNA gets packed up towards the top of these DNA curtains. And so HP1 proteins are really good in compacting DNA. And the question that I had is: how this is happening at then molecular level?
Serena Sanulli (00:14:02):
We have some good understanding of how this protein works. And one of the models that had been proposed has been called the nucleosome bridging model. And this model relied on the fact that HP1 proteins can actually bridge across nucleosomes. So by chemical work that show that HP1 proteins can form oligomers–they can form really these long chain of HP1 proteins that also can bind chromatin.
Serena Sanulli (00:14:28):
This led to a model in which the formation of this oligomer, could mediate a bridging across nucleosome [inaudible 00:14:35] fiber. And then more recently, what has been shown is that HP1 protein, it also can phase separate and form this condensate, these droplets. And you can see this in vitro. For combinant HP1 protein, human HP1 protein alone in a test tube, forming condensates. And you can see in vivo, this is GFP-tagged HP1α in drosophila. HP1 formed this very bright foci that are highly dynamic and they fuse and they come together. And so the behavior of HP1 as been observed in vivo is really consistent with the liquid-like property that we see in the test tube.
Serena Sanulli (00:15:19):
The observation of HP1 phase separation really raised a lot of excitement, especially in the correcting field, because this really led to a very simple, intuitive model to think about chromatin compaction.
Serena Sanulli (00:15:32):
This is a cartoon of the model. And this model, the idea is that HP1 proteins can accumulate on chromatin. And then once they reach a certain critical concentration, they will phase separate. And that chromatin, which has high affinity for HP1 proteins will be sequestered, dragged inside this condensate. That could be a mechanism that could be implicated in gene repression and compaction.
Serena Sanulli (00:16:01):
But actually how or whether HP1 phase separation is related to chromatin compaction was still an open question. And for me, that really meant trying to understand what was the role of chromatin inside this condensate and how HP1 proteins interact with chromatin. I don’t think I need to tell this audience about phase separation and remind that the formation of these mesoscale structures, these mesoscale droplets is actually driven by molecular interactions. And these are interaction between molecules, but specifically multivalent interactions that are weak and transient.
Serena Sanulli (00:16:44):
If we think about HP1 proteins and what we knew from previous biochemical work is that HP1 proteins can oligomerize. They provide multivalency and we know that this oligomerization is actually essential for phase separation. If you rake it, you also prevent phase separation. So this made sense. The question is still open, and I think that question I’ve been mainly interested in, in trying to understand what’s going on with the chromatin? What’s the role of chromatin when it’s dragged inside this HP1 condensate?
Serena Sanulli (00:17:21):
To understand HP1-chromatin interaction, I decided to start with the simplest modest possible heterochromatin. I used one nucleosome as shown here in this cartoon in association with HP1 protein. And I used the simplified system to really try to understand the complex, but by using a combination of in solution methods. And I used hydrogen/deuterium exchange coupled to mass spec and I’ve referred to this as HDX-MS, I use NMR, and then I use cross-linking mass spec.
Serena Sanulli (00:17:56):
And the reason I choose in solution method is because I really wanted to learn about the conformational changes and dynamics of the complex in solution and know what was going on also on the nucleosome itself. But also these were a choice that was driven by the fact that the conventional structural methods, such as cryo-EM and crystallography had failed to solve high resolution structure. And this was a really indication that this was a highly dynamic complex. And we also knew that a large portion of HP1 protein was formed by disorder region, disorder protein regions making it a very difficult target for structural studies.
Serena Sanulli (00:18:44):
I’m going to spend some time now to describe the HDX-MS experiment, so you can start to understand how this technique works, but also the kind of information that can give us in terms of conformational changes of a complex. In the experiment that I did, I incubated a nucleosome alone or a nucleosome in complex with HP1 in a solution that contained deuterium.
Serena Sanulli (00:19:09):
What happened is that over time, the hydrogen of the backbone of proteins can exchange with the deuterium in solution. And we can monitor this deuterium uptake by mass spectroscopy. Basically you can think of this deuterium uptake as a proxy of solving accessibility. You would expect the regions that are on the surface of the nucleosome will exchange faster than regions that are buried in the core. Those will be region that will show faster deuterium uptake because they’re more exposed to the solvent.
Serena Sanulli (00:19:43):
But you would also predict that the binding of a protein such as HP1 on top of the nucleosome would cause a reduction in deuterium uptakes, with lower deuterium uptake at the region of the interface. Especially when I performed this experiment, I was really expecting to see some level of protection because of HP1 protein binding the nucleosome, but also because of the idea that we had of HP1 protein as a group of protein that create a more compact and less accessible chromatin state.
Serena Sanulli (00:20:15):
But actually what we saw was exactly the contrary. To guide you through this slide, on the top here, you see the four different histones with their secondary structure mapped here, and then all these different bars are peptide and were sequenced by mass spec. And the color code is telling us how much deuterium uptake is taken up in the nucleosome-bound state, so basically the differential deuterium uptake of the free versus bond nucleosome state.
Serena Sanulli (00:20:51):
We can take all this peptide and then map them on the structure of the nucleosome. And what we immediately start to see is that the increase in deuterium uptake is really widespread across the whole nucleosome. And these mean that all this blue region that you see in the crystal structure here becomes more exposed, more accessible to the solvent when HP1 is bound. And I say this was pretty surprising, given the idea that we had of HP1 protein as a group of protein that mediate chromatin compaction.
Serena Sanulli (00:21:26):
I also want to show you some specific regions of this nucleosome to highlight how dramatic this exposure was. Those are regions that are now highlighted in orange and red. Those are regions that become dramatically exposed when HP1 is bound. And if you look now on the surface representation, you can see that those are really buried inside the folded core of the nucleosome. And those are really hydrophobic regions.
Serena Sanulli (00:21:51):
This meant, the HP1 proteins are binding on top of the nucleosome, and they’re really opening it up, exposing all these hydrophobic regions. This was very surprising data, and I was glad that I had complimentary methods that I mentioned before about cross-linking, mass spec, and NMR to support this major rearrangement observed in the nucleosome structure. Just to briefly tell you about the NMR experiment, I’m not going to go into detail. But actually NMR is a very powerful technique that can give us information at the atomic scale resolutions or residue level resolution about motions, conformational changes in that specific residues.
Serena Sanulli (00:22:35):
And what I was able to see with NMR experiment is that some of these residues, which are shown here as the red spheres, undergo major dynamics as a consequence of HP1 binding. And those are, again, residues buried inside the nucleosome core indicating, and again, HP1 is binding on the top of the nucleosome and inducing motion inside the core.
Serena Sanulli (00:23:02):
This observation, and also the kind of changes that we observe on the NMR data indicated that there were dynamics on the millisecond-microsecond timescale occurring within the specific residues inside the nucleosome core. And this data really suggested that it was a conformational change occurring in the folded core of the nucleosome.
Serena Sanulli (00:23:26):
Together NMR, HDX, but also cross-linking mass spec, they show major rearrangement in term of cross-link contacts when the HP1 was bound, really indicated there was something major going on in the nucleosome core and that the binding of HP1 opened up and reshaped the nucleosome structure. That’s how I started to think about nucleosome and the idea that there is a canonical state, which is likely what we see when we look at the crystal structure, but also the fact that the binding of proteins, such as HP1 could increase the conformational dynamics of nucleosome. They can sample conformations that are different from the one that we used to think about when we look at the crystal structure.
Serena Sanulli (00:24:10):
Now the question of course is why this is happening and why HP1 is inducing such conformational change in the nucleosome and is there any function of this in the context of chromatin compaction? To look at chromatin compaction, I decided to use an assay, a self-association pelleting assay that has been used in the chromatin field for a long time. It’s a very simple assay. You obtain a chromatin fiber [inaudible 00:24:40] is a chain of nucleosome of 12 nucleosome and this fiber, under normal conditions, are soluble in solution. But if you add a protein or something that promotes fiber self association, what will happen is that larger assemblies will form. It will be heavier and they will tend to sediment at the bottom of the cube upon centrifugation.
Serena Sanulli (00:25:07):
That’s exactly what happened when I started to add HP1 protein. You can see on the x-axis, you have increasing concentrations of HP1. On the y-axis, you have the amount of chromatin soluble in solution. When you start adding HP1, the chromatin in solution tend to decrease. This indicates that the HP1 proteins are really promoting fiber-fiber self association.
Serena Sanulli (00:25:31):
That was the time we started to think about phase separation and HP1 phase separation was discovered. That was why I decided to take these chromatin HP1 assemblies and look under the microscope. And that’s when I observed the formation of this liquid droplet of condensate. You have here on this condition HP1 alone in a test tube, which is in solution. Here you have chromatin, but when you start to put together chromatin and HP1, you start to see the appearance of this condensate. And the formation of these condensates really correlated with the pelleting that was detected in the pelleting assay.
Serena Sanulli (00:26:11):
This really indicated, this was the first evidence that HP1 was mediating phase separation of chromatin. What I also did was to label chromatin, and this is a Cy5 labeled histone. What I realized is that chromatin is really highly enriched inside these condensates, indicating that HP1 is really concentrating chromatin in there. This was really the first evidence that compaction of chromatin mediated by HP1 at least was coupled to its phase separation.
Serena Sanulli (00:26:45):
Now the question is what is the role of reshaping of the nucleosome core in this context? And what I did was to repeat the same assay, but this time I prevent the nucleosome conformational change by locking the nucleosome core with site specific disulfide bond. When we use this chromatin fiber where the nucleosome cannot breathe anymore, we can see a defect in the pelleting assay mediated by HP1 shown here in this green dataset, as well we see corresponding defects in the formation of these condensates.
Serena Sanulli (00:27:19):
This was exciting because it meant that the conformational change in the nucleosome core was important for chromatin compaction, but also meant to me that the behavior of the nucleosome itself, of the single monomer, could really influence and regulate the behavior and the folding of an entire chromatin fiber in the mesoscale.
Serena Sanulli (00:27:45):
I’m not going to go into the detail of the in vivo data. But what I want to tell you just when I learned is that if you look in cells, and I told you at the beginning, HP1 formed these very bright foci, which are highly dynamic. And then if we start to introduce mutations in HP1 that either abrogate phase separation in a test tube or alter phase separation properties, we start to see the corresponding changes also in cells, in term of formation of the foci, but we also start to see defects in gene silencing.
Serena Sanulli (00:28:20):
This indicates that the interaction between chromatin and HP1, which mediate phase separation in the test tube, are also relevant for proper heterochromatin formation and function inside the cell. Okay, so what we’re learning in terms of HP1 heterochromatin using this combination by physical method that I told you about. We knew from previous work that HP1 proteins bind chromatin containing methylation of lysine 9 on histone H3 and they have the ability to oligomemorize and potentially bridge across nucleosome. But what we learn now is also by binding the nucleosome, HP1 induces a conformational change. And this conformational change in the nucleosome core opens up nucleosome and expose regions that are normally buried inside the hydrophobic core of the nucleosome. And this opening up of the nucleosome is actually important to drive chromatin compaction and the formation of these condensates.
Serena Sanulli (00:29:22):
What we propose is that the exposure of these hydrophobic regions in the nucleosome core can now be available on the surface to template nucleosome-nucleosome interactions. And these interactions, dynamics and weak transient, are the kind of interaction that can drive compact kind of chromatin and phase separation.
Serena Sanulli (00:29:45):
You can think of the exposure of this hydrophobic region of the nucleosome as just a way to increase the opportunities for inter-nucleosome interaction. And now this increased accessibility at the level of one single nucleosome starts to make sense, because we realized that by increasing the accessibility, the exposure of one single nucleosome, we’re actually creating a chromatin fiber that is overall more compacted.
Serena Sanulli (00:30:13):
I’d like now to zoom out a little bit and get out of the HP1 heterochromatin context to think more broadly how this observation in the context of heterochromatin can help us to rethink some of the longstanding concepts of chromatin organization. Based on that, I really propose what I call a liquid-like model for chromatin organization that revises and introduces some new concepts compared to the hierarchical model I showed you at the beginning.
Serena Sanulli (00:30:42):
In this model, there is of course, nucleosome conformational dynamics. You probably can see, inside this condensate nucleosome would have to form structure. And I told you today that this conformational change in the nucleosome are actually important to regulate chromatin compaction. But we can really start to think of nucleosome conformational dynamics as a potential new layer of chromatin regulation that can regulate many different aspects of chromatin biology.
Serena Sanulli (00:31:11):
You can start to think of this as, for example, an extra way, a new code, a nucleosome conformational code that can function in addition, for example, to post-translational modification and co-regulate chromatin structure and function. And of course, an aspect of particular interest in my lab is to investigate the extent of this conformational changes and the function that they might have in cells.
Serena Sanulli (00:31:39):
The second concept, of course, and this is the relevant for the group of people that I’m talking with today is that the concept of phase separation as the possible principle that can contribute to chromatin functional organization. I still think there’s a lot to be done to address to study in a quantitative way, phase separation in the nuclear context and understand what’s the role and whether it regulates, how it regulates, transcription, and many other processes in the nucleus. But even from a very early days in terms of understanding this process, what I think is extremely exciting, is just the perspective that phase separation provides in this context.
Serena Sanulli (00:32:21):
And this perspective is the fact that chromatin can still be organized in cell in absence of this highly ordered structure that I talked about at the beginning. We don’t really see at 30-nanometer fiber in cell, but this doesn’t mean that chromatin is a messy ball of spaghetti. So we can still get some level of organization, but through structuring and interactions that are much more dynamic than we previously thought. And we can start to think of a group of interactions that are weak and transient and can regulate chromatin function and organization on the mesoscale. I really start to think about chromatin as a much more fluid polymer and really investigate a different type of interaction in terms of weak and transient interactions.
Serena Sanulli (00:33:10):
I also want to spend a couple of minutes to think about chromatin itself. We tend to think of chromatin as a really passive occupants that sits there and doesn’t do anything. We used to think of transcription factor HP1 proteins coming on chromatin doing their job and just chromatin being in some kind of inert. But what this conformational change in the nucleosome is highlighting is that actually chromatin contributes to multivalency. And that also mean that the intrinsic property of a chromatin can really fine tune and regulate the process of chromatin phase separation on mesoscale organization.
Serena Sanulli (00:33:50):
So chromatin composition can self-regulate its own organization. And this has been also supported by some other recent data. This is a work that has been published recently from Mike Rosen’s Lab in the context of chromatin. What they did was to start to look at a series of chromatin intrinsic property that have been shown to be important to regulate chromatin compaction. What they were able to show is that there were all these elements were also important in regulating chromatin phase separation in the test tube.
Serena Sanulli (00:34:25):
This is an example. You have the formation of chromatin condensate in a test tube when you have normal nucleosome. If you cut out, if you chop out the nucleosome tail, you really lose the formation of these condensates. This is telling us that the tails of nucleosome are important [inaudible 00:34:41] to show the post-translational modifications on the tails of the nucleosome, specifically, acetylation, fine tune the process as well as the spacing of nucleosome out far from each other the nucleosome are in a chromatin fiber.
Serena Sanulli (00:34:56):
This is telling us that there are really intrinsic property of chromatin, including the nucleosome core plasticity can fine tune and regulate chromatin mesoscale organization, but also is telling us that chromatin is actively participating and providing multivalency. I also want to spend a couple of minutes to think about chromatin as a polymer and thinking about phase separation in the context of very long polymers.
Serena Sanulli (00:35:26):
There is work here again from Mike Rosen’s Lab to show that the length of the chromatin polymer in a test tube can fine tune and change phase separation. So you need, of course, a minimum length of monomers of nucleosomes to be able to observe enough multivalency to see phase separation. But there’s also been a recent report that showed how the length of the DNA can also regulate the properties of the condensate observed, in this case, in the context of HP1.
Serena Sanulli (00:35:57):
Here you have condensates that are formed in the presence of 2.7 kbps of DNA. And here you have the same experiment for four in the presence of 9 kbps of DNA. You can see that the length of the DNA is really changing the formation and the properties of the condensate. And this work really started to dissect what were the difference in term of dynamics and this kind of elastic property of these condensates.
Serena Sanulli (00:36:26):
This is really making us think that the length of this polymer is not surprisingly it’s really regulating, it has a role in fine tuning phase separation. But I think is interesting to start to think how the chromatin long polymer in the cell is actually regulated and how we can have different condensate or different type of chromatin form within the same long polymer. I think this is an exciting venue for research and just think about this for what that means in terms of chromatin organization in the nucleus of the cell.
Serena Sanulli (00:37:00):
Okay, just to summarize, an important message that I want to try to put out there today is really to start to think chromatin in a different way, and not just think about chromatin as a passive occupant of HP1 droplets as transcription factor, but really something that contribute to the self-organization and 3D organization. I mentioned that it’s really interesting to start to think how a monomer, the nucleosome within the polymer, within the chromatin fiber can contribute to the organization of the entire polymer of the entire chromatin fiber, and start to think how the different flavors of nucleosome can contribute and add an extra layer of regulation in this process.
Serena Sanulli (00:37:44):
I talked about the conformational change in the nucleosome itself, so atomic scale conformational changes and how much this is linked to the mesoscale organization of chromatin. I think it’s really interesting in the future, and it has been started to show in different examples of condensates, start to think about conformational switches and how understanding conformational changes in protein and in complex can help us understand their regulation and the formation of these mesoscale assemblies.
Serena Sanulli (00:38:16):
In the context of chromatin, it’s also very interesting to notice that there are both different types of interactions that are involved in the formation of the condensates. There are electrostatic interactions, which are driven, which involves interaction with the DNA. But it also start to have hydrophobic interaction that involve the hydrophobic core of the nucleosome. I think the chromatin, from this perspective, is a very interesting polymer when we start to think about their respective role in hydrophobic and electrostatic interactions in regulating phase separation.
Serena Sanulli (00:38:51):
And then of course, in the context of HP1 heterochromatin, we start to see that we have both homotypic and heterotopic interactions. We have interaction between HP1 protein, so HP1-HP1 interactions. We have interaction between nucleosomes, inter-nucleosome interactions, and also we have interaction between HP1 and the chromatin. I think this is just, I like the complexity of the flavors of phase separation can have in a context of a complex system, such as chromatin and chromatin binding factors.
Serena Sanulli (00:39:24):
And to end this talk, I just want to make a remark about the HP1-heterochromatin system that I’ve talked about today. And the fact that if we start to look closely, we start to see that all the ingredients that are known to be involved and be important for phase separation are really part of the mix.
Serena Sanulli (00:39:43):
We have the role of disordered protein regions that we know are often involved in phase separation processes. And you can think of the histone tail. Histone tails are disordered, flexible regions. HP1 protein has multiple regions that are disordered and structured, there is macromolecular crowding, and this is an aspect that still needs to be addressed, but the nucleus is actually very crowded environment.
Serena Sanulli (00:40:10):
And then there is the role of weak multi-valent interactions that are known to drive the formation of these condensates. And then there is the polymer. You can think of chromatin as a very long polymer. And also HP1 protein can oligomerize and form these long chains. If we look at all the ingredients in the context of HP1 heterochromatin, I think is not so surprising that we observe phase separation, but the exciting and the challenge for the future is really try to understand how this physical-chemical principle, in the context of chromatin, can regulate its properties and the function, and really trying to understand, learn from these different fields to be able to better understand genome packaging and genome regulation.
Serena Sanulli (00:40:58):
Okay, I’m going to end here and just spend a few seconds to thank my supervisor, Geeta Narlikar and John Gross at UCSF. Those were my mentors during my post-doc, my collaborators, and this is us, me and the first members of my lab, unpacking boxes and trying to set up the lab. If you want to help us open boxes, you can reach out to me and I’ll be happy to chat more also about science. I’m happy now to take any questions. And I hope that will be a fun discussion.
Mark Murcko (00:41:31):
Yeah, thank you, Serena. And there are lots and lots of questions coming in, so really stimulating talk. Everybody loved it.
Serena Sanulli (00:41:38):
Mark Murcko (00:41:38):
Maybe we could do this in all kinds of different ways. I would say maybe let’s start with Phil’s question towards the end of the chat window. Phil’s asking about evidence for the dynamic nucleosome conformation isn’t also present in euchromatin.
Serena Sanulli (00:41:57):
This is a good question. Yeah, there is no evidence in the sense that we don’t really have enough data. It’s particularly challenging to look at nucleosome conformational changes. I think in the last two, three years that people are starting to look at it. And my suspicion is that there are nucleosome conformational changes in multiple chromatin context. And one question is, do you have specific conformation in heterochromatin versus euchromatin, active versus repressed regions? And this is one possibility, but we need to do more. I really think of nucleosome as something very squishy that tends to be highly dynamic and sample multiple conformations.
Serena Sanulli (00:42:41):
And then one possibility is that any factor that engage nucleosome core can bias this conformation and stabilize one of these conformations or you can think that the binding of this factor can also push and drive a very different type of conformation.
Serena Sanulli (00:42:57):
Those are all questions that we don’t know. With the techniques that I mentioned, HDX and NMR, we are able to see things that are moving, but we don’t really have a snapshot picture of how this is structure looks like. And this is some of the question that I’m interested. And some developing tools, such as, for example, nanobodies is to be able to trap and study some of these conformations.
Serena Sanulli (00:43:19):
But the most recent cryo-EM structure of nucleosome bound to factors are showing that, we’re starting to see even with cryo some conformational changes. We have missed this partially because technically, it has been challenging, and we didn’t know that something was going on there, but also because people tend to push for high resolution, and so they discard everything that they’ve gained from the most stable conformation.
Serena Sanulli (00:43:46):
I suspect that more and more will be coming out in the next couple of years. But for now, we just have [inaudible 00:43:52] I suspect that we’ll see more and try to determine more of what has to do with euchromatin versus heterochromatin.
Mark Murcko (00:44:00):
Yeah. And actually that all makes total sense. And it also leads to another question that Rosana asked, where she’s also asking about … You can see it in the chat window. This idea of nucleosome breathing and other protein changes to the way the octamer is assembled. Again, it’s all part and parcel of the same need to really be thinking about the subtleties of the dynamic motions of these. Obviously-
Serena Sanulli (00:44:25):
Yeah, I’m not sure if I understood the question right. In term of thinking the process of assembly and disassembly of nucleosome on chromatin, we know that our intermediaries that has to do with, nucleosome are put on chromatin and how they’re moved around. For example, there was another work [inaudible 00:44:45] that showed that if you want to push, slide nucleosome along chromatin in an ADP-dependent manner, you need to also have a flexible nucleosome core. I believe this is a process that has to do with intrinsic property of chromatin that that can be exploited in different contexts as well as assemblies assembly.
Serena Sanulli (00:45:09):
But I was particularly surprised in the context of HP1 protein is that there is binding energy involved, but there is no real energy that I was thinking of involving the context of chromatin remodeling when we saw that the remodeler was pushing the nucleosome along DNA using ATP. We saw distortion there, but we thought it’s a motor, it’s pushing nucleosome around. That’s why you need distortion. But when we think about HP1 protein just sitting there, the idea that I had, and they were not going to induce such a huge conformational change, which suggest that many of these changes might really occur broadly in chromatin processes. It’s just that we haven’t thought about that and also haven’t been able to see that technically.
Mark Murcko (00:45:56):
That’s great. There’s so many questions. Another one that I think is interesting comes from Bede, who’s asking, and I maybe could ask Bede to unmute to build on his question. But he’s asking about the acidic patch on the nucleosome and whether that’s a docking site, whether that can be interacting with euchromatin and then whether you see evidence from HDX or other methods.
Serena Sanulli (00:46:24):
Yes. This is a good question. The acidic patch is this region on the interface on top of the nucleosome formed by histone H3-H2B, and it’s basically the only acidic side in the whole nucleosome, and has been shown to being the anchor site for many transcription factor, chromatin binding factor that interact with chromatin. I definitely think that the acidic patch has to do with this process. Some work has been done by my Mike Rosen’s lab in this context, and also because the old chromatin compaction model based on the 30-nanometer fiber in the test tube were showing interaction between one tail of the nucleosome H4 and this acidic patch. And those were interactions that mediate packing. I do think that these interactions are likely involved also inside a condensate and this is one of the things I want to do in my lab is to address better what are the interaction involves? And this will be one of them.
Serena Sanulli (00:47:21):
One interesting aspect in term of HP1 is that HP1 does not seem to use their acidic patch. I did a bunch of experiments and looked at the acidic patch because that was the suspect that I thought of when I started to think of the interaction between HP1 and nucleosomes. And surprisingly, HP1 does not use the acidic patch. It doesn’t care at all. For me, it is interesting because you can start to think that maybe HP1 binds will shape the nucleosome and deform the acidic patch in a way that can promote interaction with one factor versus another on chromatin. You can start to think about the later regulation that can be down the hill. Yeah, that’s exciting. Yeah.
Mark Murcko (00:47:57):
Bede Portz (00:48:00):
Stereo view of condensation basically.
Serena Sanulli (00:48:02):
Bede Portz (00:48:04):
Yeah. Cool. Thank you.
Mark Murcko (00:48:06):
Yeah. So Jeremy, you had some questions, if you want to jump in. I think maybe you’ve been unmuted or Susanne.
Jeremy Schmit (00:48:18):
Yeah. Okay. My question was, let’s see if I can remember. The claim was that you get the binding on there and it creates some conformational change inside the nucleosome and that this reveals new interactions that allow them to come together. And I was just thinking that an alternative model is just that the conformational change in the nucleosome just allows a higher affinity of the HP1. What you’re seeing is just an enhancement of the HP1 binding effectively just a higher equilibrium constant there. Does that explain the data or can you rule that out some other way?
Serena Sanulli (00:49:01):
Let me see if I reframe the question and let me know if I get it right. Basically what I was trying to do. You have the two side of the thermodynamic box, right? There is one way where you can think that nucleosome is just highly dynamic and sampling this conformation, and that HP1 recognize one of these alternative state bind that, and stabilize that.
Jeremy Schmit (00:49:20):
Serena Sanulli (00:49:21):
Other options that the nucleosome is sitting there and then the binding of HP1 induce such a conformational change. The outcome of the thermodynamics so there are two side of this thermodynamic box is the same where you have an opening up and the stabilization of more open nucleosome state. I can’t rule out which one of the two pathway are in place. I don’t-
Jeremy Schmit (00:49:43):
But you seem to be pushing a third model there, where that once you rearrange the nucleosome that exposes some new interaction sites and those facilitate the compaction.
Serena Sanulli (00:49:56):
Yeah. That is what happened after this conformational change has happened right. In the [inaudible 00:50:01], when you have a chromatin fiber present there, when those region are exposed and now it can be available for the interaction with chromatin or potentially other factors that are interacting with chromatin itself. Those are the downhill effect of the stabilization of conformational change that is going on. I also want to-
Jeremy Schmit (00:50:19):
How do you know that it’s not just the HP1 effect that explains everything?
Serena Sanulli (00:50:28):
I can tell you, if I get it right, that this conformational change is HP1 specific. Is that what you’re trying to say?
Jeremy Schmit (00:50:35):
Well, I’m just saying, how do you know that it’s not just enhanced binding of HP1 that’s facilitating the compaction? How do you know that there’s other stuff that’s coming in? I believe that there has to be some other stuff that comes and sticks to it, but how do you know that that are the stuff that sticks to the exposed parts of the nucleosome is actually facilitating compaction?
Serena Sanulli (00:50:54):
Yeah. To really understand what those hydrophobic region exposed are doing, we need to do extra experiment to figure out what are they interacting with? To say something about what you say that maybe can help them understand is that if you do induce compaction of chromatin in a different way, not using HP1 but using high-salt and divalent salt and you prevent these conformational changes, you also start to see defects in the compaction.
Serena Sanulli (00:51:24):
What I’m thinking is that what HP1 is doing is really promoting, taking advantage of an intrinsic property of chromatin and just facilitating the interactions between nucleosomes.
Jeremy Schmit (00:51:35):
Okay. Thank you.
Serena Sanulli (00:51:37):
But I also want to make clear that I don’t think of nucleosome as something sitting there and being studied in one state, I think of nucleosome as highly dynamic itself. I just really need to think that whenever the nucleosome is interacting with a protein, is not just simply sitting there and being inert. But when you engage with the nucleosome, there is a response and a conformational change that occurs there. And this is not surprising if you think about any protein complex or a group of protein coming together, just for some reason, chromatin people assume that there was nothing interesting going on on that portion of the nucleosome.
Mark Murcko (00:52:15):
That’s great. There was an interesting question from Sue Swalley, if she’s still on.
Susanne Swalley (00:52:22):
Yeah, I’m here, Mark.
Mark Murcko (00:52:24):
All right. Why don’t you go ahead?
Susanne Swalley (00:52:25):
Yeah, fantastic talk. I was really struck by that canonical image of chromatin. And then when you actually looked in vivo in the cell, how different it actually looked. And in the talk where you’re showing Rosen’s work, where they cut the tails off and that affected the droplets and the ability to form droplets, what does that actually look like in the cell? Do you see a difference in the cell when you do those sorts of experiments?
Serena Sanulli (00:52:53):
I don’t think that the chopping off of the tail has been done in cells. In general, manipulating histones in a cell is very challenging technically because you have multiple copies. In some cases you have 10, 20 copies of each single histone. So doing this kind of modification is really challenging in cell. If I recall it well, all the experiment that Mike Rosen did in cells to test some of the in vitro regulation processes in term of phase separation was to play with the acetylation of the histone tail.
Serena Sanulli (00:53:23):
Acetylation is a charged residue put there on the histone tail has been shown to unpack chromatin opening up, preventing the interaction between the acidic patch on the tail that we talked about before. What he was able to do was to induce acetylation and acetylated chromatin into cell and see that that would form condensate, puncta, when it’s not acetylated and when you introduce acetylation you dissolve these condensates.
Serena Sanulli (00:53:53):
If I recall it well, that’s the only interactions involving chromatin or regulation that has been fully explored in vivo. And I don’t think anyone has ever chopped the tail of the nucleosome to look in cell, yeah.
Mark Murcko (00:54:09):
Great. And now we’re going to run out of time, although Serena, you said you can hang around for a bit, is that right?
Serena Sanulli (00:54:14):
Yes, yes. And the light is on in San Francisco. Now it’s daylight, so it’s much, much easier for me to be awake and chat more with you.
Mark Murcko (00:54:25):
You mean it’s not sunset over the Golden Gate Bridge?
Serena Sanulli (00:54:27):
No, it’s not. But before it was really night. Yes.
Mark Murcko (00:54:32):
Maybe we’ll go with one more question before we … I know a lot of folks will have to leave at the top of the hour. Charlotte had a really interesting question about the idea of aging or hardening. So Charlotte, are you unmuted?
Yeah, I think so. Can you hear me?
Mark Murcko (00:54:48):
Okay, great. Thanks for a really exciting talk, Serena. My question is that, so many other condensates have been demonstrated that if you let them just sit around for a while, they start to age and harden. And I was wondering if heterochromatin might also undergo this process and if not, what’s helping to keep it dynamic so that I can then switch between being open and closed?
Serena Sanulli (00:55:20):
Yeah. The hardening phenomenon in a test tube has also been observed in the context of HP1 and chromatin. If you start to wait longer, the fusion events will just be harder. They droplets will not be able to relax as we see in this video that I showed you today. So the aging and the hardening process seems to also occur in the context of HP1-chromatin. I haven’t really explored that further to understand what that means. I agree that the question is, so how is this regulated in the cell? Why don’t we got this hard body or gel or everything forming all the time in the cells? And this is, I think it’s an opportunity to remind that heterochromatin, everything in the cell is more complex, but specifically heterochromatin is a very complex platform. And HP1 protein not only binds chromatin, but it has also domain that is known to recruit specific, it is really a hub for recruiters of other proteins on chromatin.
Serena Sanulli (00:56:25):
This means that what we observe in the test tube with simple composition, just HP1 and chromatin could be very different when we are in condensate with different proteins. All this protein can contribute to maintain the dynamic property of the interaction, of the condensate, in cells. It does happen in test tubes, I don’t know, in vivo, but again, a lot of components are dynamically regulated in terms of chromatin itself, heterochromatin itself.
Serena Sanulli (00:56:53):
And I want to use this as an opportunity to just share with you the way I think about HP1 and phase separation. And this is not really supported by data, but it’s just the way I think about it. I don’t think of like phase separation and phase transitions of different material states to happen all the time when you have HP1 into the cell or a chromatin. I really think of it as an extra layer of regulation that can be in place and can be really dynamically fine-tuned and modulated in the cell. The material state, and likely it’s going to be coupled with the composition and the rest of the complex environment that you have in the nucleus and within heterochromatin.
Mark Murcko (00:57:39):
That’s great. Let’s go to Tanzeem next, if he’s still on. If not, let’s go to Ozgur. I think we have lost Ozgur.
Ozgur Oksuz (00:57:58):
Mark Murcko (00:57:58):
There you go.
Ozgur Oksuz (00:57:58):
Yeah. Very nice talk. I wonder if you try this reshaping on a different type of heterochromatin proteins, such as MeCP2 or even the mammalian version of Hp1α.
Serena Sanulli (00:58:14):
No, this is a good question. And I think now that we have this setup, which is a combination of NMR, HDX and cross-linking mass spec, that’s the next thing, right? Trying to figure out how the different factors can affect the nucleosome itself and can we find a pattern? Can we find specific changes associated with activation/repression? Or can we check, can we find, can we identify hotspot regions within the nucleosome that are subjected to these kind of changes?
Serena Sanulli (00:58:47):
This hasn’t been done, and this is mainly a technical reasons. Performing the experiment I told you about is actually very challenging. Just to give you an idea, to perform the NMR experiment set up, it will take six to eight months to make a sample, to be able to do one NMR experiment. And it’s similar for doing HDX. Those are not high-throughput experiments that you can perform and use different proteins so easily, but it can be done with any protein you can think of, but you’ll need to really understand the system also very well.
Serena Sanulli (00:59:18):
In the context of the experiment I’ve done, I use the easier version of HP1. I had a very good understanding of all the moving parts of the system, let’s say. I was able to perform this very difficult biophysical experiment that I talked about.
Ozgur Oksuz (00:59:37):
That’s great. Yeah, I think it will be very exciting to also know about how histone, H1 influences this reshaping the chromatin by Hp1α or maybe other heterochromatin proteins.
Serena Sanulli (00:59:51):
Yeah. There’s some data out there in the context of H1. H1 is the linker histone that sits there at entry, exit of the DNA. And it’s like putting a collar on the nucleosomes. It’s known to promote compaction. We know from Mike Rosen’s Lab that the presence of the linker in H1 promotes compaction and phase separation. And there’s been past work of NMR in the presence of the histone H1 that they did not detect any conformational change driven by HP1 in the nucleosome itself. It doesn’t seem that HP1 seems to use a conformational change in one nucleosome itself. The conformational change may be a consequence of stacking of nucleosome which is promoted by H1.
Serena Sanulli (01:00:35):
This made me think that in order to see a conformational change, the nucleosome induced by chromatin factor, my way of thinking about is that the factor needs to engage with the nucleosome core to really sit not just from the tail, but really engaging the folded core up the nucleosome. And the linker histone is very small. It’s just sitting up there and they’re not really at the exit of the DNA, is really sitting on the DNA. The way I think about it is not really contributing enough in term of binding to push, to change the conformation of the folded core.
Mark Murcko (01:01:12):
Great. Tanzeem is next.
Tanzeem Rafique (01:01:17):
Hi. Very nice talk. I was wondering what would be the dynamics of HP1 mediated chromatin compaction would be depending on the cell cycle of the cell?
Serena Sanulli (01:01:30):
Are you talking about dynamics in terms of conformational change of HP1 itself or you’re talking about binding and release from chromatin? Which scale of dynamics?
Tanzeem Rafique (01:01:42):
I was just wondering that it has been known that H3K9 trimethylation can change depending on the cell cycle of the cell. And what I understood from your talk is the compaction of the chromatin by HP1 is very much dependent on H3K9 trimethylation. I was wondering what would be the dynamics in that perspective?
Serena Sanulli (01:02:13):
Yeah. This is basically discussing how is H3K9 deposited throughout the life of the cell as a cycle, as you’re mentioning after replication? This hasn’t been explored and just to add an extra what you just said, another key regulator of HP1 phase separation and HP1 in general is the phosphorylation of HP1 itself, which is seems to be cell cycle regulated. And this conformational change in HP1 driven by phosphorylation and controlled by phosphorylation is also affecting interaction with chromatin.
Serena Sanulli (01:02:49):
Those are very good questions and exploring the … I think this is more broader than phase separation. It’s really exploring the assembly and disassembly of heterochromatin in cell. And there is a lot of work out there on this. And I’m not an expert on that, but I think is a very good question, and it will be fun to think more about that from the perspective or the knowledge that we have coming from phase separation.
Tanzeem Rafique (01:03:16):
Okay, thank you.
Mark Murcko (01:03:18):
Great. Maybe we’ll go to Rosanna. Great. Thanks.
Rosana Collepardo (01:03:29):
Hi. Yeah. I already asked my question, but I was saying to Jeremy regarding his point about, how do you know that is the nucleosome the formation that actually promotes liquid-liquid phase separation and not enhanced binding of HP1, for instance? We have done some simulations in which we have only used chromatin. And we do observe that similarly to what you just described, just nucleosome breathing enhances the multivalency of the nucleosomes, so it promotes these enhanced liquid network connectivity and also enhanced flexibility of the chromatin system. It’s exactly how you described it. We have tested just with more than-
Serena Sanulli (01:04:10):
Yeah. And I think I started to read that paper carefully. This was exciting. The changes that we see are somehow specific for HP1. There is some level of specificity. I’ve tried a few factors in test tubes and see if they care about cross-linking of nucleosome, like locking the nucleosome, and I didn’t see any changes. But I haven’t looked broadly to all the transcription factor or factor you can think of on one side. And on the other side, there is another thing that when we think about specificity is that the conformational change in the core of the nucleosome is in the context of a methylated nucleosome. If you remove the methyl part of the nucleosome and HP1 still binds, but it’s not able to induce that conformational change.
Serena Sanulli (01:05:02):
I think there is some level of specificity that is regulated by HP1, but I don’t think it’s an exclusive thing that’s happened in the context of HP1. And in multiple aspects, even intrinsic, or you can think about the histone variants, right? And change the composition of the chromatin itself, how much can just … It will be fun to put it in the modeling that you did, based on variance and see how that would affect the process.
Rosana Collepardo (01:05:26):
Yes. And also DNA sequence variations as well, and yes. Thank you.
Serena Sanulli (01:05:33):
Yeah. All my work has been done in a context of this optimized, strong sequence positioning. So you can think that if you have a less strong, the dynamics and the ability of the nucleosome to breath will be increased as well. And the spacing between nucleosome and chromatin fiber seems to be determined from Mike Rosen’s work. And it was fun to see that the spacing that seems to correlate in vivo with euchromatin was a spacing that was not favoring phase separation in the test tube versus spacing of nucleosome that tend to be found in heterochromatin or compacted region, is the spacing that favors chromatin and phase separation. Yeah. Interesting.
Rosana Collepardo (01:06:15):
Thank you. Thanks.
Mark Murcko (01:06:17):
Let’s go next to David and then Gaurav, and then Patrick. David, you’re up next.
Serena Sanulli (01:06:26):
We can’t hear you. I think you’re muted or something else.
Mark Murcko (01:06:30):
Yeah. He was on a second ago. Ah there you are. Great David.
David Kuster (01:06:35):
Okay. Yes. Okay. Sorry. Yeah. I was just wondering if there’s quantitative data in terms of dissociation constants between nucleosomes, so the inter-nucleosome affinity for different histone variants also maybe in presence of HP1?
Serena Sanulli (01:06:51):
I don’t think so. I’m not aware of any of this information out there. It’s a good point, yeah.
David Kuster (01:07:01):
Mark Murcko (01:07:02):
So Gaurav, you’re up.
Gaurav Chauhan (01:07:05):
Hi, thanks for a great talk. It was really amazing. I have a question about, the way I’m thinking about chromatin is as this polymer. And when you show these phase separated, condensed states then I think sort one length of the polymer has been phase separated by these HP1, molecules. I’m wondering, what is limiting that size of the length of the chromatin, which gets phase separated, if you will?
Serena Sanulli (01:07:38):
It’s a good question. I think this was that the kind of thought that I was trying to provoke when I mentioned at the end of my talk, that the initial data that we have in the context of HP1 and length of DNA and the number of nucleosome. I think this is a good question. The chromatin it’s very long in the cells. How that gets organized, I think you have some regulation, what is the boundary to create all the … They regulate their process. When do you stop phase separation? How do you determine which nucleosomes get included and which ones get excluded?
Serena Sanulli (01:08:12):
I don’t have an answer for that but I think it’s fun to start to think about that and connect that what we know. Just thinking about boundaries and transition from one chromatin, not phase state, but just chromatin state as we used to think in the past, kind of type of chromatin, it’s not well understood. What is the boundary that prevent heterochromatin to spread? There is a lot of work that has been looking at what prevent heterochromatin to spread, where it’s not supposed to go?
Serena Sanulli (01:08:44):
I think it would be fun to start bringing that knowledge in the context of phase separation. But yeah, I don’t have an answer for that, but I think it’s exciting, something exciting to think about for the future.
Gaurav Chauhan (01:08:58):
Mark Murcko (01:08:59):
Let’s go to Patrick and then I think we’ll close with a question from Allen and they will have to cut it off. Patrick, you’re next.
Patrick McCall (01:09:06):
Cool. Thank you. Thanks, Serena. Really, really interesting talk that brought a lot of different details of a very complicated problem to bear. I was particularly interested in the NMR data that you showed, identified lots of specific, but also distant from what, spatially distance residues showed conformational changes on timescales you were probing. Do you think that those are cooperative in some sense, or should we think of them as all independent? I guess it’s coming from a standpoint of, if you think about the energetics involved in the conformational changes, how much energy is required and how much can you actually be liberated from HP1 binding?
Serena Sanulli (01:09:42):
Yeah, that’s a good question. I don’t think I have any experimental data to answer this. Just in a speculative way, I tend to think of this as a cooperative process. And the reason is that I haven’t done NMR experiments with different mutants and breaking specific interaction between the nucleosome and HP1, so I can’t tell you that. If we do easier experiments and we look at affinity, or we look at phase separation or compaction, if you start to break some of these interactions, you start to see defects. And there are multiple domains of HP1 that engage different regions of the nucleosome.
Serena Sanulli (01:10:27):
When you interfere with some of that, for example, you start to see defects in phase separation or defects in binding. I haven’t tested, actually there is an experiment I could do to test this. I tend to think of this as a cooperative process, because for example, I was telling you if you remove the methylation of the histone tail, which you’re basically just losing one interaction, right, from the tail to one specific domain of HP1, you lose the importance of the conformational change in the process because the abrogation blocking the conformational change does not cause any defects in the process.
Serena Sanulli (01:11:06):
I think there’re few of these interactions seems to be cooperative. Yeah, I think actually now that I think about it, it seems to be cooperative. And I think about that in a cooperative way, at least in the context of HP1 and the fact that you really need to hold nucleosomes from the different parts.
Patrick McCall (01:11:25):
I think to me, that suggests then that you have a relatively small number of adapted or non-canonical states of the chromatin then. Because if you imagine that all of them could be happening independently, then you have lots of different combinations of conformational changes. But if it’s cooperative, then you really have a switch between two states in the simplest limit.
Serena Sanulli (01:11:46):
Patrick McCall (01:11:46):
That has implications for the cooperativity.
Mark Murcko (01:11:48):
Or some discrete number of states, but not a huge number, yeah.
Patrick McCall (01:11:51):
Absolutely. It’s a continuum. Absolutely. But I think it’s helpful to know which end of that you’re on. Avogadro’s number or a countable-on-my-hand number.
Serena Sanulli (01:11:58):
Yeah. I see what you’re saying. Yeah. I didn’t know that yet.
Patrick McCall (01:12:00):
Serena Sanulli (01:12:00):
Patrick McCall (01:12:01):
Thanks so much.
Serena Sanulli (01:12:04):
We can do now also an NMR experiment, CPMG experiment, on the nucleosome itself alone in absence of HP1, that’s easier, where we can start to really look and determine some of these discrete state and their dynamic range of that conformational changes. And these are some of the experiment I’m planning to do if I find a person crazy enough to do NMR on nucleosomes.
Mark Murcko (01:12:30):
We’ll go to Allen next.
Congrats on your appointment. Really nice work.
Serena Sanulli (01:12:35):
I can’t recall the phenotype from HP1 α, β, γ knockout cells, but in the [inaudible 01:12:40 Suv] 39-H12 knockout cells where you lose H3K9 trimethylation and heterochromatin, you don’t recruit HP1 anymore. You don’t see chromatin decompaction phenotype as far as I know. I’m just trying to reconcile what you’re seeing in vitro with chromatin compaction, and what actually happens in a cell.
Serena Sanulli (01:13:04):
I know that when you really remove all the HP1 proteins, you lose complete gene silencing and you have the repression, but you also lose compaction. You don’t lose it, you say you’re not losing compaction when you remove all the HP1?
Well, in a mouse cell, chromocenters usually persist, and there’s actually only very few proteins that when removed from the cells cause those structures to actually disperse in mice, at least. And just the overall idea about how heterochromatin organization varies across species, as well is difficult to reconcile with just HP1 behavior in general just because HP1 itself is so well conserved, but-
Serena Sanulli (01:13:43):
Yeah, it’s well conserved, but at the same time, really small changes seems to have huge impact in the way HP1 interact with nucleosome and induce phase separation. For example, there’s been work that compare HP1 α, β, γ in human from different sequences of the domain that have small differences that seems to really affect the interaction with DNA, their phase separation ability, and so on.
Serena Sanulli (01:14:11):
I think that small changes might have large impact. But we think cause small changes might actually have large impacts on the way they interact on chromatin and fine tune phase separation. I don’t know in vivo how to reconcile the data and I don’t think I know well enough the data you’re talking about to comment on that. But I just think that we should not underestimate small changes in sequences and differences. This is true for HP1, but also for histones, for example. Sometimes you have one point mutation which seems to dramatically affect it, completely change the function of chromatin [inaudible 01:14:52] especially for processes that are so linked to conformational dynamics and HP1 has a huge conformational dynamic in itself. I think small changes can actually dramatically change the type of conformational dynamics on the timescale of these dynamics.
Mark Murcko (01:15:13):
Well, this has been wonderful. And the fact that so many people have hung around for an extra 20 minutes, how interesting of a topic this is? Serena, thanks again from all of us. Wonderful lecture.
Serena Sanulli (01:15:24):
Thank you so much for inviting me and for the fun discussion, it was worth to wake up so early.
Mark Murcko (01:15:30):
Good. Glad you feel that way. Thank you. And thanks to everybody for joining.
Serena Sanulli (01:15:34):
Thank you so much. Have a good day.
Mark Murcko (01:15:36):
Thank you. Bye-bye.
Question from Ziad Ibrahim: Have you used methylates nucleosomes in your HDX experiments? Does HP1 bind DNA as well or only methylated Histones?
Serena’s Response: The experiments I showed were all performed with H3K9 methylated nucleosomes. HP1 also binds non-methylated nucleosomes, but with weaker affinity. In absence of H3K9me, HP1 is not sensitive to disulfide locked nucleosomes, suggesting conformational change in the nucleosome core is couple to H3K9me recognition…
Question from Bede Portz: Is there a defined stoichiometry for HP1:nucleosomes in the in vitro condensates? If you titrate HP1 vs the nucleosome array, does the stoichiometry change after you have crossed the phase boundary, or is it fixed in the two phase regime?
Serena’s Response: This is a great question, we have measure HP1-nucleosomes stoichiometry by AUC in one phase regime, but not after crossing the phase boundary.
Question from Ziad Ibrahim: Have you tried to FRAP the foci you have seen in vitro or in vivo?
Serena’s Response: No, I haven’t. In vivo FRAP experiments were previously done (some examples Cheutin et al, 2004; Schmiedeberg et al. 2004; Strom et al. 2017) and in vitro FRAP has been done recently in the context of HP1-DNA.
Question from Bede Portz: You mentioned the importance of polymer length, what about linker length between nucleosomes? Have you tried varying this and seeing what happens to condensation with HP1 as a function of altered linker length?
Serena’s Response: No, I haven’t tried this yet – It can affect nucleosome-nucleosome interactions (see Gibson et al. 2019) as well as HP1-nucleosome interactions (see Canzio et al. 2011).