Hi, this is Douglas Koshland from the University of California at Berkeley,
and today I want to tell you about the mysterious biological function of chromosome loops.
Today's lecture will not be something you can find in a textbook because this area of
chromosome biology is very active and changing and I wanted to give you a little sense of a
problem in biology, which is unresolved, but exciting.
So, we start with the fact that segregating chromosomes acquire two essential structural
features needed for their segregation. In interphase, the chromatin is diffuse.
During S phase each DNA molecule of each chromosome is replicated to generate two copies,
which are called sister chromatids. And these replicated copies are tethered together
to generate sister chromatid cohesion. Once cells enter mitosis these sister chromatids
undergo a process of condensation that leads to a highly compacted form of the sister chromatids,
which are visualized in many micrographs of mitotic chromosomes.
So, in 1990s one of the really sort of big questions in the field was, what are the factors
that promote higher order chromosome structure that are needed for chromosome segregation
in a eukaryotic cell. And my laboratory and others discovered that these processes of chromosome
organization were carried out by a magical family or SMC family of protein complexes.
So, SMC stands for structural maintenance of chromosomes
and it turned out that pretty remarkably they were not just found in eukaryotes but they were also
found in prokaryotes. So, these are in all forms of life that need to organize their chromosomes.
Furthermore, what made them particularly intriguing was that they had an unusual structure;
they were long ,50 nanometers in length due to long coil coils and they were flexible which
allowed them to achieve multiple confirmations which we will see is important, but at this time
it was very intriguing is the why were they so big and why were they so long and so flexible.
So, one of the first SMC complexes that was discovered was cohesin, and its role is to tether
together sister chromatids to generate sister chromatid cohesion that I just told you about.
Cohesion is bound to the newly replicated sister chromatids as they are being formed during S phase
such that by the end of DNA replication the
two replicated molecules are cohesed along their length.
So, what is the importance of sister chromatid cohesion? Well it promotes proper
bipolar attachment by both promoting sterically constraining the two replicated chromosomes to
attach to microtubules from opposite poles of the mitotic spindle. And once attached, it stabilizes
these sort of bipolar attachments because the consequence of those bipolar attachments
generates tension as the microtubules try to pull apart the sister chromatids.
But they cannot because they are tethered together by cohesin.
Clearly the importance of achieving this bipolar attachment is in fact
to make sure that the newly replicated molecules segregate to opposite poles during mitosis
such that upon cytokinesis each daughter cell receives one and only one copy of each of the
initial chromosomes in the initial cell. And that is clearly important for the proper
function of the cell and subsequent cell divisions.
So, condensin is another SMC complex and its job is to tether DNA in regions within
a sister chromatid to generate loops that compact the DNA. And these loops can be
seen in micrographs of meiotic chromosomes, here shown are the lampbrush chromosomes
of the salamander but you can also see these loops if you partially denature the mitotic chromosomes.
So, why do you condense the chromosomes? Well, one is to allow the cell to move them around in
mitosis without entangling with each other. And the second is that the chromosomes have
to achieve a short enough length such that when they get moved to the poles,
they are not the piece of DNA does not extend over the plane of cytokinesis,
which would then cleave the DNA and this is indeed observed if the DNA only partially condenses.
So, this was all sort of our understanding of chromosome structure as the result of being able
to visualize them in the microscope. However, a new method came to visualize chromosome structure
and that method was a very nice method developed originally in Job Dekker's lab.
It is called 3C- chromosome conformation capture. So,
in this really ingenious method what happens is that you take a cell and in that cell the
nucleosomes may be organized such that two particular nucleosomes are in close proximity
to each other. You can capture that by adding a cross-linking reagent, and now you digest,
you break the cells open, and you digest the chromatin with the restriction enzyme; the result
of this is the two nucleosomes and the associated DNA that have been cross-linked, the DNA ends will
be in close proximity and can be ligated together to generate a chimeric piece of DNA. With the DNA
stitched together from the two nucleosomes; you then generate paired end reads by DNA sequencing
and the result of that is you get the chromosome coordinates of the contacting nucleosomes.
So, how do you interpret, how do you organize this information to make it
interpreted in interesting ways? Well, what you do is, let us imagine a situation where
you have unstructured chromatin you most likely will cross-link two neighbouring nucleosomes.
And those two nucleosomes let us just say for example come from chromosome one
at position 1000 and 1200. So, those are the two coordinates then you plot them on a graph
and you will generate a contact map. And so, for those particular two nucleosomes, because their
two coordinates are very close to each other they will lie close to the diagonal. In contrast if the
chromatin forms a loop, then the two nucleosomes that are crosslinked from each to each other
may be very far apart from each other let us say at position 1000 on chromosome 1 and 5000
on chromosome 1. So, those two coordinates 1000 and 5000, will plot off the diagonal as a spot
and because these graphs are actually sort of a mirror, there is mirror symmetry,
the same coordinates will appear as a second spot off the diagonal in a mirror symmetric position.
So, once you have this technology, you apply it, in this case was applied to metazoan chromosomes
and in the first surprise came that in interphase
they observed loops 550 to 1000 kbs in size and also topologically associated domains, which
essentially are like a loop except that we just scrunched the looping DNA together.
So, this was a surprise, we think of interphase chromosomes as sort of
being diffused and disorganized but in fact they were actually organized into loops of
distinct positions that we could detect on our contact map.
Second surprise came was that the formation of these loops depended upon our SMC complexes;
maybe not surprisingly it depended on condensin-like complexes,
which what I have already told you were thought to tether together loops in cis for to give you the
kind of loops that you need to compact chromosomes for mitosis but it turned out that cohesin also
is used in some cells to generate these loops in cis. So, cohesin cannot only tether together the
two replicated DNA molecules in trans, but also DNA sequences within a sister chromatid in cis.
Third surprise and this is the biggest surprise was for years we just thought that these SMC
complexes were sort of like a little napkin ring that sort of held together two pieces of DNA
within, or between two DNA sequences. But what was discovered is that these Smc complexes are much
more complex in their activity, that once bound the DNA they can begin to extrude a loop of DNA.
So, they are active molecules and this may explain there very complicated
and long structure, they are long sort of long length and flexibility is that is the kind of
features you might expect if you are going to generate a machine that can move something.
And this discovery was first made in vitro, by the Haering and Dekker labs. And here we have
a movie of that and what you are going to see here is we have a piece of DNA that has been
tethered to a piece of glass. And we have added in this case a
condenser molecule that solution and you will see that spot of bright spot is where the condensin
is binding and it starts to extrude a loop and the loop is actually sort of collapsed on itself. So,
it will appear sort of as a line instead of an actual loop. But you can see that the loop
gets bigger and bigger as the condensin moves down the DNA towards the two tethered ends.
And this was a remarkable in vitro activity, really stunned the field.
And beautiful experiments done by David Rudner's laboratory showed that this in vitro activity
was also happening inside the live cells, in this case using the kind of technology
that you know we described for the chromatin capture
to in fact visualize that these loops were happening in vivo along tens of kilobases of DNA.
So, why would you have these kinds of looping activity, what would be the purpose of organizing
the interface chromosomes in the loops? We understand why you do it mitosis, you want
to compact them. Why would you do an interphase where you do not really need to compact the
DNA? And the answer came with the idea that maybe they help control the proper gene expression. So,
you can imagine along the length of a chromosome, you have an enhancer and a promoter that are
important for the expression of a gene in the heart and another enhancer and promoter they are
important for the expression of a gene and the kidney, happen to be neighbouring each other.
And it is very important that the proper enhancer talk the proper promoter. So, if
for example enhancer two here inappropriately talk to promoter one, you might get the expression of a
heart protein or a kidney protein in the heart, and that is something that could be very bad.
And so, it was important to make these sort of loops at positions where such
that you could you know organize the gene expression units into the proper way.
And in mammalian cells there was a factor that helped the cohesin called CTCF, which is the DNA
binding protein which seems to help generate stops to position where the loops will occur.
So, we know these loops form but what is the evidence they actually
are important for gene expression? So, it turns out that you can look in
cell types and in particular, there was known to be a DNA inversion, which caused polydactyly in
mice, which is an extra digit and when they looked at that by this chromosome capture technology it
turns out that that inversion also changed the TAD and loop associated with that particular
gene that is needed for preventing polydactyly. So, that sort of said, that you need a proper
loop to get the proper expression the gene to control the number of digits,
at least correlated with an observation. We also saw that if you remove the function of CTCF or
cohesin, these loop determining factors, that you can lose the TADs and loops throughout the genome
of a human cell line and that would change the expression of a thousand genes.
So, again there was correlation between perturbing TAD and loop formation
and gene expression and finally we could inactivate the dosage compensation complex, which
was a condensin like complex. And that complex normally is important for specifically controlling
gene expression of genes on the X chromosome. Loss of the DCC, caused the loss of TADs
and caused the loss the proper loss of the dosage compensation. So,
again there is a correlation between perturbing the gene products that cause loops and perturbing
the actual TADs or loops that are formed on chromosomes and the change in gene expression.
Further sort of an evolutionary argument, which fit this whole concept was
the yeast was thought to be a very poor model for mitotic interphase chromosome loops and that is
because here are the 16 mitotic chromosomes; in budding yeast they do not look very condensed.
So, it does not look like you know yeast has in some sense a lot of looping activity. And indeed,
you from first principle you would think well loops will not be needed in interphase either
because budding yeast the enhancer the promoters are very close to each other. So, you can control
proper gene expression just by proximity of enhancers and promoters to each other.
Furthermore, if you do the direct experiment you can say well can we see loops in yeast by our
chromosome conformation capture? And on the left, you see a mammalian cell and on the right you see
a yeast cell. And you can see that while you can see the off-diagonal spots diagnostic of the loops
in the mammalian cells, you cannot see them very well at
all along the length of a yeast chromosome. So, we have got a model, everything seems
to fit together, but you know one of the very exciting things about active research is you get
more information and that makes you realize that things are not quite what you thought.
And so, let us go back through these observations
and you can see that the first was well yes we had an inversion that causes the loss of TAD and
polydactyly and mice but more careful experiments showed that the inversion the cause of polydactyly
in this inversion was really because you were moving together two sequences that were only far
apart close together. And it did not correlate with in fact the subsequent loss of the TAD.
So, the TAD was just sort of an innocent bystander that got changed when the real effect was bringing
together in close proximity an enhancer and a promoter that were not necessarily normally next
to each other. The second was that while sure in those human cell lines we change the expression of
a thousand genes, but loops and TADs are present throughout the human genome. So, there were seven
thousand other genes whose loops and tabs were dramatically altered by the loss of CTCF and
cohesin and yet their gene expression did not change. And finally, you know while it was true
that inactivation of this condensin complex caused the loss of loops and TADs and also the loss of
proper gene expression on the X chromosome in worms, when did the experiment actually where you
altered the TADs and the loops by mutating the cis acting sequences,
now what happens is that dosage compensation acts fine. So it implies that you need this condensin
complex but it does not has to act to generate specific loops in order to change or to properly
control the gene expression of the X chromosome in worms. Contrary to the whole concept that
the purposes of these complexes to you know act at specific sites to control the interaction
of particular enhancers with promoters. So I bring this all up just to show you
that you know when we get new observations we change our view of we have to change our
models and have to rethink a little bit about what things might be happening.
So with this in mind, we decided to reinvestigate the formation and function of chromatin loops in
yeast. And we in this case was Lorenzo Costantino and Rebecca Lamothe, a talented postdoc and
graduate student from my laboratory, who got together with Stanley Hsieh who was a postdoc
in Xavier Darzacq's lab and we recently published a paper in eLife, which describes this research.
So, what we did differently from what had been done before was, Stanley had
invented a slightly altered version of this chromosome conformation capture technology. So,
we cross-linked the cells but instead of digesting with the restriction enzymes,
the chromatin was digested with micrococcal nucleus which generates a
much smaller piece of DNA, gives much higher resolution of the contacting nucleosomes. And
then you after you ligate and purify the DNA you do the paired end reads as we have done before.
So, the outcome experiment is shown here and what you can see
are human and yeast cells side by side. And the blue dots or the purple purplish circles indicate
loops that are found closer to the axis which means they are
relatively small loops that are found both in wild type human and yeast cells.
And then what you can see is, we have a factor that we, in fact Peter's Lab originally
identified, which is called Wapl/Wpl, which has been shown to prevent expansion of loops,
of these positioned loops. And so, when you get rid of Wpl, bigger loops can be formed
and they can be seen as spots that are further off the diagonal and those are circled in black.
And you can see that we see that in both yeast and human cells when we remove Wpl we see the
expansion of loops to new positions along the chromosome. So, what I would like to point out
here is that if I took off the label at the top of these columns and asked you which is
a yeast and which is a human cell you could not tell because the patterns are very identical.
Loop smaller loops along the length of the chromosome in humans and yeast;
bigger loops when you get rid of this factor that inhibits loop expansion.
And so, that similarity now turns the table on what we think about yeast. So,
it now goes from being a poor model to understand chromosome loops to actually a very good model.
And why is that? Well, we are just at the beginning to understand how the biochemical
activity that people have identified in vitro translates into loop formation in vivo. And
turns out that yeast are really cheap to grow, they have a small genome size,
which makes the DNA sequencing cheap and the computational analysis really easy.
And finally if we want to dissect all the features
that control loop formation how active the complex the SMC complex is what makes it stop, why does it
form let us say in particular parts of the cell cycle and not other, all those kinds of things
will require mutations specifically inactivates specific aspects of the SMC complex or the cell,
and yeast is ideal for that and we in fact already have lots of mutations that we can plug into to
correlate what biochemical functions and how they translate into the vivo production of loops on
chromosomes. So, we are very excited to use yeast as a model system to study chromosome looping.
Now when you look at a contact map, if I were to show you some soon, they are usually pretty
complex in which you can have a single spot plus some smaller spots, all in these diagonals etc
or triangles that kind of off the length of the contact map. And these can be explained
by suggesting for example in the cartoon on the left that the sequence at position one
interacts with a sequence at position two to generate spot position three and position four.
So how can you have a sequence interacting with three different sequences further downstream of
the chromosome? Well, that could happen if the chromosome actually folds into
a quaternary structure. And so, when you are throwing in the cross-linker it can crosslink
one to two one three or one to four, and this has been a common interpretation of what these
complex chromatin contact maps might indicate.
But you know one of the features of again the value of the yeast because of our very high
resolution sequencing and quantitative measures we can get because of the abundance of the material
is some new visions of how a complex contact map might be generated. And this comes in this
case from information about knowing where cohesin binds along the length of chromosomes using this
technique called chromatin immunoprecipitation, in which the size of the peak reflects how much/ the
percentage of cells, which have cohesin bound at that site. And this technology was used many years
ago to define where cohesin bound on chromosomes. In fact, Shikha Laloraya, former postdoc of mine
and a faculty in India was one of the pioneers to first use this technology to map cohesin's
binding to chromosomes in yeast and subsequently by others it was used in mammalian cells.
So, we show those peaks of cohesin binding and what you can see is that
the off-diagonal spot arises from the cohesin bound for example the first one at position two
and bringing together the sequence associated with cohesin and bound at position three.
Now one of the things we knew about the cohesin binding at these sites was it was stably bound.
So, the cohesin bound at the so-called cohesin associated regions or CARs is stable, but we
also knew there was a second population of cohesin that was dynamic, that was
binding and coming on and off chromosomes. And we assumed that that was likely the looping cohesin.
And the third thing we knew in this study was that or we revealed in our study in eLife was that
stably bound cohesin in CARs occurs prior to loop formation.
So, with these observations we came up with a model,
which is you first lay down the stably bound cohesin, which is probably doing the cohesion,
and then this dynamic cohesin gets on and starts pumping out loops.
And it goes along until it runs into this stably bound cohesin and the activity stops.
And that would explain how you could generate a specific loop at specific positions.
Now, we have to incorporate that model with the fact that the peaks
of cohesin binding are not the same along the length of a chromosome.
And so, we interpret that to mean that at some sites in a chromosome, cohesin is bound on that
DNA in every cell in the population. While at other sites shown on the left here, the little
red dumbbells, cohesin is bound at that particular site in only a subfraction of the total cells.
And you can see if you sort of place those kinds of stops along the length of the chromosomes in
different cells with different patterns, you are going to get different looping patterns.
So, each individual cell if we could do a contact map on individual cell, would actually have
a different contact map. And if you take all these contact maps of the individual cells and
mix them together as a population, which is like the actual experiment we are doing here,
you can generate a contact map, which is identical to the to the one that is previously
or often interpreted as being reflecting some specific tertiary folded structure.
So, this implies that the folding is much more dynamic in individual cells,
and the pattern of loops is is different between cells.
So, now these observations lead us to the idea that
there must be some additional conserved biological function other than controlling gene expression.
So, first of all as I said, we have these genomic loops that exist genome-wide in
an organism like yeast where there is no reason to invoke a function in gene expression because
it is not really needed, the promoter enhancers are close together.
Second, there is heterogeneous loop formation in individual cells in yeast,
when you want to try to accomplish homogeneous gene expression. So, you know, how are you going
to get all cells to- if they are- if all cells are expressing a particular gene but only a subset of
them have loops, does not make a lot of sense if that is what the loop is doing is controlling or
ensuring the proper expression that gene. And third, you know in human cells as I
explained earlier, there are loops all throughout the genome and yet only a subset of genes are
causing changes in gene expression. So, what are the other 7000
loops that are existing that are not causing gene expression, what are they doing?
So, we came up with this idea that maybe it is involved in DNA repair. So, imagine that you
have two paired sister chromatids through cohesion and you get a double strand break in one of those
chromatids in a repetitive sequence, labelled one and two here. If the break in one repairs
off the sort of repeat out of the identical position on the other sister chromatid,
all will be good, you will just regenerate the two initial sister chromatids, as expected.
In contrast, if the break in one repairs off a repeat that is further down along the chromosome
arm that can generate, depending upon how the repair happens, duplications and deletions,
and that can be bad for lots of reasons.
So, thinking about that in our model we can think about how looping activity might actually,
by generating loops along the two sister chromatids, bring the broken piece of DNA
in contact with the proper repeat, as opposed to the improper repeat. And therefore,
directing repair of the broken DNA off the repeat that's not going to
change the DNA sequence and restore the proper chromosome structure.
So, that is our model at the moment, may not be right, but
I think the bigger question of what loops they are doing in chromosomes remains mysterious.
And I want to say that positioned loops is a conserved feature of all eukaryotes, and in fact,
there are positioned loops in prokaryotes as well. And the SMC complexes act
to actively form loops in all eukaryotes. So, this concept of motors is like
a real wild factor for us in the field. The position of loops in the genome differ
by the presence of stops; and there is a slight difference so, in mammalian cells we think the
stops are made by this factor called CTCF, while in yeast it may be the stably bound
cohesin. Although some recent experiments with mammalian cells that stably bound cohesin in
the mammalian cells may be a second way of generating stops for loop formation. The
existence of positioned loops throughout the yeast genome and in fact the human genome, indicates
that they likely perform a universal function distinct from controlling gene expression.
So, I hope you will sort of appreciate this is a really active and interesting area of research and
stay tuned to understand when the mystery gets solved in our little detective story some years
down the line about what chromosome loops are really doing and how they are forming.
And so, with that I want to thank again everyone in my lab who helped us and allowed
this research to happen, particularly Rebecca and Lorenzo, and thank you for your attention.