Hi, I'm Abby Dernburg,
and I'm gonna tell you in this segment
about our characterization
of special chromosome regions
that play a critical role during meiosis
to ensure proper chromosome segregation.
So, I'll remind you that during meiosis
the goal is to separate homologous chromosomes
-- the copy of the same chromosome inherited from the mother
and the father --
and that in order to do that,
during meiosis chromosomes have to pair with each other,
they have to undergo synapsis,
the formation of the synaptonemal complex,
and they have to undergo recombination,
in which DNA is cut and repaired
to give rise to these crossovers,
which old chromosomes together
until they're ready to segregate.
So we have many open questions in this field.
Our lab, and many other labs,
are very interested in understanding,
how are these processes of
pairing, synapsis, and recombination
executed and regulated?
And also, these are really dynamic processes
that are happening on very different temporal and spatial scales.
So how is it that all of these different processes
are coordinated so that they occur in the proper order,
and to ensure that they occur as faithfully as possible?
So, as I've told you in my introductory segment,
we study these processes in the nematode C. elegans,
and it's a great system
because we can visualize a lot of what's happening
by looking in the microscope.
And this is the region of the C. elegans reproductive tract,
the gonad, in which chromosomes undergo pairing.
And just to show you again what you're seeing,
these are individual nuclei,
each sphere is a nucleus stained with a DNA dye,
and as nuclei enter this transition zone region
where pairing and synapsis occur,
they take on a different appearance
and you can kind of get the sense that chromosomes
are moving around in the nucleus,
which they obviously must do to go from being unpaired to paired.
One of the things that happens in
all organisms, as far as we know,
during meiosis, is that chromosomes are reorganized
in a way that we think facilitates the process of pairing.
So, in a typical cell during interphase,
the period of active protein expression between mitoses,
chromosomes are sort of diffuse in the microscope,
and they are kind of globular,
they tend to kind of ball up into little territories
within the nucleus.
And they're organized by many proteins.
One of the more important proteins that organizes chromosomes
are the cohesin complexes,
and those play many roles.
Their most famous role is to hold chromosomes together
after DNA replication,
but they also serve to organize chromosomes into loops.
So, when chromosomes replicate
and now there's two copies,
the cohesin complexes
hold them together until mitosis or meiosis.
And what happens during meiotic prophase is really interesting,
and we don't really understand it yet.
Somehow, these cohesin complexes
get reorganized into a linear structure
that we call the chromosome axis,
and one thing that this probably does is
it causes the chromosomes to
adopt an elongated conformation,
which probably facilitates pairing simply by
laying out the chromosome in a linear way,
because trying to pair up globular things
is much harder than trying to pair up linear things.
So, we know that this process
of pairing and synapsis has multiple steps,
and we know that the formation of this matrix,
the synaptonemal complex,
is a stepwise process
that begins with the formation of the axes,
as I just described.
So that happens even before chromosomes pair,
and then only after they pair
is the formation of this complete complex triggered,
and we sort of draw it
as if it's a zipper zippering up the chromosomes,
and we have some evidence that it actually behaves that way.
So, how does pairing and synapsis occur?
When I started my lab
we wanted to explore this question,
and we started by investigating these special regions
that we knew existed on
the 6 chromosomes in C. elegans.
So, these are the 6 chromosomes,
and on each chromosome
I've just illustrated with a dark box
this region that we knew was important
for the process of pairing and synapsis.
We knew that these regions were important
from studies of chromosome rearrangements.
So for example,
if you have a worm that has a normal copy of chromosome IV,
but then it has this chromosome,
which has a little piece of chromosome IV,
but is mostly chromosome V,
these two chromosomes are mostly non-homologous
-- not related to each other --
but because they share homology at that end,
that we call the pairing center region,
they will undergo pairing and synapsis,
and they'll undergo recombination
and segregate away from each other.
So we didn't really know the molecular nature
of these pairing centers,
but we knew they were important for determining
which chromosome a particular chromosome would pair with.
And we also could show that in a situation like this,
where the two X chromosomes
have both been deleted for that region
that contains this pairing center activity,
the X chromosomes would fail to pair and synapse.
That's what we're looking at here.
So these are individual nuclei,
again each ball is one nucleus,
and these nuclei are at the pachytene stage of meiosis.
So, in a wild type animal,
if we stain these nuclei with antibodies
that recognize the axis, in red,
and the synaptonemal complex, in green,
what we see is 6 stretches
that stain with both antibodies,
indicating that the 6 chromosomes
are fully paired and synapsed.
Whereas in this mutant,
what we see is 5 synapsed chromosomes
and two little red squiggles,
which are the unpaired X chromosomes
that have failed to pair and synapse.
So we knew that this region was important,
and we wanted to understand, at a molecular level,
what is it?
And how does it mediate chromosome pairing and synapsis?
Our first major clue came from analysis
of a different kind of mutation,
and this was a mutation called him-8.
So I told you that 'him' stands for high incidence of males,
and him-8 was a mutation identified in a screen
for mutants that give lots of males.
It was an unusual mutant because,
whereas most mutations that affect meiosis
affect all the chromosomes,
this mutation only seems to affect the behavior of the X chromosomes,
and yet him-8, the gene,
mapped to a chromosome that was not the X chromosome.
So we were very interested
in understanding what it was about this him-8 mutation
that messed up segregation of the X chromosomes.
When we looked cytologically
at chromosomes in him-8 mutants,
what we saw was very similar to what we see
in the absence of the X chromosome pairing centers.
Again, the X chromosome specifically failed
to undergo pairing and synapsis,
and so him-8 seemed like a really interesting molecule
to get to know a little bit more about.
So what we did is we cloned the gene,
which simply means we identified the genetic locus
that was affected by the him-8 mutation,
and it turned out to encode a protein
that we raised an antibody against,
and when we stained the germline tissue
with this antibody, this is what we saw.
So, on top is again a whole gonad,
a reproductive organ within the worm,
stained with a DNA dye,
so you see the individual nuclei.
And then below is antibody staining for the HIM-8 protein,
and what you can see is there are little tiny dots
throughout the gonad,
and if I blow up this region of the gonad, the transition zone,
where the chromosomes are undergoing pairing and synapsis,
you get a little more detailed picture.
What we see is in the premeiotic nuclei,
there are two spots of HIM-8 in each nucleus,
and then in this transition zone region
we see a mixture of nuclei with one spot
and occasionally two spots,
and then all of the spots fuse to form a single spot.
So, this very intriguing localization pattern
suggested that HIM-8 might interact directly
with the pairing center region of the X chromosome,
and we could test that idea
by performing a dual labeling experiment
where we label the HIM-8 protein
with an antibody, shown in yellow,
and we labeled specific regions of the genome
by in situ hybridization.
So here we've used a probe that recognizes
the left end of the X chromosome in pink,
and the middle of the X chromosome in green.
And I hope you can see that in these meiotic nuclei,
HIM-8 colocalizes with the left end of the X chromosome,
where we knew this pairing center activity was.
So, that's great: it looks like we have a bona fide protein
that binds to the X chromosome
and is required for its pairing and synapsis
during meiosis.
The other thing that we learned by localizing HIM-8
is shown here,
where we've also stained these nuclei
for the nuclear lamina,
so we've used an antibody against the lamin protein,
which forms sort of a matrix
underlying the nuclear membranes,
and that's just to mark the nuclear periphery.
And what I hope you can see is that the HIM-8 spot
in each of these nuclei
is plastered right up against the edge of the nucleus.
And we wondered, of course,
what is this apparent interaction
with the nuclear envelope about?
And in my next segment,
I'll tell you more about that.
First, I'll just tell you that
after identifying HIM-8 as this protein
that binds specifically to the X chromosome,
we realized there must be other factors
that interacted with the other chromosomes,
and we speculated they might be similar to HIM-8.
It turns out that HIM-8
is one of four genes in the C. elegans genome
that are highly related to each other.
They all have two zinc fingers,
which are DNA binding domains,
and we were able to show that ZIM-2,
as we named it for zinc finger in meiosis,
specifically recognizes the pairing center region
on one of the autosomes, on chromosome V.
But then there are two other proteins
that are shared by autosomes:
so, ZIM-1 binds chromosomes II and III,
and ZIM-3 binds chromosomes I and IV.
This is a sort of intriguing setup,
where there are four proteins, six chromosomes...
we don't think it's just that the protein
is somehow identifying the chromosome.
We actually think that these proteins probably had a common ancestor,
and at one time there was probably only one protein
for all of the chromosomes,
but we do think that maybe the diversification
of this protein family has contributed some cues
that are used in the process of pairing and synapsis.
One thing we really wanted to know, of course,
when we identified these pairing center proteins
is, what is it that targets them to individual chromosomes?
And to address this,
we took advantage of an interesting feature of C. elegans biology,
which is that when you inject DNA
by microinjection into the nematode,
what happens is that injected DNA gets sort of ligated together
to make these very high-copy extrachromosomal arrays,
and they can be maintained through a population.
So what we did is we took chunks of genomic DNA
from the region of the X chromosome that recruits HIM-8,
injected them into worms,
formed these extrachromosomal arrays,
and then asked whether these arrays
contained anything that could recruit the HIM-8 protein.
And so this is an example of one such experiment
where we've taken a FISH probe
to mark the array in red,
and we've stained for the HIM-8 protein in yellow,
and in this case the big extrachromosomal array
clearly can recruit the HIM-8 protein.
So what we did is we sort of whittled down
from very large pieces of DNA,
down and down,
until we identified a much smaller
of only 500 basepairs,
that was sufficient to recruit the HIM-8 protein in this assay.
And this 500 basepair fragment looks like this,
I've color-coded the bases here,
just to highlight the very obvious repetitive structure
in the middle of it.
So we identified this motif
that was repeated
within this short fragment that's sufficient to recruit HIM-8,
and we speculated that this motif
was potentially a HIM-8 binding motif.
That idea was definitely reinforced
when we looked at the distribution
of this motif in the genome.
It's pretty much restricted to the X chromosome,
and it's highly enriched on the left end of the X chromosome,
where this pairing center activity is.
And we were able to show that,
indeed, this motif is bound by HIM-8
both in vitro and in vivo,
and that's what targets it to the X chromosome.
And we wanted to know, though,
whether there's something else in this region
that is important for pairing center activity,
or is it simply the recruitment of HIM-8
through this simple DNA motif?
So, to ask that question,
what we did is we took these worms
that I described that had these extrachromosomal arrays
that could recruit the HIM-8 protein,
and we crossed them to worms
that had X chromosomes that lacked their natural pairing center,
and then we used the trick where
we irradiate the animals with ultraviolet light,
and we could find cases where
the array was inserted back into the X chromosome
that lacked its natural pairing center.
And when we homozygosed those chromosomes,
we could ask whether these artificial
HIM-8 recruiting arrays
were sufficient for pairing center function.
And to our delight, we saw that
the insertion of these HIM-8 binding
repetitive elements onto the X chromosome
was in fact sufficient for pairing and synapsis.
And we could score that cytologically here
by showing that the X chromosomes
are now pairing and synapsing,
and we can also score it genetically
by showing that the very high rate of X chromosome missegregation,
reflected as the number of male progeny
in the absence of the X chromosome pairing center,
is rescued when we inserted this array
into the chromosomes.
And here's just one more assay where we could show that
the X chromosomes are undergoing crossover recombination,
because they're not hanging out as individual univalents
at the end of meiotic prophase,
they are still paired.
Okay, so now we have a zinc finger protein
that is recruited to the X chromosome and,
through similar kinds of assays,
we were able to show that there are similar motifs
on the other chromosomes
that recruit these other zinc finger proteins.
And so now we wanna understand
how it is that these pairing centers
and their associated zinc finger proteins
contribute to pairing and synapsis.
And that's what I'll describe in the next segment.
Thanks!