I've discussed two mechanisms of transcriptional precision in the early Drosophila embryo;
shadow enhancers and paused polymerase.
These two mechanisms ensure robust, rapid and uniform patterns of gene activation,
and, when used together, very rapid rates of RNA synthesis.
I want to finish up be discussing mechanisms of transcriptional repression.
And I've already alluded to the importance of repression in the early Drosophila embryo.
For example, repressors form the two borders of the Eve-Stripe 2 pattern.
The Snail repressor establishes the boundary between the mesoderm and neurogenic ectoderm.
And now I want to address how does this repression work.
And I will turn specifically to Snail repressor.
So here, once again, we're looking at the expression of the Sog gene in the early embryo.
As I've mentioned a few times, Sog is activated by low levels of the Dorsal gradient
in the lateral neurogenic ectoderm.
It's kept off in the ventral mesoderm by the Snail repressor
and of course Sog is one of those magic genes
that has both paused polymerase and a shadow enhancer.
The enhancers aren't shown here, but earlier I showed you that
Sog has both a primary and intronic enhancer and a remote, 5' shadow enhancer.
So Sog, like Snail, comes on quite quickly and uniformly in the early embryo.
Now, Sog, in fact, beats Snail to the punch
and is turned on a little bit before there are critical levels of the Snail repressor.
So, initially, Sog is turned on in both the ventral regions that will form the mesoderm as well as
the lateral regions that will form the neurogenic ectoderm where Sog becomes stably expressed.
And a graduate student in the lab, Jacques Bothma has done high-resolution imaging
of this loss of Sog expression. (although it's only transiently expressed).
This loss of transient expression of Sog in the ventral mesoderm due to the Snail repressor.
So that's what I'm going to show you.
The experimental design is summarized here.
So this summarizes the Sog transcription unit.
It's a fairly large gene (20 kilobases) with a larger 5' intron and 3' intron.
Jacques made separate fluorescent probes for the 5' intron and the 3' intron.
The two probes are separated by 10 kilobases.
And then he mixed these together and hybridized them to staged embryos.
When the Sog gene first comes on, you only see green hybridization signals
and that's because the polymerase is only located within the first intron
so the only nascent transcripts present at this early stage of activation of Sog
contain only 5' intron sequence.
But after a delay, the polymerase enters the 3' intron
and as it does so, the 5' intron is spliced out.
That's indicated by this little lariat.
Then, the polymerase extends into the 3' intron and
now the 3' intronic hybridization probe cross reacts with these nascent transcripts.
So after a delay, the Sog gene is stably expressed.
You see yellow hybridization signals because the nuclei contain a mixture
of nascent transcripts containing either 5' intron sequences or 3' intronic sequences.
So now the question is: How does Snail work as a repressor?
The Snail repressor kicks in and will switch the gene off in the ventral mesoderm as I mentioned.
And there are at least two possible mechanisms.
One possibility is that Snail disrupts polymerase elongation.
The polymerase is stopped right in its tracks
so the polymerase falls off and the gene is immediately shut off.
And so according to this view,
we should go from a yellow hybridization signal when the gene is on
to no hybridization signal at all, just a blue, unlabeled nuclei.
So that's if Snail works at the level of polymerase elongation.
A second mechanism is if the polymerase works at the level of initiation
by blocking the release of the paused polymerase from the promoter.
According to this mechanism, once Snail kicks in, there's no new synthesis.
There's no new release of polymerase
but the elongating polymerases that were released
prior to the onset of Snail repression are able to complete transcription.
And if so, we should see an intermediate stage of hybridization
which we call the red flash.
These are the elongating polymerases completing transcription of the gene
that had been released from the promoter prior to the onset of Snail repression.
So according to the first mechanism (inhibition of elongation), we should go from yellow to off.
According to the second mechanism of blocking the release of polymerase
or transcription initiation, we should go from yellow to red to off.
And this is what we see.
So, we obtained evidence that Snail works at the level of transcriptional initiation,
blocking the release of polymerase, but the polymerase that had already been released,
completes transcription even after the onset of Snail repression.
And some evidence for that is shown here.
So here you're looking at progressively older nuclear cleavage cycle 14 embryos.
These embryos were double-labeled
with the green 5' intronic probe and the red 3' intronic probe
looking at Sog expression and repression.
When Sog is first switched on, you see only green hybridization signals.
Activation's fairly synchronous as we would expect for a gene containing paused polymerase.
And it's very rapid as we would expect for a gene that
has both paused polymerase and a shadow enhancer.
And you see expression of both lateral regions where the gene will be stably expressed
and ventral regions where it's going to get shut off.
At this early stage, the only nascent transcripts contain hybridization with the 5' intronic probe.
The polymerase has not yet entered the 3' intron.
After a ten minute delay, the nuclei stain yellow
and that's because now the polymerases have entered the 3' intron
and we get hybridization with both the green 5' intronic probe and the red 3' intronic probe.
And this delay of ten minutes, going from green to yellow,
is consistent with the rate of polymerase elongation of 1kb a minute
as I mentioned before and was shown previously in John Lis's lab.
OK, so now Sog is stably on. The Snail repressor kicks in.
And you see that Sog is getting gradually repressed in the ventral mesoderm over time.
And it takes a long time to switch off Sog expression; about 20 minutes between
the onset of the Snail repressor and the elimination of Sog expression in the ventral mesoderm.
And you see an intermediate stage of the red flash right here
and that's the elongating polymerases completing the transcription of the 3' intron
that had been released prior to the onset of Snail repression.
so this red flash is very good evidence that Snail works
at the level of initiation not elongation.
Elongating polymerases complete transcription even after the Snail repressor has kicked in.
But this mechanism of repression, blocking the release of polymerase from the promoter,
blocking initiation, means that there's going ot be a repression lag.
And that lag is commensurate with the size of the gene.
Sog is 20 kilobases in length and it takes 20 minutes to fully repress Sog expression,
to completely eliminate its expression in the ventral mesoderm.
And that's due to the elongating polymerases completing transcription
even after the onset of Snail repression.
We estimate that during the repression lag of 20 minutes
as many as 50 to 100 unwanted Sog mRNAs could be made in the mesoderm
where they're not needed.
Moreover, those ectopic Sog mRNAs may cause developmental problems.
And so if you want to get rid of transcripts that are produced after the onset of repression,
that arise due to this repression lag,
you might have to rely on post-transcriptional mechanisms such as microRNAs.
And Sog is indeed regulated by a specifically expressed microRNA
possibly getting rid of, cleaning out, those transcripts made after the onset of Snail repression
due to this repression lag.
Large genes, really large genes, have a more severe problem with repression lag.
There are Hox genes in the Drosophila genome, like Antennapedia and Ubx,
which are on the order of 100 kilobases in length.
It literally takes an hour and a half to repress these genes.
Both Antennapedia and Ubx are subject to extensive regulation by microRNAs
possibly due to this repression lag issue.
So now, once Snail represses Sog in the ventral mesoderm,
paused polymerase is once again established at the Sog promoter.
Although, Sog is never going to get turned on in the ventral mesoderm.
It's turned off in the ventral mesoderm by 3 hours after fertilization.
No more Sog transcription in the mesoderm.
And it will never come back on in the mesoderm throughout the lifecycle of the fly.
Never again.
Over the next several hours of development, the paused polymerase
at the promoter regions of Sog in the mesoderm, is lost.
And so you go from a gene that's repressed but poised
it's poised by virtue of containing the paused polymerase
to repressed and silent which lacks a paused polymerase.
So this is a more stable form of gene silencing.
I want to discuss this transition between repression achieved by Snail
but you still have the paused polymerase
to the silencing of this gene in the mesoderm
because you've now removed the paused polymerase
and the gene will never again come back on in the mesoderm.
This transition from repressed but poised or paused to silenced
is mediated by a special group of repressors called the Polycomb repressors.
And an example is shown on the next slide.
This shows a Polycomb mutant called Esc or Extra Sex Combs.
And what you're looking at is the cuticle
or the secretion of the ventral skin in late staged embryos.
In Polycomb mutants such as Esc, all the body segments
acquire the phenotype of the posterior most eighth abdominal segment or A8.
So in Polycomb mutants the anterior abdomen, the thorax and even some of the head segments
are transformed into A8.
That's what you see in the Esc mutant.
This transformation is due in part to the misexpression of Hox gene called Abdominal B.
Abdominal B is normally transcribed at the highest levels in the developing A8 segment
but in Polycomb mutants, Abdominal B is transcribed at high levels throughout the embryo.
And that transforms all the body segments to an A8 identity.
Now, I mentioned in the very beginning an anterior Hox gene called Antennapedia
and there are others like Ultrabithorax and Sex Combs Reduced.
Antennapedia is transcribed normally in the middle thorax in T2.
But in the Esc mutants, AbdB is transcribed everywhere
and AbdB is a repressor and it switches Antennapedia off.
And that leads to this transformation of the thorax into extra A8 posterior segment identity.
So a postdoc in the lab, Vivek Chopra, did whole genome Pol2 ChIP binding assays
to look genome-wide at the distribution of RNA polymerase
in advanced staged wild type embryos and Esc mutants.
And he found a significant increase in the amount of paused polymerase
in Esc mutants at a couple of thousand different genes.
So an example is shown here.
This is the Antennapedia gene itself.
Antennapedia has several promoters. This is proximal most promoter.
In wild type embryos, you see a little bit of polymerase binding at the promoter,
a little bit of paused polymerase.
But in the Esc mutant, you see a significant increase
in the amount of paused polymerase at the promoter.
And this was surprising to us because, as I said,
in Esc mutants AdbB is transcribed everywhere and Antennapedia is shut off.
So you see more polymerase binding in the Esc mutant
at the Antennapedia promoter even though the gene is off in these mutants.
And this trend of increased polymerase binding, increased paused polymerase,
in Esc mutants, as I said, is seen for couple of thousand genes.
A few more examples are shown in the next slide.
So here are two patterning genes; Poxmeso, Twin of Eyegone.
In advanced staged wild type embryos these genes contain little or no paused polymerase
but in Esc mutants you start seeing significant paused polymerase at the promoters.
So, this is the trend: In Polycomb mutants, there is an increase in paused polymerase.
And our model for what's going on
(we have some additional evidence for this which I'm not going to show right now)
is summarized in this last model slide.
So, here's a gene that's repressed but poised because is contains paused polymerase.
Again, think about the Sog gene in the mesoderm of a 3 hour embryo
after Snail has finally shut it down after the repression lag.
The gene is off but you have the paused polymerase.
The Sog gene will never come back on in the mesoderm
and so eventually the Polycomb repressors take off the paused polymerase at the promoter
and replace it with a positioned nucleosome.
So what we believe the Polycomb repressors are doing
is to convert repressed genes that are poised into repressed genes that are silenced
that now have a nucleosome at the promoter instead of paused polymerase.
And that's a more permanent form of repression.
It's harder to turn this gene back on than this type of gene.
So then if we take away Polycomb, as seen in the Esc mutants,
of course there is an increase in the paused polymerase
because we haven't achieved this reaction of replacing
the paused polymerase at repressed genes with the positioned nucleosome.
So that's our model.
So I'm going to just end by acknowledging the associates in the lab who did the work.
The first part on shadow enhancers and robustness in response to changes in temperature
that was done by a graduate student, Mike Perry.
The studies on paused polymerase
promoting rapid and synchronous patterns of gene activation
and paused polymerase working with shadow enhancers
to give very high rates of synthesis such as the rapid synthesis of Snail transcription
this was the work of a graduate student named Alistair Boettiger.
The visualization of repression and the repression lag I discussed
regarding the inhibition of Sog expression in the ventral mesoderm by Snail
that was the work of a graduate student named Jacques Bothma.
And finally, this last part that the Polycomb mutants such as Esc
replace the paused polymerase at repressed genes with a positioned nucleosome
to convert them to a silent state
that was the work of a postdoc named Vivek Chopra.
The studies on paused polymerase were launched in collaborative studies
done initially with Julia Zeitlinger and Rick Young at MIT.
Julia was a postdoc at that time
and she now has her own lab at the Stowers Institute at Kansas City.
And more recently, we've collaborated with a postdoc named Leighton Core
in John Lis's laboratory at Cornell
on the paused polymerase and that fact that the bound polymerase
is in an active state based on whole genome nuclear run-on assays.
And with that I will thank all of you out there in the ether for your time.