Lecture 19 (RR7): Mechanisms of transcriptional activation/inhibition Flashcards
How do all the proteins that control transcription interact?
- the proteins that control transcription interact with the DNA in such a way that they enhance the recruitment of the general transcription factors.
- Many thought that transcription occurs in a linear manner.
- We now know that it does not happen in a linear manner. Everything is probably bound up in these topologically interesting chromatin loops based on protein-protein interactions that drive the transcription reaction forward.
- Modulators and other molecules are essential to bridge and sit POL II at the right spot.
- A lot of the general transcription factors do not stay with an elongating RNA polymerase. In fact, they don’t even stay on the promoter. The only one that stays with the promoter is TFIID (or TBP). Everything else leaves the DNA once the RNA polymerase takes off into elongation
Transcription in Prokaryotic cells
- Streams of RNA polymerase make their way down along the DNA template, generating RNA as they proceed. I
- n this case, we can see it because they become associated with ribosomes immediately (not the case in eukaryotes).
- Initially, we thought that once we activate or initiate transcription, then pol II will make lots and lots of RNA until some signal tells it that the promoter has to be shut down and closed.
How does a “highly transcribed” gene look like?
Gene X = reporter
We want to see the production of the RNA that corresponds to gene X. To do this:
1) add on a little sequence or a couple of sequences at the beginning of Gene X transcript so that you could detect it when the transcript was being generated. This is based on the idea that RNAs form into specific secondary structures (as shown in picture).
2) These secondary structures are recognized by some proteins that are very devoted to each one of those structures. So by introducing one of the stem loops or hairpins that interact strongly with a well known RNA binding protein, you can put an RNA handle on that transcript so that you can quantify how much of that RNA is being synthesized at any giving time (would be driven as well by a promoter that we understand how it functions and with an enhancer).
3) In addition to making this construct, you also have to make an RNA binding protein that recognizes that secondary structure (STEM loop or hair pin) just based on sequence. Like a lock and key, you chose a hairpin or stem loop structure that corresponds to a known protein that can interact with it (it does not have to be a protein present in the organism you’re working with - it can be heterologous = comes from a different organism).
By now u need two things:
1. an RNA binding GFP transgene — you express this in the cell
2. A reporter that is going to make an RNA target for that GFP
4) if it works (9/10 it wont). It will interact with that structure and it will give you a means of identifying how much RNA is being made in the cell.
How does the transcription of that “highly transcribed gene” look like?
You have a transcriptional construct with:
1) enhancer, promoter will tell RNA pol II and all the transcription factors where to go
2) RNA Pol II will proceed through the target (Gene X) and make an RNA. In this case there is only one RNA Pol II but there could be more (each RNA pol II would make an RNA).
3) The RNA will have a structure at the 5’ end (in this case) that will be recognized by the GFP that recognizes exclusively that structure (the green part - stem loop or hair pin) .
They were able to quantify the amount of RNA that was being produces in the nucleus of these cells that has both of the transgenes.
This was tried previously with no success because the GFP attached to it soaked up the system so it was hard to be able to see these types of regions where the threshold of being was higher than the background. With new machine/techniques this was solved (got rid of background using computational subtraction).
*the sequence you added to Gene X theoretically should not add to the amount that will be transcribed. It might affect how well it gets translated.
Fly Embryonic Development
Early embryonic development of the fly has many steps:
1) The nuclei go through synchronous divisions (nuclear divisions) that look like a dance. They undergo these divisions for 13-14 rounds and then the cells start to move.
2) They start to ingress and go through gastrulation. These cells are marked on the membrane with GFP tagged proteins and you get a signal during development that tells them that they have to start migrating in a very specific manner and then they ingress.
3) After that, the morphological features start to appear in a fly embryo.
Transcription occurs in…
Transcription occurs in Bursts
* High-resolution video microscopy revealed recently that many genes expressed at high level are not continuously transcribed at high rates.
* Rather, transcription initiation from highly transcribed genes occurs in bursts of multiple initiation events separated by periods of no transcription between transcriptional bursts.
What experiment led to the discovery that transcription occurs in bursts.
This was observed in Drosophila embryos containing constructs of reporter genes that were attached to multiple copies of engineered stem loops in their untranslated sequence. The stem loops bind to bacteriophage coat proteins with very high affinity and specificity.
**Experiment: **
- snail requires a particular enhancer to be activated at that time (shadow enhancer).
- They made 3 variants:
1. Eliminated the enhancer entirely
2. Variant where they placed the shadow enhancer of the snail gene upstream in a 5’ region that would drive the transcription of a downstream gene. In this case the downstream gene contains 24 copies of an MS2 RNA structure (a stem loop).
3. Enhancer at the 3’ region so downstream of this gene.
- They took the 3 variants and introduced them into drosophila cells (living animals). Then they imaged the animals as they started going into gastrulation during embryogenesis.
** Results: **
- Looking at the images, you can see that the GFP starts to accumulate in the various puncta in case 2 and 3. **Therefore, you can conclude that an enhancer is needed to drive the expression strongly of the transgene. **
- The 5’ positioning of the enhancer works very well - you get very strong activation of the genes.
- The 3’ end positioning of the enhancer works equally well (maybe not quite as strong). What is evident, especially in this variant, is that the GFP corresponds to the RNA that is being made. The RNAs that are being made do not just accumulate overtime but rather they blink. This suggests that RNA Pol II is not constantly driving through these genes and making tons of RNA. Somehow there is a periodicity to transcription, there are bursts of transcription.
- This means that there is a periodicity to those general transcription factors that are going on the promoter and allowing for RNA Pol II to take off the gene body.
Transcriptional efficiency and bursts
- Increased transcriptional efficiency is associated with increased burst frequency. It is not necessarily the amplitude of the amount of polymerase that goes through but in fact the frequency that seems to be important (and it is not exclusive to flies).
- Burst frequency drives transcriptional efficiency. We know this because by using a shadow enhancer which is a strong enhancer for snail, you get specific frequency of transcription that is not shared by the same kind of transgene that is driven with an enhancer that is considerably weaker (rho enhancer).
- **The transcription efficiency is due to the bursting frequency that occurs at a given promoter during transcriptional initiation. **
Tony Hyman and Cliff Brangwynne
- Interested in understanding how the very early cell divisions take place in the sea elegans embryo and the asymmetry that arises following those cell divisions.
- The asymmetry is to eventually distinguish from those two relatively similar cells. One cell will give rise to the immortal lineage (germ lineage) and there is some developmental determinants called P granules that always seem to follow this lineage.
- Granules made their way from being uniform in the early zygotic embryo to all being segregated to the one site (presumptive posterior) where the germ cells will eventually form.
P-granules
- Turns out that P-granules are not granules at all… they are droplets (liquid). We thought they were solid entities for very long. They are droplets that are both liquid but they don’t mix together (like olive oil and balsamic).
- P-granules are examples of liquid-liquid condensates.
- This demixing of proteins and macromolecules is likely the basis of a number of things (primordial world).
- After a few decades, we found these concentrates in a number of situations as a mean of concentrating and compartmentalizing key macromolecules that very often work together.
Intrinsically disordered domains
- Intrinsically disordered domains can mediate liquid-liquid phase separation
- They used to be referred to as a “flexible domain structure” that somehow were able to link these very important entities in DNA binding transcription factors. Now we know that these flexible protein domains are in fact intrinsically disordered regions that play a critical role in defining where those proteins will function.
- Very often they are sufficient to put them into liquid-liquid condensates.
Transcriptional regulators
- Transcriptional condensates greatly increase the rate of transcription initiation.
- Transcriptional regulators can form lquid-liquid condensates.
Example: BRD4 - a protein with 4 bromodomains that stimulates transcription elongation in promoter proximal regions wheres histones are acetylated.
- This bromodomain protein is needed for the activation of some genes.
- GFP labbeling of Mediator suggest that it forms large complex liquid-like structures in cells.
-Their formation is dependent on intrinsically disordered regions (IDRs) of nuclear proteins, including: transcriptional activators, repressors and co-activators.
Experiment studying the formation of protein condensates.
These proteins and domains confer some information to contribute to the formation of condensates. The experiment was driven by the idea that looking at some key transcriptional activators (in this case MED1- a subunit mediator) and other transcriptional activators BRD4 had these intrinsically disordered domains.
- If you expressed a MED1 that’s been tagged with mCherry then it includes the intrinsically disordered domain and you put that into the embryonic stem cells it will make this beautiful droplet like puncta.
- If you express this with a regular GFP molecule, the GFP doesn’t go where the red droplet is.
- However, if you take the intrinsically disordered domain BRD4. This bromodomain protein recognizes acetylated histones (regions of the chromatin that are associated with transcriptionally active chromatin). If you take the BRD4 domain and then co-express that with MED mCherry, the two go together into a droplet. Therefore, the intrinsically disordered domain of BRD4 bromodomain protein is sufficient to bring it into a condensate with mediator (MED1).
- These domains seem to be critical in a number of cases for bringing critical proteins into these compartmentalized environments.
- When IDRs required for formation of transcriptional condensates are deleted from an activator, the transcriptional activation function of the mutant activator is greatly diminished.
What is critical for the formation of liquid-liquid condensates?
- Concentration (concentration of macromolecules)
Number of macromolecules
-DNA
-RNA (sticky - presumed to be one of the condensates that seed these droplets)
-Protein
-other…
- Valency
- Electrostatic interactions (occur between macromolecules)
- Post-translational modifications (contribute charge if conformational change- makes them more or less sticky)
- Intrinsically disordered proteins (often sufficient to drive the formation of condensate)
Mediator and RNA Pol II form liquid-like structures in the cell during transcription
They showed by tagging mediators and RNA pol II and then examining the static micro graphs, that mediators and RNA pol II form these large droplets.
- They were liquid liquid like so that they changed their shape typical to droplets.
- Their interactions were dynamic - sometimes they come together, sometimes they leave.
If you use drugs to block transcription, you block the interaction and the formation of these large complexes.
Dynamic kissing model: a mediator condensate was forming in these chromatin loops that would every once in a while kiss RNA Pol II around its transcriptional start site. But this was transient, it would go away. And then repeat…
The dynamic kissing model would be consistent with the bursts!! This may be the explanation to the peaks. The frequency of the bursts that are associated with the efficiency of the transcription reaction.
Condensate formation vs Condensate dissolution
Model that suggested that when this kissing takes place, the seeding of the formation of a condensate is preceded by the formation of RNA in the transcription reaction. When you initiate transcription and form a little RNA locally, this is enough to act favourably through the electrostatic interactions that are typical of RNAs. They are really sticky and act like glue to bring in all other proteins. So by seeding the reaction, you form these condensates to increase overtime, thereby increasing the efficiency of transcription. But, at some point, because of the electrostatic interactions between RNA molecules, they increase their concentration and start to repel each other. SO when they arrive at a threshold concentration where the electrostatic reaction starts to act negatively, they fall apart (=the other side of a peak) and then all recommences.
Transcription occurs in bursts
High-resolution video microscopy recently revealed that many genes expressed at high level are not continuously transcribed at high rates. Rather, transcription initiation from highly transcribed genes occurs in bursts of multiple initiation events separated by periods of no transcription between transcriptional bursts.
helpful added info:
This was seen in Drosophilia embryos. In the experiment below, 24 copies of the binding site for the bacteriophage MS2-coat protein were inserted immediately downstream of the promoter region of the Drosophila snail (sna) gene, which is expressed in the early Drosophila embryo.
When transgenic flies were generated containing this reporter inserted into one site on one chromosome, no expression of the transgene was detected. But transcription was detected when the inserted DNA contained an enhancer downstream of the reporter gene, an enhancer that activates expression of sna in the early Drosophila embryo (Figure 8-40b). Transcription was detected by expressing MS2-coat protein fused to GFP (green fluorescent protein) in the same cells in which the reporter gene construct was inserted.
**A video of images of the embryo during the 14th nuclear cycle ( min long) revealed one region of GFP fluorescence in each cell at the site of the transcribed, inserted reporter gene. But the intensity of the GFP signal in each cell was not continuous. Rather, the intensity of each spot of fluorescence increased and decreased over several minutes.
**
Relationship between transcriiptional efficienty and bursts
- Transcripional efficiency correlates with increased burts frequency
- RNA dependent formation and dissolution of transcriptional condensates may explain the burt phenomenon.
