What types are there?
Acidic ADs – the
strongest AD’s (GAL4P)
Glutamine rich ADs – (SP1,
requires many copies, not so strong by itself)
Proline rich ADs – CEBP
VP16 AD (acidic) – strong
activation, tegument protein from HSV, does not bind DNA. IE HSV genes contain
many OCT1 binding sites, recruits HCF1 (co activator), the OCT1-HCF1 complex is
mostly inactive – VP16 is able to bind this complex and mediates the
activation.
Yeast 2 Hybrid (Y2H) –
AD – activation domain
BD – (DNA) binding domain
Prey – The protein fused
to the AD
Bait – The protein fused
to the BD
Cells are transfected
with plasmids containing these modified proteins and can be used to investigate
protein-protein interactions and DNA-protein interactions by using various
combinations of prey/bait combinations. If the proteins interact with
each-other there will be transcription.
However, sometimes there
are false positives, special proteins that cannot be used in the Y2H method – bait
proteins which contain activation domains!
In the article Meir
presented researchers studied transcriptional activators in yeast, they
subdivided these groups into 3 (weak medium and strong activators). There was
an inverse correlation between the strength of the activation with the
abundance of the proteins. All three
groups showed lower isoelectric points (pI) which means they are acidic (acidic
ADs).
These newly identified
activators’ amino acid composition showed a general negative charge.
Most activation domains are largely “unstructured, when a crystallized
protein doesn’t display beta sheets or alpha helices. However, when the AD
binds its target it becomes structured.
Natural ADs are usually composed of unstructured
peptides with potential to form amphipathic helices.
Many ADs form an amphipathic α-helix upon interaction with the transcriptional
machinery, with the hydrophobic face of the helix contacting the binding
partner
A lot of strong mammalian
transcription factors such as E2F-1, Jun, Fos, STAT1, Myc are short lived,
meaning they undergo proteolysis very fast, there might be a correlation
between the activation strength and their short life span.
When GAL4-fusion proteins
(acidic ADs) were compared with proline-rich (CTF) and glutamine-rich
(Q18,Q19,SP1) ADs without the presence of a proteolysis suppressor
(PS1) they were readily degraded.
However, when treated with PS1 all ADs displayed similar protein expression
levels.
Degron – a sequential
motif within the protein which serves as a binding site for ubiquitin, leading
to degradation. Degrons are usually found within the transcription activation
domain, there are some exceptions such as beta-catenin… it undergoes
phosphorylation by GSK3-Beta and only then it is degraded.
Another look at AD
strength correlated with degradation:
When there are more GAL4
copies, transcription becomes more pronounced, degradation however, also
becomes an issue (LLnL is a protease inhibitor), 3XM is a non-functional GAL4
fusion protein acting as an extra negative control.
Next, the researchers wanted to demonstrate that degradation is mediated via ubiquitinylation:
On the left: cells were
transfected with GAL4-fusion constructs with and without his-tagged protein, GAL4
was blotted.
On the right: His-tagged proteins were blotted and only 3X/6X fusion proteins
appeared, just like in the previous figure, 1 copy of GAL4-fusion protein was
not enough to induce ubiquitinylation.
“The model”
Transcription factors possess repressive domains, it binds the activation domain. The repressive domains inhibit the activation domains and are only “opened” under certain circumstances.
In the previous lecture
Meir gave us an example of CEBP which has a repressive domain and an activation
domain, when fused to VP16, it was also repressed.
Ubiquitin’s role in transcription activation
Meir shows an article
dealing with this issue
Met30 – a protein that
ubiquitinylates VP16
LexA – protein containing a DNA binding domain, like GAL4
Myc/Cln3/VP16 – common ADs
Upon Met30 depletion,
VP16-LexA levels are stable à
However, when transcription
comes into play, it appears that Met30 is necessary for activation!
Next, the researchers
wanted to elucidate Met30’s role in transcriptional activation. They
transfected cells with several plasmids:
1. Δ –
LexA
2. Δ-Ub – ubiquitin linked LexA
3. Δ-VP16 – VP16 linked LexA
4. Δ-Ub-VP – both factors together
As previously shown, LexA-VP16 transcription in the presence of Met30 is functional. Ubiquitin-VP16 linked LexA, however, displays near identical levels of transcription with and without Met30 – it was essentially “rescued”.
The proteasome’s involvement in
transcriptional activation (another article)
remember: the proteasome exists not only in
the cytoplasm but within the nucleus as well.
Researchers
performed a ChIP-assay (chromatin immuno precipitation) – an assay used to
display the interaction between proteins and DNA.
The
proteasome is comprised of two subunits (20S and 19S, 26S together). There are
two 19S subunit which act as regulatory caps (literally like two “hats”) and a
20S subunit which acts as a hollow “pipe” between them.
Galactose
is an inducer of Raffinose
Sug1/Rpt6/Rpt5 = 19S
(A) As
soon as 10 minutespost induction, 19S was recruited onto the GAL1-10 promoter
(Gal4 is a positive control). The 20S subunit, however, was not recruited even
after 2 hours.
(B) Is
the binding GAL4 dependent ? WT and ΔGAL4 were tested and the interaction was
shown to be GAL4 dependent.
Conclusion: ADs also interact with
proteasomes!
The model shown below suggests that even when a transcription activator binds to DNA, it still cannot activate it without the help of recruited elements and ubiquitinylation
From the
slide:
a | Target gene activation starts
with oestrogen-bound ER undergoing a phosphorylation event that promotes E3
ligase binding and facilitates binding of the complex to oestrogen response
element (ERE) or to AP1 or SP1 sites at a target gene promoter.
b | ER ubiquitylation might alter the receptor conformation to
facilitate pre-initiation complex assembly.
c | When transcription initiation is complete, ubiquitylation marks ER
for degradation and might help to disassemble the transcription initiation
complex, which facilitates the transition to a productive elongation complex
and elongation of transcription.
d | ER clearance by proteolysis would permit promoter recharging and
thereby allow the next round of promoter firing.
This
suggested approach is a bit different, and might explain why the acidic ADs are
so unstable, if they require ubiquitinylation just so transcription can start…
it would require their eventual degradation!
CREB (cyclic AMP responsive element
binding protein) recruitment
CBP/p300
– These are two proteins very similar in both structure and function:
Several
viral factors (E1A, SV40 large T, LANA) are able to bind the HAT (histone
acetyl transferase) binding domain, interestingly, many transcription factors
bind the exact same region.
The KIX
region binds KID (kinase inducible domain), KID is a domain which exists in
CREB. Other proteins which are phosphorylation dependent (like CREB, Ser133)
can also bind CBP/p300 via the same domain (KID), this creates competition
between factors for CBP/p300 proteins.
Rubinstein-Taybi
syndrome (RTS) is a genetic disorder (cognitive dysfunction), this caused due
to haploinsufficiency (one of the alleles has a mutated copy of CBP/p300) –
this causes lower levels of the working protein to be available.
E1A is
able to bind p300/CBP in its KIX domain thus inhibiting transcriptional factor
binding. PCAF is another co-activator with similar function to CBP/p300
There
are also proteins that help recruitment of CBP/p300 such as Tax (a viral
protein from HTLV).
Tax is able to phosphorylate CREB and thus activates CREB further, leading to
transcriptional activation.
NFKB pathway
Few
notes:
The
alternative pathway is completely dependent on IKKa.
How do
viruses handle the NFKB pathway ? On one hand it causes cells to proliferate
and on the other it causes an anti viral response!
Answer:
they do both, depending on their type
For
example: viruses with latency/lytic modes will (upon infection) inhibit the
NFKB pathway and then later will activate it.
p105 –
ubiquitous, it’s everywhere
p100 – expressed mostly in B-cells
REL65 – ubiquitous
RELB – thymus, lymph nodes, important for B cell development regulation
c-REL - lymphocytes
Activation of NFKB pathway
Green
boxes – viral proteins
Red boxes- bacterial proteins
Many
viral proteins are able to activate the pathway, most of them do so in the
beginning phases of infection, though some do not (HBV’s HBX for example).
Inhibition of NFKB pathway
Most viral proteins inhibit the pathway by inhibiting phosphorylation of
IKBa.
There’s a protein with similar structure and function to IKBa which cannot
be degraded, it sequesters p105/RELA and keeps them within the cytoplasm
Some proteins inhibit the actual translocation from the cytoplasm to the
nucleus.
And some (like E1A) inhibit the binding of p65 (RELA) to the DNA.
Regulation of NFKB pathway
As mentioned earlier, in herpesviruses such as EBV and KSHV there are two
phases, lytic and latent.
Apparently, NFKB pathway activation is critical in maintaining the latent
phase (activated by various proteins, vFLIP, LMP1 and more). This activation is
vital in two ways:
1. Causing the cells to proliferate
2. Inhibiting the lytic cycle
If we inhibit NFKB and allow for a “viral-friendly” environment, the virus
will go into the lytic cycle.
When the virus expresses certain proteins (ZTA/RTA) it is able to
reactivate its lytic cycle.
The RTA/ZTA promoters have AP1 binding sites.
When AP1 binds to the promoters it activates it, if the NFKB pathway is active
it inhibits AP1s and does not allow transcription. Upon repression of
NFKB, AP1 is able to activate RTA/ZTA.
important: he asked which factors bind AP1, no one knew the
answer [this might be a good question for the test. The answers are Jun and
Fos, they create the AP1 complex.
This slide is meant to show that KSHV is able to activate the NFKB pathway in both the classical pathway and the alternative pathway.
What happens if we inhibit NFKB? Another article
An EMSA (electrophoretic mobility shift assay) was performed.
If the NFKB pathway is inactivated, no NFKB will be found within the
nuclear extract.
When KSHV containing B cells were
treated with Bay 11 (an NFKB inhibitor), no NFKB was found within the nuclear
extract.
Black – Bay 11 treated
White – Untreated
As mentioned before, a lack of a proliferation signal due to NFKB inactivation leads to apoptosis.