September 07, 2016
Cell phones have evolved in amazing ways. What started as a “dumbphone” that only let you make calls and, somewhat awkwardly, text, has now become a smartphone – a marvelous mini-computer.
What this means is that even if a smartphone’s SIM card goes bad so that you can’t make a call or send a text, you still have something that can do a lot. In fact, since it can still connect to Wi-Fi, you can even text or call! This is obviously different than a “dumbphone” which, if it can’t be used as a phone, is pretty much only useful as a hurled weapon. (Which is just what Dr. Drakken threatened Ron Stoppable with at 8:27 of this video.)
In a new study out in GENETICS, Thurtle-Schmidt and coworkers show that the histone deacetylase Sir2p is more like a smartphone than a dumb one. Even when you knock out its ability to deacetylate histones, Sir2p can, in the right background, still silence the genes it is supposed to.
Sir2p is the founding member (#APOYG!) of the important sirtuin enzyme family. It silences genes by first deacetylating acetylated lysines on histone tails (H3 and H4 specifically). This then allows the Sir-protein complex (which includes Sir2p, Sir3p, and Sir4p) to bind the nucleosome Sir2p just deacetylated. Now the Sir-protein complex deacetylates nearby histone tails and so on until the Sir-protein complex has spread across a gene, silencing it.
Obviously the ability of Sir2p to deacetylate is important in this scenario! But these researchers found that like a smartphone without a SIM card, Sir2p can sometimes do its job even without its deacetylase powers.
But instead of going around a carrier and using Wi-Fi, Sir2p needs for a second histone deacetylase, Rpd3p, to be gone. Without Rpd3p, Sir2p can now silence genes. Not as well as it could before, but some.
To find this out the researchers set up a suppressor screen. They used a reporter that replaced the a1 open reading frame (ORF) at HMR with the URA3 gene. The a1 ORF is normally silenced by Sir2p. Basically, if this gene is silenced, the yeast can grow in the presence of 5-fluoroorotic acid (5-FOA).
Next they added a mutant Sir2p that lacked its catalytic ability to this reporter strain. Finally they mutagenized this strain and looked for mutants that could again silence genes. They got 1500 5-FOA resistant mutants.
Of course many of these may have been the result of mutations in the URA3 gene. They did a secondary screen that used mating as a way to rule out this possibility. In the end they had four mutants.
One of these four was a mutation that had been found in previous screens – SUM1-1. This mutant appears to bypass the need for any of the SIR genes by setting up a different kind of silenced chromatin.
The other gene that came out of the screen was RPD3. They found three different mutations in this deacetylase that all partly restored Sir2p’s ability to silence genes and found that deleting the gene had the same effect. Follow-up work showed that this effect was indeed Sir2p-dependent. Now they had to figure out how eliminating a second deacetylase frees this mutant Sir2p to do its job.
In some ways it isn’t surprising to get RPD3 out of a screen like this. It seems to be important in keeping the Sir-protein complex from spreading too far (who wants the whole chromosome shut down?) and deleting it affects various silenced genes.
Rpd3p is found in two different complexes imaginatively named large (Rpd3L) and small (Rpd3S). When the authors deleted genes specific to either complex, their sir2 mutant did not regain its ability to silence genes. Only when they deleted a gene involved in both complexes, SIN3, were they able to mimic the effects of deleting RPD3 (“phenocopied the rpd3Δ”).
One possible idea is that since Rpd3p keeps the Sir-protein complex from spreading, its deletion might allow for increased spreading even in the absence of Sir2p’s histone deacetylase activity. This is what they found.
Using Chromatin ImmunoPrecipitation (ChIP) against Sir4p, one of the proteins in the Sir-protein complex, the authors repeated the result that in the absence of Sir2p’s histone deacetylase activity, there is less Sir4p at silenced regions. When they looked at the same strain deleted for RPD3, they found an increase of Sir4p at silenced genes. Not to the levels seen in the wild type strain, but enough to probably explain the partial silencing seen in the strain.
Makes sense so far but it isn’t the whole story. Nicotinamide (NAM) is competitive inhibitor of sirtuins like Sir2p. As such, we might predict that it should have no effect on the silencing of a gene by a catalytically inert Sir2p. We would be wrong.
Turns out NAM does affect silencing in this strain which suggests that some other sirtuin might be playing a role. There are four homologs of SIR2: HST1, HST2, HST3, and HST4. A bit of work including creating a triple mutant strain deleted for RPD3 and HST3, and containing the mutant Sir2p, showed that Hst3p is involved in this silencing.
Whew, that was a lot! So mutating away the deacetylase activity of Sir2 unsilences genes. And deleting RPD-3 from this strain restores some of that silencing. And the restored silencing in this strain is at least partly dependent on Hst3p.
So there you have it. Like a cell phone without a SIM card using Wi-Fi, Sir2p can still do its job if Rpd3p isn’t around to interfere. As long as Hst3p, like turning the phone’s Wi-Fi on, is there to help.
by Barry Starr, Ph.D., Director of Outreach Activities, Stanford Genetics
Categories: Research Spotlight
May 01, 2014
When a character is asked to show his badge in the movie The Treasure of the Sierra Madre, he famously says something along the lines of, “Badges? We don’t need no stinkin’ badges!*” If histones in yeast heterochromatin could talk they might say something similar, except instead of badges they’d bring up modifications. Maybe something along the lines of, “Modifications? We don’t need no stinkin’ modifications for activation!” At least, they’d say this if a new study by Zhang and coworkers holds up.
In this study, the authors show that two different genes in the yeast S. cerevisiae are activated in heterochromatin in the absence of any significant changes to the surrounding chromatin. This result is surprising because most researchers think activation and changes in chromatin always go hand in hand. Apparently, in at least some situations they do not.
This isn’t to say that chromatin didn’t do anything here…it most certainly did. It served as a general damper on transcription. But in this study chromatin was by no means the major player; it had a relatively small influence on the levels of basal and activated gene expression. The authors suggest that this may be true for other genes in the more transcriptionally active euchromatin as well.
In the first set of experiments, Zhang and coworkers used a model system where the heat inducible gene HSP82 is flanked by the HMRE silencer from the HMR mating type cassette. These silencers cause a 30-fold reduction in transcription of this hsp82-2001 transgene.
Using chromatin immunoprecipitation (ChIP) the authors show that their transgene is indeed embedded in heterochromatin. They see a lot of Sir3p around the promoter, a high density of histones that lack any of the telltale modifications of euchromatin, and very little RNA polymerase II (Pol II) or the mRNA capping enzyme Cet1p around the promoter. These are all hallmarks of heterochromatin in yeast.
Things change when the yeast is subjected to heat shock. Consistent with the observed 200-fold increase in transcription, they suddenly see lots of Pol II and Cet1p around. But there is not a big change in the number of histones around the gene nor in their modifications.
When HSP82 is in its normal place in the genome, its activation is accompanied by specific acetylation and methylation of H3 and H4 histones. In heterochromatin, despite significant induction, there is none of this. The histones remain looking the same whether there is significant transcription or not.
One trivial explanation for this might be that the chromatin is unaffected because the levels of transcription are lower than normal. In other words, the lower final activity in the induced state is affecting histone modification.
Zhang and coworkers rule this out by using a TATA-less HSP82 gene in euchromatin and show that all the appropriate histone modifications still happen. This is true even though the damaged gene has 5-fold less activity compared with their transgene. The low level of transcription does not appear to explain activation in the absence of histone modification.
Of course another reason for this unexpected observation might be that this pretty artificial construct isn’t representative of natural genes. This doesn’t change the fact that its transcription is activated in the absence of histone modification, but it does question its relevance in the real world.
To address this issue, the authors looked for an inducible gene in natural heterochromatin and with a little bit of detective work, found the subtelomeric YFR057W gene. No one knows what this gene does, but a close look showed a possible Stb5p binding site in its promoter.
When Stb5p heterodimerizes with Pdr1p, the resulting dimer activates genes involved in pleiotropic drug resistance. Indeed the authors found that YFR057w was induced 150-fold with a small amount of cycloheximide. And when they used ChIP to compare the induced and uninduced states, they again found almost no changes in the chromatin around this gene despite an increase in the amount of Pol II and Cet1p.
Taken together these results suggest that activation doesn’t always have to come with chromosomal changes. Which, while a bit surprising today, wouldn’t have turned any researchers’ heads a few decades ago.
In the old days (1980’s and 1990’s), a lot of focus was on how transcriptional activators might affect the ability of Pol II to load onto the DNA and to pry it open and start transcribing. A lot of this was based on prokaryotic work where there really isn’t very much in the way of chromatin and a lot of activation depends on improving the ability of the polymerase to transcribe.
These days when people think about turning up a gene, they think about changing nearby chromatin. Various enzymes work to modify histones at specific places, which both loosens up the chromatin to allow access by Pol II and serves as a way for various coactivators to recognize the DNA.
As usual, reality is probably a combination of the two. Activators can activate transcription in lots of different ways, some of which probably include chromatin changes while in others chromatin changes are simply a consequence of activation. Not all transcription activation needs stinkin’ histone modifications.
by D. Barry Starr, Ph.D., Director of Outreach Activities, Stanford Genetics
Categories: Research Spotlight