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