New & Noteworthy

More Than Just a Brick

September 07, 2016

A catalytically damaged Sir2p, like a smartphone without a SIM card, can still do its job in the right environment. Image from Wikimedia Commons.

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.

Sir spreading.gif

by Barry Starr, Ph.D., Director of Outreach Activities, Stanford Genetics

Categories: Research Spotlight

Tags: H4K16, HST3, sirtuin, heterochromatin, SIR2, RPD3, silencing

The Sounds of Silencing

June 17, 2015

For centuries, we thought of the universe as an empty, eerily silent place. Turns out we were dead on when it came to the emptiness, not so much when it came to the silence.

Despite more and more powerful equipment, SETI has yet to find any meaningful radio signals coming from the stars. Yeast research is in a better position: new techniques applied to telomeric gene expression now make sense of the signals. Image by European Southern University (ESO) via Wikimedia Commons

Once we invented devices that could detect electromagnetic radiation—starting with the Tesla coil receiver in the 1890s—we began to realize what a noisy place the universe really is. And now with modern radio telescopes becoming more and more sensitive, we know there is a cacophony of signals out there (although the Search for Extraterrestrial Intelligence has yet to find any non-random patterns).

The ends of chromosomes, telomeres, have also long thought to be largely silent in terms of gene expression. But a new paper in GENETICS by Ellahi and colleagues challenges that idea. 

Much like surveying the universe with a high-powered radio telescope, the researchers used modern techniques to make a comprehensive survey of the telomeric landscape–and saw that the genes were not so silent. Their work revealed that there’s a lot more gene expression going on at telomeres than we thought before.

It also gave us some fascinating insights into the role of the Sir proteins, founding members of the conserved sirtuin family that is implicated in aging and cancer.

Telomeres are special structures that “cap” the ends of linear chromosomes to protect the genes near the ends from being lost during DNA replication, something like aglets, those plastic tips that keep the ends of your shoelaces from fraying. They have characteristic DNA sequence elements that we don’t have space to describe here (but you can find a short summary in SGD).

Classical genetics experiments in Drosophila fruit flies showed that telomeres had a silencing effect on the genes near them, and early work in yeast seemed to confirm this. Reporter genes became transcriptionally silenced when they were placed near artificial constructs that mimicked telomere sequences.

This early work was solid, but had a few limitations.  The artificial telomere constructs were, well, artificial; some of the reporter genes encoded enzymes that had an effect on overall cellular metabolism, such as Ura3; and the studies tended to look at just one or a few telomeres.

To get the whole story, Ellahi and colleagues decided to look very carefully at the telomeric universe of S. cerevisiae. First, they used ChIP-seq to look at the physical locations of three proteins, Sir2, Sir3, and Sir4, on chromosomes near the telomeres.

These proteins, first characterized and named Silent Information Regulators for their role in silencing yeast’s mating type cassettes, had been seen to also mediate telomeric silencing. Scientists had hypothesized that they might be present at telomeres in a gradient, strongly repressing genes close to the chromosomal ends and petering out with increasing distance from the telomere. 

Ellahi and coworkers re-analyzed recent ChIP-seq data from their group to find where the Sir proteins were binding within the first and last 20 kb regions of every chromosome. These 20 kb regions included the telomere and the so-called subtelomeric region where genes are thought to be silenced. They found all three Sir proteins at all 32 natural telomeres.

However, the Sir proteins were not uniformly distributed across the telomeres, but rather occupied distinct positions. Typically, all three were in the same position, as would be expected since they form a complex. And they were definitely not in a gradient along the telomere.

Next the researchers asked whether gene expression was truly silenced in that subtelomeric region. They used mRNA-seq to measure gene expression from the ends of chromosomes in wild type or sir2, sir3, or sir4 null mutants.

They found that contrary to expectations, there is actually a lot of transcription going on near telomeres, even in the closest 5 kb region. The levels are lower than in other parts of the genome, but that can be partly explained by the fact that open reading frames are less dense in these regions. And only 6% of genes are silenced in a Sir-dependent manner.

The sensitivity of mRNA-seq allowed Ellahi and colleagues to uncover new patterns of gene expression in this work. They were able to detect very low-level transcription from some of the telomeric repetitive elements. Also, because the SIR genes are involved in mating type regulation, the mRNA-seq data from the sir mutants revealed a whole new set of genes that are differentially expressed in different cell types (haploids of mating types a and α, or a/α diploids).

The researchers point out that their work raises the question of why the cell would use the Sir proteins to repress transcription of a few subtelomeric genes. Wouldn’t it be more straightforward if these genes just had weaker promoters to keep their expression low?

They hypothesize that Sir repression could actually be part of a stress response mechanism, allowing a few important genes to be turned on strongly when needed. This idea could have intriguing implications for the role of Sir family proteins in aging and cancer in larger organisms. 

So, neither the universe nor the ends of our chromosomes are as silent as we thought. But unlike the disappointed SETI researchers, biologists studying everything from yeast to humans can now build on this large quantity of meaningful data from S. cerevisiae telomeres. 

by Maria Costanzo, Ph.D., Senior Biocuration Scientist, SGD

Categories: Research Spotlight

Tags: Saccharomyces cerevisiae, silencing, telomere