December 07, 2016
For an election to go smoothly, people cannot stay too long in the voting booth. If a lot of people stayed in the booth and answered emails, sent texts, etc., after they finished voting, then the whole process would grind to a halt.
There is some evidence that activating genes may work similarly. The transcription factors (TFs) that bind DNA and turn up the expression of nearby genes can’t stay too long. If they do, the activation starts to peter out.
What is thought to happen with these sorts of TFs is that they bind their preferred DNA, and then once they have attracted the cellular machinery needed to read the gene, they are targeted for destruction. Then a new TF can bind and repeat the process.
In a new study in GENETICS, Akhter and Rosonina set out to investigate the process by which the yeast transcription activator Gcn4p is removed after it has bound DNA and done its job. Gcn4p activates a number of genes in response to amino acid starvation.
They found that a key step in the process is the addition of SUMO proteins to DNA-bound Gcn4p, which gets the ball rolling on the destruction of Gcn4p. Imagine a sumo wrestler settling in next to a voter once he enters the booth and then throwing him out if he tarries too long.
Their model is that once Gcn4p binds DNA, it is sumoylated. Then the DNA-bound, sumoylated GCN4 is further modified by kinases like Cdk8p, a component of the mediator complex which acts as a bridge between TFs and the cellular machinery responsible for reading a gene. This modified TF is then sent off to the 26S proteasome where it is degraded making room for an unmodified Gcn4p.
Previous research had shown that sumoylation of GCN4 required DNA binding. The first thing these authors did in this study was to determine if Gcn4p had to bind to its target DNA sequence in order to be sumoylated. It did not.
When they fused a mutant Gcn4p that could not bind DNA to the DNA binding domain of Gal4p, they found that this molecule was sumoylated at the correct places on the Gcn4p part of the fusion protein, lysines 50 and 58, when bound to a Gal4p binding site. Therefore, Gcn4p does not need to occupy its own DNA binding site in order to be sumoylated.
Another set of experiments showed that while DNA binding was required for sumoylation, interaction with RNA polymerase II (RNAP II), the enzyme that reads the genes that Gcn4p activates, does not appear to be necessary. For one of these experiments they used a temperature sensitive mutant of the largest subunit of RNAP II, Rpb1p, and showed that even at higher temperatures when RNAP II is inactive in these cells, DNA-bound Gcn4p is still sumoylated. In the other experiment they showed that DNA-bound
Gcn4p was still sumoylated when they used the “anchor away” technique to drag Rpb1p out of the nucleus and into the cytoplasm.
So DNA binding is sufficient, and the specific site is not important. And Gcn4p doesn’t have to be activated in order to be sumoylated.
Of course, turnover like this is a delicate thing. If Gcn4p is pulled off too soon, then it can’t activate as much as it might otherwise be able to do. This might affect the cell’s response to starvation just as much as Gcn4p staying put too long. Sort of like the sumo wrestler throwing a voter out of the voting booth before they could finish their voting can muck up the election.
Akhter and Rosonina created a fusion protein of Gcn4p and the yeast SUMO peptide Smt3p. Unlike Gcn4p, this protein is sumoylated before it binds DNA.
They found that yeast expressing this fusion protein fared less well under starvation conditions compared to yeast cells that expressed the wild type version of GCN4. And using chromatin immunoprecipitation (ChIP) analysis they showed that at least at the ARG1 gene, this was because there was less of the fusion protein bound under activating conditions.
So cells need for TFs to stay at the right place for the right amount of time. If they are pulled off too early or stay too long, the levels of activation can fall below what is best for the cells.
Unfortunately, we don’t have time to go over other experiments that tease out which kinases are important and when, but I urge you to read about them for yourselves. They take full advantage of the genetic tools available in yeast to make this sort of study possible…#APOYG!
Integrating all of this gives the following model:
Gcn4p is only a dimer when bound to DNA and this dimerization may be the signal for sumoylation by Ubc9p. A preinitiation complex forms through its interaction with the DNA-bound, sumoylated Gcn4p which brings in the enzyme RNAP II to transcribe the gene. Once the polymerase has left the nest, the kinase Cdk8p comes in and phosphorylates Gcn4p which signals Cdc4p/Cdc34p to ubiquitinate Gcn4p. The ubiquitinated Gcn4p is then degraded by the 26S proteasome opening the upstream activator sequence (UAS) up to a fresh, new Gcn4p.
Here, with the help of our super hero Saccharomyces cerevisiae, Akhter and Rosonina have dissected out what happens to a transcription factor once it binds to DNA (at least ones that bind for short times). It will be fascinating to see if this translates to other TFs in other beasts. While I love yeast for all it can do for us for bread, wine, beer, human health, helping solve world problems like climate change, and so on, I think my favorite use is still that it allows us to better understand the basic biology of how our cells work.
by Barry Starr, Ph.D., Director of Outreach Activities, Stanford Genetics
Categories: Research Spotlight
Tags: Cdk8 , Gcn4 , gene activation , sumoylation , transcription