April 24, 2012
A new study by Lickwar and coworkers suggests that many transcription factors fidget on and off the DNA, waiting for some signal to get to work. Once they get that signal, they clamp down and start affecting the activity of nearby genes.
If true this would help explain some perplexing results researchers have been getting with chromosomal immunoprecipitation (ChIP) assays. Transcription factors appear to be bound at many places where they are not affecting any nearby genes. Now we might have an idea why.
These researchers came up with this model through the use of an elegant, in vivo competition study. What they did was to set up a yeast strain that contained two different versions of the transcription factor Rap1p. One version was tagged with a FLAG epitope and was under the control of RAP1’s endogenous, constitutive promoter. The other version was tagged with a Myc epitope and was under the control of an inducible promoter.
They started out seeing where Rap1p was bound in the absence of the inducer by using an antibody against FLAG. This is the equivalent of a typical ChIP experiment. They found Rap1p was bound in many places throughout the genome including sites where it did not appear to affect any nearby genes.
Then they added the inducer galactose and at various time points repeated the ChIP experiment with antibodies against either FLAG or Myc. They were basically looking for how quickly the Myc-tagged Rap1p replaced the FLAG-tagged Rap1p with the idea that less stably bound transcription factors would be replaced more quickly.
They indeed found that some sites were better able to withstand the onslaught of Myc-tagged Rap1p. And more importantly, that these sites were near genes most influenced by Rap1p. In other words it appears that the more stably bound the Rap1p, the bigger the effect it has on nearby genes.
They then went on to show that more stable binding correlated with lower nucleosome occupancy and stronger in vitro binding. From this data they propose a model where the level of the effect on transcription is the result of a competition between nucleosome and transcription factor binding. Stronger transcription factor binding keeps nucleosomes away so transcription can proceed.
They took the model one step further and proposed that transcription factors are idling on the DNA, waiting for a signal to bind more tightly and influence the activity of nearby genes. In other words, transcription factors are ready to have an effect at a moment’s notice.
This part of the model still has to be proven though. All that has been shown so far is that a slow off rate is required for effective transcription activation by Rap1p. What we don’t know is whether this translates to other transcription factors or if idling Rap1p is ever more stably bound.
by D. Barry Starr, Ph.D., Director of Outreach Activities, Stanford Genetics
Categories: Research Spotlight
Tags: ChIP, chromosomal immunoprecipitation, competition, off rate, stable binding, transcription factor, yeast