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Puzzling Out Gene Expression

February 21, 2013

Have you ever put together a million piece puzzle that was all blue? That is sort of what it sometimes feels like figuring out how genes are turned on or off, up or down.

jigsaw puzzle

There are hundreds or even thousands of proteins called transcription factors (TFs) controlling gene expression. And there is a seemingly simple but frustratingly opaque string of DNA letters dictating which TFs are involved at a particular gene. Figuring out which sets of proteins bind where to control a gene’s expression can be a baffling ordeal.

Up until now most of the ways of identifying which TFs are bound at which genes have been incredibly labor intensive to do on a large scale. With all of the current techniques, researchers need to construct sets of reagents before they even get started. For example, to be able to immunoprecipitate TFs along with the DNA sequences they bind, you need to insert epitope tags in all the TF genes so an antibody can pull them down. Other techniques are just as involved.

What the field needs is a quick and dirty way to find where TFs bind in the genome. And now they just might have one.

In a new study, Mirzaei and coworkers used a modification of the well-known technique mass spectrometry (mass spec) to identify TFs that bind to a specific piece of DNA. With this technique, called selected reaction monitoring, the mass spec looks only for specific peptide sequences. This not only makes it much more sensitive and reproducible than ordinary mass spec, but it should also be relatively straightforward to do if a lab has access to the right sort of mass spec. They haven’t worked out all the bugs and it is definitely still a work in progress, but the technique looks promising.

Mirzaei and coworkers set up assays to detect 464 yeast proteins that are known or suspected to be involved in regulating RNA polymerase II transcription. Then they tested their assay on a 642 base pair piece of DNA known to contain signals that affect the levels of FLO11 transcription. They found fifteen proteins (out of the 222 they searched) that bound this piece of DNA. Of these, only one, Msn1p, had been previously identified as regulating the FLO11 gene. The other fourteen had not been found in any previous assays.

The authors next showed that two of these fourteen proteins, Mot3p and Azf1p, represented real regulators of the FLO11 gene. For example, deletion of MOT3 led to a threefold increase in FLO11 expression under certain conditions. And when AZF1 was deleted, FLO11 could not be activated under a different set of conditions. So Mot3p looks like a repressor of FLO11 and Azf1p looks like an activator.

This was a great proof of principle experiment, but much more work needs to be done before this will become a standard assay in the toolkit of scientists studying gene expression. They need to figure out why some known regulators of FLO11 (Flo8p, Ste12p, and Gcn4p) were missed in the assay and whether the other twelve proteins they discovered play a role in the regulation of the FLO11 gene.

Having said this, it is still important to note that even this early stage model of the assay identified two proteins that scientists did not know controlled FLO11 gene expression. At the very least this is a quick and easy way to quickly identify candidates for gene expression. We may not be able to use it to see the whole picture on the puzzle, but it will at least get us a good start on it.

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