How I found the silver lining in my cloud.

I still remember that day very vividly. It was just a routine experiment where I was checking the binding of silencing proteins at the silent loci in a number of yeast mutants. Being the control freak, I also checked for Sir1, a protein that was expected to bind only at the silencing initiation sites through its interactions with the replication complex ORC, but not through out the silenced domains.  To my surprise, Sir1 bound just like the other silencing proteins.  Having my share of premature excitements about new findings, I repeated the experiment multiple times and got the same result each time. Not only did this silencing protein, but its recruiter ORC complex bound the entire silenced region as well. 

For these experiments, I used a well established technique for showing protein-chromatin associations, named Chromatin Immuno-Precipation (ChIP). The same technique was used over and over in our lab and others to show the binding and spreading of the silencing proteins. This technique involves a step where chromatin is physically sheared and it was widely assumed that the shearing was uniform at different kinds of chromatin. However, a simple southern experiment that I did as a control (I told you I was a control freak, didn't I?) showed this was not the case. The heterochromatin was more resistant to shearing, resulting in larger fragments and in return giving the illusion that the studied protein was binding across a wider region. I think that was the day I couldn't hold onto my tears, feeling devastated that all the data I have collected over the last 3 years was just an artifact of the method I was using. 

As it was usually the case, my boss, Jasper, took me out of the darkness. He said this would make a great story and change the interpretation of many experiments in the literature that used this very same technique. Being the great scientist he is, he also noticed that the signal we got was more significant than the shearing artifact could contribute. Luckily we showed that when the silenced region was decoupled from the silencers using an in-vivo recombinase, ORC still bound through the silenced region. So with this work, not only did we unveil an artifact in a widely used technique, but also showed that ORC could bind regions that are further away from its binding site in a silencing dependent fashion. 

My take home message from this work though was that every cloud has a silver lining, and I should try to see that after I wipe away my tears...

Copyright: © 2013 Bilge Ozaydin. The above content is licensed under the Creative Commons Attribution License (CC-BY), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.