University of Arkansas
Name: Alan Tackett
Position: assistant professor, University of Arkansas for medical sciences, department of biochemistry and molecular biology, 2005-present.
Background: PhD, University of Arkansas for medical sciences, 2002; postdoc at The Rockefeller University, 2002-2005.
Tackett was co-correspondent with David Allis at The Rockefeller University on research that identified a member of a protein complex that appeared to “read” how other proteins are modified and then react by “writing” other modifications.
According to Tackett, this finding, published in the Dec. 8 issue of Molecular Cell, is among the first proof that proteins can read and write a “protein code.”
As part of his work, Tackett used I-DIRT, or Isotopic Differentiation of Interactions as Random or Targeted, a method he and colleagues at The Rockefeller University developed last year to distinguish contaminants from bona fide interactors in immunopurifications.
ProteoMonitor spoke with Tackett this week about the research into protein-histone interactions.
Tell me about your work.
Basically, what we did was we used some new proteomic technology that we published about a year ago called I-DIRT to identify all the in vivo members of a protein complex, and furthermore, to show that this protein complex has the ability to read and write histone modifications. And that’s important because histone modifications drive cellular events like transcription. And there’s a whole idea of a histone code that’s been proposed in the last few years where certain proteins in theory should be able to read these modifications and then induce some type of function in the cell.
What we found was actually one of the first examples of a protein complex that has the ability to, first, read one of these modifications on a histone, and then write an additional modification which induces a cellular event, in this case transcription. So this is really one of the initial examples of a protein complex that can read and write a histone code.
This has been a hypothesis and there’s been a lot of evidence in support of this, but it’s still really a working hypothesis ultimately.
What have been the challenges in trying to prove this hypothesis? Has there been a lot of work done on this?
There’s been a lot of work put into this the last few years. In terms of what we’re interested in — the mass spectrometry behind it — the instrumentation has just become advanced enough in the last few years to be able to study these histone modifications, and more importantly, multiple modifications on a single peptide.
We had two major uses. First of all, we used this technique, I-DIRT. It basically relies on the ability to measure isotopes in proteins and to determine which proteins are real interacting members of a complex. That was kind of our first step, that we had an idea that this protein complex would be involved in this reading and writing [of] the histone code. And the first thing that we wanted to do was to find all the cellular components of this protein complex. So, mass spectrometry and proteomics came into play there.
Second of all, we used a chemical labeling strategy combined with mass spectrometry to determine exactly where this protein complex put on its second modification, the writing part of the histone code.
You were able to see a protein Yng1 interacting with a histone, H3. What did you find?
The whole idea was this protein Yng1 has a particular region, or domain of that protein, that binds to a specific modification on histone H3. And that modification is a trimethylation of lysine 4. That’s the reading component of this. The writing component is once this Yng1 protein binds, it also brings in interacting proteins, and these proteins are part of a protein complex called an acetyltransferase. What it does is it then writes more of this histone code by putting an acetylation on lysine position 14.
Was that the only instance of protein-histone interaction you saw, or were there others?
In this work, this was the only evidence of this. It was this component, this Yng1 component, of a multi-component complex that actually bound to the trimethylation. And then another component of the complex put on the acetylation.
Why has it been so difficult to see this kind of interaction?
It’s just been recently that the domain, or the region of Yng1 that binds to these modifications, [was] described to bind in this modification. So once that information was found, we first confirmed that it does bind, and then we determined that it puts on this acetylation writing.
There was this domain or part of Yng1 that folds in a particular manner. And nobody really knew what it did, but that information came out a few months ago. With that information we were able to hypothesize that if it binds, this member of this protein complex which puts on acetylation, likely acetylates something near by. And we just found what it does acetylate.
Is this novel or are you verifying the findings of someone else’s work?
This is novel in [these] respect[s]: … it’s the first application of this I-DIRT technology. And second of all, it really is one of the first examples of a protein complex that can read and write a histone code. Now, there have been examples of the ability to read or to write, but direct evidence of reading and writing, this is really one of the first cases for that. So it gives us more confidence that the histone code [hypothesis] is correct.
Even if we get to the stage where the histone code is verified, deciphering these codes would be a massive undertaking.
Yes, it would. It’s a very combinatorial code, and that really is one of the challenges because depending on where these histones are in a chromosome, they’ll be modified in a different manner. And there are multiple modification sites on each one, so it becomes a very combinatorial problem.
In your research were you able to decipher the code of this Yng1-H3 interaction?
Well, what we were able to do is, I’d like to call it deciphering maybe at a local code. What it is, is this Yng1 protein, what it helps to do is regulate transcription of a certain set of genes. And at those regions we were able to at least partially define the local code so there has to be this trimethylation, followed by this acetylation before gene transcription can happen. But on the larger scale, this was not defining this histone code. That would be a very tremendous undertaking.
What we’re doing now is looking at other members of this Yng1 containing protein complex, and determining if they also help recognize histone modifications. And then we’re also following up on similar types of protein complexes on how they bind to certain histone modifications and then induce some type of cellular output.
Are you looking for something specific, a specific type of interaction, or whether they interact?
Yes, on both. What we really want to do [is] find the proteins that bind certain modifications that drive gene transcription in some type of [disease state]. You have transcription occurring when you don’t want it to occur. Well, maybe you can then come back and try to prevent this specific protein posttranslational modification interaction to prevent transcription of that gene which might be causing a disease state. So that’s kind of the ultimate goal.
[What we’re doing now] is really more of interactions that are for general transcriptions. This is kind of like setting the groundwork before you move into that direction.