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Stowers Joan Conaway on Using Proteomics to Study Transcriptional Regulatory Complexes

Joan Conaway
Stowers Institute for Medical Research

At A Glance

Name: Joan Conaway

Position: Investigator, Stowers Institute for Medical Research, since 2001.

Background: Interim head, program in molecular and cell biology, Oklahoma Medical Research Foundation, 2000. Assistant member, associate member and member of foundation 1989-2000.

Adjunct professor, department of biochemistry and molecular biology, University of Oklahoma Health Sciences Center, since 1998. Faculty at university since 1991.

PhD in cell biology, Roger Kornberg's laboratory, Stanford University School of Medicine, 1987.

At this week's Seventh International Symposium on Mass Spectrometry in the Health and Life Sciences, held in San Francisco, Joan Conaway, an investigator at the Stowers Institute for Medical Research, gave a talk on studying transcriptional regulatory complexes using a proteomic approach. ProteoMonitor caught up with Conaway before her presentation to find out more about her background and work. Seventh International Symposium on Mass Spectrometry in the Health and Life Sciences, held in San Francisco, Joan Conaway, an investigator at the Stowers Institute for Medical Research, gave a talk on studying transcriptional regulatory complexes using a proteomic approach. ProteoMonitor caught up with Conaway before her presentation to find out more about her background and work.

You're using proteomics to study transcriptional regulation?

Yes, we're a lab that's using proteomics in collaboration with great colleagues here to try to address transcription mechanisms. Our lab has been working on the biochemistry of transcription for a long time. I guess we purified things and identified them before there was proteomics. But now that this technology is here we can try to take advantage of it. We've been interested for a long time in the etymology of transcription.

How long have you been at Stowers?

Stowers is a brand new place — it's been open only since the late fall of 2000. We came here in the summer of 2001.

Before that, we (we meaning my husband and I — we work together very closely) were at a place called the Oklahoma Medical Research Foundation. We started working on transcription when I was a graduate student and my husband was a postdoc. This was back in 1984. At that point, basically what was known about mechanisms of RNA polymerase II transcription was that the enzyme couldn't initiate from a promoter by itself. It needed help from several transcription factors that had been identified. People had shown that there was more than one activity required to be able to initiate transcription, but nobody knew what those were. So we decided that we would see whether we could develop methods for preparation of transcriptionally active fractions from rat liver, because it was a tissue that you could get in large quantities. Up until that time, most people had been working with HeLa cells as a source for fractionation. The problem was we figured you would need probably anywhere from half a kilogram to kilograms of material to be able to purify these activities, which translates into thousands of liters of HeLa cells, which we were not in the position to be able to get at that point.

So we took an old-fashioned approach of purification by complementation of activities, and classical biochemical fractionation methods to resolve and purify the proteins that were required to get Pol II to initiate. Back then was the time when once you had purified an activity and you wanted to find out what it was, you'd send it off for Edman degradation, and then screen libraries using degenerate primers. It was a time-consuming effort. At that point, we really didn't get into the cloning business ourselves very much. As Edman degradation and mass spec methods got better and better, and as expressed sequence tag databases became available, we started to use those approaches, and they've been very powerful.

What got you interested in the basic question of transcription?

Well, when we got started in 1984, it was one of the major unanswered questions in molecular biology or biochemistry — exactly what is the machinery that controls mRNA synthesis in mammalian cells? And really the only way one could tackle that problem at that point was biochemically.

We didn't really get involved in the sequencing or cloning of any of the factors that are needed for the initiation by RNA polymerase II. We purified a number of transcription factors that control elongation by RNA polymerase II — initially in '92. The initial stuff we did was still in collaboration with Bill Lane. We were in Oklahoma and Bill Lane was at Harvard. We had a long-distance collaboration with him for about a decade. We would send things back and forth, and communicated by telephone and email and fax. It as a terrific collaboration.

Then once we moved here to the Stowers Institute, as the technology was improving and seeming to be more accessible to people who were not necessarily mass spectrometrists, the institute here decided that it wanted to invest in proteomics. Initially, we got a Finnigan ion trap machine, learned how to do LC/MS/MS nanospray stuff , with help from Bill Lane who advised us as we were setting all this up. Shortly thereafter, we were successful in recruiting Mike Washburn and Laurence Florens there — a terrific couple who both trained with John Yates. Since they've arrived, we've been collaborating with them to use MudPIT (Multidimensional Protein Identification Technology) approaches to analyze a collection of multiprotein complexes that have roles as coactivators for RNA polymerse II, and complexes that are involved in chromatin remodeling and modifications.

What kind of proteins have you identified?

We've been trying to define the composition of a complex called the mediator complex. The mediator is a huge, multi-subunit complex needed for communication between DNA-binding transcriptional activators and RNA polymerase II. The mediator complex, because of its enormous size, has a tendency to fall apart — it's a very large and fragile complex. The mammalian mediator complex had been difficult to study. What we ultimately wound up doing was generating a whole series of cell lines that express different epitope tag subunits of this complex. Then by combining affinity purification of complexes through epitope tag subunits with MudPIT, it was possible to resolve a lot of confusion about the composition of the subunits of the mediator complex.

Did you already know before what were the subunits of the mediator complex?

The yeast mediator complex had been purified in Roger Kornberg's lab, and they had done a spectacular job of defining that complex biochemically. There were a variety of mammalian-like mediator complexes that had been purified in various labs. A comparison of these mediator-like complexes from different labs left quite ambiguous what the exact composition was, and also whether there was a significant number of different forms of this complex, or whether there was one or a few different forms of the complex.

Is there anything new or unusual that you use in terms of proteomics techniques?

I think the affinity purification is a pretty standard process. There are very few labs using the MudPIT approach for characterization of complexes. It's an incredibly powerful technology developed by Yates' lab.

Most people in our business purify the complex, run it out on a gel, cut out the band, and identify individual polypetides in individual gel slices. You take losses when you try to elute material out of gels. And if you have 30 subunits, that's 30 gel slices you have to cut out.

In the case of MudPIT, one can do a medium/high-throughput analyses. It's feasible to generate hundreds of cell lines that express different tags and analyze them. Whereas if we had to do all of that by cutting bands out of gels, it wouldn't be possible.

What kind of cells are you using for your research?

We're using HeLa cells. We're using various human cell lines.

When you did come up with a picture of the mediator complex, was it different from what you expected?

The data suggested that there were likely a limited number of forms of the complex. I think the most important contribution of that work was, it probably provided a fairly simple picture of this very complicated complex and provided essentially a catalogue of all the subunits of the complex that can be a foundation for future studies of how all those different subunits function together to regulate transcription.

Now parts of the lab are trying to build on that information to try to understand the function of individual subunits of the complex, and how they work in transcription. We also are continuing related types of experiments to define the compositions of a variety of other large, multi-subunit complexes with functions in transcriptional regulation, in particular ones that modulate chromatin structure.

What are you looking to do in the future?

From a proteomics standpoint, we're continuing to try to define multi-protein complexes with roles in transcriptional regulation. We also have a significant interest in the family of ubiquitin ligases and we're trying to see whether we can use similar proteomic approaches to define the repertoire of a particular family of ubiquitin ligases, and to try to use these approaches to define substrates.

Ubiquitin is a small protein that becomes covalently conjugated to other proteins and acts as a sort-of molecular tag, either to target them for degradation by a proteosome, or there's growing evidence that modification by ubiquitination can act essentially like any other post-translational modification to regulate their activities.

On the transcription side, the proteomics information has given us more of a catalogue of complexes that we can identify, and we're now working to both exploit existing biochemical assays and develop new ones for exploring how these complexes work together to control transcription.

Do you see clinical or medical applications to this research?

Yes. There's not an immediate medical or clinical application, but transcription is one of the key control points in cellular regulation. Many genes that, when mutated, give rise to diseases of one kind or another encode proteins with key roles in transcriptional regulation.

For example, one of the proteins that we study in the lab is called ELL. ELL controls the rate of transcription elongation by RNA polymerase II. It was originally discovered by Janet Rowley and her colleagues as a translocation partner with a gene called MLL. And so the translocation of this MLL and ELL gives rise to acute myeloid leukemias. And MLL itself is now known to be a subunit of a histone methyl transferase complex that modulates transcription.

So in that sense, the work that we do in our lab has medical importance in that it illuminates basic biochemical mechanisms that underlie a lot of different disease states, and the hope is that by defining in greater details these fundamental biochemical mechanisms, one will identify additional therapeutic targets.

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