Spotlight

Mikhail Kashlev, Ph.D.
Head, Molecular Mechanisms of Transcription Section
Gene Regulation and Chromosome Biology Laboratory

Spotlight Archive

“Just imagine” seems to be Dr. Mikhail Kashlev’s favorite expression, often said with awe and enthusiasm as he contemplates taking a well-known premise one step further or finding a simpler, more efficient way to do something. Dr. Kashlev and his staff have begun to more precisely identify transcription elongation factors through studies of TFIIS (a transcript cleavage factor that rescues elongation complexes arrested within the genes), RSC (a 15-protein complex), and FACT (the nucleosome chaperone).

Imagine: A Simple System for a Complex Study
Imagining inspired Dr. Kashlev to replicate in vitro in a simple E. coli system, the transcription factors that he and other researchers had seen in vivo.

A simple bacterial model has many advantages. It has only one RNA polymerase, responsible for all transcription in the cell. Polymerase can be purified from bacteria cells in just half a day, compared to 2 to 3 days for eukaryotic yeast cells. “Quantity of the protein is also incomparable—from E. coli you can get large amounts of polymerase. From eukaryotic cells, it is much less,” Dr. Kashlev noted. “Besides being quick, E. coli has a sophisticated genetic system for selection and analysis of mutations in RNA polymerase and associated factors. Finally, there are only a few transcription initiation and elongation factors in bacteria, compared to eukaryotes. That’s why I considered this E. coli polymerase as a good starting point for our biochemistry and genetics experiments; we could then use this knowledge to move to address similar questions in RNA polymerase II,” he said.

Using E. coli RNA polymerase as a starting point, Dr. Kashlev focuses his research on more complex eukaryotic transcription machinery. Researchers know that when eukaryotic polymerase starts transcription in vivo, the compact gene structure, known as chromatin, unfolds, permitting polymerase to go through, and then closes behind polymerase in seconds. Chromatin is made from the nucleosomes and associated factors. “Imagine,” he said, “how difficult that must be for RNA polymerase to transcribe through the compacted DNA and get the structure back to the compact form after transcription. There is a mechanism, not very well understood, in the cell: imagine this—forget high-ordered structures; go to the nucleosomal level where DNA’s wrapped tightly around the protein—somehow the polymerase can easily open it up, go through it, and then get it to close up again.”

Dr. Kashlev’s laboratory includes Dr. Maria Kireeva, who works on the FACT chaperone, RSC, and elongation factors for eukaryotic RNA polymerase; Yury Purtov, who also studies the FACT chaperone; and Drs. Lucyna Lubkowska and Vitaly Chasov, who research E. coli RNA polymerase and factors. Dr. Kashlev focuses on quantum dots. He thinks it may be more effective to combine two drugs that each inhibit more than one kinase; some companies are already moving in this direction.

The group is interested in what determines the efficiency of transcription through chromatin. Recent research indicates that many genes in eukaryotes are regulated at levels by proteins affecting the rate of transcription elongation rather than initiation. Two groups of proteins have been identified in this mechanism: one improving the catalytic activity of Pol II (elongation factors) and, another, affecting “transparency” of nucleosomes to transcription (histone modification and chromatin remodeling enzymes). Acetylation, phosphorylation, and methylation of the histones and ATP-dependent chromatin remodeling are universal components of gene regulation.

Large protein ensembles, known as chromatin remodeling complexes, and histone chaperones alter nucleosomes or their higher order arrays to make chromatin more accessible to transcription. RNA polymerase itself and transcription elongation factors such as TFIIF, TFIIS, and elongin may also work to establish and maintain the “opened” state of the chromatin. Thus, transcription elongation factors and chromatin modifiers/re-modelers are considered promising targets for new anti-cancer drugs, which may help to re-gain transcription control of growth-promoting genes, as well as to overcome silencing of genes with the tumor suppressor functions.

Dr. Kashlev’s group’s primary goal is to establish an in vitro system for dissection of the chromatin remodeling mechanisms associated with transcription elongation and investigation of a “crosstalk” between transcription elongation factors and chromatin re-modelers.

Stripping down the yeast system and gradually adding one protein factor at a time gave them some remarkable insights. First, they determined that TFIIS was “absolutely crucial” to transcription in the nucleosome, Dr. Kashlev said. Without it, transcription arrest occurs. However, they also noted that “even in the presence of TFIIS transcription from the nucleosome in vitro was very slow,” he said.

So they began testing other proteins in their in vitro system. “We were able to increase the efficiency, which hadn’t been done before we began this work,” Dr. Kashlev noted. From the family of nucleosome remodeling complexes, they identified another factor: RSC (pronounced “risk”), a large protein complex with 15 proteins. “The combination of these two factors—TFIIS and RSC—makes transcription through the nucleosome highly efficient,” he said.

“We also want to know what helps to rebuild the nucleosome,” Dr. Kashlev continued. “Polymerase has a direct role, although we don’t know exactly what it is yet; another group of proteins helps polymerase to do its job more effectively: FACT, a nucleosome chaperone, helps to reassemble nucleosomes. FACT proteins bind to elongating polymerase, which moves and transcribes the gene. We believe that this protein is a part of the mechanism of nucleosome rebuilding behind polymerase,” he explained. FACT may also help to transcribe chromatin by unfolding nucleosomes in front of RNA polymerase.

In the case of cancer, this opening and closing could be a negative event. “Just imagine that the process is unbalanced in the cell, which is what usually happens with genes which are expressed at a high level, or up-regulated, in the tumor cell. Usually the compact structure of DNA in nucleosomes represses the genes. When this structure is stably formed on the gene, it is not transcribed or expressed. But imagine polymerase moving in this structure, and this structure’s not closed behind it. Now the entire gene is opened and overexpressed or elevated, and we get the wrong protein in the wrong time,” he said. “Some proteins help to open up nucleosomes in front of polymerase, and some proteins help to close the nucleosomes behind, so they’re like a moving factory. Polymerase has its own role in that, but the major role in opening up nucleosomes of DNA and closing it behind belongs to the proteins which attach to the polymerase,” he continued.

Although Dr. Kashlev’s group has been studying the way transcription encounters a single nucleosome, next they will move multi-nucleosomal templates into the system. “We’ll look for multi-nucleosomes—more than a single particle sitting in front of polymerase,” Dr. Kashlev said.

Nanobiochemistry: A Quantum Leap

The second major project, which started only about a year ago, is a collaboration with Dr. Amos Oppenheim’s group at the Hebrew University in Jerusalem, Israel, in nanobiochemistry to monitor basic biochemical processes in the fluorescent microscope. Dr. Kashlev is writing a patent on this, and received a 2006 Directors’ Intramural Innovation Award in February from NIH for the project.

Because each biochemical reaction involves billions of protein and nucleic acid molecules, studying this transcription in vitro takes enormous numbers of individual molecules, and the responses are usually averaged. However, once again using that word “imagine…” Dr. Kashlev and his team searched for an efficient, economical way to study individual molecules and small populations of molecules for these same reactions. The result? Quantum dots.

“Imagine,” Dr. Kashlev said, “if you label all the components—polymerase, TFIIS, RSC, FACT, nucleosome—with very bright fluorophores, special nanometer-sized molecules that emit light and are called quantum dots; you can attach small nanoparticles to your separate molecules in vitro, in a bulk biochemical reaction. Now each molecule which has a fluorophore with a nanoparticle attached to it, can be seen in the fluorescent microscope with a millisecond resolution.”

Since biological molecules in solution are constantly moving, by adding hexa-histidine tags to the molecules and immobilizing the complexes, the researchers were quickly able to get the results they needed. “It took almost a year to develop this system, but now, in fifteen minutes, you can see your labeled protein in the microscope. You look at a very small section of your slide, but your entire field will have 100,000 molecules. Imagine you want to know how the polymerase binds to elongation factors. You can look for intensity, zoom in to look for the individual level of each molecule; zoom out, look for a piece of the intensity of light, which indicates that more and more molecules of polymerase have arrived at the cluster. You can see which cluster is first, second, what is the rate, et cetera; and after everything comes to a steady state, you can start a second part of the experiment, looking for dissociation constants for each individual cluster. That’s why this method has a huge potential to be automated, to put many different types of molecules in clusters. You can collect an enormous amount of information,” he said.

Dr. Kashlev earned his PhD at the Moscow Institute of Molecular Genetics in 1990, and was a postdoctoral fellow at Columbia University, then a research associate at Public Health Research Institute, and came to NCI-Frederick in 1996 to found the Molecular Mechanisms of Transcription Section. The staff includes four researchers; in addition, frequent visitors and several visiting scientists often work there, as he currently has collaborations with 10 labs across the country and in Jerusalem.

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