Transcription is the first step in gene expression; the majority of genes are regulated at the levels of transcription initiation and/or elongation. Our research concentrate on analysis of the mechanisms and regulation of initiation and elongation by prokaryotic and eukaryotic RNA polymerases using highly purified in vitro systems and approaches in vivo (described in detail below). This work is currently supported by NIH RO1 and NSF grants.
Since defects in gene regulation are the leading causes of many human cancers, our research has direct implications for medicine. There are also very direct connections between players involved in chromatin remodeling and in carcinogenesis (1, 2). Many enhancer-regulated genes are directly involved into carcinogenesis (3, 4). Numerous human diseases are caused by mutations in various transcript elongation factors (5).

References
1. Schafer, S. & Jung, M. (2005) Arch. Pharm. 338, 347-57.
2. Sims, R. J., 3rd & Reinberg, D. (2004) Nat. Cell. Biol. 6, 685-7.
3. Zhang, Y. & Gordon, G. B. (2004) Mol. Cancer Ther. 3, 885-93.
4. Lincoln, D. W., 2nd & Bove, K. (2005) Front. Biosci. 10, 506-11.
5. Conaway, J. W. & Conaway, R. C. (1999) Annu. Rev. Biochem. 68, 301-19.

I. TRANSCRIPTION IN CHROMATIN
All eukaryotic DNA is tightly condensed within small nuclei in nucleoprotein complexes called chromatin. The nucleosome represents the first level of DNA compaction; the nucleosome consists of the histone octamer which organize ~150 bp DNA in one and 2/3 superhelical coils on the surface of the octamer. It has become clear that chromatin is an ultimate player in eukaryotic transcription and therefore mechanisms involved in eukaryotic transcription and its regulation should be analyzed in context of properly assembled chromatin. During the last few years, we made several fundamental and novel discoveries in this direction:

1. Using RNA polymerase III (Pol III) and short nucleosomal templates as a simple experimental model, we have discovered that during transcription through the nucleosome the histone octamer never leaves the DNA, but rather steps around the polymerase through formation of an intranucleosomal DNA loop [1,2]. We have found that the DNA loop (but not DNA-histone interactions themselves) constitutes the major part of the nucleosomal barrier to transcription [3]. Structures of the transfer and pausing intermediates were identified using biochemical techniques and cryo electron microscopy [4]. Pol III and ATP-dependent chromatin remodelers use very similar mechanisms for chromatin remodeling [5]. Currently the mechanism of chromatin remodeling by Pol III is actively studied in the lab.

2. Using a novel experimental system [6] supporting transcript elongation in a solid phase (immobilized Pol II) we have discovered a novel mechanism of pol II transcription through chromatin [7-9] remodeling of chromatin that could be involved in epigenetic inheritance [5,10]. This experimental system allowed us to evaluate the mechanism of action of protein complex FACT that can facilitate chromatin transcription [11] and the role of histone variants in transcription through chromatin [12-14]. The nature of the high regulated nucleosomal barrier to transcription and possible ways of its regulation by Pol II was also analyzed [9].
These data allowed us to propose possible mechanisms operating during transcription of nucleosomal templates in vivo [5,10].

References
1. Studitsky, V.M., Clark, D.J. and Felsenfeld, G. (1994) A histone octamer can step around a transcribing polymerase without leaving the template. Cell 76, 371-82.
2. Studitsky, V.M., Kassavetis, G.A., Geiduschek, E.P. and Felsenfeld, G. (1997) Mechanism of transcription through the nucleosome by eukaryotic RNA polymerase. Science 278, 1960-3. [pdf]
3. Studitsky, V.M., Clark, D.J. and Felsenfeld, G. (1995) Overcoming a nucleosomal barrier to transcription. Cell 83, 19-27. [pdf]
4. Bednar, J., Studitsky, V.M., Grigoryev, S.A., Felsenfeld, G. and Woodcock, C.L. (1999) The nature of the nucleosomal barrier to transcription: direct observation of paused intermediates by electron cryomicroscopy. Mol. Cell 4, 377-86. [pdf]
5. Studitsky, V.M., Walter, W., Kireeva, M., Kashlev, M. and Felsenfeld, G. (2004) Chromatin remodeling by RNA polymerases. Trends Biochem. Sci. 29, 127-35. [pdf]
6. Walter, W., Kireeva, M.L., Tchernajenko, V., Kashlev, M. and Studitsky, V.M. (2003) Assay of the fate of the nucleosome during transcription by RNA polymerase II. Methods Enzymol. 371, 564-77.
7. Kireeva, M.L., Walter, W., Tchernajenko, V., Bondarenko, V., Kashlev, M. and Studitsky, V.M. (2002) Nucleosome remodeling induced by RNA polymerase II. Loss of the H2A/H2B dimer during transcription. Mol. Cell 9, 541-52. [pdf]
8. Liu, Y.V., Clark, D.J., Tchernajenko, V., Dahmus, M.E. and Studitsky, V.M. (2003) Role of C-terminal domain phosphorylation in RNA polymerase II transcription through the nucleosome. Biopolymers 68, 528-38. [pdf]
9. Kireeva, M.L., Hancock, B., Cremona, G.H., Walter, W., Studitsky, V.M. and Kashlev, M. (2005) Nature of the nucleosomal barrier to RNA polymerase II. Mol. Cell 18, 97-108. [pdf]
10. Studitsky, V.M. (2005) Chromatin remodeling by RNA polymerase II. Mol. Biol. 39, 639-54. [pdf]
11. Belotserkovskaya, R., Oh, S., Bondarenko, V.A., Orphanides, G., Studitsky, V.M. and Reinberg, D. (2003) FACT facilitates transcription-dependent nucleosome alteration. Science 301, 1090-3. [pdf]
12. Angelov, D., Verdel, A., An, W., Bondarenko, V., Hans, F., Doyen, C.M., Studitsky, V.M., Hamiche, A., Roeder, R.G., Bouvet, P. and Dimitrov, S. (2004) SWI/SNF remodeling and p300-dependent transcription of histone variant H2ABbd nucleosomal arrays. EMBO J. 23, 3815-24. [pdf]
13. Angelov, D., Bondarenko, V.A., Almagro, S., Menoni, H., Mongelard, F., Hans, F., Mietton, F., Studitsky, V.M., Hamiche, A., Dimitrov, S. and Bouvet, P. (2006) Nucleolin is a histone chaperone with FACT-like activity and assists remodeling of nucleosomes. EMBO J. 25, 1669-79. [pdf]
14. Doyen, C.M., An, W., Angelov, D., Bondarenko, V., Mietton, F., Studitsky, V.M., Hamiche, A., Roeder, R.G., Bouvet, P. and Dimitrov, S. (2006) Mechanism of polymerase II transcription repression by the histone variant macroH2A. Mol. Cell. Biol. 26, 1156-64. [pdf]

II. MECHANISM OF ENHANCER AND INSULATOR ACTION
Enhancers are regulatory DNA sequences, controlling gene expression when positioned over a distance up to 60 kb from regulated promoters [1]. I have proposed that the mechanisms of enhancer action over a short and over a long distance are different [2]. Recently we have developed novel experimental techniques and very efficient experimental system in vitro for analysis of the mechanism of enhancer action [3].

Using these approaches, we have shown that indeed transcriptional enhancers use a different mechanism for action over a large distance. DNA supercoiling was identified as a primary factor responsible for efficient enhancer-promoter communication [4-6]. DNA supercoiling brings enhancer and promoter together and thus eliminates a problem of communication over a large distance [4,7]. We have also eliminated all alternative models for enhancer action proposed previously [5].

Currently we are investigating how DNA supercoiling facilitates enhancer-promoter communication and whether similar mechanism of enhancer action operates in vivo and in chromatin environment in vitro.

Insulators are recently discovered DNA sequences that form the borders functional units of gene regulation (chromatin domains) in eukaryotes. In particular, they can block enhancer action over a large distance. The mechanism of insulator action is unknown. Recently we have recapitulated insulator activity in an enhancer-dependent system in vitro using a bacterial sequence-specific DNA-binding/looping protein [7]. Surprisingly, this rationally designed, inducible regulatory element recapitulates many properties characteristic for insulators in vivo [7]. The mechanism of inhibition of enhancer-dependent transcription by this novel regulatory element is currently analyzed.

Finally, new approaches for analysis of communication between distantly positioned regulatory sequences on DNA organized into chromatin have been developed. The effects of histone modifications and changes in overall structure of the 30 nm chromatin fiber on communication are currently under analysis.

References
1. Bondarenko, V.A., Liu, Y.V., Jiang, Y.I. and Studitsky, V.M. (2003) Communication over a large distance: enhancers and insulators. Biochem. Cell. Biol. 81, 241-51. [pdf]
2. Studitsky, V.M. (1991) Allosteric mechanism of enhancer action? FEBS Lett. 280, 5-7 [pdf]
3. Bondarenko, V., Liu, Y.V., Ninfa, A.J. and Studitsky, V.M. (2003) Assay of prokaryotic enhancer activity over a distance in vitro. Methods Enzymol. 370, 324-37.
4. Liu, Y., Bondarenko, V., Ninfa, A. and Studitsky, V.M. (2001) DNA supercoiling allows enhancer action over a large distance. Proc. Natl. Acad. Sci. U.S.A. 98, 14883-8. [pdf]
5. Bondarenko, V., Liu, Y., Ninfa, A. and Studitsky, V.M. (2002) Action of prokaryotic enhancer over a distance does not require continued presence of promoter-bound sigma54 subunit. Nucleic Acids Res. 30, 636-42. [pdf]
6. Atkinson, M.R., Blauwkamp, T.A., Bondarenko, V., Studitsky, V. and Ninfa, A.J. (2002) Activation of the glnA, glnK, and nac promoters as Escherichia coli undergoes the transition from nitrogen excess growth to nitrogen starvation. J. Bacteriol. 184, 5358-63. [pdf]
7. Bondarenko, V.A., Jiang, Y.I. and Studitsky, V.M. (2003) Rationally designed insulator-like elements can block enhancer action in vitro. EMBO J. 22, 4728-37. [pdf]

III. TOPOISOMERASES, TRANSCRIPTION AND ANTI-CANCER DRUGS
DNA topoisomerases are nuclear enzymes regulating the topology of DNA by introducing transient breaks in DNA strands (1). Eukaryotic topoisomerases are primary targets for numerous highly potent anti-cancer drugs (2). Recently we have recapitulated several important aspects of topo-targeted drugs action in vitro (manuscript in preparation) and currently are actively involved in analysis of the mechanism of this process.

References
1. Corbett, K. D., and Berger, J. M. 2004. Annu. Rev. Biophys. Biomol. Struct. 33, 95-118.
2. Li, T. K., and Liu, L. F. 2001. Annu. Rev. Pharmacol. Toxicol. 41, 53-77.

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