Faculty Summaries
Vasily M. Studitsky, PhD
Vasily M. Studitsky, PhD
Professor
Vasily.Studitsky@fccc.edu
Office Phone: 215-728-7014
Office: W209
  • Unraveling the Nucleosomal Cycle: Mechanism of Histone Survival During Pol II Transcription

    Han-Wen Chang , Artem Demidenko & Olga Studitskaia (Kulaeva)
    Nucleosomal Cycle
    “Nucleosomal Cycle”: Mechanism of Histone Survival during Pol II Transcription

    In 2002, we have discovered a novel, Pol II-specific pathway of transcription through chromatin (1) that is now referred to as a “nucleosomal cycle”. Nucleosome forms a strong barrier to transcribing Pol II (2); as Pol II overcomes the barrier, it proceeds through the nucleosomal cycle. The nucleosomal cycle involves multiple partial and reversible uncoiling of nucleosomal DNA from the octamer behind and in front of the enzyme (Fig. 1 (3)); as a result, histones remain constantly bound with DNA (4) and nucleosomes survive transcription, remaining at the original location after this process (1). Thus during transcription by Pol II only H2A/H2B histones are exchanged; histones H3/H4 carrying specific covalent modifications survive transcription. Since the entire eukaryotic genome is transcribed at a certain frequency, the Pol II-type mechanism is likely to be important for maintaining the histone marks across the genome (3).

    The rate of transcription through chromatin by pol II and the fate of nucleosomes are largely determined by the structures of the critical intermediates formed during this process and by the rates of interconversion between them (3,5,6). Our current goal is to describe the “nucleosomal cycle” in terms of the changes in DNA-histone interactions during transcription through chromatin by Pol II and the rates of interconversion between the intermediates. Dynamics of transcription through chromatin and structures of specific complexes will be analyzed using time-resolved footprinting, FRET, single-particle (sp) FRET, electron cryo-microscopy (ECM), structural modeling and computational analysis.

    References

    1. Kireeva, M. L., Walter, W., Tchernajenko, V., Bondarenko, V., Kashlev, M., and Studitsky, V. M. (2002) Mol. Cell 9, 541-552
    2. Bondarenko, V. A., Steele, L. M., Ujvari, A., Gaykalova, D. A., Kulaeva, O. I., Polikanov, Y. S., Luse, D. S., and Studitsky, V. M. (2006) Mol. Cell 24, 469-479
    3. Kulaeva, O. I., Gaykalova, D. A., Pestov, N. A., Golovastov, V. V., Vassylyev, D. G., Artsimovitch, I., and Studitsky, V. M. (2009) Nat. Struct. Mol. Biol. 16, 1272-1278
    4. Kulaeva, O. I., and Studitsky, V. M. (2010) Transcr. 1, 85-88
    5. Kulaeva, O. I., Hsieh, F. K., and Studitsky, V. M. (2010) Proc. Natl. Acad. Sci. USA 107, 11325-11330
    6. Hsieh, F. K., Fisher, M., Ujvari, A., Studitsky, V. M., and Luse, D. S. (2010) EMBO Rep. 11, 705-710
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  • Role of histone chaperones and other proteins during transcription through chromatin

    Artem Demidenko & Olga Studitskaia (Kulaeva)
    cell differentiation and carcinogenesis
    FACT is a regulatory hub in cell differentiation and carcinogenesis

    Our “minimal” experimental system in vitro (1) recapitulates qualitative aspects of Pol II transcription through chromatin (2). However in the “minimal” system a nucleosome presents a high barrier to transcribing enzyme and nucleosome survival is only 50-90% efficient;  these observations suggest that other factors may facilitate transcription through chromatin and nucleosome survival in vivo (3). Over the years, we have identified multiple protein factors that facilitate transcription through chromatin in vitro: chromatin remodelers (4), histone chaperones FACT (Fig. 2) and nucleoline (5-7), and elongation factor TFIIS (8). Recently we have identified several other proteins that facilitate transcription through chromatin and nucleosome survival in vitro (unpublished). The goal of the current projects is to determine the mechanisms of action of these protein factors during transcription through chromatin in terms of their effects on the structures of the intermediates and the rate constants.

    References

    1. Gaykalova, D. A., Kulaeva, O. I., Pestov, N. A., Hsieh, F. K., and Studitsky, V. M. (2012) Methods Enzymol. 512, 293-314
    2. Kireeva, M. L., Walter, W., Tchernajenko, V., Bondarenko, V., Kashlev, M., and Studitsky, V. M. (2002) Mol. Cell 9, 541-552
    3. Kulaeva, O. I., Hsieh, F. K., Chang, H. W., Luse, D. S., and Studitsky, V. M. (2013) Biochim Biophys Acta, in press.
    4. Gaykalova, D. A., Nagarajavel, V., Bondarenko, V. A., Bartholomew, B., Clark, D. J., and Studitsky, V. M. (2011) Nucleic Acids Res. 39, 3520-3528
    5. 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) EMBO J. 25, 1669-1679.
    6. Belotserkovskaya, R., Oh, S., Bondarenko, V. A., Orphanides, G., Studitsky, V. M., and Reinberg, D. (2003) Science 301, 1090-1093
    7. Bondarenko, V. A., Steele, L. M., Ujvari, A., Gaykalova, D. A., Kulaeva, O. I., Polikanov, Y. S., Luse, D. S., and Studitsky, V. M. (2006) Mol. Cell 24, 469-479
    8. Kireeva, M. L., Hancock, B., Cremona, G. H., Walter, W., Studitsky, V. M., and Kashlev, M. (2005) Mol. Cell 18, 97-108

    People associated with the project: Artem Demidenko, Fu-Kai Hsieh, Olga Kulaeva

  • Mechanisms of chromatin-specific DNA repair

    Recently our studies of transcription through chromatin have resulted in discovery of a novel, chromatin-specific pathway of transcription-coupled DNA repair (unpublished). We found that ss DNA breaks (SSBs) in certain positions within non-transcribed DNA strand induce nucleosome-specific arrest of transcribing Pol II. Although SSBs in non- transcribed DNA strand are efficiently repaired in vivo, previously it was impossible to recapitulate this process in vitro. Therefore we have likely discovered a first step of a novel, truly chromatin-specific DNA repair pathway. This pathway and its interface with other, better studied pathways of DNA damage response (DDR) will be studied in the future. Since DDR deficiencies in tumor cells are popular targets for anti-cancer drug development there is a strong potential for developing of this project into a clinically relevant study.

  • Development of FACT- and PARP1-targeted anti-cancer drugs

    Artem Demidenko
    chromatin structure, protein factors, histone
    The role of chromatin structure, protein factors and histone modifications
    During the past several years, FACT and Poly(ADP-ribose)Polymerase (PARP) proteins have become popular targets for anti-cancer treatment. However, most of FACT- and PARP-targeted inhibitors cause severe side effects. Recently we have discovered novel molecular mechanisms of FACT and PARP1 action during transcript elongation through chromatin (unpublished) and have established experimental systems that allow development of novel inhibitors targeting FACT- and PARP-related pathways. This project is dedicated to further analysis of the mechanisms of FACT and PARP action and development of novel small molecule inhibitors, which in future could be used for anti-cancer treatment.
  • The role of chromatin structure, protein factors and histone modifications in gene regulation over a distance

    Ekaterina Nizovtseva & Olga Studitskaia (Kulaeva)
    Long-Distance Communication in Chromatin
    Long-Distance Communication in Chromatin: Chromatin Structure & Flexibility

    Distant regulation of gene expression by enhancers and insulators is mediated by direct interaction between proteins bound at communicating DNA elements and involves looping of intervening DNA/chromatin regions. Recent genomic studies have revealed widespread occurrence and unique aspects of distant regulation of gene expression (1). We have developed relatively simple, highly purified and efficient experimental systems for quantitative analysis of enhancer action over a distance in vitro on DNA and in chromatin (2-4). This simple system allowed us to identify supercoiled DNA and chromatin structure as primary devices supporting efficient communication over a distance in pro- and eukaryotes, respectively (2,3) and led to discovery of the mechanisms of distant action of DNA regulatory elements (“slithering” and “slithering barrier” mechanisms for enhancers and insulators, respectively (3,5)). The first transcriptional insulator blocking enhancer action over a distance in vitro was rationally designed, constructed and analyzed (5).

    More recently, we have developed defined chromatin templates (4) allowing detailed analysis of the mechanisms of distant communication in chromatin (Fig. 3 (6)). Using this system, histone “tails” were identified as factors that are essential for distant enhancer action (6); it was shown that distant communication is a novel, mechanistically distinct rate-limiting step in gene regulation (6,7). Currently this project develops towards identification of chromatin elements (DNA sequences and histone modifications) and chromatin-bound factors modulating the efficiency of distant communication.

    References

    1. Kulaeva, O. I., Nizovtseva, E. V., Polikanov, Y. S., Ulianov, S. V., and Studitsky, V. M. (2012) Mol Cell Biol 32, 4892-4897
    2. Rubtsov, M. A., Polikanov, Y. S., Bondarenko, V. A., Wang, Y. H., and Studitsky, V. M. (2006) Proc. Natl. Acad. Sci. USA 103, 17690-17695.
    3. Liu, Y., Bondarenko, V., Ninfa, A., and Studitsky, V. M. (2001) Proc. Natl. Acad. Sci. USA 98, 14883-14888.
    4. Polikanov, Y. S., and Studitsky, V. M. (2009) Methods Mol. Biol. 543, 563-576
    5. Bondarenko, V. A., Jiang, Y. I., and Studitsky, V. M. (2003) EMBO J. 22, 4728-4737.
    6. Kulaeva, O. I., Zheng, G., Polikanov, Y. S., Colasanti, A. V., Clauvelin, N., Mukhopadhyay, S., Sengupta, A. M., Studitsky, V. M., and Olson, W. K. (2012) J. Biol. Chem. 287, 20248-20257
    7. Mukhopadhyay, S., Schedl, P., Studitsky, V. M., and Sengupta, A. M. (2011) Proc. Natl. Acad. Sci. USA 108, 19919-19924
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