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This article is part of the supplement: Epigenetics and Chromatin: Interactions and processes

Open Access Poster presentation

Pausing as a mechanism of nucleosome recovery

Han-Wen Chang1*, Olga I Kulaeva12, Alexey Shaytan2, M Kibanov2, K Severinov3, David J Clark4 and Vasily M Studitsky12

  • * Corresponding author: Han-Wen Chang

Author Affiliations

1 Department of Pharmacology, UMDNJ-Robert Wood Johnson Medical School, Piscataway, NJ 08854, USA and Faculty of Biology

2 Moscow State University; Moscow, Russia

3 Waksman Institute of Microbiology and Department of Molecular Biology and Biochemistry, Rutgers, State University of New Jersey, Piscataway, NJ08854, USA

4 Program in Genomics of Differentiation, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA

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Epigenetics & Chromatin 2013, 6(Suppl 1):P14  doi:10.1186/1756-8935-6-S1-P14

The electronic version of this article is the complete one and can be found online at: http://www.epigeneticsandchromatin.com/content/6/S1/P14


Published:18 March 2013

© 2013 Chang et al; licensee BioMed Central Ltd.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Background

Nucleosome survival during transcription is important for the maintenance of chromatin integrity, gene regulation, cell survival, and aging. In according to the studies in vitro and in vivo, Pol II pauses at positions inside of a nucleosome and nucleosome survives after Pol II transcription through chromatin [1]. A key intermediate, Ø-loop (EC+49), formed at +45 region inside of nucleosome and Pol II pausing at +45 nucleosomal region positively correlated to nucleosome survival [2,3]. A putative electrostatic interaction between Pol II and histone octamer has also been predicted to stabilize the Ø-loop intermediate by structural modeling[3]. In this study, we will provide more evidences to link those characteristics together.

Materials and methods

Protein purification [3][4]

Computational structure modeling [3]

Transcription assay, Nucleosome fate and DNase I footprinting [3,5]

Results

In structural modeling, three negatively charged surfaces (regions 1, 2, 3) were identified on Rpb1, a large subunit of yeast Pol II, and plausibly interacted to histone octamer, especially H2B N-tail region, by the electrostatic force. Results of transcriptions by Thermus thermophilus (T.th.) and Thermus aquaticus (T.aq.) RNAPs which contained less net negative charges at all three regions showed lower +45 pausing, no Ø-loop formation and nucleosome displacement. Finally, lower nucleosomal barrier was also shown during the transcription by mutated yeast Pol II which contains less negatively charged at region 2 of Rpb1.

Conclusions

Collectively, we demonstrated the positive correlations of higher negative net charge of the interacting region of RNAP, the stronger +45 barrier, more efficient nucleosome survival and more efficient Ø-loop intermediate formation during transcription.

References

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    Biochim Biophys Acta 2012. OpenURL

  2. Hsieh FK, Fisher M, Ujvari A, Studitsky VM, Luse DS: Histone Sin mutations promote nucleosome traversal and histone displacement by RNA polymerase II.

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  3. Kulaeva OI, Gaykalova DA, Pestov NA, Golovastov W, Vassylyev DG, Artsimovitch I, Studitsky VM: Mechanism of chromatin remodeling and recovery during passage of RNA polymerase II.

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  5. Gaykalova DA, Kulaeva OI, Pestov NA, Hsieh FK, Studitsky VM: Experimental analysis of the mechanism of chromatin remodeling by RNA polymerase II.

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