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Dynamics of tobacco DNA topoisomerases II in cell cycle regulation: to manage topological constrains during replication, transcription and mitotic chromosome condensation and segregation

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The topoisomerase II expression varies as a function of cell proliferation. Maximal topoisomerase II expression was tightly coupled to S phase and G2/M phase via both transcriptional and post-transcriptional regulation. Investigation in meiosis using pollen mother cells also revealed that it is not the major component of meiotic chromosomes, it seems to diffuse out once meiotic chromosomal condensation is completed.

Abstract

Synchronized tobacco BY-2 cell cultures were used to study the role of topoisomerase II in various stages of the cell cycle. Topoisomerase II transcript accumulation was observed during the S- and G2/M- phase of cell cycle. This biphasic expression pattern indicates the active requirement of topoisomerase II during these stages of the cell cycle. Through immuno-localization of topoisomerase II was observed diffusely throughout the nucleoplasm in interphase nuclei, whereas, the nucleolus region exhibited a more prominent immuno-positive staining that correlated with rRNA transcription, as shown by propidium iodide staining and BrUTP incorporation. The immuno-staining analysis also showed that topoisomerase II is the major component of mitotic chromosomes and remain attached to the chromosomes during cell division. The inhibition of topoisomerase II activity using specific inhibitors revealed quite dramatic effect on condensation of chromatin and chromosome individualization from prophase to metaphase transition. Partially condensed chromosomes were not arranged on metaphase plate and chromosomal perturbations were observed when advance to anaphase, suggesting the importance of topoisomerase II activity for proper chromosome condensation and segregation during mitosis. Contrary, topoisomerase II is not the major component of meiotic chromosomes, even though mitosis and meiosis share many processes, including the DNA replication, chromosome condensation and precisely regulated partitioning of chromosomes into daughter cells. Even if topoisomerase II is required for individualization and condensation of meiotic chromosomes, it seems to diffuse out once meiotic chromosomal condensation is completed.

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Acknowledgements

This work was supported in part by internal grants of ICGEB in terms of a research grant. B. N. Singh thanks Council of Scientific and Industrial Research (CSIR) for awarding the Senior Research Fellowship. The award of Research Associateship by the Department of Biotechnology (DBT) India, to V. M. M. Achary is also acknowledged.

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  1. Badri Nath Singh

    Present address: Waksman Institute, Rutgers, The State University of New Jersey, Piscataway, NJ, USA

Authors and Affiliations

  1. Crop Improvement Group, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi, Delhi, 110067, India

    Badri Nath Singh, V. Mohan Murali Achary, Varakumar Panditi, Sudhir K. Sopory & Malireddy K. Reddy

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Contributions

BNS performed major experiments including expression analysis, microscopy studies and analyzed the data. VMMA a part of expression analysis, microscopy and drafted the manuscript. VP carried out the microscopic studies. SKS and MKR designed all the experiments, wrote and revised the manuscript.

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Correspondence to Malireddy K. Reddy.

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Electronic supplementary material

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11103_2017_626_MOESM1_ESM.tif

Supplementary Fig. 1 (a) The 5’ flanking sequence of the Nt topo II gene. The Nt topo II coding sequence is shown in bold. The arrow indicates the transcriptional start site, and the ‘C’ is numbered +1. The cell cycle responsive E2F and ICE regulatory elements present in the promoter region are shown in bold. (b) Sequence analysis of the 3’ UTR region of the Nt topo II mRNA. The lower case letters represent the coding region. Various regulatory elements identified within the 3’ UTR region. The polyadenylation signal (PolyA), cytoplasmic polyadenylation element (CPE) and U-rich element (ARE) are shown in bold and underlined. (TIF 1383 KB)

11103_2017_626_MOESM2_ESM.tif

Supplementary Fig. 2 Western blot analysis of nuclear extracts prepared from tobacco and pearl millet with tobacco anti-topoisomerase II antibodies. Intact nuclei were isolated from tobacco and pearl millet seedlings. The soluble proteins were extracted from these nuclei by gently lysing in the presence of 200 mM KCl and the extracted polypeptides were size fractionated on a 10% polyacrylamide gel and transferred onto a nitrocellulose membrane. Western analysis was carried out using anti tobacco topo-II antibodies. Lane M: molecular weight markers, lanes 1 and 6 no protein was loaded; lanes 2 and 3 tobacco nuclear extract loaded and lanes 4 and 5 pearl millet nuclear extracts loaded. The numbers shown on the extreme left represents the protein marker size in kDa. The arrow on the right showing the western positive topoisomerase II polypeptide. (TIF 392 KB)

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Singh, B.N., Achary, V.M.M., Panditi, V. et al. Dynamics of tobacco DNA topoisomerases II in cell cycle regulation: to manage topological constrains during replication, transcription and mitotic chromosome condensation and segregation. Plant Mol Biol 94, 595–607 (2017). https://doi.org/10.1007/s11103-017-0626-4

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