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HMGB1 gene knockout in mouse embryonic fibroblasts results in reduced telomerase activity and telomere dysfunction

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Abstract

Telomere repeats are added onto chromosome ends by telomerase, consisting of two main core components: a catalytic protein subunit (telomerase reverse trancriptase, TERT), and an RNA subunit (telomerase RNA, TR). Here, we report for the first time evidence that HMGB1 (a chromatin-associated protein in mammals, acting as a DNA chaperone in transcription, replication, recombination, and repair) can modulate cellular activity of mammalian telomerase. Knockout of the HMGB1 gene (HMGB1 KO) in mouse embryonic fibroblasts (MEFs) results in chromosomal abnormalities, enhanced colocalization of γ-H2AX foci at telomeres, and a moderate shortening of telomere lengths. HMGB1 KO MEFs also exhibit significantly (>5-fold) lower telomerase activity than the wild-type MEFs. Correspondingly, enhanced telomerase activity is observed upon overexpression of HMGB1 in MEFs. HMGB1 physically interacts with both TERT and TR, as well as with active telomerase complex in vitro. However, direct interaction of HMGB1 with telomerase is most likely not accountable for the observed higher telomerase activity in HMGB1-containing cells, as revealed from the inability of purified HMGB1 protein to stimulate telomerase activity in vitro. While no transcriptional silencing of TERT is observed in HMGB1 KO MEFs, levels of TR are diminished (~3-fold), providing possible explanation for the observed lower telomerase activity in HMGB1 KO cells. Interestingly, knockout of the HMGB2 gene elevates telomerase activity (~3-fold) in MEFs, suggesting that the two closely related proteins of the HMGB family, HMGB1 and HMGB2, have opposite effects on telomerase activity in the cell. The ability of HMGB1 to modulate cellular activity of telomerase and to maintain telomere integrity can help to understand some aspects of the protein involvement in chromosome stability and cancer.

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Acknowledgments

Plasmids pBluescript-mTR Native/short(noT7), HA-TERT-pcDNA3.1-Zeo and mHMGB2-Flag-pcDNA3 were kindly obtained from Scott J. Garforth (Einstein College of Medicine, NY, USA) and Lea A. Harrington (Department of Medical Biophysics, University of Toronto), respectively. Wild-type and HMGB1 −/− mouse embryonic fibroblast cell lines (SV40-T) were kindly provided by Marco E. Bianchi (San Raffaele Research Institute, Milan). We also thank Thomas R. Cech (University of Colorado, Boulder) for providing plasmids phTERT-HA2 and phTR, and Joachim Lingner (UPLIN, Laussane) for providing plasmids encoding hTERT and its truncated forms. Antibodies to γH2AX and POT1 were kindly provided by Emilie Lukášová and Eva Bártová (Institute of Biophysics, Brno). We are also grateful to Emilie Lukášová and Pavel Matula (Faculty of Informatics, Masaryk University, Brno) for help with confocal microscopy and colocalization analysis using Acquiarium software (http://cbia.fi.muni.cz/acquiarium.html), respectively. This research was supported by grants to M.Š. from the Grant Agency of the Czech Republic (P301/10/0590 and P305/12/2475). JF was supported by the Grant Agency of the Academy of Sciences of the Czech Republic (IAA500040801) and by the project Central European Institute of Technology (CEITEC; CZ.1.05/1.1.00/02.0068) financed from European Regional Development Fund.

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Correspondence to Jiří Fajkus or Michal Štros.

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Communicated by Jan Karlseder

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Polanská, E., Dobšáková, Z., Dvořáčková, M. et al. HMGB1 gene knockout in mouse embryonic fibroblasts results in reduced telomerase activity and telomere dysfunction. Chromosoma 121, 419–431 (2012). https://doi.org/10.1007/s00412-012-0373-x

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