2D gel electrophoresis reveals dynamics of t-loop formation during the cell cycle and t-loop in maintenance regulated by heterochromatin state

Zepeng Zhang, Tianpeng Zhang, Yuanlong Ge, Mengfan Tang, Wenbin Ma, Qinfen Zhang, Shengzhao Gong, Woodring E. Wright, Jerry Shay, Haiying Liu, Yong Zhao

Research output: Contribution to journalArticlepeer-review

Abstract

Linear chromosome ends are capped by telomeres that have been previously reported to adopt a t-loop structure. The lack of simple methods for detecting t-loops has hindered progress in understanding the dynamics of t-loop formation and its function in protecting chromosome ends. Here, we employed a classical two-dimensional agarose gel method (2D gel method) to innovatively apply to t-loop detection. Briefly, restriction fragments of genomic DNA were separated in a 2D gel, and the telomere sequence was detected by in-gel hybridization with telomeric probe. Using this method, we found that t-loops are present throughout the cell cycle, and t-loop formation tightly couples to telomere replication. We also observed that t-loop abundance positively correlates with chromatin condensation, i.e. cells with less compact telomeric chromatin (ALT cells and trichostatin A (TSA)-treated HeLa cells) exhibited fewer t-loops. Moreover, we observed that telomere dysfunction-induced foci, ALT-associated promyelocytic leukemia bodies, and telomere sister chromatid exchanges are activated upon TSAinduced loss of t-loops. These findings confirm the importance of the t-loop in protecting linear chromosomes from damage or illegitimate recombination.

Original languageEnglish (US)
Pages (from-to)6645-6656
Number of pages12
JournalJournal of Biological Chemistry
Volume294
Issue number16
DOIs
StatePublished - Apr 19 2019

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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