TY - JOUR
T1 - 2D gel electrophoresis reveals dynamics of t-loop formation during the cell cycle and t-loop in maintenance regulated by heterochromatin state
AU - Zhang, Zepeng
AU - Zhang, Tianpeng
AU - Ge, Yuanlong
AU - Tang, Mengfan
AU - Ma, Wenbin
AU - Zhang, Qinfen
AU - Gong, Shengzhao
AU - Wright, Woodring E.
AU - Shay, Jerry
AU - Liu, Haiying
AU - Zhao, Yong
N1 - Publisher Copyright:
© 2019 Zhang et al.
PY - 2019/4/19
Y1 - 2019/4/19
N2 - 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.
AB - 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.
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U2 - 10.1074/jbc.RA119.007677
DO - 10.1074/jbc.RA119.007677
M3 - Article
C2 - 30819801
AN - SCOPUS:85064855639
SN - 0021-9258
VL - 294
SP - 6645
EP - 6656
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 16
ER -