The interaction of ZSCAN4 with shelterin complex members: A possible role for telomere regulation in cancer cells

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2014-05

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Abstract

Telomeres are repetitive sequences at the ends of chromosomes protected by DNA binding proteins of the shelterin complex that form capping structures. Some of these proteins play a role in the regulation of telomere length through their interaction with other proteins. Recently, the newly identified Zinc finger and SCAN domain containing 4 gene (ZSCAN4), which play a key role in genomic stability by regulating telomere elongation, was shown to co-localize with TRF1 foci. This suggests that the interaction of ZSCAN4 with TRF1 or the shelterin complex functions in regulation of telomere elongation. Based on these results, I hypothesized that ZSCAN4 binds to one or more of the shelterin complex members to function in regulating telomere length in cancer cell lines. Co-immunoprecipitation results demonstrated that ZSCAN4 binds with TRF1 in vitro. Pull-down assay results confirmed this interaction was indirect. The ZSCAN4-mediated cooperative binding of TRF1 to telomeric DNA has important implications for understanding the role of ZSCAN4 to telomere extension. To further investigate whether direct interaction existed between ZSCAN4 and other shelterin complex members, a pull-down assay with purified shelterin proteins was performed. Here, I demonstrate that ZSCAN4 directly binds with RAP1 in vitro. Localization and visualization of ZSCAN4 interaction with RAP1 and TRF1 was performed in situ to observe how ZSCAN4 is involved in telomere maintenance. Using a bimolecular fluorescence complementation (BiFC) assay, I show that the interaction between ZSCAN4with RAP1can be directly visualized in living cells. In this study, I demonstrate for the first time, ZSCAN4 directly associates with RAP1 (physical association protein) and indirectly with TRF1 (functional association protein) in cancer cells. Furthermore, I show that ZSCAN4 plays an important role in telomere maintenance in both conventional and ALT pathways for telomere maintenance, independent of telomerase activity.

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Keywords

Zscan4, Rap1, Cancer

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