Characterization the role of telomeric RNA (TERRA) in telomeric DNA double-strand break (DSB) repair in human ALT (telomerase independent) cells
Date
2019
Authors
Alturki, Taghreed Mohammed, author
Bailey, Susan, advisor
Argueso, Lucas, committee member
Wiese, Claudia, committee member
Yao, Tingting, committee member
Journal Title
Journal ISSN
Volume Title
Abstract
Telomeres are specialized nucleoprotein complexes that protect natural chromosomal termini from degradation and prevent their detection as of DNA damage. Therefore, telomeres play critical roles in maintaining genomic stability. Telomeres are composed of tandem arrays of conserved repetitive sequences (TTAGGG in vertebrates), bound by a suite of proteins collectively termed "shelterin". Shelterin proteins are essential for telomere length regulation and end-capping structure/function. Due to their repetitive nature and together with telomeres possessing an abundance of heterochromatic marks, telomeres have long been regarded as silenced, non-transcribed features of the genome. The relatively recent discovery of telomeric RNA (TElomere Repeat- containing RNA; TERRA) opened many new avenues of investigation. TERRA is a long, noncoding RNA (lncRNA) that serves a structural role at telomeres, as well as function in regulation of telomere length and telomerase activity, the specialized reverse transcriptase capable of elongating telomeres de novo. Further, TERRA participates in telomeric recombination in tumors that maintain telomere length in a telomerase independent fashion via the Alternative Lengthening of Telomeres (ALT) pathway. Emerging evidence also supports telomeric "DNA- TERRA hybrids" as indispensable for end protection and capping function; e.g., RNA interference mediated depletion of TERRA induced telomeric DNA damage responses (DDRs) and aberrations. Thus, TERRA participates in facilitating telomeric recombination and in preventing inappropriate telomeric DNA damage responses. We hypothesized that TERRA plays a critical role in the repair of telomeric DNA damage. To address the intriguing possibility that TERRA plays a role in the telomeric DNA damage response, we evaluated the colocalization of TERRA and γ-H2AX, a well-accepted marker of double-strand breaks (DSBs), at broken telomeres in human Osteosarcoma U2OS-ALT cells in different phases of the cell cycle. To test our hypothesis, we generated U2OS-ALT cells that stably expressed FUCCI green fluorescent signals to label cells in G2 phase. Telomeric DSBs were then induced in FUCCI-U2OS cells utilizing the ENT endonuclease fused to Telomere Repeat Factor 1 TRF1 (ENT-TRF1), and validated via colocalization of telomeres with γ-H2AX-FLAG. Forty-eight hours following transfection, FUCCI-U2OS cells were also treated with EdU to label cells in S phase. Cells negative for both FUCCI and EdU identified cells in G1 phase. Using this powerful strategy to distinguish cells in G1, S and G2 phases of the cell cycle, we showed that FUCCI-U2OS cells accumulate in G2 phase following transient transfection with ENT-TRF1. We validated that expression of ENT-TRF1 generates telomeric DSBs in U2OS-ALT cells through detection of telomere- γ-H2AX-FLAG colocalization events. Importantly, our data revealed that telomeric DSB induction triggers enrichment of TERRA in G2 phase. Taken together, these observations suggested that TERRA is increased in cells transfected with ENT-TRF1; i.e., in U2OS cells harboring telomere-specific DSBs. TERRA recruitment to telomeric DSB damage sites in G2 was validated by assessing co-localization between TERRA and ENT (FLAG). Similarly, TERRA recruitment to telomeric DSBs in G1/S was also evaluated. Futhermore, non-denaturing Telomere DNA FISH was employed to visualize G-rich and C-rich single-stranded (ss)telomeric DNA. Treatment of U2OS ENT transfected cells with Rnase A and Rnase H to remove TERRA, uncovered elevated levels of resected 5' C-rich (ss)telomeric DNA (complementary TERRA sequence), suggesting a potential role for TERRA in protecting resected telomeric DNA prior to cells entering G2 phase where Homologous Recombination (HR)-mediated elongation/repair would be possible. Consistent with published reports, telomeric DSBs were also significantly induced in cells transfected with TRF1-only (positive control). However, although TERRA co-localized with FLAG/broken telomeres, resected (ss)telomeric DNA was not detected upon removal of TERRA. Therefore, our results further support induction of telomeric DSBs with overexpression of TRF1, and additionally indicate that they are repaired via a different pathway than those induced by ENT, potentially alternative End Joining (alt-EJ), as previously proposed. In conclusion, our work revealed for the first time that the telomeric RNA TERRA, is enriched specifically at telomeric DNA DSB sites in U2OS (ALT) cells.