title: TODRA, a lncRNA at the RAD51 Locus, Is Oppositely Regulated to RAD51, and Enhances RAD51-Dependent DSB (Double Strand Break) Repair.
abstract: Expression of RAD51, a crucial player in homologous recombination (HR) and DNA double-strand break (DSB) repair, is dysregulated in human tumors, and can contribute to genomic instability and tumor progression. To further understand RAD51 regulation we functionally characterized a long non-coding (lnc) RNA, dubbed TODRA (Transcribed in the Opposite Direction of RAD51), transcribed 69bp upstream to RAD51, in the opposite direction. We demonstrate that TODRA is an expressed transcript and that the RAD51 promoter region is bidirectional, supporting TODRA expression (7-fold higher than RAD51 in this assay, p = 0.003). TODRA overexpression in HeLa cells induced expression of TPIP, a member of the TPTE family which includes PTEN. Similar to PTEN, we found that TPIP co-activates E2F1 induction of RAD51. Analysis of E2F1's effect on the bidirectional promoter showed that E2F1 binding to the same site that promotes RAD51 expression, results in downregulation of TODRA. Moreover, TODRA overexpression induces HR in a RAD51-dependent DSB repair assay, and increases formation of DNA damage-induced RAD51-positive foci. Importantly, gene expression in breast tumors supports our finding that E2F1 oppositely regulates RAD51 and TODRA: increased RAD51 expression, which is associated with an aggressive tumor phenotype (e.g. negative correlation with positive ER (r = -0.22, p = 0.02) and positive PR status (r = -0.27, p<0.001); positive correlation with ki67 status (r = 0.36, p = 0.005) and HER2 amplification (r = 0.41, p = 0.001)), correlates as expected with lower TODRA and higher E2F1 expression. However, although E2F1 induction resulted in TPIP downregulation in cell lines, we find that TPIP expression in tumors is not reduced despite higher E2F1 expression, perhaps contributing to increased RAD51 expression. Our results identify TPIP as a novel E2F1 co-activator, suggest a similar role for other TPTEs, and indicate that the TODRA lncRNA affects RAD51 dysregulation and RAD51-dependent DSB repair in malignancy. Importantly, gene expression in breast tumors supports our finding that E2F1 oppositely regulates RAD51 and TODRA: increased RAD51 expression, which is associated with an aggressive tumor phenotype (e.g. negative correlation with positive ER (r = -0.22, p = 0.02) and positive PR status (r = -0.27, p<0.001); positive correlation with ki67 status (r = 0.36, p = 0.005) and HER2 amplification (r = 0.41, p = 0.001)), correlates as expected with lower TODRA and higher E2F1 expression. However, although E2F1 induction resulted in TPIP downregulation in cell lines, we find that TPIP expression in tumors is not reduced despite higher E2F1 expression, perhaps contributing to increased RAD51 expression. Our results identify TPIP as a novel E2F1 co-activator, suggest a similar role for other TPTEs, and indicate that the TODRA lncRNA affects RAD51 dysregulation and RAD51-dependent DSB repair in malignancy.
authors.affiliation: Human Genetics, Hebrew University Medical School, Jerusalem, Israel; Medical Genetics Institute, Shaare Zedek Medical Center, Jerusalem, Israel.
authors.forename: Inbal
authors.initials: I
authors.lastname: Gazy
authors.affiliation: Medical Genetics Institute, Shaare Zedek Medical Center, Jerusalem, Israel.
authors.forename: David A
authors.initials: DA
authors.lastname: Zeevi
authors.affiliation: Medical Genetics Institute, Shaare Zedek Medical Center, Jerusalem, Israel.
authors.forename: Paul
authors.initials: P
authors.lastname: Renbaum
authors.affiliation: Medical Genetics Institute, Shaare Zedek Medical Center, Jerusalem, Israel.
authors.forename: Sharon
authors.initials: S
authors.lastname: Zeligson
authors.affiliation: Department of Genetics, Alexander Silberman Institute of Life Sciences, Hebrew University of Jerusalem, Jerusalem, Israel.
authors.forename: Lital
authors.initials: L
authors.lastname: Eini
authors.affiliation: The Mina and Everard Goodman Faculty of Life Science, Bar Ilan University, Ramat Gan, Israel.
authors.forename: Dana
authors.initials: D
authors.lastname: Bashari
authors.affiliation: Genomic Data Analysis Unit, Hebrew University Medical School, Jerusalem, Israel.
authors.forename: Yoav
authors.initials: Y
authors.lastname: Smith
authors.affiliation: Department of Family Medicine, Hebrew University Medical School, Jerusalem, Israel; Clalit Health Services, Jerusalem, Israel.
authors.forename: Amnon
authors.initials: A
authors.lastname: Lahad
authors.affiliation: Department of Genetics, Alexander Silberman Institute of Life Sciences, Hebrew University of Jerusalem, Jerusalem, Israel.
authors.forename: Michal
authors.initials: M
authors.lastname: Goldberg
authors.affiliation: The Mina and Everard Goodman Faculty of Life Science, Bar Ilan University, Ramat Gan, Israel.
authors.forename: Doron
authors.initials: D
authors.lastname: Ginsberg
authors.affiliation: Human Genetics, Hebrew University Medical School, Jerusalem, Israel; Medical Genetics Institute, Shaare Zedek Medical Center, Jerusalem, Israel.
authors.forename: Ephrat
authors.initials: E
authors.lastname: Levy-Lahad
mesh_terms: D004249:DNA Damage; D004260:DNA Repair; D006801:Humans; D062085:RNA, Long Noncoding / Q000235:genetics*; D051135:Rad51 Recombinase / Q000235:genetics* / Q000502:physiology
chemical_list: D062085:RNA, Long Noncoding; C495153:RAD51 protein, human; D051135:Rad51 Recombinase