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. 2017 Jun 20;7:3896. doi: 10.1038/s41598-017-04248-w

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© The Author(s) 2017

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Induction of an upstream gene correlates with the downregulation of a tandem oriented downstream promoter. (A) Outline of the tandem oriented reporter gene construct. The ZMYND10 - RASSF1A promoter including the ZYMND10 transcriptional end site (TES) and the RASSF1A promoter and transcriptional start site were cloned in a tetracycline inducible vector system (Tet -On/TO). The EGFP-ZYMND fusion gene was under the control of the pCMV 2xTETO₂ promoter and the 500 bp RASSF1A promoter including 5′-UTR and 17 bp of Exon1α was ligated in frame to Myc tagged Renilla luciferase (RLuc). (B) UCSC genome browser view of the 2.3. kb ZMYND10 - RASSF1A promoter locus. Black boxes represent exons. Outline of the genetic structure of the tandem reporter TO-EGFP-2,3-RLuc. In the construct TO-EGFPpA-2,3-RLuc a 300 bp SV40 poly A site (pA) was inserted at the 3′-end of EGFP. To generate the tandem reporter TO-EGFP-0,5-RLuc a 1.8 kb fragment of ZYMND10 was deleted. (C) Luciferase assay of the TO-EGFP-2,3-RLuc construct. The tandem reporter was stably transfected into TREx293 cells and induced for four days with the indicated concentration of doxycycline (Dox). RLuc activity was analyzed as described in the methods section. (D) Time dependent downregulation of RLuc activity. TREx293 cells stably transfected with TO-EGFP-2,3-RLuc were induced with 4 mM Dox (+) or uninduced (−) for the indicated days (d) and RLuc activity was analyzed. (E) Analysis of different tandem constructs by western blot and luciferase assay. Two TREx clones (B7 and A7) of the stable transfected TO-EGFP-2,3-RLuc were induced with 4 mM Dox for four days and the protein levels were subsequently analyzed by western blot. Protein lysates were separated on SDS PAGE and blotted. Full-length blots are included in the supplementary information file. For detection of the indicated proteins primary antibody against myc, GFP and GAPDH were utilized. In parallel RLuc activity was analyzed with a luciferase assay. RLuc luciferase activity was normalized to transient transfected firefly luciferase. Additionally the expression of RLuc and EGFP were analyzed in two TO-EGFPpA-2,3-RLuc TREx clones (A1 and A2) and the C2 clone of TO-EGFP-0,5-RLuc by western blot and luciferase assay. *p < 0.05, **p < 0.01 and ***p < 0.001.