![Fig. 1. Effects of PLZF and PLZF-RARα on VDR, VP-16, and RARα-mediated transactivation. / (A) Transient overexpression of PLZF or PLZF-RARα represses 1,25(OH)2D3-dependent activation of a VDR responsive reporter (lanes 1-3). This repression was not affected by the addition of 150 ng/mL TSA to the culture medium (lanes 4-6). Plasmids containing complementary DNAs for PLZF, PLZF-RARα, VDR, and a minimal VDR responsive reporter containing 2 copies of a VDRE from the OPN gene, cloned approximately 100 bp upstream of the E1b TATA box and luciferase gene, were used to electroporate U937 cells with the indicated amounts of DNAs. The cells were left for 2 hours before the addition of 1,25(OH)2D3(10−8 M), TSA, or vehicle (ethanol). Data are from a representative experiment done in triplicate. (B) VP16-dependent activation is not affected by PLZF expression. Transfection of U937 cells was carried out as described in the legend for panel A. A GAL4-VP16 fusion was coexpressed with PLZF and a GAL responsive reporter, with the indicated amounts of DNA. (C) PLZF did not repress ATRA-dependent activation of an ATRA responsive reporter by RARα. The reporter used here contained the RARE from the RARβ gene promoter positioned upstream of the Tk promoter and luciferase gene. Transfection was done as described above, and the cells were left for 2 hours before the addition of ATRA (10−6 M) or vehicle (dimethyl sulfoxide [DMSO]). (D) PLZF associates with VDR but not RARα in a ligand-independent manner. In vitro–translated35S-labeled PLZF was incubated at 4°C for 2 hours with affinity-purified GST-VDR or GST-RARα in the presence (+) or absence (−) of 1,25(OH)2D3 or ATRA (10−7M and 10−6 M, respectively). The resulting complexes were washed, separated by SDS–polyacrylamide gel electrophoresis (PAGE), dried, and exposed to film. Lane 1 represents 10% of the labeled PLZF input (I). PLZF did not interact with GST alone (lane 2). PLZF interacted with GST-VDR independent of ligand (lanes 3 and 4) but not with GST- RARα (lanes 5 and 6). All transfections were done at least 3 times in triplicate each time. The histograms show results from a representative experiment from this series. LUC indicates luciferase reporter gene; TK, thymidine kinase promoter; RLU, relative light unit.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/98/12/10.1182_blood.v98.12.3290/6/h82311804001.jpeg?Expires=1735501094&Signature=Y4yXZT4uCwhx33Uz5DVdBvxOUu0VPaXGsqMVIvqkQYOQFU14D7Vs1n~xuN4LUH~UWKOrk7~jx-DDrdvD51kbDnO4H41~Ax88gCXIWJAYx4oMk7z-wkSP1HnazxHOHSoknAAJjGdiL8UFSmk074Ykmf0OAsaOCJrFbhmTDBtRyq-jIkAvQ3el1EyZQad5-JYbbfXgdszHFoaPUv90s3wmnEtvDLcNdBI56-LVDkvbZ6Rj3sqae6kYFTas~QLX-cf3C2Te4NG4KMyriTsmzW3jZDO17ZmK9nWJX4YBe8goajrTfJ0hOBy4wLFjpn4wt8ftfilK01hoDvgiPUVNzwmVOQ__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Effects of PLZF and PLZF-RARα on VDR, VP-16, and RARα-mediated transactivation.
(A) Transient overexpression of PLZF or PLZF-RARα represses 1,25(OH)2D3-dependent activation of a VDR responsive reporter (lanes 1-3). This repression was not affected by the addition of 150 ng/mL TSA to the culture medium (lanes 4-6). Plasmids containing complementary DNAs for PLZF, PLZF-RARα, VDR, and a minimal VDR responsive reporter containing 2 copies of a VDRE from the OPN gene, cloned approximately 100 bp upstream of the E1b TATA box and luciferase gene, were used to electroporate U937 cells with the indicated amounts of DNAs. The cells were left for 2 hours before the addition of 1,25(OH)2D3(10−8 M), TSA, or vehicle (ethanol). Data are from a representative experiment done in triplicate. (B) VP16-dependent activation is not affected by PLZF expression. Transfection of U937 cells was carried out as described in the legend for panel A. A GAL4-VP16 fusion was coexpressed with PLZF and a GAL responsive reporter, with the indicated amounts of DNA. (C) PLZF did not repress ATRA-dependent activation of an ATRA responsive reporter by RARα. The reporter used here contained the RARE from the RARβ gene promoter positioned upstream of the Tk promoter and luciferase gene. Transfection was done as described above, and the cells were left for 2 hours before the addition of ATRA (10−6 M) or vehicle (dimethyl sulfoxide [DMSO]). (D) PLZF associates with VDR but not RARα in a ligand-independent manner. In vitro–translated35S-labeled PLZF was incubated at 4°C for 2 hours with affinity-purified GST-VDR or GST-RARα in the presence (+) or absence (−) of 1,25(OH)2D3 or ATRA (10−7M and 10−6 M, respectively). The resulting complexes were washed, separated by SDS–polyacrylamide gel electrophoresis (PAGE), dried, and exposed to film. Lane 1 represents 10% of the labeled PLZF input (I). PLZF did not interact with GST alone (lane 2). PLZF interacted with GST-VDR independent of ligand (lanes 3 and 4) but not with GST- RARα (lanes 5 and 6). All transfections were done at least 3 times in triplicate each time. The histograms show results from a representative experiment from this series. LUC indicates luciferase reporter gene; TK, thymidine kinase promoter; RLU, relative light unit.
