Viral and cellular factors that target the promyelocytic leukemia oncogenic domains strongly activate a glucocorticoid-responsive promoter

Abstract

Promyelocytic leukemia (PML) oncogenic domains (PODs) accumulate the transcriptional cofactor named CREB binding protein (CBP) and have been suggested to function as centers of transcription. Transcriptional activation by nuclear hormones, such as glucocorticoids, is augmented by the key constituent of PODs, the PML protein, and decreased by the POD-associated Tax protein of human T-cell leukemia virus type 1 (HTLV-1). This led to the hypothesis that intact PODs might play a positive role in the activation of these promoters. We report here that transiently expressed E4orf3 protein of adenovirus type 5, immediate-early protein 1 of human cytomegalovirus, and the PML-retinoic acid receptor fusion protein from leukemia cells each redistribute CBP within the nucleus. However, unlike the Tax protein of HTLV-1, these factors did not inhibit a glucocorticoid-inducible promoter but strongly enhanced its activity. Thus, at least glucocorticoid-induced transcription does not depend on POD integrity.

FIG. 1

Relocalization of the CREB binding protein by E4orf3, CMV IE1, and PML-RAR. (A) PML⁺⁺ HeLa cells (30) were induced to express PML by removal of tetracycline and infected with Ad (multiplicity of infection [MOI] of 5) 24 h later or mock infected (g to i). The virus employed was either the wild type (termed wt300) (a to c) or a mutant lacking E4orf3 expression (E4inorf3) (d to f). Fourteen hours after infection, PML (a, d, and g) and CBP (b, e, and h) were stained with antibodies to these proteins, followed by secondary antibodies coupled to fluorescent dyes. The nuclei were visualized using 4′,6′-diamidino-2-phenylindole (DAPI) stain (c, f, and i). (B) PML⁺⁺ HeLa cells were induced to express PML and then transfected using Superfect (Qiagen) with expression plasmids for a PML-RAR fusion protein (a to c), CMV IE1 (d to f), or Ad E4orf3 (g to i). Twenty-four hours after transfection, the cells were fixed and stained with antibodies against PML (a), the hemagglutinin tag (d), or the FLAG tag (g). Simultaneously, CBP was stained in all cells (b, e, and h) and the nuclei were visualized with DAPI (c, f, and i). Note that in part e, the pattern of CBP distribution is shown in a nontransfected cell next to a transfected cell.

FIG. 1

Relocalization of the CREB binding protein by E4orf3, CMV IE1, and PML-RAR. (A) PML⁺⁺ HeLa cells (30) were induced to express PML by removal of tetracycline and infected with Ad (multiplicity of infection [MOI] of 5) 24 h later or mock infected (g to i). The virus employed was either the wild type (termed wt300) (a to c) or a mutant lacking E4orf3 expression (E4inorf3) (d to f). Fourteen hours after infection, PML (a, d, and g) and CBP (b, e, and h) were stained with antibodies to these proteins, followed by secondary antibodies coupled to fluorescent dyes. The nuclei were visualized using 4′,6′-diamidino-2-phenylindole (DAPI) stain (c, f, and i). (B) PML⁺⁺ HeLa cells were induced to express PML and then transfected using Superfect (Qiagen) with expression plasmids for a PML-RAR fusion protein (a to c), CMV IE1 (d to f), or Ad E4orf3 (g to i). Twenty-four hours after transfection, the cells were fixed and stained with antibodies against PML (a), the hemagglutinin tag (d), or the FLAG tag (g). Simultaneously, CBP was stained in all cells (b, e, and h) and the nuclei were visualized with DAPI (c, f, and i). Note that in part e, the pattern of CBP distribution is shown in a nontransfected cell next to a transfected cell.

FIG. 2

Activation of a glucocorticoid-responsive promoter by E4orf3, CMV IE1, and PML-RAR. (A) HeLa cells were transfected with expression plasmids (2 μg) for β-galactosidase (beta gal), PML-RAR, CMV IE1, Ad5 E4orf3, and HTLV-1 Tax (0.5 μg), together with a reporter plasmid (1 μg) containing the MMTV 5′ LTR regulating the expression of luciferase. Twenty-four hours after transfection, the cells were treated with dexamethasone (1 μM) for another 24 h (open bars) or left without further treatment (filled bars). Subsequently, the cells were harvested and luciferase activity was determined from at least five independent experiments. The mean values are shown along with the standard error. (B) HeLa cells were transfected and processed as described for panel A, but instead of the MMTV LTR reporter construct, an NFκB-responsive reporter plasmid (Stratagene) was used and no dexamethasone was added. (C) HeLa cells were transfected with the same MMTV LTR-luciferase reporter construct as described for panel A (3 μg) along with an expression plasmid conferring neomycin resistance (pCIN4 [26]; 100 ng), and a pool of stably transfected cells was selected with G418 (0.4 μg/ml). These cells were then transiently transfected with expression plasmids for β-galactosidase, PML-RAR, CMV IE1, and Ad5 E4orf3, as indicated. Reporter activities in the absence (filled bars) or presence (open bars) of dexamethasone are shown as in panel A. (D) The same pool of stably transfected HeLa cells was infected (MOI = 1) with wild-type Ad or a mutant lacking E4orf3 or mock infected as indicated. Immediately after infection, dexamethasone was added in one set of experiments (open bars) or omitted (filled bars), and luciferase activity was determined 18 h after infection.

FIG. 3

Induction of the cellular glucocorticoid-responsive gene for metallothionein IIa by E4orf3 during Ad infection. HeLa cells were infected (MOI = 1) with Ad expressing (wt300, lanes 1, 3, 5, and 7) or lacking E4orf3 (E4inorf3, lanes 2, 4, 6, and 8). Twenty-four hours after infection, RNA was extracted from the cells and the metallothionein and GAPDH mRNA levels were determined by quantitative reverse transcription-PCR as described in the text. The reaction was stopped at 20 (lanes 1, 2, 5, and 6) or 25 (lanes 3, 4, 7, and 8) amplification cycles, and the PCR products were visualized by ethidium bromide staining of an agarose gel. In control reactions, the reverse transcription step was omitted (lanes 5 to 8).