PML/RARalpha fusion protein transactivates the tissue factor promoter through a GAGC-containing element without direct DNA association

Abstract

A severe coagulopathy is a life-threatening complication of acute promyelocytic leukemia (APL) and is ascribable mainly to the excessive levels of tissue factor (TF) in APL cells regulated in response to the promyelocytic leukemia/retinoic acid receptor alpha (PML/RARalpha) fusion protein. The underlying molecular mechanisms for this regulation remain ill-defined. With U937-PR9 cell lines stably expressing luciferase reporter gene under the control of different mutants of the TF promoter, both luciferase and ChIP data allowed the localization of the PML/RARalpha-responsive sequence in a previously undefined region of the TF promoter at position -230 to -242 devoid of known mammalian transcription factor binding sites. Within this sequence a GAGC motif (-235 to -238) was shown to be crucial because deletion or mutation of these nucleotides impaired both PML/RARalpha interaction and promoter transactivation. However, EMSA results showed that PML/RARalpha did not bind to DNA probes encompassing the -230 to -242 sequences, precluding a direct DNA association. Mutational experiments further suggest that the activator protein 1 (AP-1) sites of the TF promoter are dispensable for PML/RARalpha regulation. This study shows that PML/RARalpha transactivates the TF promoter through an indirect interaction with an element composed of a GAGC motif and the flanking nucleotides, independent of AP-1 binding.

Fig. 1.

Stimulatory effect of PML/RARα on the transcription of the TF gene in U937 cells through an interaction with the TF promoter. (A) pSG5 eukaryocytic expression vectors encoding wild-type PML, RARα, and PML/RARα or its mutant forms, PML/RARαΔR and PML/RARαΔCC, were transfected into U937 cells. Total RNAs were isolated from the transfectants, TF transcription was determined by RT-PCR, and the samples were normalized based on GAPDH expression. (B) Total RNAs were isolated from U937-PR9 cells treated with 100 μM of ZnSO₄. The transcription of the TF and genes was examined by RT-PCR which was normalized by GAPDH expression. (C) Sonicated chromatin fragments of U937-PR9 cells were immunoprecipitated with antibodies specific for RARα (RARα-Ab), PML (PML-Ab), RNA polymerase II (POL II-Ab and POL II-Ab*), or a nonspecific antibody (NS-Ab). PCR was carried out with primers corresponding to the genomic TF promoter sequences except for the POL II-Ab–precipitated sample (POL II-Ab*), in which primers corresponding to GAPDH were applied. (D) Nuclear proteins extracted from U937-PR9 or U937-MT cells induced with or without ZnSO₄ were electrophoresed with 8% polyacrylamide gels and blotted with a polyclonal anti-RARα antibody. Lamin B served as a control.

Fig. 2.

Critical role of the sequences between −229 and −247 of the TF promoter in its transactivation by PML/RARα in U937-PR9 cells. (A) U937-PR9 cells stably expressing the luciferase gene under the control of full-length TF promoter (−2174) were incubated with ZnSO₄ for the indicated period. Luciferase data are expressed as the mean and SD of triplicate tests. (B) Nuclear proteins of the cells were analyzed by Western blotting with an anti-RARα antibody. (C) Schematic presentation of the TF promoter shows the putative transcription factor binding sites and the different truncation mutations used in this study. Numbering is indicated with respect to the transcriptional start site (bent arrow). (D) Luciferase analysis (mean and SD of triplicate tests) for the truncated TF promoters was performed; −247* represents U937-MT cells expressing TF promoter truncated at −247. (E) Designing strategies for the luciferase vectors and the exogenous TF promoter-specific primers are shown schematically. (F) ChIP assays were performed as described in the legend of Fig. 1. (G) Protein expression of PML/RARα and RARα in stable sublines (−247, −229, and −216) was examined by Western blotting.

Fig. 3.

Identification of the region within TF promoter pivotal for its transactivation regulated by the interaction with PML/RARα. (A) The sequence homology between the nucleotides −232 to −242 of the TF promoter (noncoding strand) and the consensus sequence of the matrix I$GAGAFACTOR_Q6 corresponding to the putative Drosophila melanogaster GAGA factor reactive element. The matrix I$GAGAFACTOR_Q6 was originated from TRANSFAC MATRIX TABLE (Release 9.2). The P0 column is the position index of the matrix. The A, C, G, and T columns indicate the occurrence of nucleotide at the corresponding positions. The consensus sequence of I$GAGAFACTOR_Q6 using IUPAC alphabets (S = C/G; W = A/T; M = A/C; N = A/C/G/T; R = A/G) is indicated by a hollow arrow, and the noncoding strand of the TF promoter is indicated by a solid arrow. The asterisked nucleotides constitute the core of the consensus sequence. (B) Schematic presentation of the TF promoter shows the mutations in TF promoter used in this study. U937-PR9 cells expressing the TF promoter containing truncations at −242 or at −238, a deletion from −235 to −238 (Δ−238∼−235) or a mutation from −235 to −237 (M−237∼−235) were established. Luciferase (C) and ChIP (D) data of these mutants are shown. (E) A schematic diagram that delineates a proposed model for the interaction of PML/RARα with the TF promoter. PML/RARα forms a complex through the coiled-coil domain of the PML moiety with the intermediary/partner protein(s) which interact with the−232∼−242 nucleotides (boxed) of the TF promoter with a crucial GAGC core (underlined). This interaction may influence the chromatin remodeling that alters the RNA polymerase II-related transcription initiation complex and thus the transactivation of the promoter.

Fig. 4.

Effect of the mutated AP-1 sites on the regulation of the TF promoter by PML/RARα. EMSA analysis for the interaction of PML/RARα with the TF promoter was performed using radiolabeled DNA probes spanning from position −250 to −196 of the TF promoter. (A) Nuclear extracts from U937-PR9 cells were incubated with −250∼−224, −232∼−211, and −216∼−196 probes. The upper (hollow) arrow indicates the position of sample wells. (B) The in vitro translated PML/RARα or RARα proteins were incubated with the −250∼−224 and −229∼−199 probes. The DR5 probe containing a putative RARE was used as a positive control; NS indicates nonspecific bands. (C) Nuclear extracts from U937-PR9 cells or in vitro translated PML/RARα or RARα proteins were electrophoresed and blotted with an anti-RARα antibody. (D) EMSA was performed with probes containing wild-type or mutated AP-1d (−232∼−211 or −232∼−211m) and AP-1p (−216∼−196 or −216∼−196m) sites. (E) U937-PR9 cells stably expressing luciferase gene under the control of TF promoter with simultaneously mutated AP-1d and AP-1p sites were assessed for luciferase activity. (F) ChIP assays were carried out with the same cells per the foregoing procedures.