c-sis/Platelet-derived Growth Factor-B Promoter Requirements for Induction during the 12-0- Tetradecanoylphorbol-1 3-acetate-mediated Megakaryoblastic Differentiation of K562 Human Erythroleukemia Cells’

Platelet-derived growth factor (PDGF), a powerful mitogen and chemoattractant, is composed of two subunits, A and B, which are synthesized by normal megakaryocytes. We have studied the transcriptional regulation of the c-sis/PDGF-B gene in human K562 erythroleukemia cells that have been induced to undergo megakaryoblastic differentiation by treatment with 12-O-tetradecanoylphorbol-13-acetate. Upon differentiation of these cells, c-sis/PDGF-B transcription is increased 50-100-fold. We show here that a minimal c-sis/PDGF-B promoter region, spanning nucleotides -64 to +6, retains full inducibility. Linker scanning mutagenesis within this minimal region identified four segments that were important for expression in differentiating K562 cells: a previously defined sis proximal element (SPE; -64 to -45), the TATA box, the 10 bp immediately downstream of the TATA box [TATA neighboring sequence (INS); -24 to -15], and the mRNA start site region. Combined mutation of the SPE and TNS resulted in a greater impairment of induction than did mutation of either sequence alone. In contrast, combined mutation of the SPE and the start site or of the TNS and the start site did not lower induction beyond that displayed by the least inducible single mutants. The combination of the SPE and the TNS was sufficient to confer wild-type levels of inducibility to a heterologous promoter. Both the SPE and the TNS were sensitive to alterations in the helical spacing between these elements and the TATA box. Using the electrophoretic mobility shift assay, we demonstrated Received 5/1/97; revised 6/30/97; accepted 6/30/97. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to mdicate this fact. 1 This work was supported by Grant BE-268 (to W. E. F.) from the American Cancer Society and Grants CA09135 (to G. C. K.) and CA07175 from the National Cancer Institute. 2 To whom requests for reprints should be addressed, at McArdle Laboratory for Cancer Research, University of Wisconsin, 1400 University Avenue, Madison, WI 53706. Phone: (608) 262-1275; Fax: (608) 262-2824; E-mail: [email protected]. binding of Sp family members and of two additional unidentified nuclear factors to the TNS in both 12-0tetradecanoylphorbol-13-acetate-treated and untreated cells. The TNS, therefore, appears to represent a target for a constitutively bound factor(s) that is required for cooperation with a differentiation-specific factor bound at the SPE to drive efficient c-sis/PDGF-B transcription in TPA-treated K562 cells. Introduction PDGF3 is the major mitogen in human serum for cells of mesenchymal origin. It is composed of two disulfide-linked subunits, PDGF-A and PDGF-B, which are encoded by separate genes on different chromosomes (1 , 2). The two subunits can pair to form homoor heterodimers that elicit biological responses through binding to cell surface receptors. There are two types of PDGF-Rs, PDGF-Ra and PDGFR , which also homoor heterodimerize upon ligand binding to initiate signal transduction (reviewed in Refs. 3 and 4). Responses to PDGF binding include not only mitogenesis but also chemotaxis, differentiation, actin filament reorganization, and membrane ruffling (5). PDGF has been implicated in a variety of neoplastic conditions. The PDGF-B gene is the cellular homologue of the v-sis gene found in the simian sarcoma and Parodi-Irgens feline sarcoma retroviruses (6-8), which cause fibrosarcomas and astrocytomas in their hosts (9). c-sis/PDGF-B can transform immortalized rodent cell lines (1 0, 1 1) and confer anchorage independence upon diploid human fibroblasts (12). Recombinant retroviruses carrying the entire c-sis/ PDGF-B chromosomal locus cause fibrosarcomas in mice (1 3). Expression of PDGF-A and/or PDGF-B is commonplace in many human tumor cell lines and tumor biopsies (reviewed in Ref. 14); such expression is often in contrast to their normal untransformed cell counterparts (1 5-1 9). Moreover, Wang et aL (20) showed that the degree of PDGF-B expression in PDGF-R positive soft tissue (mesenchymal) tumors strongly correlated with increasing tumor grade. Additional evidence implicating PDGF signaling pathways in human cancer is provided by the observation that a dominant negative form of PDGF-A (which can inhibit the activity of both 3 The abbreviations used are: PDGF, platelet-derived growth factor; PDGF-R, PDGF receptor; WA, 12-O-tetradecanoylphorbol-13-acetate; SPE, sis proximal element; TNS, TATA neighboring sequence; SDE, sis distal element; DH, DNaseI hypersensitivity; TdT, terminal deoxynucleotidyl transferase; Inr, initiator element; EMSA, electrophoretic mobility shift assay; TBP, TATA-binding protein; Egr-1 , early growth response-i. 964 TPA-inducible PDGF-B Promoter Elements 4 H-M. Jin and W. E. FahI, unpublished observation. PDGF-A and PDGF-B) causes the tumonigenic phenotype of some astrocytomas expressing PDGF-A, PDGF-B, and PDGF-Rf3 to revert (21). We are interested in understanding how expression of the c-sis/PDGF-B gene is regulated. Megakaryocytes are the presumed precursors to platelets and are one of the natural sites of PDGF synthesis, because platelets lack any transcniptional apparatus. Megakaryocytes, however, make up only -0.5% of all nucleated cells in the bone marrow (22), so it is difficult to obtain large numbers of these cells for study. Instead, we have chosen the K562 human erythroleukemia cell line as a model system for the study of c-sis/PDGF-B transcriptional regulation. These cells, which are derived from a chronic myelogenous leukemia, exhibit erythroid characteristics but can be induced to differentiate along a megakaryoblastic pathway by treatment with phorbol esters (for review, see Ref. 23). Such differentiation is manifested by morphological changes, decreased expression of erythroid markers, and up-regulation of some megakaryocyte-specific genes, such as c-sis/PDGF-B (24), PDGF-A (25), and gpllb and gpII!a integnins (26). The steady-state level of c-sis/ PDGF-B mRNA is induced in TPA-treated K562 cells by at least 200-fold, and much of this up-regulation takes place at the transcriptional level (24, 27). Previous studies using this system have revealed that a region extending from approximately 250 bp upstream to about 50 bp downstream of the c-sis/PDGF-B mRNA start site is sufficient to reproduce a majority of the mnducibility of the c-sis/PDGF-B gene (27, 28). These studies have identified the following regions as important for the expression of the c-sis/PDGF-B gene in differentiating K562 cells: a silencer region at -372 to -343 relative to the mRNA start site (27); the SDE at 103 to -93, which was part of a larger, positive regulatory region running from 103 to -60 (27); the SPE at -64 to -45 (28); and the region around the TATA box (-34 to 1 5; Ref. 28). The localization of functionally important regulatory sequences within the first 1 00 bp upstream of the c-sis/PDGF-B start site corresponds well to the presence of a DH site from approximately -90 to +60 that is present in TPA-treated K562 cells but not in untreated K562 cells (29). However, no DH in either TPA-treated or untreated K562 cells has been observed for the region of the promoter conresponding to the silencer element (29). Additional DH sites centered at +1 .9 and +4.0 kb are present in untreated but not in TPA-treated K562 cells and are capable of downregulating c-sis/PDGF-B promoter (1 12 to +43) reporter activity (29). These sites may, therefore, be contributing to transcriptional silencing of the endogenous c-sis/PDGF-B promoter in untreated K562 cells. Last, a combined silencer/ enhancer region located 7-1 1 kb upstream of the promoter seems to have a net positive effect on c-sis/PDGF-B reporter activity in both TPA-treated and untreated K562 cells (30). It has been argued that in untreated K562 cells, the + 1 .9/+4.0 kb DH sites serve to counteract this -1 1/-7 kb upstream enhancer region and that the combined activities of the upstream enhancer region and the proximal -1 00 bp of the c-sis/PDGF-B promoter can account for the total increase in c-sis/PDGF-B mRNA levels observed in TPA-treated K562 cells (30). Within the proximal promoter, nuclear factors that bind to the SPE and to the 1 03 to -60 positive regulatory element have been reported (27, 28), and some of these are specific for TPA-treated K562 nuclear extracts. The promoter elements identified thus far, however, are not sufficient to account for all of the induction displayed by the proximal c-sis/ PDGF-B promoter. For example, the SPE was unable to confer induction upon a heterologous promoter when it was tested in differentiating K562 cells, either individually or in combination with the SDE.4 Furthermore, the activity of the silencer region was observed in one study (27) but not in another (28). Therefore, we decided to identify sequences within the c-sis/PDGF-B promoter that were both necessary and sufficient for the transcriptional activation observed upon differentiation of K562 cells by TPA. Results Nested Deletions Define the Minimal, TPA-responsive PDGF-B Promoter. Previously, we have used linker scanning mutational analysis to identify sequences within the c-sis/PDGF-B promoter that are important for the transcriptional induction that occurs when human K562 erythroleukemia cells undergo TPA-mediated megakaryoblastic differentiation (28). The two important regions of the promoter identified from this analysis were the SPE, at -64 to -45 relative to the transcriptional start site, and the region encompassing the TATA box. The SPE, however, proved to be insufficient to confer TPA inducibility when it was placed upstream of a heterologous promoter (TI-Luc) containing the TATA region from the adenovirus major late promoter and the initiator region from the murine TdT gene4 (see also Fig. 3). We therefore decided to undertake a search for the additional element(s) within the c-sis/PDGF-B promoter that, in combination with the SPE, is required for full TPA inducibility. To define a minimal promoter region that retained TPA mnducibility, we constructed a series of 5’ and 3’ nested deletion mutants of the human c-sis/PDGF-B promoter. We knew from earlier work that a CAT reporter construct contaming the region from -257 to +75 of the c-sis/PDGF-B promoter retains approximately the same level of induction as a larger -4-kb promoter-reporter construct when transfected into K562 cells (28). We extended this analysis by deleting additional sequences from the 5’ and 3’ ends of the promoter (Fig. 1A). These nested deletion mutants were transfected into K562 cells, followed by treatment with either TPA or solvent (ethanol); hereafter, solvent-treated cells are referred to as “untreated.” In this experiment, deletion of -403 to -257 (NA-Luc versus pRA-Luc) reduced the level of induction by nearly 50% (Fig. 1B). However, the degree of induction displayed by NA-Luc was still far greater than that observed for any of the “uninducible” promoters (e.g. , 44CLuc or TI-Luc; see below). A previously identified silencer region maps to the region deleted in NA-Luc, yet this promoter showed no increase in activity compared to the larger pRA-Luc, which does contain the putative silencer element.