MOLECULAR AND CELLULAR BIOLOGY, July 1992, p. 3107-3116 0270-7306/92/073107-10$02.00/0
Vol. 12, No. 7
Copyright ©D 1992, American Society for Microbiology
Oct2 Transactivation from a Remote Enhancer Position Requires a B-Cell-Restricted Activity T. WIRTH`* Zentrum fOir Molekulare Biologie Heidelberg, Im Neuenheimer Feld 282, D-6900 Heidelberg, Gennany, and Institut fiir Molekularbiologie II der Universitat Zurich, CH 8057 Zurich, Switzerland2 A. ANNWEILER,1 M. MULLER-IMMERGLUCK,2
Received 16 January 1992/Accepted 17 April 1992
Previous cotransfection experiments had demonstrated that ectopic expression of the lymphocyte-specific transcription factor Oct2 could efficiently activate a promoter containing an octamer motif. Oct2 expression was unable to stimulate a multimerized octamer enhancer element in HeLa cells, however. We have tested a variety of Oct2 isoforms generated by alternative splicing for the capability to activate an octamer enhancer in nonlymphoid cells and a B-cell line. Our analyses show that several Oct2 isoforms can stimulate from a remote position but that this stimulation is restricted to B cells. This result indicates the involvement of either a B-cell-specific cofactor or a specific modification of a cofactor or the Oct2 protein in Oct2-mediated enhancer activation. Mutational analyses indicate that the carboxy-terminal domain of Oct2 is critical for enhancer activation. Moreover, this domain conferred enhancing activity when fused to the Octl protein, which by itself was unable to stimulate from a remote position. The glutamine-rich activation domain present in the amino-terminal portion of Oct2 and the POU domain contribute only marginally to the transactivation function from a distal position.
Regulation of gene expression in higher eucaryotes is governed both by promoter elements localized just upstream of the gene and additional enhancer elements which can function over long distances. These cis-acting elements interact with a diverse set of transcription factors that often show a preference for either promoter (i.e., Spl or CCAATbinding transcription factor/nuclear factor I) or enhancer elements (i.e., factors interacting with the E-box motifs in immunoglobulin enhancers or the bovine papillomavirus E2 transactivator) (12, 23). However, several transcription factors have been shown to function from both proximal promoter and remote enhancer positions (2, 30, 42). The octamer motif ATGCAAAT plays a central role in immunoglobulin gene expression. This site is conserved in the heavy-chain enhancer elements as well as in virtually all heavy- and light-chain promoter elements (7, 29). It is also found in the promoter regions of other genes specifically expressed in B cells (15), and slightly aberrant octamer motifs have been identified in the intronic enhancer of the immunoglobulin K light-chain gene (3, 26). In addition, it is present in the regulatory regions of a variety of genes that do not show B-cell-restricted expression (7). Mutational analyses have confirmed the importance of this motif for specific promoter and enhancer functions (10, 19, 21, 44). Several genes that encode octamer-binding proteins have been identified, and all these proteins show homology in the domain responsible for specific DNA binding, the so-called POU domain (la, 24, 27, 31, 34, 38, 39). The POU domain consists of a conserved POU-specific (75 to 80 amino acids long) subdomain and a POU-homeo (60 amino acids long) subdomain which are separated by a less well-conserved linker of variable length (16). The lymphoid cell-specific functions of the octamer motif have been attributed to the Oct2 transcription factor, a lymphoid cell-specific protein which is coexpressed in B cells with the ubiquitous Octl protein (20, 34, 35). Mouse and human B cells express *
multiple Oct2 isoforms which arise from a singular Oct2 gene by an alternative splicing mechanism (43). These Oct2 isoforms all contain the conserved POU domain, and they are coexpressed at all stages of B-cell development (43). Ectopic expression of Oct2 in HeLa cells or NIH 3T3 fibroblasts has been shown to activate a promoter containing an octamer motif (9, 24, 25, 40, 43). Those experiments identified two regions in the Oct2 protein contributing to full transactivating potential, a glutamine-rich domain in the amino terminus and a region carboxy terminal to the POU domain (9, 25, 40). In those experiments, a multimerized octamer motif functioning as an efficient B-cell-specific enhancer could not be stimulated in HeLa cells by coexpression of Oct2 (25). Two alternate hypotheses could explain this result: (i) as only a single Oct2 isoform had been tested, it was possible that one of the other recently identified variants could be responsible for enhancer transactivation; (ii) alternatively, Oct2 could require a B-cell-specific component for enhancer activation that is missing in the HeLa cell line. We have tested these two models experimentally by cotransfecting a variety of Oct2 isoforms with a reporter bearing multimerized octamer motifs as enhancer. Both nonlymphoid cells and a B-cell line containing very low levels of endogenous Oct2 were transfected. We show that the enhancer can be activated by several Oct2 isoforms but that this activation can be achieved only in a B-cell background. In contrast, a fusion protein of Oct2 with the very strong acidic transactivation domain of the herpes simplex virus VP16 protein can readily stimulate the enhancer in non-B cells, too. Furthermore, our analyses indicate that the C terminus of Oct2 is crucial for efficient enhancer transactivation. MATERIALS AND METHODS
Plasmid constructions. The pCL plasmid containing the chicken lysozyme promoter from -579 to +15 upstream of the luciferase gene was supplied by A. Hecht. The six copies
Corresponding author. 3107
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of either the wild-type or mutated enhancer fragment have been described previously (10). They were inserted into a unique SmaI site downstream of the luciferase gene. All inserts had the same orientations. The octamer promoter constructs were derived from the pTATA plasmid (obtained from A. Hecht), which contains the herpes simplex virus thymidine kinase promoter from -38 to +52 upstream of the luciferase gene. The wild-type octamer motif ATGCAAAT and a motif with a point mutation, ATGAAAAT, were used. Expression vectors carrying the Oct2 isoforms were the described cytomegalovirus expression constructs (43), pGD retroviral vectors (4), or Rous sarcoma virus-based expression vectors (vector obtained from R. Renkawitz). The different expression vectors yielded identical results and were used interchangeably. The Oct2 deletion mutants and the Octl-Oct2 fusion proteins have been described previously (9, 25, 40). The Oct2-VP16 fusion was constructed as follows. The VP16 transactivation domain was isolated by polymerase chain reaction amplification from the murine sarcoma virus VP16 expression plasmid. The primers used were 5' CCATCGATACACACGCGCAGACTGTCGACG 3' (positions 2054 to 2073) and 5' CGTCTAGAACCCACCGTACT CGTCAATTCC 3' (positions 2287 to 2307) (28). The amplified fragment corresponds to amino acids 406 to 488 of the VP16 protein. The fragment was cut with ClaI and XbaI, filled in with the Klenow fragment of DNA polymerase, and inserted into the filled-in EcoRI site of a pBluescript plasmid containing the Oct2.6 isoform which had the C terminus just downstream of the POU domain deleted (partial SmaI deletion). The fusion was then transferred to the Rous sarcoma virus expression vector. Cell culture and DNA transfections. S194 cells were kept in Iscove's modified Dulbecco modified Eagle medium and transfected with DEAE-dextran as described elsewhere (1). Conditions for culturing and transfection of NIH 3T3 fibroblasts (43) and chicken HD11 macrophages were described previously (37). Whole-cell extracts for mobility shift assays were prepared by freeze-thaw lysis in buffer C (6). Conditions for band shift assays with the cleared lysates and the octamer fragment from the heavy-chain enhancer were as described previously (42). Whole-cell extracts for enzyme assays were obtained by freeze-thaw lysis in 0.1 M potassium phosphate-1 mM dithiothreitol, pH 7.8. Luciferase assays were performed as described previously by using a Berthold Lumat 9501 (5). Analysis of 3-galactosidase activity was performed by following standard protocols (14). All luciferase expression values were corrected for 3-galactosidase enzyme levels. RESULTS Oct2 isoforms can activate an octamer-containing enhancer in B cells. It had been shown previously that six copies of a short fragment from the mouse intronic heavy-chain enhancer encompassing the octamer motif and the ,uE4 motif can function as an efficient B-cell-specific enhancer element (10). We therefore generated reporter plasmids bearing this multimerized fragment about 2 kb downstream of the transcriptional initiation site of the luciferase gene (Fig. 1). We chose the chicken lysozyme promoter as the proximal promoter element because it showed fairly low activity in B cells on its own (about threefold higher than a reporter construct containing only a TATA box; unpublished observations), and it was stimulated to high levels of activity by
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the multimerized wild-type enhancer fragments (Fig. 1B). A reporter construct bearing the P-galactosidase gene under the control of a strong enhancer (cytomegalovirus lacZ or Rous sarcoma virus lacZ) was included in all transfections. All luciferase expression values were normalized for ,-galactosidase activity in the same cell extracts. Consistent with previous findings, the enhancer activity was observed only in the S194 plasmacytoma cell line and not in fibroblasts or in a chicken macrophage cell line, in which the promoter showed significantly higher background activity (Fig. 1A and data not shown). In agreement with previous observations, mutation of the ,uE4 motif reduced enhancer activity only marginally, whereas the octamer mutation led to a strong reduction of enhancer activity, confirming the importance of the octamer motif for the activity of this enhancer element (10). Interestingly, the residual enhancer activity was still B cell specific (Fig. 1A and data not shown) (10). A double mutation of the ,E4 motif and the octamer site abolished this residual enhancer activity (data not shown) (10). To examine whether any of the Oct2 isoforms could activate the enhancer, we performed cotransfections with NIH 3T3 fibroblasts. However, none of the isoforms was capable of stimulating the enhancer to a measurable level (data not shown). As a positive control, we constructed a fusion protein encompassing the amino terminus and the POU domain of Oct2 and the transactivating domain of the herpes simplex virus VP16 transactivator (Fig. 2B), and this construct activated the wild-type enhancer about 20-fold in NIH 3T3 fibroblasts. Similarly, only this Oct2-VP16 fusion could activate the enhancer in a chicken macrophage cell line (data not shown). These results showed that the multimerized octamer enhancer could be activated in non-B cells but that Oct2 alone was unable to do so. From these results, the second of the models, namely, the involvement of a B-cell-specific activity required for Oct2 function from a remote position, became increasingly attractive. To test this idea, we cotransfected the various reporter constructs with the Oct2 expression vectors into the mouse S194 plasmacytoma cell line, which is characterized by low levels of endogenous Oct2 proteins (Fig. 2C, lane 1). Despite these low levels of endogenous Oct2, the multimerized enhancer fragment yielded already significant octamerdependent activity (Fig. 1A). Cotransfection with the Oct2 expression vectors resulted in a clear stimulation of this baseline activity (Fig. 2A). There was considerable variation in the transactivation potentials of various Oct2 isoforms. Three of the isoforms (Oct2.1, Oct2.2, and Oct2.6) gave a clear stimulation, whereas the others (Oct2.4, Oct2.5, and Oct2.3) had little effect or even led to a slight reduction of the baseline activity. This variation was observed over a range of expression vector concentrations (data not shown). The analysis of Oct2 proteins by mobility shift assays revealed that with the exception of the Oct2.4 isoform, all isoforms accumulated to comparable levels (Fig. 2C). Western (immunoblot) analyses confirmed that the levels of expression determined by the mobility shift assays correlated with the amount of stable protein (data not shown). Under these conditions, the transfected cells contained much more exogenous Oct2 protein than endogenous Oct2 factors (Fig. 2C, compare lane 1 with lanes 2 to 7). The assumption that endogenous and exogenous Oct proteins compete for binding sites in the enhancer is supported by the finding that inactive Oct proteins repressed the activity of the enhancer constructs to levels of the reporters bearing mutated octamer motifs (see below). Therefore, the measured activity is almost exclusively contributed by the transfected isoform.
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FIG. 1. Activity of the various reporter constructs in S194 and NIH 3T3 cells. (A) Plasmids were transfected into either S194 cells or NIH 3T3 cells, and enzyme activity was determined 24 h after transfection. The activity of the enhancerless pCL plasmid was arbitrarily set to 10 for each cell type. The letters in parentheses identify the enhancer present in the constructs. Abbreviations: ED, wild-type E4 and octamer motifs; eD, mutant E4 motif, wild-type octamer motif; Ed, wild-type E4 motif, mutant octamer motif. Transfection efficiencies were controlled by inclusion of a ,B-galactosidase expression construct. Standard deviations are indicated by error bars. (B) Schematic representation of the reporter construct. The chicken lysozyme promoter from -579 to +15 is represented by the stippled box; luciferase gene and the rabbit P-globin splice and poly(A) region are shown as empty rectangles. The black box indicates the position of the multimerized enhancer when present. The blowup shows the orientation of the E4 and octamer motifs in the constructs.
Differential contribution of the Oct2 transactivation domains to enhancer stimulation. The low activity of the Oct2.4 isoform could be attributed to low protein accumulation. The other two Oct2 isoforms that do not yield efficient stimulation show normal stability, however. The differences be-
tween Oct2.3 and Oct2.5 compared with the more active
Oct2 isoforms are localized in different regions of the Oct2 protein. The Oct2.5 isoform is the largest isoform that carries an extended C terminus due to an out-of-frame insertion of a miniexon (Fig. 2B). The Oct2.3 isoform
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O tci2 -V P l1 6 I FIG. 2. Cotransfection of Oct2 isoforms can stimulate the octamer-containing enhancer in S194 cells. (A) Three micrograms of the respective reporter constructs was cotransfected with 2 ,ug of expression vectors carrying the different Oct2 isoforms into S194 cells. All expression values are expressed in relation to the activity of the pCL(ED) construct cotransfected with the parental expression vector (bar 1). This value was set to 100. Bars: 1 to 7, cotransfections with the pCL(ED) reporter plasmid; 8 to 14, cotransfection with the pCL(eD) reporter plasmid; 15 to 21, cotransfection with the pCL(Ed) reporter plasmid. The parental expression vector (bars 1, 8, and 15), a vector expressing the Oct2.1 isoform (bars 2, 9, and 16), the Oct2.2 isoform (bars 3, 10, and 17), the Oct2.3 isoform (bars 4, 11, and 18), the Oct2.4 isoform (bars 5, 12, and 19), the Oct2.5 isoform (bars 6, 13, and 20), or the Oct2.6 isoform (bars 7, 14, and 21) was included in the cotransfections. The insert shows the stimulation of the different reporter constructs by the Oct2-VP16 fusion in S194 cells. Bars: 1, 3, and 5, cotransfection with the parental expression vector; 2, 4, and 6, cotransfection with the Oct2-VP16 fusion. Standard deviations are indicated
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contains a short insert of 22 amino acids in front of the glutamine-rich transactivation domain (Fig. 2B; for details, see reference 43). From these considerations, the contribution of the two described transactivation domains of the Oct2 protein to enhancer transactivation remained unclear. To directly address the question of whether both transactivation domains are equally important for enhancer stimulation, we cotransfected the reporter plasmids with Oct2 mutants lacking the whole region either N terminal or C terminal of the POU domain (Fig. 3B). The parental Oct2 isoform was the human homolog of the mouse Oct2.2 form. Expression of these mutants in HeLa cells efficiently stimulated an octamercontaining promoter. The N-terminal deletion had shown a moderately reduced transactivating potential (25). These deletion mutants had previously been characterized in non-B cells only, and we therefore determined whether they showed similar promoter transactivation features in the B-cell line. We cotransfected them together with a luciferase reporter carrying a single wild-type or mutant octamer motif upstream of the herpes simplex virus thymidine kinase TATA box into the S194 cell line. As it had been shown that overexpression of Octl is also capable of activating an octamer-containing promoter in HeLa cells (18), we included Octl in our analyses, too. The results closely resembled those of the HeLa cotransfection experiment. Deletion of either transactivation domain did not significantly affect the stimulatory potential, confirming the previous notion of a functional redundancy of the two transactivation domains. Consistent with the results with HeLa cells, Octl also weakly stimulated the octamer-containing promoter (data not shown). However, a strikingly different picture emerged when these expression vectors were cotransfected with the reporter constructs bearing the multimerized octamer enhancer element (Fig. 3A). Whereas removal of the glutamine-rich N-terminal transactivation domain had essentially no effect on the transactivating potential, deletion of the C-terminal region turned out to be a strong down mutation. With increasing amounts of cotransfected expression vector, this difference became increasingly obvious. The intact Oct2 isoform showed somewhat lower levels of transactivation at low concentrations of expression vector due to reduced protein accumulation (Fig. 3C). At elevated expression vector concentrations, this effect was obliterated (data not shown). Most surprisingly, cotransfection of the Octl expression vector resulted in repression of the enhancer construct. The level of expression seen with cotransfected Octl was similar to the one obtained with the reporter construct bearing the multimerized octamer mutation. This result confirmed the above speculations that endogenous and exogenous Oct proteins compete for available binding sites and that at a high concentration of exogenous protein the contribution of the endogenous proteins becomes negligible. The repression of octamer-dependent enhancer activity by Octl allowed us to reassess the importance of individual domains of the Oct2 protein by using Octl-Oct2 fusions. Both proteins are very similar in the POU domain; their
POU-specific domains are virtually identical (at 74 of 75 positions), and their POU-homeo domains are also well conserved (53 of 60 positions) but nevertheless show some functional differences with respect to protein-protein interactions (36). We cotransfected a set of Octl-Oct2 fusion proteins (Fig. 4B) (40) with the enhancer reporter, and the results are shown in Fig. 4A. For enhancer transactivation, the Oct2 carboxy-terminal domain was absolutely required. Whenever the Octl C terminus was present in the fusion proteins, the expression level was repressed. Neither the Oct2 N-terminal transactivation domain nor the POU domain of Oct2 had a measurable stimulatory effect in the presence of the Octl C terminus. In all our transfections, the hybrid construct 2-1-2 was somewhat more active than the 1-1-2 hybrid. We cannot rule out that this difference is due to slight differences in the accumulation of the respective proteins. The transactivation by the wild-type Oct2 protein was always higher than that of the fusion protein, in which only the Oct2 POU domain was replaced by the respective domain from Octl. This difference remained even when increasing concentrations of the fusion protein expression vectors were used and was not due to different expression levels of the respective proteins (data not shown). Finally, we wanted to map the region within the Oct2 C terminus required for remote activation more precisely. Analysis of the contribution of the Oct2 C terminus to promoter activation in HeLa cells had revealed that the C-terminal 18 amino acids were important for transactivation (9). In addition, the Oct2 C-terminal domain contains a putative leucine zipper motif (la, 24, 31). Mutation of this motif did not affect promoter transactivation in HeLa cells (9, 40). We therefore tested several C-terminal mutations for the capacity to activate from a distance. Consistent with the previous observation (Fig. 3A), the deletion of most of the C terminus (A377 to 463) reduced activity 2.5-fold and therefore essentially abolished enhancer activation (Fig. 5). Interestingly, deletion of almost the complete leucine zipper motif had no effect on the observed transactivation. The region C terminal to the leucine zipper was necessary for full enhancing activity, however. Both an internal deletion (A377 to 443) and the removal of the 18 C-terminal amino acids (A443 to 463) resulted in a partial reduction of activity (Fig. 5). No significant differences in the stabilities of the various proteins with deletions were observed (data not shown). DISCUSSION Oct2-mediated enhancer activation is B cell specific. We have shown that the Oct2 transcription factor can efficiently stimulate from a promoter position and also from a distant enhancer position. In contrast, the ubiquitous Octl transcription factor can stimulate only from a proximal promoter position. Whereas promoter transactivation can be achieved in a variety of cell types (9, 24, 25, 40), activity from a remote position requires an activity that is present in B cells but absent from several non-B-cell lines. There are several possible explanations for this observation. We favor the idea that Oct2 needs to interact with a
by error bars. Abbreviations, same as for Fig. 1A. (B) Schematic outline of the various Oct2 isoforms and the Oct2-VP16 fusion. Oct2 transactivation domains TA-1 and TA-2 are indicated. For details on the Oct2 isoforms, see reference 43. (C) Aliquots of the transfected cells were analyzed by electrophoretic mobility shift assay with a labeled octamer-containing probe. S194 cells were transfected with the parental expression vector (lane 1) or expression vectors containing the cDNAs coding for Oct2.1 (lane 2), Oct2.2 (lane 3), Oct2.3 (lane 4), Oct2.4 (lane 5), Oct2.5 (lane 6), or Oct2.6 (lane 7).
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