Cell, Vol. 71, 231-241,

October

16, 1992, Copyright

0 1992 by Cell Press

A Novel B Cell-Derived Coactivator Potentiates the Activation of lmmunoglobulin Promoters by Octamer-Binding Transcription Factors Yan Luo, Hiroshi Fuji,’ Thomas Gerster,t and Robert G. Roeder Laboratory of Biochemistry and Molecular Biology The Rockefeller University New York, New York 10021

Summary A novel B cell-restricted activity, required for high levels of octamer/Octdependent transcription from an immunoglobulin heavy chain (IgH) promoter, was detected in an in vitro system consisting of HeLa cellderived extracts complemented with fractionated B cell nuclear proteins. The factor responsible for this activity was designated Ott coactivator from B cells (OCA-B). OCA-B stimulates the transcription from an IgH promoter in conjunction with either Ott-1 or Ott-2 but shows no significant effect on the octamer/Octdependent transcription of the ubiquitously expressed histone H2B promoter and the transcription of USFand Spl-regulated promoters. Taken together, our results suggest that OCA-B is a tissue-, promoter-, and factor-specific coactivator and that OCA-B may be a major determinant for B cell-specific activation of immunoglobulin promoters. In light of the evidence showing physical and functional interactions between Ott factors and OCA-8, we propose a mechanism of action for OCA-B and discuss the implications of OCA-B for the transcriptional regulation of other tissue-specific promoters. Introduction The B cell-specific expression of immunoglobulin genes reflects the presence of both B cell-specific promoters and B cell-specific enhancers (for review see Staudt and Lenardo, 1991). The B cell specificity of some immunoglobulin promoters is due solely to an octamer (consensus AllTGCAT) element (Dreyfus et al., 1987; Wirth et al., 1987) although this same element is also implicated in the ubiquitous expression of small nuclear RNA genes (for review see Parry et al., 1989) the cell cycle regulation of the histone H2B gene (LaBella et al., 1988) and the VP1 6dependent expression of herpes virus intermediate early genes (Gerster and Roeder, 1988; O’Hare and Goding, 1988; Stern et al., 1989). Octamer-binding factors that are presumptive transcriptional activators include the ubiquitous Ott-1 , the tissue-specific Ott-2, and other developmentally regulated factors (for review see Scholer, 1991). All are members of the POU domain family of regulatory factors containing conserved POU-specific and * Present address: Department of Biochemistry, Niigata University School of Medicine, Niigata 951, Japan. TPresent address: Siozentrum der Universitlt Basel, Abteilung Zellbiologie, 4066 Basel, Switzerland.

POU homeodomains (Herr et al., 1988; for review see Rosenfeld, 1991). Consistent with their presumed roles as transcriptional activators, purified Ott-1 and Ott-2 were shown both to bind to and stimulate transcription from H2B and immunoglobulin promoters, respectively (Fletcher et al., 1987; Scheidereit et al., 1987). In addition, ectopic Ott-2 was shown to stimulate transcription of reporter genes with artificial B cell-specific promoters in nonlymphoid cells (Gerster et al., 1990; Tanaka and Herr, 1990; Miiller et al., 1988; Miller-lmmergluck et al., 1990). Along with the earlier demonstrations that Ott-2 is largely B cell restricted (Staudt et al., 1988; Landolfi et al., 1986) and that octamer-dependent transcription of immunoglobulin promoters is markedly more efficient in B cell extracts than in nonlymphoid cell extracts (Mizushima-Sugano and Roeder, 1986; Poellinger et al., 1989), these observations led to the presumption that Ott-2, rather than Ott-1, is involved in and, indeed, responsible for the B cell specificity of immunoglobulin promoters. More recently, the view that Ott-2 is the major or sole determinant for tissue-specific immunoglobulin promoter function has come into question on the basis of several observations. First, despite its effect on artificial octamercontaining promoters, ectopic Ott-2 did not efficiently activate normal immunoglobulin promoters in cotransfection assays (Gerster et al., 1990). Second, the levels of Ott-2 are not strictly correlated with immunoglobulin expression in various cell types (Cockerill and Klinken, 1990; Hatzopoulos et al., 1990). Third, comparative studies of homogeneous preparations of Ott-1 and Ott-2 showed that the binding of these factors to immunoglobulin (and H2B) promoters was quantitatively, as well as qualitatively, the same and, most significantly, that they effected equivalent levels of octamer-dependent transcription from immunoglobulin promoters (and from an H2B promoter) in reconstituted Ott-dependent systems (Pierani et al., 1990; see also LeBowitzet al., 1988). Along with the fact that the level of Ott-l- or OctQ-dependent immunoglobulin promoter transcription observed in these systems was far less than that observed in B cell extracts, these observations suggested that another B cell-specific factor(s) or a B cellspecific Ott modification was responsible for the high levels of octamer-dependent transcription that typify B lymphoid cells. Initial evidence for such a factor was provided by previous studies (Pierani et al., 1990) showing that oligonucleotide affinity column-bound factors (including Ott-1 and Ott-2) from lymphoid cells restored high levels of immunoglobulin transcription, equivalent to those observed with B cell extracts, to Ott-depleted HeLa extracts. Here we report the isolation and functional characterization of a novel tissue-specific coactivator that facilitates efficient immunoglobulin promoter transcription by either Ott-1 or Ott-2. These results have significant implications regarding the proposed role of a number of site-specific DNA-binding proteins as the major determinants Of tissuespecific gene expression.

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1. Activation

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(A) Diagrams of IgH and reference promoter templates. The IgH construct (Poellinger et al., 1989) contains an IgH (BCL,) promoter segment (-73 to -16) cloned into the Sacl-Pstl sites of the OVEC vector (Westin et al., 1987) immediately upstream of the rabbit 8-globin coding sequence. The end-labeled probe for Sl mapping of IgH transcripts spans nucleotide positions -18 to +75 of the hybrid gene and creates a 75 nt signal from specifically initiated transcripts. The reference promoter (Re9, which monitors basal activity independently of upstream elements, contains a human immunodeficiency virus minimal promoter fused to a shorter version of the p-globin coding sequence and reveals a 60 nt signal after Sl mapping with the same probe employed for the IgH template. The wild-type (WT) and mutant (0.) octamer (OCTA) sequences in the IgH templates are shown at the bottom. (B) Effect of isolated B cell factor OCA-B on IgH promoter transcription in a HeLa cell extract. Aliquots (5 ul) of BC109, 250 uglml BSA (minus), or fractions of OCA-B (F7, FE, F9) from the lectin-agarose chromatographic step were used to complement HeLa extracts (12 ul) using IgH and reference (Ref) promoters as templates. (C) A scheme showing the purification of various factors. The numbers indicate the molar concentrations of KCI in the BC buffers. For detail see Experimental Procedures.

Results Identification of a B Cell-Specific Coactivator for Ott-1 and Ott-2 Our previous study (Pierani et al., 1990) showed that octamer oligonucleotide affinity-bound components from B cells, in contrast with purified Ott-1 or Ott-2, fully stimulated immunoglobulin promoter transcription in Octdeficient HeLa extracts. These results were consistent either with the presence of a secondary factor bound with Ott-1 or Ott-2 or with a cell-specific modification of Ott-1 or Ott-2 that was lost upon further purification. Consistent with the first possibility, further fractionation of the oligonucleotide matrix-bound fraction by ion-exchange chromatography resulted in the separation of a novel B cell factor (coactivator) that, along with purified Ott-1 or Ott-2, stimulated immunoglobulin promoter transcription in Oct-ldepleted HeLa extracts (H. F. and R. G. R., unpublished data). Subsequent studies described here employed preparations of the new B cell factor that was separated from

Ott-1 and Ott-2 by differential elution from a lectin affinity column. In this procedure (Experimental Procedures and Figure lC), Ott-2 and other general factors eluted in the 0.1 M KCI flow-through fraction while all Ott-1 and most of the new B cell factor remained bound. The tightly bound Ott-1 was resistant to KCI concentrations up to 1 M but was eluted with 0.4 M N-acetyl-glucosamine (Pierani et al., 1990). In contrast, the new B cell factor was eluted from the column (prior to Ott-1) by 0.5 M KCI, and the peak fractions were free of Ott-1 and Ott-2 as monitored by electrophoretic mobility shift assay (EMSA). Based on studies to be described below, the factor was designated OCA-B for Octcoactivator from B cells. As assayed with the templates described in Figure 1A, this factor markedly increased immunoglobulin heavy chain (IgH) promoter transcription in HeLa extracts (Figure 1 B) to a level equivalent to that observed in B cell extracts (see Figures 28 and 3 below). A comparison of the intact (WT) and mutant octamer (0-) IgH promoter templates revealed that both the basal and the OCA-B-dependent activities were dependent upon an intact octamer element, whereas an oc-

Cell-Specific 233

Coactivators

tamer-independent reference promoter (described in Figure 1A) was not affected by peak fractions (F8 and F9) of OCA-B (Figure 1 B). A nonspecific activity (Figure 1 B, F7) that slightly stimulated the transcription from the reference promoter was consistently observed to elute just ahead of the peak OCA-B fractions but did not complicate subsequent analyses with pooled (F7, F8, and F9) fractions. Moreover, this activity appeared to be separable from OCA-B upon further fractionation on a DE-52 column (see below). These results indicate function of OCA-B in HeLa cell extracts through the endogenous Ott-1 . To compare directly the ability of Ott-1 versus Ott-2 to mediate activation by OCA-B, antibodies specific for Ott-1 or Ott-2 were used to deplete HeLa extracts of Ott-1 and Namalwa (B cell) extracts of either Ott-1 or Ott-2. The efficacy and selectivity of Ott removal was demonstrated by EMSAs (Figure 2A) and by Western blots (data not shown) of the depleted extracts. As shown in Figure 28, Ott-depleted HeLa extracts showed only a very low level of IgH transcription (Figure 28, lane 2) that was stimulated slightly by purified Ott-1 (Figure 28, lane 3) consistent with the previous demonstration of a low level of octamerdependent transcription of the IgH promoter in HeLa extracts(Poellinger et al., 1989). In striking contrast, the addition of OCA-B with Ott-1 effected a very large increase in IgH transcription (Figure 28, lane 4) while OCA-B alone had no effect (Figure 2B, lane 5). Moreover, as shown by this analysis (Figure 28, compare lanes 1 and 4) and a similar study with unfractionated extracts (see Figure 3, lanes l-3), the presence of OCA-B (with Ott-1) in HeLa extracts elevated IgH promoter transcription to a level comparable with that observed in B cell extracts. To compare directly the cellular specificity of the coactivator activity, high salt eluates of lectin columns loaded separately with HeLa and Namalwa nuclear extracts were assayed in Ott-l-depleted extracts with exogenous Ott-1 The results in Figure 28 (lanes 8 and 7) clearly show the absence of a HeLa activity comparable to OCA-B. The parallel fractionation of HeLa and Namalwa extracts by more conventional means (ion-exchange chromatography) also failed to reveal a HeLa activity comparable to OCA-B (H. F. and R. G. Ft., unpublished data). These results demonstrate the 6 cell specificity of OCA-B relative to HeLa cells, although a more extensive analysis of the cell-type specificity of OCA-B remains to be performed. A direct comparison of the OCA-B-independent and OCA-B-dependent activities of purified Ott-1 and Ott-2 is shown in Figure 28. In agreement with previous studies, Ott-1 and Ott-2 stimulated IgH promoters to comparable but low extents (Figure 28, lanes 8, 9, and 11) and, importantly, each effected an equivalent large increase in IgH promoter transcription in the presence of a more highly purified preparation of OCA-B (Figure 28, lanes 8,10, and 12). Quantitation by densitometry consistently revealed a 2- to 3-fold stimulation of IgH promoter by either Ott-1 or Ott-2 and an additional 5 to 1O-fold stimulation by OCA-B in Ott-depleted HeLa nuclear extracts (Figure 2B and see below). The stimulation level of IgH transcription effected by OCA-B in HeLa extracts in conjunction with Ott-1 or Ott-2 approximates that observed in B cell extracts when

normalized to an octamer-independent promoter (Figure 28; see also Figure 3). These and other functional studies (data not shown) have consistently failed to show any difference in the ability of Ott-2 or Ott-1 from either B cells or HeLa cells to mediate IgH transcription in the presence of OCA-B. As can be seen in Figure 2C, the stimulation of IgH promoter transcription by OCA-B in HeLa nuclear extract (Figure 2C, lanes 3-9) was roughly linear over a certain range and appeared to be saturable at a level of transcription comparable to that observed in Namalwa nuclear extracts (Figure 2C, lane 1) or in Ott-P-depleted Namalwa extracts (Figure 2C, lane 2; see also Figure 5). An Ott factor dose-dependent stimulation of the IgH promoter in Ott-depleted HeLa nuclear extracts is shown in Figure 2D. In agreement with the results of Figure 28 and Pierani et al. (1990) Ott-1 stimulated the IgH promoter to only a small extent (maximum 3-fold) even at a saturating concentration of 50 fmol of Ott-1 to 10 fmol of template. Very similar results were observed with Ott-2 alone (Pierani et al., 1990; data not shown). However, activation by OCA-B was equally prominent (,5-fold) for Ott-1 and Ott-2 over the entire range of Ott-1 and Ott-2 tested, including the lowest ratio of 0.4 fmol of Ott-1 or Ott-2 per 10 fmol of template. With the more purified OCA-B (DE-52 fraction) used in these two experiments, the activity that was present in the cruder fraction and that slightly stimulated the reference template (see above and Figure 3) was not observed under any conditions (see Figure 2D). Promoter- and Activator-Dependent Specificity of OCA-B In view of the demonstration that the B cell-specific coactivator potentiates the ability of either Ott-1 or Ott-2 to stimulate the IgH promoter, it was important to investigate further both the promoter specificity and the activator specificity of OCA-B. To test the promoter specificity of OCA-B, parallel experiments were conducted with both IgH and H2B templates (shown in Figures 1A and 3, respectively). The H2B template is an important control since it contains an octamer element through which the S phase activation is mediated by a mechanism involving Ott-1 (Fletcher et al., 1987; LaBellaet al., 1988). In agreement with our previous study (Pierani et al., 1990), the depletion of Ott-1 in HeLa extracts (see Figure 2A) effectively eliminated H2B transcription, whereas purified Ott-1 and Ott-2 both effected substantial and equivalent increases in H2B transcription (Figure 3, lanes 4, 5, and 8). However, in contrast with the marked effect of OCA-B on transcription of the IgH promoter in HeLa extracts(Figure 3, lanes 1 and 2; preceding results), the addition of OCA-B had no effect on H2B transcription in unfractionated HeLa extracts (Figure 3, lanes 10 and 11) or in Ott-l-depleted HeLa extracts in response to either Ott-1 (Figure 3, lanes 5 and 8) or Ott-2 (Figure 3, lanes 8 and 9). As anticipated, OCA-B alone had no effect on the basal H2B activity (Figure 3, lane 7) although a small effect on the reference promoter (due to a contaminating activity; see above) was observed (Figure 3, lanes 4 and 7). Previous studies (Pierani et al., 1990)

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1234567 Stimulation fold -W l : Corresponding

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2. Stimulation

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(A) Selective immunodepletion of Ott-1 or Ott-2 from HeLa and B cell (Nam) nuclear extracts. The extent of Ott depletion in anti-Ott-1 or anti-Ott-2 depleted (plus) and mock-depleted (minus) extracts were determined in parallel using an EMSA with a 40 bp octamer-containing probe. Ott-I and Ott-2 indicate, respectively, specific Ott-l-DNA and Ott-e-DNA complexes whose specificities have been verified by oligonucleotide competition and footprint analyses (Pierani et al., 1990). (6) Effect of OCA-B and purified Ott-1 or Ott-2, alone or combined, on IgH promoter activation in HeLa-derived extracts. Aliquots (5 nl) of BCIOO, 250 pg/ml BSA (minus), purified Ott-1 or Ott-2 (about 10 ng in BCIOO, 250 nglml BSA), and OCA-B were added to O&depleted HeLa chromatographic step, while nuclear extracts (12 ~1) as indicated. The OCA-B used in lanes 1-7 (5 td, - 15 ug protein) was from the lectin-agarose by OCA-B is indicated in lanes 6-12 only the OCA-B used in lanes 9-12 (3 nl, -3 ng protein) was from the DE-52 column. The fold stimulation and roughly reflects the differences between the intact HeLa and Namalwa (Nam) nuclear extracts in transcribing the IgH promoter in vitro (compare lane 1 with lane 2 in Figure 5 and lane 1 with lane 3 in Figure 3). Note that in contrast with an equivalent lectin-agarose chromatographic fraction from a HeLa extract (lane 6) the OCA-B fraction from an Ott-Bdepleted Namalwa extract markedly stimulates the transcription (lane 7). (C) OCA-B dose-dependent activation of IgH promoter. Increasing amounts of the DE-52 column-derived OCA-B fraction were used to complement complete HeLa nuclear extracts (12 nl; lanes 3-9). Complete (lane 1) or Ott-2-depleted Namalwa nuclear extracts (lane 2) were included in the assay as positive controls. The intensities of individual bands from lanes 3-9 were measured by densitometry. Relative transcription activity was calculated accordingly. The upper part of the panel shows an autoradiogram of the analysis, while the lower part shows a plot of the quantitated data. The activity in HeLa extracts was considered I. (D) Activity of the IgH promoter at variable Ott factor concentrations. Ott-depleted HeLa nuclear extracts (12 ul) and increasing amounts of Ott factors were used to transcribe either IgH or reference templates, in the absence or presence of a constant amount of OCA-B (2 nl of an active fraction from the DE-52 column). Relative transcription activity was calculated as above. The lower panel shows the autoradiogram, while the upper panel shows a plot of the titration. The activity with the Octdepleted HeLa extract was taken as 1.

indicated that Ott-l- or Ott-e-dependent transcription of H2B, like that of IgH, is dependent upon an intact octamer element. These results demonstrate clearly the specificity of OCA-B for the IgH promoter relative to another promoter (H2B) whose activity is dependent upon the same activators. Apart from an effect of OCA-B on transcription of an

immunoglobulin K promoter by Ott-1 or Ott-2 (H. F. and Ft. G. R., unpublished data), the possible specificity of OCA-B for other octamer-containing promoters is not yet known. To analyze possible effects of OCA-B on other upstream activators, human immunodeficiency virus core promoter

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The reference template and either IgH (lanes l-3) or H2B (lanes 4-l 1) templates were transcribed along with combinations of OCA-B, Ott-1, and Ott-2 in Namalwa, HeLa, or Ott-depleted HeLa nuclear extracts as indicated. Reactions contained 10 ng of Ott-1 or Ott-2 (when present), 5 ul (15 trg of protein) of the lectin-agarose fraction of OCA-B, and compensating volumes of BCIW, 250 &ml BSA. The upper panel shows the H2B template (Pierani et al., 1990) that contains an H2B gene segment (-59 to +229) cloned into Hindlll-EcoRI sites of vector pEMBL19 (Den& et al., 1965). The end-labeled probe for the Sl mapping spans nucleotide positions -23 to +70 of the gene and produces a 70 nt signal from specifically initiated transcripts.

templates with recognition sites for the upstream stimulatory factor (USF) (Gregor et al., 1990) and Spl (Kadonaga et al., 1987) were analyzed in HeLa extracts selectively depleted of USF and Spl , respectively, by affinity chromatography (Pognonec and Roeder, 1991; Meisterernst et al., 1991). As shown in Figure 4, transcription of the corresponding templates was effectively reduced upon removal of Spl (Figure 4, lane 2 versus lane 8) or USF (Figure 4, lane 8 versus lane 12). Readdition of purified Spl (Figure 4, lane 4) or USF (Figure 4, lane 10) effectively restored transcription to the corresponding templates, whereas OCA-6 had no substantial effect on transcription of either template when added alone (Figure 4, lanes 3 and 9) or in conjunction with Spl (Figure 4, lane 4 versus lane 5) or USF (Figure 4, lane 10 versus lane 11). Therefore, OCA-6 appears to be an activator-specific coactivator.

Preferential Association of OCA-B with Ott-1 in B Cells Although the previous studies have demonstrated the ability of OCA-B to function with either Ott-1 or Ott-2 on an immunoglobulin promoter, it is relevant to ask whether OCA-B might preferentially function or associate with one factor. To address this question, B cell extracts selectively and quantitatively depleted of Ott-1 or Ott-2 (see Figure 2A) were tested for IgH promoter activity (Figure 5). Somewhat surprisingly, the removal of Ott-2 (Figure 5, lane 8

Figure 4. SplOCA-B

2

3

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6

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6

7

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Promoters

S

Are Not Affected

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Spl- and USF-depleted HeLa nuclear extracts, along with two control (mock depleted) extracts (HeLa NE(l) and HeLa NE(2), 12 ul each), were employed to transcribe either template A (USF regulated) or template B (Spl regulated). OCA-B (5 ul), partially purified Spl (1 ul), and recombinant 43 kd USF (10 ng) were added (plus) as indicated. The templates used are diagrammed in the upper panel. Templates A and B contain two copies of USF- or Spl-binding sites, respectively, inserted in front of a hybrid gene consisting of a human immunodeficiency virus DNA segment (-41 to +60) fused to chloramphenicol acetyltransferase (CAT) coding sequences (Pognonec and Roeder. 1991). The transcripts from these templates were measured by primer extension. The end-labeled, CAT-specific primer is homologous to transcripts from both templates and gives rise to a 127 nt signal on specifically initiated transcripts.

versus lane 2) had no significant effect on IgH transcription, whereas removal of Ott-1 markedly reduced the IgH transcription (Figure 5, lane 6 versus lane 2) to a level equivalent to that observed in normal HeLa extracts (Figure 5, lane 1) or in Ott-ldepleted HeLa extracts supplemented with Ott-1 (Figure 5, lane 4) or Ott-2 (Figure 5, lane 5). These results support the conclusion from the studies described above that Ott-2 is not essential for optimal IgH transcription in response to OCA-B, and they further indicate that OCA-6 is preferentially associated with Ott-1 in nuclear extracts and presumably in the cell. This conclusion is consistent with the lectin affinity chromatography results described earlier. Thus, Ott-1 in crude or partially purified form binds actively to a lectin column, presumably as a result of glycosylation since it is selectively eluted by N-acetyl-D-glucosamine, whereas Ott-2 does not bind under any conditions tested. In contrast, OCA-B is retained on the lectin column only when loaded in the presence of Oct.1 and not after separation from Ott-1 by selective elution (0.5 M KCI) from the same column or by the direct fractionation of B cell extracts by conventional means (data not shown). Altogether, these results indicate that OCA-B interacts directly (in the absence of DNA) and preferentially with Ott-1 . A further analysis revealed that purified Ott-2 had no

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123466789 Figure tracts

5. Preferential

Association

of OCA-B

with Ott-1

in 0 Cell Ex-

Transcription of the IgH reference promoters in the various unfractionated and Ott-depleted nuclear extracts (12 ul) indicated and in the absence or presence (plus) of purified Ott-1 or Ott-2 (- 10 ng) as indicated. The Ott-depleted extracts were the same ones analyzed in Figure 2A. Transcription levels were measured by Sl nuclease protection analysis.

noticeable effect on the residual (normal) IgH transcription in Ott-Pdepleted B cell extracts (Figure 5, lanes 8 and 9) whereas purified Ott-1 enhanced significantly (restoring transcription to 70% of the control level) the basal level of IgH transcription in an Ott-l-depleted B cell extract (Figure 5, lanes 6 and 7). The latter result was somewhat surprising in view of the near complete loss of activity upon removal of Ott-1 and the apparent coimmunoprecipitation (with Ott-1) of most of the functional OCA-B (which otherwise should function with Ott-2; see above). However, in related experiments it was observed that the addition of saturating amounts of either purified Ott-1 or purified Ott-2 to a B cell extract depleted of Ott factors by an octamer affinity column restored high levels of transcripK promoters (Piertion from both IgH and immunoglobulin anietal.,1990;Y.L.,H.F.,T.G.,andR.G.R.,unpublished data). Thus, the low IgH promoter activity in Oct-ldepleted B cell extracts that still contain Ott-2 and the restoration of activity by a purified exogenous Ott-1 may reflect a residual level of OCA-B, a lower affinity of OCA-B for an Ott-P-DNA complex relative to an Ott-1 -DNA complex (see below), and an ineffective (subsaturating) level of endogenous Ott-2. In a further analysis of the mechanism of action of OCA-B, its ability to interact stably with Ott-promoter complexes was analyzed by EMSAs with an IgH promoter fragment containing an intact octamer element. As shown in Figure 6A, Ott-1 (lane 2) and Ott-2 (lane 8) showed independent binding to the probe while OCA-B (lane 14) did not. As anticipated, the addition of OCA-B resulted in the dose-dependent formation of a novel complex with Ott-1 (Figure 6A, lanes 3-7), although an analogous complex was not observed with Ott-2 (Figure 6A, lanes 9-l 3). The specificity of the Ott-1 and Ott-1 -OCA-B promoter complexes was verified by oligonucleotide competition (data not shown) and antibody supershift (Figure 6B) experiments, which demonstrated requirements for both the octamer element and for Oct.1 . In the analysis of Figure 6B,

anti-Ott-1 immune serum dramatically reduced the Ott-1 DNA complex and eliminated the Ott-1-OCA-B-DNA complex(Figure6B, lanes3-5), generating corresponding supershifted complexes, while preimmune serum was without effect on either (Figure 6B, lanes 6-8). Although the exact basis for the failure to see an Ott-2-OCA-B promoter complex is not clear (see Discussion), the present observations most likely reflect a greater affinity of OCA-B for Ott-l-DNA complexes relative to Ott-2-DNA complexes. This is in accord with the preferential association of OCA-B with Ott-1 in nuclear extracts. From these data we propose that a putative Octinteracting cofactor, although able to function in conjunction with either Ott-1 or Ott-2 in reconstituted assays, preferentially interacts with Ott-1 in the natural state and probably exists in stoichiometric excess over Ott factors in B cells. Discussion As summarized in the Introduction, several recent studies have questioned the original view that specific activation of immunoglobulin promoters is mediated by Ott-2, whose levels and in vitro function do not correlate strictly with immunoglobulin promoter activity. The present study provides the first direct evidence for an additional B cell factor that could be a major determinant of immunoglobulin promoter function in lymphoid cells. This factor, designated OCA-B, markedly enhances octamer-dependent transcription of immunoglobulin promoters in conjunction with either Ott-1 or Ott-2, does not stimulate octamer-dependent transcription of an H2B gene via Oct.1 or Ott-2 or the transcription of other tested promoters through different activators, has no independent transcriptional activity, and is found in lymphoid cells but not HeLa cells. Hence, it appears to be a cell-, promoter-, and activator-specific coactivator. These results have important implications both for the mechanism of action of site-specific DNA-binding proteins and their assumed roles, in the case of developmentally regulated genes, as the major determinants of cell type-specific gene activation and cell determination and differentiation. Role of the Coactivator OCA-B and Ott-1 Versus Ott-2 in lmmunoglobulin Gene Activation The preeminent role of the immunoglobulin promoter octamer element in high level expression in lymphoid cells, originally indicated by various in vitro studies (Introduction), has been confirmed and extended by studies in transgenic mice (Jenuwein and Grosschedl, 1991). Although the contributions of Ott-1 versus Ott-2 to lymphoid-specific activation via this site have not yet been tested in mice, the present results indicate that either Ott-1 or Ott-2 suffices for high level promoter activation in vitro, and potentially in vivo, in conjunction with OCA-B. These results are consistent with the transcription of immunoglobulin genes in two acute lymphoblastic leukemic cell lines (or derived extracts) apparently deficient in Ott-2 (Johnson et al., 1996). Despite the equivalence of Ott-1 and Ott-2 in effecting high level transcription of immuno-

Cell-Specific 237

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123456

globulin promoters in the presence of OCA-B, we have pointed out elsewhere (Pierani et al., 1990) that there could be a discrimination between these factors in vivo as a result of other natural constraints (e.g., chromatin structure). However, the preferential association of OCA-6 with Ott-1 (versus Ott-2) in nuclear extracts, as well as the greater aff inity of OCA-B for promoter-bound Ott-1 relative to promoter-bound Ott-2, favor the view that Ott-1 is the preferred activator for OCA-B in vivo and is consistent with the lack of a good correlation between Ott-2 levels and immunoglobulin gene transcription (for review see Jenuwein and Grosschedl, 1991). The present study has emphasized the function of OCA-B in conjunction with Ott-1 or Ott-2 at the promoter, but the lymphoid-specific intragenic IgH enhancer also contains a functional octamer element (Staudt and Lenardo, 1991). Although an apparent functional redundancy of control elements in the intronic enhancer renders the intronic octamer nonessential for heavy chain gene expression in lymphoid tissues in transgenic mice assays

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of OCA-B

with

(A) Dose-dependent formation of an OCA-BOtt-I-DNA complex. EMSA was carried out with a “P-labeled, octamer-containing DNA probe (-73 to -29 of the BCL, heavy chain promoter, with the heptamer mutated) in the absence (minus) or presence (plus) of either 5 ng of Ott-1 (lanes l-7) or 5 ng of Ott-2 (lanes 6-14) and with the amounts of OCA-B indicated. All OCA-B fractions were adjusted to 2.0 ul, with the corresponding chromatographic fraction from HeLa ceils (see Figure 28, lane 6). (8) Effect of Anti-Ott-1 antibody on Ott-I -DNA and OCA-B-Ott-I-DNA complexes. EMSA was performed with the same DNA probe descrtbed in (A), in the absence (minus) or presence (plus) of 5 ng of Ott-I (in 1 pi of BCIOO, 256 @ml BSA) and 0.5 ul of OCA-B. Reactions contained in addition either 1 pl of BCl 66,250 pg/ml BSA (lanes 1 and 2) or 1 ul aliquots of immune (lanes 3-5) or preimmune (lanes s-s) antisera diluted with BClOO, 250 &ml BSA (dilution indicated). The band below the Ott-1 DNA complex was not sensitive to antibody challenge (lane 5) and was obvious only under themodifiedEMSAconditions(see Experimental Procedures) that enabled detection of the OCA-B-O&l-DNA complex. The relative intensity of the band also varied in different experiments. Thus, it most likely reflects nonspecific binding to the probe by a contaminating protein in the O&l preparation.

(Jenuwein and Grosschedl, 1991), it can mediate lymphoid-specific enhancer function assays with appropriate enhancer segments (Lenardo et al., 1987; Gerster et al., 1987). Thus, it remains an interesting possibility that OCA-B might be required under physiological circumstances in which the intronic enhancer octamer is important for enhancer function. A related question is whether Ott-2 (or one of the various Ott-2 family members; Wirth et al., 1990) might be specifically required in such a situation or, possibly, for the function of octamercontaining promoters or enhancers in other lymphoidspecific genes. This possibility would provide an explanation for the existence and preferential location of Ott-2 in lymphoid tissues. Another related possibility is that Ott-2 might be required under conditions where Ott-1 function is down-regulated but immunoglobulin promoter (or enhancer) function maintained. Given that the H2B promoter is cell cycle regulated via the octamer element and the associated Ott-1 (active only in S phase), such a situation might obtain in various nondividing lymphoid cells such as

plasma cells or circulating B lymphocytes stimulation (De France, 1987).

prior to antigenic

Mechanism of Action of OCA-B and Cognate Activators Previous studies have indicated that Ott-1 and Ott-2 bind the same quantitatively and qualitatively to octamer elements in immunoglobulin promoters and effect comparable but low levels of activation in the absence of OCA-B (Pierani et al., 1990). Consistent with these results, activation domains outside the DNA-binding domains of Ott-1 and Ott-2 are evident from cotransfection assays into nonlymphoid cells (Gerster et al., 1990; Miller-lmmergluck et al., 1990; Tanaka and Herr, 1990). In contrast with Ott-1 and Ott-2, OCA-B shows no intrinsic DNA binding or transcriptional activity but markedly potentiates the activity of both Ott-1 and Ott-2 on immunoglobulin promoters. These results are compatible with several mechanisms. First, OCA-B may, by stoichiometric or catalytic interactions, enhance the intrinsic activation domains of Ott-1 or Ott-2. Second, OCA-B may have independent activation domains that function as a result of being tethered to the promoter by Ott-1 or Ott-2, a situation analogous to that proposed for the Ott-l-dependent binding of the viral activator VP1 6 to target promoters (see below). This possibility is consistent with preliminary studies (data not shown), suggesting that the POU domain of Ott-1 suffices for OCA-B binding and that mutations in the Ott-2 activation domains, while lowering the basal activity, still effect a stimulation by OCA-B. However, given that the Ott activation domains are essential for full activity, it seems probable that domains in OCA-B and Ott-1 or Ott-2 may function cooperatively. The situation proposed for Ott factors and OCA-B may resemble that described for other activators and coactivators. In yeast, the activity of the DNA-binding protein GAL4 is potentiated by GAL1 1, which shows no activity in the absence of GAL4 but contains an independent activating function that operates in concert with the GAL4 activation domains (Himmelfarb et al., 1990). Mammalian cells provide several examples involving octamer-binding Ott factors: first, promoter activation by Ott-4 from distal octamer elements is potentiated by an adenoviral protein (El A) that interacts with Ott-4-octamer complexes, and known activation domains in both proteins are required (Schiiler et al., 1991); second, activation of herpes virus immediate early promoters involves stable interactions of the viral protein VP16, which shows independent activation potential in other assays, with Ott-1 bound at distal sites containing both octamer elements and essential flanking sequences (Gerster and Roeder, 1988; O’Hare and Goding, 1988; Stern et al., 1989); third, activation of the mammalian 7SK promoter through the octamer element and proximal sequence element requires interactions between bound Ott-1 and the proximal sequence element transcription factor, neither of which show transcriptional activation when assayed independently (Murphy et al., 1992); and, fourth, activation of the IL-2 promoter involves an inducible factor interacting with Ott-1 and adjacent sequences (Ullman et al., 1991). The mammalian studies

raise the possibility that promoter-bound Ott factors in higher eukaryotes maygenerallyfunction with stably interacting factors (coactivators) that are not part of the basal transcriptional machinery. Consistent with this model and with indications of physical interactions between Ott-1 and OCA-B in the absence of DNA, an interaction of OCA-B with Ott-1 -promoter complexes was demonstrated. Despite the ability of OCA-B to function with Ott-2, a similar interaction of OCA-B with an Ott-2 promoter complex was not demonstrable by mobility shift assays. This could reflect a greater instability of such a complex (relative to that observed with Ott-1) brought about by the specific electrophoretic assay conditions (compare Schdler et al., 1991) or compensating interactions of other factors involved in preinitiation complex formation, such that an intrinsically weaker Ott-2-OCA-B complex is not distinguished in a functional assay. The inability to see adifference in OCA-B-dependent transcription at equivalent limiting concentrations of Ott-1 and Ott-2 isconsistent with thisobservation. Asimilar situation has been observed with TFIID mutants that fail to show TATA-binding in gel shift assays but that show normal levels of transcription and comparable abilities to bind to DNA in the presence of TFIIA (Yamamoto et al., 1992; D.-K. Lee, M. Horikoshi, J. DeJong, and R. G. R., submitted). Related to this issue is the question of additional sequence requirements, beyond the octamer element, for OCA-B function. Previous studies suggested that nothing more than an octamer element is required for B cell-specificfunction, butthisquestiondeservesacareful reexamination by mutagenesis of natural promoters that are OCA-B dependent in vitro. Another unresolved question concerns the lack of an OCA-B effect on Ott-l- or Ott-2dependent transcription of the H2B promoter. One possible explanation is the shorter (8-9 bp) and highly conserved spacing between the octamer and TATA elements in the vertebrate H2B promoters (Harvey et al., 1982; Fletcher et al., 1987) versus the larger and more variable (818 bp) spacing between these elements in various immunoglobulin promoters (e.g., Scheidereit et al., 1987; Poellinger et al., 1989). The shorter spacing may obviate the need for a coactivator, with direct activation of H2B promoters by an S phase-activated Ott-1 . Asecond possibility is that Ott-l-dependent activation of H2B promoters involves a distinct S phase-activated coactivator. In either case, extracts from the rapidly dividing nonsynchronized HeLacells employed would have been expected to contain the corresponding S phase-specific components that would render Ott-1 maximally active on the H2B promoter and independent of OCA-B. Implications of OCA-B for Other Homeodomabn-Activated and/or Developmentally Regulated Genes As discussed above, efficient promoter activation through octamer elements and associated Ott factors may generally require coactivators that interact with Ott factors directly (as demonstrated for OCA-B) or in conjunction with additional promoter sequences. Ott factors are members of both the POU domain (for reviews see Rosenfeld, 1991;

Cell-Specific 239

Coactivators

Scholer, 1991) and the broader homeodomain (for review see Scott et al., 1989) families of regulatory proteins. Many of these factors are developmentally regulated and implicated directly or indirectly in the lineage-specific transcription that is largely responsible for specifying cellular phenotypes. However, the expression patterns of the target genes are often more restricted than the expression patterns of the regulatory factors that are presumed to determine their activities (Rosenfeld, 1991; Ruvkun and Finney, 1991; Hayashi and Scott, 1990). This suggests that there are additional selectivity factors that are essential for, and thus restrict, the function of these more broadly distributed factors. The identification of OCA-6 is especially significant in this regard as it provides the first example of a tissue-restricted coactivator that appears to fulfill such a function. Thus, it may represent a new class of coactivators whose members may play major roles in restricting the activity of presumptive DNA binding activators to impose sharper spatial and temporal boundaries for gene control and cell differentiation. It may be expected that distinct classes of activators (e.g., homeodomain proteins versus helix-loop-helix proteins) will employ distinct classes of such coactivators. Experlmental Selective

Procedures Factor

Depletlon

of Nuclear

Extracts

Nuclear extracts were prepared from HeLa cells or Namalwa cells (Pierani et al., 1990) as described (Dignam et al., 1983). Extracts were maintained in BC buffer at the KCI concentrations indicated; thus, BClOO is EC buffer plus 100 mM KCI. BC buffer itself contained 20 mM Tris-HCI (pH 7.9). 20% glycerol, 0.25 mM EDTA, 0.125 mM EGTA, 0.025% Nonidet P-40 in addition to the following freshly added components: 0.5 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 2.5 @ml each of antipaln, leupeptin, aprotinin. chymostatin, and pepstatin A. For the selective immunodepletion of Ott-1 or Ckt-2. 1 ml of nuclear extract (in BClOO) was mixed with 200 pl of affinity-purified anti&t-l IgG or anti-m-2 IgG in BCl 00 containing 250 pg/ml bovine serum albumin (BSA). The mixtures were incubated on ice for 1 hr and then passed twice through protein A-Sepharose columns equilibrated in BCIOO. 250 &ml BSA. The flow-through fractions sewed as factordepleted nuclear extracts. (Mock-depleted extracts were prepared by passing equivalent mixtures of nuclear extracts and BClOO, 250 ug/ ml BSA through the same types of columns.) When normalized to the ditutton factor, the flow-through fractions were as active as untreated extracts with respect to DNA binding and transcription. Spldepleted and USF-depleted HeLa nuclear extract were made as described, respectively, by Meisterernst et al. (1991) and by Pognonec and Roeder (1991). The rabbit antibodies employed here were raised against nearly full-length Dct-1 encoded by a cloned cDNA (Tanaka and Herr, 1990) and against an N-terminal part (residues 1-183) of the Ott-2 encoded by a cloned cDNA (Scheidereit et al., 1987). These proteins were expressed in and purified from bacteria. Anti-O&i! is very specific for Ott-2, consistent with the divergent N-terminal sequences in Ott-1 and Get-2, whereas anti-O&l cross-reacts with Ott-2 (presumably the conserved POU domain) at high concentrations. However, at the lower concentrations employed here (the optimum determined by titration), anti-O&l reacts specifically and quantitatively with Ott-1 (Figure 2A).

Fractionation

of Namaiwa

Nuclear

Extracts

Fifty milliliters (- 500 mg of protein) of Namalwa nuclear extract (in BClOO) was loaded onto a 10 ml iectin-agarose column equilibrated in BClOO, 250 &ml BSA. After extensive washing with BClOO, the column was eluted with two column volumes of BC500 followed by two column volumes of EC500 containing 400 mM N-acetyl-o-glucosamine. CCA-B activity eluted with the protein peak in the first elution step (500 mM KU), which gave an -25fold puriflcatton. Ott-2 was recovered in the 100 mM KCI flow-through fraction, while Dct-1 , as well

as Spl, eluted with the protein peak in the second elution step; this resulted in a -2OO-fold purification of the latter components. The pooled fractions from this step were also used as a source of partially purified Spl (Figure 4). The parallel fractionations of HeLa- or Ckt-2depleted Namalwa extracts on iectin-agarose columns were conducted essentially as just described, except that the extract and MIumn volumeswere reduced accordingly. Further purification of OCA-B involved pooling the active OCA-B fractions(7.5 ml, - 20 mg of protein) obtained by lectin-agarose chromatography, dialysis to BClOO, and fractionation on a 5 ml DE-52 column. Subsequent to sample loading, the DE-52 column was washed extensively with BClOO and developed with five column volumes of a 0.1-0.5 M KCI gradient in BC buffer. Fractions containing OCA-B activity, which eluted at about 0.25 M KCI with a 4- to 5-fold purification, were pooled, dialyzed to BClOO, and analyzed.

Purlficatlon

of Oct.1,

Ott-2,

and USF

Ott-1 was purified from HeLa extracts to - 95% homogeneity using the combined lectin and oligonucleotide affinity method described by Pierani at al. (1990). Recombinant Ott-2 was obtained as follows. The full-length Ott-2 cDNA (Gerster et al., 1990) was inserted as an Xhol fragment into the Sall site of vaccinia recombinant vector pATA(Stunnenberg et al., 1988). Plaque-purified, Ott-2-containing recombinant viruses were used to infect 1 liter spinner culture of HeLa cells. Nuclear extracts were prepared from the infected, Oct.P-overproducing cells essentially as described by Dignam et al. (1983). After removal of nucleic acids by passage over a DE-52 column, the extracts were dialyzed to BClOO and loaded onto a heparin-agarose column. Ott-2, monitored by DNA binding activity, was eluted at 0.25 M KCI and was further purified (to >50% homogeneity) by chromatography on an octamer DNA affinity column. Recombinant USF was expressed in and purified from bacteria as described (Pognonec and Roeder, 1991).

DNA Binding

and In Vitro

Transcrlptlon

Assays

EMSAs with an H2B octamer-containing fragment, in vitro transcrip tion assays, and Sl nuciease mapping were as described by Pierani et al. (1990). Primer extension was according to Luo et al. (1991).

EMSA Analysis

of OCA-B-O&l-DNA

Complexes

In addition to the BClOO, 250 pglml BSA buffer system components introduced by the addition of Ott-1 or Ott-2, DCA-8, and antisera (2.5 PI), the components of the binding buffer were 12.5 mM HEPES-KOH (pH 7.9) 8% glycerol, 100 pg/ml BSA, 31.25 mM KCI, 1 mM MgCI,, 0.5 mM dithiothreitol, 0.25 mM phenyimethylsulfonyl fluoride, 0.025% Nonidet P-40, 0.2 pg of poiy(dl-dC):poly(dl-dC), 5 x lo4 cpm 32Plabeled probe in a total volume of 12.5 pl. Incubation was for 30 min at 30°C. Sample aliquots (10 pl) were loaded onto a 8% 19:l acrylamide: bisacrylamide gel, which had been prerun at 10 mA for 3 hr. Both the gel and running buffers were 0.25x TGE (8.25 mM Tris, 47.5 mM glycine, and 0.125 mM EDTA) plus 1 mM MgC&, 0.5 mM dithiothreitol, and 0.025% Nonidet P-40. The gel also contained 5%-8% glycerol. After loading, the gel was run for another 4-5 hr. Owing to the very low ionic strength of the running buffer, it was changed once after the prerun and once in the middle of the run. Alternatively, the buffer was recirculated.

Acknowledgments The order of the first two authors is arbitrary. We thank Drs. Hiroyuki Kato, Philippe Pognonec, and Jong-Bok Yoon for generous gifts of some reagents used in this study, Drs. Philippe Pognonec and Bernhard Kirschbaum for critical reading of the manuscript, Dr. Thomas Gutjahr for generous assistance in the preparation of the manuscript, and members of the Roeder lab for their suggestions and critical comments during the course of this work. T. G. was supported by a fellowship from Schweizerischer Nationalfonds. This work was funded by National Institutes of Health grants to R. G. R. and by general support from the Pew Charitable Trusts to the Rockefeller University. Deoxynucleotides were synthesized by the Protein Sequence Facility of the Rockefeller University. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby

Cell 240

marked “advertisement” in accordance solely to indicate this fact.

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