Coactivation by OCA-B: definition of critical regions and synergism with general cofactors

Abstract

Molecular dissection of the B-cell-specific transcription coactivator OCA-B has revealed distinct regions important, respectively, for recruitment to immunoglobulin promoters through interaction with octamer-bound Oct-1 and for subsequent coactivator function. Further analysis of general coactivator requirements showed that selective removal of PC4 from the essential USA fraction severely impairs Oct-1 and OCA-B function in a cell-free system reconstituted with partially purified factors. Full activity can be restored by the combined action of recombinant PC4 and the PC4-depleted USA fraction, thus suggesting a joint requirement for PC4 and another, USA-derived component(s) for optimal function of Oct-1/OCA-B in the reconstituted system. Indeed, USA-derived PC2 was found to act synergistically with PC4 in reproducing the function of intact USA in the assay system. Consistent with the requirement for PC4 in the reconstituted system, OCA-B was found to interact directly with PC4. Surprisingly, however, removal of PC4 from the unfractionated nuclear extract has no detrimental effect on OCA-B/Oct-1-dependent transcription. These results lead to a general model for the synergistic function of activation domains in Oct-1 and OCA-B (mediated by the combined action of the multiple USA components) and, further, suggest a functional redundancy in general coactivators.

FIG. 1

(A) A simple linear description of the OCA-B molecule. The 256-residue polypeptide is rich in proline (∼16%; for the complete sequence, see references , , and 54). Relevant putative functional domains include domain A (with its sequence alignment with an E1A segment shown) and domain B (with acidic residues marked by asterisks). The mutant forms of OCA-B used in this study are also described. The mutant proteins were made by a PCR-based strategy. (B) Protein profile of bacterially expressed, 6His-tagged wild-type OCA-B and mutant forms of OCA-B. A roughly calculated 0.1 ng each of the proteins was analyzed by immunoblotting with polyclonal anti-OCA-B antibodies. (Given that all three mutant proteins may have lost some epitopes compared to the wild type, the amounts of the mutant proteins might be slightly underestimated. Nevertheless, this underestimation would not complicate our analyses of, and the conclusions made about, these mutant proteins.) The amounts used for the in vitro transcription analyses in Fig. 2 were based on this estimation.

FIG. 2

Analyses of wild-type (Wt) OCA-B and mutant (Mut.) forms of OCA-B. (A) In vitro transcription assay. As indicated, three doses of wild-type OCA-B and mutant OCA-B (respectively, 10 ng [lanes 2, 5, 8, and 11]; 20 ng [lanes 3, 6, 9, and 12], and 50 ng [lanes 4, 7, 10, and 13]) were used in the titration experiment to complement a HeLa nuclear extract (10 μl for each reaction). The transcripts were analyzed by primer extension as described by Luo and Roeder (31). Lane 1 (−) was a control (without the addition of OCA-B or its derivatives). Transcription activation levels (by the middle point dosage of OCA-B, i.e., 20 ng; an amount equivalent to that of the endogenous OCA-B present in 10 μl of the B-cell [Nam] nuclear extract), compared to lane 1 (taken as 1), are 8, 2.7, 2.2, and 1.4, respectively, for the wild type and the B, A, and A/B mutant proteins (lanes 3, 6, 9, and 12; also, see the fold stimulation shown under each corresponding lane). (B) In vivo (transfection) assay. An IgH reporter promoter construct (with the promoter inserted in front of the luciferase gene) was cotransfected with effector constructs (expressing OCA-B or its mutants under the control of the cytomegalovirus promoter) in HeLa cells. Relative IgH promoter activation levels were scored by measuring the light units of the extracts of the transfected cells 48 h posttransfection normalized to β-galactosidase controls. Taking the level with no effector as 1, the relative levels are 12, 4, 3, and 2, respectively, for the wild type and the B, A, and A/B mutant proteins. The mutant OCA-B proteins were expressed at levels similar to that of the wild-type protein in transfected cells, as assessed by immunoblotting with anti-OCA-B antibodies (data not shown). (C) Octamer DNA–POU-1 complex supershift assay. POU-1 is a truncated version of Oct-1 containing only the POU domain, sufficient to bind OCA-B either in solution or when on DNA (e.g., reference 31). The supershift condition was the same as that described by Luo and Roeder (31), except that approximately 25-fold less OCA-B (or its mutants) was used, such that only a portion of the binary complex was supershifted. To obtain the highest possible resolution, the free probe (a labeled DNA fragment containing the IgH octamer site) was allowed to run out of the gel. As can be seen, the B mutant protein (lane 3) retained the ability to form the higher-order complex, which was even denser than that formed with the wild type (lane 2) for a reason we do not know, whereas the A mutant protein (lane 4) lost the capacity to bind the binary complex. Given that the combinatorial A/B mutant protein (lane 5) could give rise to the formation of a residual level of the ternary complex, it is possible that the introduction of the B mutant protein created a conformational change that can increase the POU–OCA-B interaction even with domain A deleted. We emphasize that such a complication in the binding assay did not complicate our in vitro transcription analyses and conclusions. (D) Test of dominant negative potentials of wild-type OCA-B and its mutant forms. Eight microliters of B-cell (Nam) nuclear extract was used for each reaction (there is ∼2 ng of endogenous OCA-B per μl in this nuclear extract [see above]). The nuclear extract was supplemented with either BC100 buffer (lane 1) or, as indicated, recombinant wild-type or mutant OCA-B (∼100 ng, for a molar ratio of ∼6:1 over the endogenous OCA-B) in BC100 (lanes 2 to 5). The transcripts were analyzed as described for panel A.

FIG. 3

PC4 as an essential component of USA and acting synergistically with PC2, to support the function of OCA-B in a reconstituted transcription system. (A) PC4 depletion (dep.) from the USA coactivator fraction (left panel) and from the nuclear extract (NE; right panel; the treated nuclear extracts were used in the experiment shown in Fig. 5). The completion of the depletion was examined by immunoblotting with anti-PC4 antibodies. (B) IgH promoter activity was analyzed in the reconstituted system with components of general factors and RNA polymerase II (top) in the presence (+) or absence of Oct-1, OCA-B, or the USA fraction, as indicated. Lanes 1 to 5 demonstrate an IgH promoter dependency on activator Oct-1, general coactivator fraction USA, and specialized coactivator OCA-B. Oct-1 is absolutely required for the promoter activity (compare lanes 2 and 3 to lane 4, which represents a complete set of components sufficient to bring about a high level of IgH promoter transcription). In an otherwise complete system, the transcription is stimulated ∼15-fold by USA and ∼8-fold by OCA-B (compare lane 1, missing USA, and lane 5, missing OCA-B, respectively, to lane 4, complete). Lanes 6 to 8 demonstrate that PC4 is an essential component of USA to support the function of OCA-B in the reconstituted transcription system. The defect of the PC4-depleted USA fraction (lane 7), compared to the complete USA fraction (lane 6), can be rescued by the addition of 50 ng of recombinant PC4 (lane 8). (C) PC4-PC2 synergism for the function of OCA-B in the reconstituted system. Components (top) were used to transcribe the IgH promoter in the absence or presence (+ or ++) of a general coactivator(s), as indicated. The levels of coactivation are (compared to lane 1) ∼15-fold by USA (lane 2), ∼1.5- to 3-fold by PC2 (lanes 5 and 3), ∼3- to 5-fold by PC4 (lanes 6 and 4), and ∼10- to 15-fold by PC2 plus PC4 (lanes 8 and 7). Amounts of factors used in the reconstituted system (for both B and C) are as follows: TFII-A, 0.5 μl; recombinant TFII-B, 50 ng; TFII-D, 2 μl; TFII-E/F/H fraction, 3.5 μl; RNA polymerase II, 0.25 μl; Oct-1 (when added), 20 ng; recombinant OCA-B, 50 ng; recombinant PC4, 25 (+) and 50 (++) ng; USA, mock-depleted USA and PC4-depleted USA, 1 μl of each; PC2, 1 (+) and 2 (++) μl.

FIG. 4

Identification of PC4 as a potential downstream target for the function of OCA-B. The rationale for this experiment is explained in the text, and experimental details are described in Materials and Methods. Note that only a portion (residues 171 to 256) of wild-type (WT) OCA-B and the B mutant (Mut.) protein was fused to GST. + and ++ represent two (lower and higher, respectively) concentrations of immobilized GST or GST fusions. Inp. NE, input nuclear extract.

FIG. 5

PC4 depletion (Dep.) from a nuclear extract (NE) does not affect the transcription of either octamer/Oct-dependent (IgH) or -independent (2×Sp1) promoters, suggesting a potential redundant activity for PC4 in the nuclear extracts. Untreated HeLa (lane 1), Namalwa (B cell; lane 2), mock-depleted HeLa (lanes 3 to 6), and PC4-depleted HeLa nuclear extracts were used to transcribe the promoters in the absence or the presence (+) of PC4 (50 ng) and/or OCA-B (20 ng), as indicated. While the transcription level of the control template (2×Sp1) is constant in all lanes, the IgH promoter responds to OCA-B in either mock-depleted (lanes 5 and 6 versus lanes 3 and 4) or PC4-depleted (lanes 9 and 10 versus lanes 7 and 8) HeLa nuclear extracts to a level similar to that observed in the B-cell extract (compare lanes 5 and 6 and lanes 9 and 10 to lane 2). The control template (2×Sp1) is described in reference .

FIG. 6

Model for OCA-B function. Mut., mutant; GTF, general transcription factors; ACT, activation domain(s) of Oct-1 and -2; Pol II, RNA polymerase II. Asterisks denote the activation domains in either OCA-B or Oct-1 and -2. The dashed lines indicate that, in addition to contacting residues within the octamer motif (2, 4), OCA-B may also contact downstream sequences, as revealed by a footprinting assay (32). See the text for a full description of the model.