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.