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. 1998 Jul;18(7):3803–3810. doi: 10.1128/mcb.18.7.3803

FIG. 3.

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.