FIG. 3.

The N-terminal 59 to 94 aa of CIITA is responsible for the inhibition of collagen α₂(I) promoter activities. (A) The collagen α₂(I) CAT reporter gene or its empty vector, pGL3-CAT, was transiently cotransfected with the N terminus or C terminus of CIITA [designated Flag CIITA(1-335) and CIITA(334-1130), respectively] or its empty vector, pcDNA3, into NIH 3T3 cells as described in the legend to Fig. 1. A portion of the cell lysate was subjected to CAT assay for collagen α₂(I) promoter activity. (B) DRA-Luc reporter gene or its empty vector, pGL3-Luc, was transfected into the same cells as those described for panel A. Luciferase (Luc) activity was used to show DRA promoter activity. CAT (A) or luciferase (B) activities were normalized based on the activity of the pGL3-CAT group (A) or the pGL3-Luc group (B), respectively. (C) A series of CIITA deletion constructs are shown. AD, acidic domain; PST, proline-, serine-, and threonine-rich domain; GTP, GTP binding site; NES, nuclear export sequence; BLS/NLS, nuclear localization sequence. (D) The experiment depicted was performed in a manner identical to that of the experiment depicted in panel A. The collagen α₂(I) CAT reporter gene or its empty vector, pGL3-CAT, was transiently cotransfected with CIITA, its empty vector control, pcDNA3, or a series of C terminus deletion mutants, as depicted in the panel C. The results are expressed as relative fold induction of CAT activity over the negative control pGL3-CAT basic vector. Error bars represent means ± standard errors. Data shown have been reproduced three times. (E) The collagen α₂(I) CAT reporter gene was transiently cotransfected with empty vector, wild-type (wt) CIITA, or CIITA(59-94)Δ.