Rj2.2.5

Modulation of Gene Expression by CIITA in Rj2.2.5 Cells

The class II transactivator (CIITA) is a master regulator of MHC class II expression. To determine whether CIITA regulates genes other than the MHC class II and I family, DNA microarray analysis was used to compare the expression profiles of the CIITA-expressing B cell line Raji and its CIITA-negative counterpart Rj2.2.5. On average, 45% and 43.5% of the genes in the chip were present in Raji and Rj2.2.5 cells, respectively, indicating no gross difference between the cell lines.

Genes found to be expressed at higher levels in Raji vs Rj2.2.5 cells were classified by function (Table 1). The HLA class II genes HLA-DRB, -DPA, -DPB, -DQB, -DMA, -DMB, and -DOA were found to be expressed at high levels in Raji and absent or low (background hybridization) in Rj2.2.5 cells. Table 1 displays the list of 48 genes up-regulated in Raji cells over Rj2.2.5 cells. Based on their actual or predicted function, the genes were grouped into sets of kinases/phosphatases, transcription factors, cell cycle- and cell structure-related factors, nuclear import proteins, RNA processing components, enzymes, receptors, cell signaling molecules, and chromatin remodelers.

Sixteen genes were found to be decreased in their expression levels in Raji vs Rj2.2.5 cells, suggesting that these genes are down-modulated in the presence of CIITA (Table 2). Those genes displaying the greatest decrease in expression levels in the Raji vs Rj2.2.5 comparison were protein tyrosine phosphatase (PTPRR), glycoprotein M6A (GPM6A), and kallikrein (KLK1).

Table 1. Genes up-regulated in Raji vs Rj2.2.5. (Nagarajan UM, et al., 2002)

Table 2. Genes down-regulated in Raji vs Rj2.2.5. (Nagarajan UM, et al., 2002)

Expression of HLA-DOB Induced by CIITA in B Cells

HLA-DO, encoded by the HLA-DOA and HLA-DOB genes, has been shown to function as a modulator of Ag presentation. DNA microarray comparisons between B cells wild-type and mutant for the master regulator of MHC class II transcription, CIITA, identified HLA-DOA and HLA-DOB as being up-regulated by CIITA.

The stained cells were analyzed by flow cytometry. In comparison to Raji cells, Rj2.2.5 showed deficient levels of HLA-DOβ (Fig. 1). SJO cells also displayed deficient levels of both HLA-DM and HLA-DOβ (Fig. 1). These data suggest that both CIITA and RFX are required for the expression of the heterodimer HLA-DO.

A 317-bp fragment spanning the WXY box of the HLA-DOB promoter was cloned upstream of a luciferase reporter gene, and the resulting construct was termed pGL3-DOB. pGL3-DOB was transiently transfected into both Raji and Rj2.2.5 cells, and the expression of the reporter assayed. The relative activity of the reporter was 12-fold decreased in Rj2.2.5 in comparison to Raji (Fig. 2). Immunoprecipitation of CIITA- and RFX5-containing chromatin using formaldehyde-fixed Raji cell lysates, followed by PCR, revealed specific bands for the HLA-DOB promoter for both Abs (Fig. 3). Thus, both RFX5 and CIITA can be found at the HLA-DOB promoter in vivo and demonstrate that CIITA can or does play a role in the regulation of the HLA-DOB gene.

Fig. 1 HLA-DOβ protein is not expressed in CIITA- and RFX5-deficient cell lines (Rj2.2.5 and SJO). (Nagarajan UM, et al., 2002)

Fig. 2 Raji and Rj2.2.5 cells were co-transfected with either HLA-DRA promoter or HLA-DOB promoter-driven luciferase reporters. (Nagarajan UM, et al., 2002)

Fig. 3 CIITA binds to the HLA-DOB promoter. (Nagarajan UM, et al., 2002)