Expression of the MHC class II transactivator (CIITA) type IV promoter in B lymphocytes and regulation by IFN-gamma

Abstract

The MHC class II transactivator (CIITA), the master regulator of MHC class II (MHC II) expression, is a co-activator that controls MHC II transcription. Human B lymphocytes express MHC II constitutively due to persistent activity of CIITA promoter III (pIII), one of the four potential promoters (pI-pIV) of this gene. Although increases in MHC II expression in B cells in response to cytokines have been observed and induction of MHC II and CIITA by IFN-gamma has been studied in a number of different cell types, the specific effects of IFN-gamma on CIITA expression in B cells have not been studied. To investigate the regulation of CIITA expression by IFN-gamma in B cells, RT-PCR, in vivo and in vitro protein/DNA binding studies, and functional promoter analyses were performed. Both MHC II and CIITA type IV-specific RNAs increased in human B lymphocytes in response to IFN-gamma treatment. CIITA promoter analysis confirmed that pIV is IFN-gamma inducible in B cells and that the GAS and IRF-E sites are necessary for full induction. DNA binding of IRF-1 and IRF-2, members of the IFN regulatory factor family, was up-regulated in B cells in response to IFN-gamma and increased the activity of CIITA pIV. In vivo genomic footprint analysis demonstrated proteins binding at the GAS, IRF-E and E box sites of CIITA pIV. Although CIITA pIII is considered to be the hematopoietic-specific promoter of CIITA, these findings demonstrate that pIV is active in B lymphocytes and potentially contributes to the expression of CIITA and MHC II in these cells.

Fig. 1

Increased levels of MHC class II and promoter IV-specific CIITA expression are observed in B cells in response to IFN-γ treatment. Equal amounts of RNA from Raji, BJAB and primary B cells, treated for 0 or 24 h with recombinant human IFN-γ, were subjected to RT-PCR using primers specific for the MHC II (HLADRA), CIITA pIII and pIV RNAs. RT-PCR using primers for human cyclophilin was performed as an internal standard for cDNA loading. This experiment has been repeated twice with similar results.

Fig. 2

A specific increase in human CIITA (MHC2TA) pIV activity is observed in B cells in response to IFN-γ. Transient transfections of Raji B cells were performed by electroporation using 10 μg of luciferase reporter constructs under the control of CIITA pIV or CIITA pIII or using 10 μg of -Control, pGL2-Basic. After a 6 h recovery period, half of the cultures were treated with 500 U/ml of recombinant human IFN-γ for 24 h before cultures were harvested. Luciferase activity was measured and normalized to Renilla luciferase activity as described above. Bars show the s.e.m., n = 3. At least three independent experiments have been performed with similar results.

Fig. 3

The response of CIITA pIV to IFN-γ in B cells requires the STAT1 and IRF-1-binding sites. Transient transfections of Raji B cells were performed by electroporation using 10 μg of a luciferase reporter construct containing unmutated CIITA pIV (pIV), pIV with a mutated STAT1-binding site (GAS) [pmIV(GAS)], pIV with a mutated IRF-1-binding site (IRF-E) [pmIV(IRF-E)] or 10 μg of -Control, pGL2-Basic. For reference, the sequence of unmutated CIITA pIV and locations of the GAS and IRF-E are shown in Fig. 6. After a 6 h recovery period, half of the cultures were treated with 500 U/ml of recombinant human IFN-γ for 24 h before harvesting and measurement of luciferase activity. Bars show the s.e.m., n = 4. At least three independent experiments have been performed with similar results.

Fig. 4

IRF-1 induces the activity of CIITA pIV in B cells. Transient cotransfections of Raji B cells were performed by electroporation using 10 μg of IRF-1 expression plasmid or the empty expression plasmid vector, and 10 μg of a luciferase reporter construct containing either unmutated CIITA pIV (pIV), pIV with a mutated STAT1-binding site (GAS) [pmIV(GAS)], pIV with a mutated IRF-1-binding site (IRF-E) [pmIV(IRF-E)] or 10 μg of the -Control, pGL2-Basic. After 24 h, cells were harvested and luciferase activity was measured. Bars show the s.e.m., n = 3. At least three independent experiments have been performed with similar results.

Fig. 5

IRF-2 induces the activity of CIITA pIV in B cells. Transient cotransfections of Raji B cells were performed by electroporation using 10 μg of an IRF-1 expression plasmid, an IRF-2 expression plasmid or the empty expression plasmid vector, and either 10 μg of -Control, pGL2-Basic or the CIITA pIV luciferase reporter construct. After 24 h, cells were harvested and luciferase activity was measured. Bars show the s.e.m., n = 3. At least three independent experiments have been performed with similar results.

Fig. 6

The in vivo footprint of CIITA pIV in B cells reveals protein-DNA contacts within the GAS, IRF-E and E box sites. The sequence of the promoter region is shown with boxes indicating the relevant cis-elements. Genomic footprints of the upper strand are shown in the lower panel. Lane 1 shows genomic DNA that was methylated in vitro to reveal the complete guanine ladder. Lanes 2 through 4 show the results of IFN-γ treatment using Raji B cells treated with DMS in culture. Open arrows indicate bases protected from modification. While a number of bases in the E box and IRF-E sites are protected both before and after treatment (indicated by arrows), one central base in the GAS site becomes protected after IFN-γ treatment (see asterisk).

Fig. 7

Binding of IRF-1 and IRF-2 to CIITA pIV is induced in B cells in response to IFN-γ. (A) EMSA analysis demonstrates that a specific protein complex is induced by IFN-γ treatment (lane 1 versus lane 2) (arrow). Nuclear extracts were from Raji B cells treated with IFN-γ (500 U/ml) for 0 (UNT) or 18 h (IFNγ). The probe spans the CIITA pIV IRF-E. Cold oligonucleotide competitors, used at 50-fold molar excess (50X), are designated in the top row. Abbreviations: WT, homologous CIITA pIV competitor; MT, CIITA pIV competitor with mutated IRF-E site; IRF-E, cold competitor with the IRF-E site of the TAP1 gene (White et al., 1996); NUC. EX., nuclear extract. (B) Incubation with anti-IRF-1 or anti-IRF-2 induces a supershifted or partially supershifted complex, respectively (see asterisk). Antibodies are indicated at the top. Abbreviations: AB, antibody; NS, normal serum.