Transcriptional regulation of HLA-A and -B: differential binding of members of the Rel and IRF families of transcription factors.

Abstract

HLA-A and -B transplantation antigens can be expressed differentiafly at the basal level and in response to interferons (IFNs). To determine which DNA control elements and nuclear factors are responsible for these differences,HLA-A and -B upstream regulatory regions were used in expression and mobility-shift analyses. The HLA-A enhancer was found to contain two Rel (KBF/NF-#cB) binding motifs, while the HLA-B enhancer has only one and is transactivated less well by overexpression of the NF-#cB p65 subunit. transcription (13, 14). Overexpression of IRF-1 has been shown to increase the basal and IFN-induced levels of class I expression (15, 16).HLA-A and -B genes are highly homologous but exhibit locus-specific differences in both their transcribed (17) andThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.upstream regions (18,19) and are not tightly coordinated in their regulation. They can differ in basal and induced levels of expression, with cortical thymocytes and related thymomas such as MOLT-4, for example, expressing low levels of HLA-A but undetectable , while HLA-B loci respond more strongly to IFNs (21,22) and are subject to stronger suppression by c-myc expression (23). We report here that some of the locus-specific differences between the ENH and IRE of HLA-A and -B have significant effects on the binding of the Rel and IRF families of transcription factors and consequently on the regulation of the two loci. MATERIALS AND METHODSCell Culture, Transfection, and Chloramphenicol Acetyltransferase (CAT) Assays. MOLT4 and YHHH (24) cell lines were maintained in Dulbecco's modified Eagle's medium, and JM and Daudi in RPMI 1640 medium, all with 5% fetal bovine serum. Induction of NF-KB in JM cells was achieved by a 2-hr treatment with phytohemagglutinin (PHA, 5 ,ug/ml; Sigma) and phorbol 12,13-dibutyrate (PBt2, 50 nM; Sigma). Transfections and CAT assays were performed as described (25). Cells were harvested 20 hr after transfection. IFN-aA (kindly provided by M. Brunda, Hoffmann-La Roche) was added after transfection at 2000 units/ml where indicated.Plasmids. CAT reporters were produced by cloning synthetic oligonucleotides into an Sph I site introduced into the Bgl II site at the 5' end of the metallothionein promoter of pMCAT3 (26) and verified by double-stranded sequencing. The Rel p50 and p65 expression plasmids (27) were kindly provided by A. Baldwin (University of North Carolina, Chapel Hill) and the CMV-GalVP16 vector (28) by P. O'Hare (Marie Curie Research Institute, Oxted, Surrey, U.K.).Electrophoretic Mobility-Shift Assays. Nuclear extracts from cell lines were prepared (29) and mobility-shift assays were performed (25) as described. Baculovirus Expression of IRF-1 and IRF-2. Human IRF-1 and IRF-2 clones were obtained from the lymphoid lines HUT78 and MOLT-4, respectively, by two rounds of amplification by polymerase chain reaction...

Locus-Specific Constitutive and Cytokine-Induced HLA Class I Gene Expression

“…Since IFN-␥ induction of CIITA requires IRF-1 (29) and is enhanced by IRF-2 (30), IRFs may indirectly activate HLA class I promoters by inducing CIITA expression. However, the results are also consistent with direct activation, because IRF-1 binds more strongly to the HLA-B ICS than the HLA-A ICS (14,31). Surprisingly, both IRFs activated the control Sp1.TK.GL3 luciferase reporter gene (ϳ50-fold), demonstrating their capacity to strongly activate certain promoters.…”

B cell activation regulates exosomal HLA production

“…Since NF-jB regulates expression of HLA molecules [33,34], one could expect that NF-jB activation simply increases the synthesis of exosomal HLA inside the cells. However, we found an increased level of exosomal HLA release in spite of an equivalent HLA expression in stimulated B cell lines (data not shown).…”

Drosophilaimmunity. A sequence homologous to mammalian interferon consensus response element enhances the activity of the diptericin promoter

“…For example, the full expression of the murine macrophage nitric oxide synthase gene (mac-NOS) depends on the presence of two regions containing NF-KB binding sites; region II which responds to interferon stimulation contains additional GAAANN motifs (23). The HLA-B genes are also induced by interferons and the presence of GAAANN sequences increases the basal level of expression of these genes which is mediated by NF-icB binding sites (24). This stimulation results of the cooperative action of NF-iKB and IRF-1 (25).…”