SummaryRecently, four genes (TAP1, TAP2, LMP2, LMP7) involved or potentially involved in the processing and transport of major histocompatibility complex class I-associated antigen to the endoplasmic reticulum have been identified. We now report the initial characterization of the bidirectional promoter for the human transporter associated with antigen processing 1 (TAP1) and low molecular mass polypeptide 2 (LMP2) genes. These genes are divergently transcribed from a central promoter region of only 593 bp. Functional analysis using a bidirectional reporter system demonstrates the minimal 593-bp promoter is sufficient for concurrent expression in both directions. There is no TATA box homology at either end but there is a prevalence of GC boxes. Transcription is initiated at multiple sites for each gene without any of the TAP1 transcripts overlapping with the LMP2 transcripts. The region proximal to the TAP1 gene is required for maximal basal level expression of not only TAP1 but also LMP2. Furthermore, this region is necessary for tumor necrosis factor oe (TNF-oe) induction of both genes. Site-specific mutations of an NF-t~B element in the TAP1 proximal region blocked induction by TNF-cx in both the TAP1 and LMP2 directions. An adjacent GC box was required for basal expression of both genes as well as augmenting the TNF-oe induction of the distal LMP2 gene. In vivo genomic footprinting of this region revealed strong protein/DNA interactions at the NF-KB and GC box consensus sequences. In vitro binding studies confirmed the capacity of the NF-KB site to bind p50/p65 and p52/p65 heterodimers and of the GC box to bind Spl. Thus, the promoter elements proximal to the TAP1 gene play a significant role in regulating basal and induced expression of both TAP1 and LMP2. The findings presented in this report clearly link LMP2 expression with TAP1 expression and provide additional suggestive evidence linking LMP2 to class I antigen presentation.

The TAP1 and LMP2 genes are central for class I MHC function and share a common promoter. Here, we analyze the molecular mechanism of IFN gamma up-regulation of TAP1 and LMP2. In vivo footprinting indicates IFN gamma up-regulates protein-DNA contacts at an IRF-E that is essential for the up-regulation of TAP1 and LMP2 by IFN gamma. Gel shift analysis indicates that this site binds IRF-1. The expression of TAP1 and LMP2 are both greatly reduced in IRF-1-deficient mice. Surface class I MHC as well as CD8+ T cells are reduced in IRF-1-/- mice. The role of IRF-1 in the regulation of TAP1 and LMP2 suggests a mechanism for the antiviral properties of IRF-1 and the unexpected deficiency of CD8+ T cells observed in IRF-1-/- mice.

Class II transactivator (CIITA) is a master regulator required for constitutive and IFN-γ-inducible expression of class II MHC genes. Although the role of CIITA is greatly appreciated, the mechanisms underlying constitutive and IFN-γ-induced expression of CIITA are not understood. The study of CIITA induction is extremely important, but has been fraught with difficulty. This study describes for the first time a large (7-kb) fragment of 5′ flanking sequences that mediates the B cell-specific, IFN-γ-induced, and TGF-β-suppressed expression of CIITA. This pattern of expression matches the authentic expression of the endogenous gene. Within the 7-kb fragment, sequences that lie between nucleotides −545 and −113 relative to the transcriptional start site are critical for constitutive promoter expression in B cells. In contrast, inducible activation of CIITA by IFN-γ requires sequences contained in an additional 4 kb of upstream DNA. This region mediates an IFN-γ response when linked to either the endogenous CIITA promoter or a heterologous promoter. A role for STAT1 in regulation of the CIITA promoter is shown by the rescue of IFN-γ induction by expression of STAT1 in STAT1-defective U3A cells. TGF-β significantly inhibits IFN-γ-mediated induction of the CIITA promoter in 2fTGH fibroblasts, which indicates that the promoter is a target for TGF-β. This inhibition is achieved by suppression of the basal promoter. This study provides a focal point for understanding the mechanism of B cell-specific, IFN-γ-induced, and TGF-β-suppressed expression of CIITA.

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