Abstract
Expression of the major histocompatibility complex (MHC) class II-associated invariant chain (Ii) is required for efficient and complete presentation of antigens by MHC class II molecules and a normal immune response. The Ii gene is generally co-regulated with the MHC class II molecules at the level of transcription and a shared SXY promoter element has been described. This report defines the proximal promoter region of Ii which may regulate Ii transcription distinct from MHC class II. In vivo genomic footprinting identified an occupied, imperfect CCAAT box and an adjacent GC box in the proximal region. These sites are bound in Ii-ositive cell lines and upon interferon-gamma induction of Ii transcription. In contrast, both sites are unoccupied in Ii-egative cell lines and in inducible cell lines prior to interferon-gamma treatment. Together these two sites synergize to stimulate transcription. Independently, the transcription factor NF-Y binds poorly to the imperfect CCAAT box with a rapid off rate, while Sp1 binds to the GC box. Stabilization of NF-Y binding occurs upon Sp1 binding to DNA. In addition, the half-life of Sp1 binding also increased in the presence of NF-Y binding. These findings suggest a mechanism for the complete functional synergy of the GC and CCAAT elements observed in Ii transcription. Furthermore, this report defines a CCAAT box of imperfect sequence which binds NF-Y and activates transcription only when stabilized by an adjacent factor, Sp1.
Highlights
One of the central steps in elicitation of an immune response is the presentation of foreign antigenic peptides by the major histocompatibility complex (MHC)1 class II molecules on the surface of cells [1, 2]
A consensus NF-Y/CCAAT box has previously been characterized in the distal region of the invariant chain (Ii) promoter and is referred to as the Y box
A similar bare promoter was observed for the MHC class II promoter DRA in Jurkat cells even though the transcription factors are available in the nucleus [54]
Summary
DNA Constructs—The invariant chain promoter, chloramphenicol acetyltransferase (CAT) reporter gene construct, 790IiCAT, contains the human Ii promoter sequences from ϩ1 to Ϫ790 base pairs relative to the transcription initiation site [49]. Mutant constructs were derived by oligonucleotide-directed, site-specific mutagenesis as described previously [50]. Expression of the transiently transfected reporter genes was assayed by quantitating chloramphenicol acetyltransferase activity in cell extracts as described previously [51]. Gel shift analysis was performed as described previously [56]. Gel shift binding reactions with increasing amounts of specific competitor DNA Half-lives of the DNA binding activities were calculated as described previously [58]. Except the same specific oligonucleotides described for gel shift analysis were used as the competitors as indicated in the figure legend. The half-life of either factor alone was determined from EMSA reactions with the IimtGC or IimtYprox probes which only bind one of the factors to simplify the calculations
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