Complex Architecture of Major Histocompatibility Complex Class II Promoters: Reiterated Motifs and Conserved Protein-Protein Interactions

Abstract

The S box (also known as at the H, W, or Z box) is the 5-most element of the conserved upstream sequences in promoters of major histocompatibility complex class II genes. It is important for their B-cell-specific and interferon gamma-inducible expression. In this study, we demonstrate that the S box represents a duplication of the downstream X box. First, RFX, which is composed of the RFX5-p36 heterodimer that binds to the X box, also binds to the S box and its 5-flanking sequence. Second, NF-Y, which binds to the Y box and increases interactions between RFX and the X box, also increases the binding of RFX to the S box. Third, RFXs bound to S and X boxes interact with each other in a spatially constrained manner. Finally, we confirmed these protein-protein and protein-DNA interactions by expressing a hybrid RFX5-VP16 protein in cells. We conclude that RFX binds to S and X boxes and that complex interactions between RFX and NF-Y direct B-cell-specific and interferon gamma-inducible expression of major histocompatibility complex class II genes.Class II genes of the major histocompatibility complex (class II) code for proteins that initiate and propagate immune system responses (19,30,31). They present antigenic peptides to the T-cell antigen receptor and direct helper T cells to appropriate target B cells (10). The end result of these interactions is to activate and differentiate antigen-specific B cells to plasma cells (2). Expression of class II is carefully regulated at the level of transcription such that high levels of these proteins are observed on the surface of B cells, activated human T cells, and many somatic and antigen-presenting cells after their induction by the T-cell lymphokine gamma interferon (IFN-␥) (1,4,8,13,24).Since the DRA gene is invariant and is expressed at the highest levels of all class II genes, its promoter has been studied extensively (1,8,24). Lessons learned with the DRA promoter hold for other class II promoters (1,4,8,13,24,48). Mapping of cis-acting elements revealed that the sequences from positions Ϫ140 to Ϫ60 are sufficient for its B-cell-specific and IFN-␥-inducible expression (44). From the 5Ј to the 3Ј direction, these conserved upstream sequences (CUS) consist of the S box (also known as the H, W, or Z box), the X box, and the Y box (1,4,8,13,24,48). Further downstream, the DRA promoter contains an octamer-binding site and an initiator element (Inr) (4,13,22,44,50). The X box and its flanking sequences have been subdivided into the pyrimidine tract and the X1, X2, and X3 boxes (24). For the purposes of this study, the X box refers to the X1 box. Whereas S and X boxes are most important for the B-cell-specific and IFN-␥-inducible expression (39, 43), the Y box, octamer-binding site and Inr sequences also determine the site of initiation of DRA transcription (4,13,22,50).A large number of proteins that interact with the CUS have been described. Regulatory factors X (RFX1 to RFX5 and p36) bind to the X box (25,29,36). Although the RFX1 homodimer binds better to the isolated ...

The S Box of Major Histocompatibility Complex Class II Promoters Is a Key Determinant for Recruitment of the Transcriptional Co-activator CIITA

“…1B) and RFX can bind weakly to this motif in gel retardation assays (48,49). Binding of two RFX molecules to this motif and the X box was found to be cooperative in these studies (48,49). Finally, it was reported that the W box can be converted into a perfect X box sequence without compromising promoter function (49).…”

“…A widespread model is that the W region could be a duplicated target site for RFX (1,52). This is based on the observation that the W region of the DRA gene contains a sequence that exhibits limited homology to the X box and can be bound weakly by RFX in vitro (44,48,49,53). However, the functional relevance of this finding is not clear because this X box-like motif does not coincide with the most conserved core S sequence.…”

Identification of Class II Transcriptional Activator-Induced Genes by Representational Difference Analysis: Discoordinate Regulation of the DNα/DOβ Heterodimer

“…In part, CIITA derives its activity through interaction with the trimeric transcription factor RFX, which is essential for transactivation from the X box and is thought to bind the W box as well (27,40). Voliva et al have shown that the X box of DO␤ can substitute functionally in the DR␣ promoter (54).…”

“…RFX is a trimeric protein composed of RFX5 (complementation group C) (22), RFXAP (group D) (23), and RFXANK/RFX-B (group B) (24,25). RFX is thought to direct B cell-specific and IFN-␥-inducible expression of MHC class II genes through binding to the X box (26) and possibly the W box (27). CIITA (group A) (28) is the only MHC class II transcription factor known to be induced in response to IFN-␥, and its expression alone is sufficient to activate expression of the MHC class II genes (29 -31).…”

Histone Deacetylation, But Not Hypermethylation, Modifies Class II Transactivator and MHC Class II Gene Expression in Squamous Cell Carcinomas

“…The general mechanisms of MHC class II expression have been reported (14). Three MHC class II molecules, HLA-DR, -DP, and -DQ, are all regulated by cis-acting promoter elements, W, X1, X2, Y boxes, and their interactions with transcriptional factors such as members of the regulatory factor X family, NF-Y, and CREB have been reported (15)(16)(17). In addition to these binding factors, MHC class II transactivator (CIITA) is also required for efficient promotion of MHC class II genes (18,19).…”