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

Abstract

Tightly regulated expression of major histocompatibility complex (MHC) class II genes is critical for the immune system. A conserved regulatory module consisting of four cis-acting elements, the W, X, X2 and Y boxes, controls transcription of MHC class II genes. The X, X2, and Y boxes are bound, respectively, by RFX, CREB, and NF-Y to form a MHC class II-specific enhanceosome complex. The latter constitutes a landing pad for recruitment of the transcriptional co-activator CIITA. In contrast to the well defined roles of the X, X2, and Y boxes, the role of the W region has remained controversial. In vitro binding studies have suggested that it might contain a second RFX-binding site. We demonstrate here by means of promoter pull-down assays that the most conserved subsequence within the W region, called the S box, is a critical determinant for tethering of CIITA to the enhanceosome complex. Binding of CIITA to the enhanceosome requires both integrity of the S box and a remarkably stringent spacing between the S and X boxes. Even a 1-2-base pair change in the native S-X distance is detrimental for CIITA recruitment and promoter function. In contrast to current models, binding of RFX to a putative duplicated binding site in the W box is thus not required for either CIITA recruitment or promoter activity. This paves the way for the identification of novel factors mediating the contribution of the S box to the activation of MHC class II promoters.Major histocompatibility complex (MHC) 1 class II molecules are heterodimeric cell surface glycoproteins that present peptides to the antigen receptor (T cell receptor) of CD4 ϩ T cells. Engagement of MHCII-peptide complexes by the T cell receptor is essential for selection of the mature CD4 ϩ T cell repertoire during T cell development in the thymus and for the initiation, propagation, and regulation of adaptive immune responses by mature T cells in the periphery. Because of these key functions in the adaptive immune response, MHCII expression is tightly controlled in a cell type-specific and inducible manner (for reviews see Refs.

New Functions of the Major Histocompatibility Complex Class II-Specific Transcription Factor RFXANK Revealed by a High-Resolution Mutagenesis Study

“…To corroborate these findings with biochemical support, we performed a promoter pull-down assay which we developed previously to assess the assembly of a stable higher-order enhanceosome complex containing RFX, NF-Y, CREB, and the S-box binding factor (23,29). Briefly, DNA fragments containing the HLA-DRA promoter region were incubated with extracts from BLS-1 cells complemented with wild-type or mutant RFXANK.…”

“…For each assay, ϳ10 g of either GST or GST-RFXANK prebound to Microspin beads was mixed with 5 l of [ 35 Promoter pull-down assays. Promoter pull-down assays with whole-cell extracts were performed essentially as described previously (23,29). Briefly, 0.6 mg of whole-cell extracts prepared from BLS-1 cells and transfectants was incubated with a magnetic-bead-bound biotinylated HLA-DRA promoter fragment, prepared by PCR using pDRsyn (45) as a template.…”

“…CI-ITA has to be recruited to MHC-II promoters by multiple protein-protein interactions with the components of a large multiprotein complex referred to as the MHC-II enhanceosome (4,16,23,37,44,49), which assembles on a characteristic promoterproximal regulatory motif located ϳ150 to 300 bp upstream of the transcription initiation site (1). This regulatory module is composed of four highly conserved sequence elements, called the S (or W), X, X2, and Y boxes, which are targets of unidentified S-box binding proteins, RFX, X2BP (which contains CREB), and NF-Y, respectively (21,27,29,38). While NF-Y and CREB are implicated in the regulation of many genes, RFX is almost exclusively specific for MHC genes.…”

ZXDC, a novel zinc finger protein that binds CIITA and activates MHC gene transcription

“…CIITA does not bind DNA, but rather is recruited to MHC class II promoters by interacting with the CREB protein and subunits of the RFX and NFY complexes (DeSandro et al, 2000;Hake et al, 2000;Scholl et al, 1997;Zhu et al, 2000). In addition, the role of the W box in recruiting CIITA to MHC class II promoters is critical, though the factor(s) that binds this element have not been identified (Brown et al, 1998;Muhlethaler-Mottet et al, 2004).…”

Regulation of major histocompatibility complex class II gene expression, genetic variation and disease

“…Although the function of the S box is modified by an unidentified factor, CIITA recruitment is highly dependent on the integrity of the S box and the precise spacing between S and X boxes. 44 Expression of the CIITA gene (also known as MHC2TA) is inducible and found to be tightly linked to coordinated MHC class II expression. The regulation of CIITA is highly complex involving four different promoters in a cell and stimulus-specific manner.…”