Two distinct domains within CIITA mediate self-association: involvement of the GTP-binding and leucine-rich repeat domains.

CIITA is the master regulator of class II major histocompatibility complex gene expression. We present evidence that CIITA can self-associate via two domains: the C terminus (amino acids 700 to 1130) and the GTP-binding domain (amino acids 336 to 702). Heterotypic and homotypic interactions are observed between these two regions. Deletions within the GTP-binding domain that reduce GTP-binding and transactivation function also reduce self-association. In addition, two leucine residues in the C-terminal leucine-rich repeat region are critical for self-association as well as function. This study reveals for the first time a complex pattern of CIITA self-association. These interactions are discussed with regard to the apoptosis signaling proteins, Apaf-1 and Nod1, which share domain arrangements similar to those of CIITA.

Transcriptional scaffold: CIITA interacts with NF-Y, RFX, and CREB to cause stereospecific regulation of the class II major histocompatibility complex promoter.

Scaffold molecules interact with multiple effectors to elicit specific signal transduction pathways. CIITA, a non-DNA-binding regulator of class II major histocompatibility complex (MHC) gene transcription, may serve as a transcriptional scaffold. Regulation of the class II MHC promoter by CIITA requires strict spatial-helical arrangements of the X and Y promoter elements. The X element binds RFX (RFX5/RFXANK-RFXB/RFXAP) and CREB, while Y binds NF-Y/CBF (NF-YA, NF-YB, and NF-YC). CIITA interacts with all three. In vivo analysis using both N-terminal and C-terminal deletion constructs identified critical domains of CIITA that are required for interaction with NF-YB, NF-YC, RFX5, RFXANK/RFXB, and CREB. We propose that binding of NF-Y/CBF, RFX, and CREB by CIITA results in a macromolecular complex which allows transcription factors to interact with the class II MHC promoter in a spatially and helically constrained fashion.

Two distinct gamma interferon-inducible promoters of the major histocompatibility complex class II transactivator gene are differentially regulated by STAT1, interferon regulatory factor 1, and transforming growth factor beta.

The major histocompatibility complex (MHC) class II transactivator (CIITA) is the master regulatory factor required for appropriate expression of class II MHC genes. Understanding the expression of CIITA is key to understanding the regulation of class II MHC genes. This report describes the independent regulation of two distinct CIITA promoters by cytokines with opposing functions, gamma interferon (IFN-gamma) and transforming growth factor beta (TGF-beta). A functional analysis of deletion mutations of the upstream promoter (promoter III) identified an IFN-gamma-responsive region located approximately 5 kb from the transcriptional start site. An in vivo DNase I hypersensitivity analysis detected a hypersensitive site in this area which supports the relevance of this region. When the downstream promoter (promoter IV) was studied by in vivo genomic footprinting, IFN-gamma-induced changes at putative binding sites for STAT1, interferon regulatory factor 1 (IRF-1), and E-box proteins were seen. Gel shift and supershift analyses for IRF-1 confirmed the in vivo footprint results. The role of the IFN-gamma-inducible transcription factor STAT1 was examined functionally. Although both promoters were controlled by STAT1, promoter-specific regulation was exhibited. The IFN-gamma response of promoter III was completely dependent on STAT1 and not IRF-1, while promoter IV was partially activated by IRF-1 in the total absence of STAT1 expression. While both promoters were affected by TGF-beta, activation of promoter III by IFN-gamma was more severely diminished by TGF-beta treatment. The differential control of CIITA promoters by TGF-beta, IRF-1, and STAT1 may be important in refining regulation of class II MHC genes in different cell types and under different stimulatory conditions.

Identification of distinct regions of 5' flanking DNA that mediate constitutive, IFN-gamma, STAT1, and TGF-beta-regulated expression of the class II transactivator gene.

Class II transactivator (CIITA) is a master regulator required for constitutive and IFN-gamma-inducible expression of class II MHC genes. Although the role of CIITA is greatly appreciated, the mechanisms underlying constitutive and IFN-gamma-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-gamma-induced, and TGF-beta-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-gamma requires sequences contained in an additional 4 kb of upstream DNA. This region mediates an IFN-gamma 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-gamma induction by expression of STAT1 in STAT1-defective U3A cells. TGF-beta significantly inhibits IFN-gamma-mediated induction of the CIITA promoter in 2fTGH fibroblasts, which indicates that the promoter is a target for TGF-beta. 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-gamma-induced, and TGF-beta-suppressed expression of CIITA.

CCAAT-binding factor NF-Y and RFX are required for in vivo assembly of a nucleoprotein complex that spans 250 base pairs: the invariant chain promoter as a model.

The events that lead to promoter accessibility within chromatin are not completely understood. The invariant chain (Ii) promoter was used as a model to determine the contribution of different DNA-binding factors in establishing occupancy of a complex promoter. Gamma interferon induction of the Ii promoter requires the cooperation of multiple cis elements including distal S, X, and Y/CCAAT elements along with proximal GC and Y/CCAAT elements. The heteromeric transcription factor NF-Y binds to both Y/CCAAT elements. Genomic footprinting was used to analyze in vivo protein-DNA contacts for integrated Ii promoters bearing mutations in each element. The results reveal a hierarchy of transcription factor loading with NF-Y binding to the distal Y/CCAAT element being required for establishing protein-DNA interactions over the entire 250 bp analyzed. Mutation of the X box disrupts binding primarily at the adjacent Y/CCAAT element along with a lesser effect on GC box binding. Importantly, this finding is verified with a cell line which lacks a functional X-box-binding factor, RFX, providing physiological validity for the strategy described here. Mutation of both the S element and the GC box results in either no or little effect on transcription factor binding. However, mutation of the proximal Y/CCAAT element disrupts binding to the adjacent GC box and partially reduces binding in the distal S/X/Y domain. The crucial role for NF-Y in establishing promoter occupancy may be related to its histone fold motif, the essential component for assembling nucleosome-like structures.

Function of NF-kappa B/Rel binding sites in the major histocompatibility complex class II invariant chain promoter is dependent on cell-specific binding of different NF-kappa B/Rel subunits.

The promoter of the human major histocompatibility complex class II-associated invariant-chain gene (Ii) contains two NF-kappa B/Rel binding sites located at -109 to -118 (Ii kappa B-1) and -163 to -172 (Ii kappa B-2) from the transcription start site. We report here that the differential function of each of these NF-kappa B/Rel sites in several distinct cell types depends on cell-specific binding of NF-kappa B/Rel transcription factors. Ii kappa B-1 is a positive regulatory element in B-cell lines and in the Ii-expressing T-cell line, H9, but acts as a negative regulatory element in myelomonocytic and glia cell lines. In vivo protein-DNA contacts are detectable at Ii kappa B-1 in cell lines in which this site is functional as either a positive or negative regulator. Electrophoretic mobility supershift assays determine that members of the NF-kappa B/Rel family of transcription factors can bind to this site in vitro and that DNA-binding complexes that contain p50, p52, p65, and cRel correlate with positive regulation whereas the presence of p50 correlates with negative regulation. Ii kappa B-2 is a site of positive regulation in B-cell lines and a site of negative regulation in H9 T cells, myelomonocytic, and glial cell lines. In vivo occupancy of this site is observed only in the H9 T-cell line. Again, in vitro supershift studies indicate that the presence of p50, p52, p65, and cRel correlates with positive function whereas the presence of only p50 and p52 correlates with negative function. This differential binding of specific NF-kappa B/Rel subunits is likely to mediate the disparate functions of these two NF-kappa B/Rel binding sites.