CIITA stimulation of transcription factor binding to major histocompatibility complex class II and associated promoters in vivo

Abstract

CIITA is a master transactivator of the major histocompatibility complex class II genes, which are involved in antigen presentation. Defects in CIITA result in fatal immunodeficiencies. CIITA activation is also the control point for the induction of major histocompatibility complex class II and associated genes by interferon-gamma, but CIITA does not bind directly to DNA. Expression of CIITA in G3A cells, which lack endogenous CIITA, followed by in vivo genomic footprinting, now reveals that CIITA is required for the assembly of transcription factor complexes on the promoters of this gene family, including DRA, Ii, and DMB. CIITA-dependent promoter assembly occurs in interferon-gamma-inducible cell types, but not in B lymphocytes. Dissection of the CIITA protein indicates that transactivation and promoter loading are inseparable and reveal a requirement for a GTP binding motif. These findings suggest that CIITA may be a new class of transactivator.

Figure 1

CIITA mediates in vivo promoter occupancy of MHC class II and associated genes. In vivo footprint of the DRA upper strand in 2fTGH and CIITA-deficient G3A cells. As indicated above each lane, the cells were induced with IFN-γ or stably express CIITA from an integrated transgene. The functional promoter elements are marked on the left. Solid arrows indicate enhancements; open arrows indicate protections; in vitro indicates control, deproteinized methylated genomic DNA in vitro. IFN-γ-induced 2fTGH cells display strong protections and enhancements at the X1 and X2 boxes and substantial protections at the Y box (lanes 1 and 2). Before IFN-γ treatment, the same, but less intense, interactions are observed [data not shown and Wright and Ting (17)]. G3A cells have a bare DRA promoter unless CIITA is introduced (lanes 3–6). In vivo genomic footprinting was done as described on at least three different DNA preparations, analyzed at least twice each. Cells were treated with recombinant IFN-γ at 500 units per ml for 48 hr before harvest.

Figure 2

DRA transactivation and promoter assembly in cells expressing mutant forms of CIITA. (A) CIITA mutants. A, acidic domain; P, proline-rich domain; S, serine-rich domain; T, threonine-rich domain; GTP indicates the three homologies to a GTP binding motif; BLS2Δ indicates the region deleted in BLS patient line BLS2. The RJ protein was isolated from the class II-negative cell line RJ2.2.5. Transactivation indicates whether that form of CIITA activated DRA transcription in a cotransient transfection assay. +, Full activation compared with wild-type CIITA; −, absolutely no activity. (B) Cell surface expression of DR in G3A cells stably expressing the indicated form of CIITA was detected by fluorescence-activated cell sorter. Solid profile shows DR expression in the test cells; open profile is the DR expression in G3A cells stably transfected with empty vector. Only the wild-type and Δ132–212 CIITA proteins were able to transactivate. Similar levels of CIITA proteins were expressed in each stably transfected cell line, as detected by Western analysis with the FLAG epitope tag antibody (IBI/Kodak). (C) In vivo footprint analysis of DRA promoter occupancy. Cell lines analyzed are as in B and p4 indicates the cell line with the empty vector. Footprinting and lane markings are as in Fig. 1. Only in the wild-type (lane 3) and Δ132–212 (lane 6) cell lines are protections and enhancements clearly found within the X1, X2, and Y boxes. The other mutant CIITA proteins do not promote factor binding.

Figure 3

Invariant chain and DMB promoter occupancy in cells expressing mutant CIITA proteins. (A) Invariant chain distal promoter region. Strong protections of the X and Y elements are seen in the cells expressing wild-type and Δ132–212 CIITA proteins (lanes 3 and 6). The contacts are the same as shown previously in B cells (18, 19) and were confirmed at least four times. (B) DMB promoter region. G3A cells expressing wild-type or GTP3(ΔSKAD) CIITA or the empty vector p4 were examined by in vivo footprinting of the DMB promoter upper strand. The strongest protections are on the X1 box at nucleotide −136 and on the Y box at nucleotide −99. The lane markings are as described in Fig. 2C.

Figure 4

CIITA does not effect promoter occupancy of the MHC-encoded, nonclass II gene TAP1. The same cell lines analyzed in Fig. 2 were examined for TAP1 promoter binding in vivo. Only the GC box is occupied in these cells, and the in vivo footprint is not altered by CIITA (lanes 2 and 3). The interferon response factor element is not occupied before or after introduction of CIITA. The contacts shown here are similar to those previously reported in HeLa cells (30).