Eur. J. Immunol. 1994. zyxwvutsrqpo 24: 2415-2420 zyxwvutsrq Gildas Rigaud, Fiorenza Paiola and Roberto S. Accolla Institute of Immunology and Infectious Diseases, School of Medicine, University of Verona, Policlinico zyxwvutsr di Borgo Roma, Verona In zyxwvutsr vivo DRA promoter occupancy in MHC class 11-negative cells 2415 In vivo modification of major histocompatibility complex class I1 DRA promoter occupancy mediated by the AIR-1 trans-activator* RJ 2.2.5 is a human B cell mutant derived from the Burkitt lymphoma Raji cell, which is defective in the AIR-1 locus function. This locus encodes a transcrip- tional trans-activator required for the constitutive expression of major histocom- patibility complex (MHC) class I1 genes. Here we show, by zy in vivo DNase I footprinting, that the AIR-1 locus defect correlates with changes in the DRA promoter occupancy. Interestingly, reexpression of human MHC class I1 genes in RJ 2.2.5 zyxwvut x mouse spleen cell hybrids is associated with partial reversion of DRA promoter occupancy to the Raji pattern. DRA promoter occupancy in other class 11-negative B cell lines, derived from patients with bare lymphocyte syndrome, is drastically different from the one observed in RJ 2.2.5 and Raji cells. Moreover, the use of the DNase I as an in vivo footprinting agent reveals that the patients’ cell lines do not display a completely “bare promoter” as previously reported using dimethyl sulfate as the footprinting agent. Thus, the use of DNase I allowed us, for the first time, to correlate the AIR-1 locus defect with class I1 promoter occupancy alterations and distinguish these alterations from the ones observed in phenotypically similar but genetically distinct MHC class II- negative cells. 1 Introduction Major histocompatibility complex (MHC) class I1 genes encode heterodimeric cell surface glycoproteins which play a crucial role in the homeostasis of the immune response as antigen-presenting receptors to regulatory CD4+ T lym- phocytes [l]. In humans, three MHC class I1 subregions, designated €&A-DR, -DP and -DQ encode the corres- ponding class I1 heterodimers. Tissue-specificexpression of the various class I1 genes is coordinately regulated and the mechanisms involved act mainly at the transcriptional level PI. Much effort has been devoted toward understanding the molecular and genetic mechanisms of class I1 gene expres- sion. Structural and functional studies have demonstrated the importance of a 300-bp sequence located 5’ upstream to the transcriptional initiation site. This sequence includes at least four boxes, designated Y, X1, X2 and W/S which are highly conserved both among distinct class I1 genes and across different species. Mutations, deletions or changes in the spacing of these boxes diminish and/or abolish the [I 130321 * This work was supported by grants from: Associazione Italiana Ricerca sul Cancro (AIRC), CNR Biotecnologie e Biostrumen- tazione, MURST 40% Aspetti Sperimentali della Riposta Immune, ARBI. G. Rigaud is an EMBO fellow. Correspondence: Gildas Rigaud, Institute of Immunology and Infectious Diseases, School of Medicine, University of Verona, Policlinico di Borgo Roma, 37100 Verona, Italy (Fax: +39 45 58 09 00) Abbreviations: LMPCR: Ligation-mediated PCR HR: Hyper- reactive Key words: Major histocompatibility complex class I1 / Gene expression / DNase I in vivo footprint / Complementation groups transcription of reporter genes when introduced in class II- expressing cells. These boxes bind a very heterogeneous series of nuclear factors. Some of them have been already cloned and may have functional importance [2]. MHC class I1 mutants generated either by in vim muta- genesis and immunoselection [3-51 or derived from patients with a severe combined immunodeficiency designated as bare lymphocyte syndrome (BLS) [6], share the common characteristic of being transcriptionally inactive. At least four groups of complementation have been defined by somatic cell fusion, suggesting the existence of at least four independent loci controlling class I1 transcriptional activity [7, 81. One of these mutants, RJ 2.2.5 [3], is defective in AIR-1 locus activity. The AIR-1 locus encodes an MHC class I1 transcriptional activator whose function is highly conserved among species [9].The mouse equivalent, desig- nated Air-1 and mapped on chromosome 16 [9], has been shown to complement the defect of the human mutant RJ 2.2.5. Transfection studies strongly suggest that the AIR-1 locus product exerts its action on the crucial 300-bp promo- ter region located just upstream of the transcription initiation site [lo, 111. Recently a cDNA designated CIITA has been cloned and shown to complement the defect of RJ 2.2.5 and other mutant cells included in the same comple- mentation group. It is likely that CIITA represents the product of the AIR-1 locus [12]. However, the mechanism of action of the trans-activator remains elusive. By a series of approaches including electromobility shift assay, in vim footprinting [lo] and in vivo dimethyl sulfate (DMS) footprinting [13] , no differences in DNA/protein interac- tions between RJ 2.2.5 and the isogenic class 11-positive Raji cell have been observed. In vivo footprinting is certainly more suitable than other techniques for assessing DNA/protein interactions taking place in physiological conditions. However, the previously used DMS may not be the ideal footprinting agent because (i) it is a highly reactive molecule that can interact with DNA involved in a DNA-protein interaction [14], but also z 0 VCH Verlagsgesellschaft mbH, D-69451 Weinheim, 1994 0014-2980/94/1010-2415$10.00 + .25/0