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Hsp 60 is a chaperonin protein, homologous to GroEL of Escherichia coli and highly conserved across species. Immune response induced by the hsp 60 equivalent of numerous microorganisms elicits in animals and man a dominantcross-reactive T lymphocyte response. Hsp 60 has been strongly implicated as an example of molecular mimicry in the pathogenicity of autoimmune diseases and, more recently, in T cell-mediated protection. Curiously, in spite of this interest, the gene encoding HSP 60 has not yet been cloned. Sequencing of numerous PCR-derived HSP 60 clones, obtained following amplification of genomic DNA revealed multiple distinct but highly related sequences. These were all different from the sequence encoding the expressed protein and all had interrupted reading frames. PCR amplification from mRNA, however, yielded only the sequence expected for the expressed hsp 60 protein. This apparent paradox was resolved by cloning and sequencing HSP 60-specific genomic clones: the majority of these clones corresponded to intronless genes having the characteristics of retro-pseudogenes and were flanked by unrelated DNA sequences. In addition, several genomic clones were isolated that corresponded to a unique functional HSP 60 gene. This gene is composed of multiple exons, some very short. The transcription start site was identified and 750 bp of 5' flanking sequence were determined. The human HSP 60 gene is induced by heat. We conclude that hsp 60 is encoded by a single highly fragmented gene, that co-exists with multiple HSP 60 retro-pseudogenes, normally not expressed

The RFX DNA binding domain (DBD) is a novel highly conserved motif belonging to a large number of dimeric DNA binding proteins which have diverse regulatory functions in eukaryotic organisms, ranging from yeasts to human. To characterize this novel motif, solid phase synthesis of a 76mer polypeptide corresponding to the DBD of human hRFX1 (hRFX1/DBD), a prototypical member of the RFX family, has been optimized to yield large quantities (∼90 mg) of pure compound. Preliminary two-dimensional 1H NMR experiments suggested the presence of helical regions in this sequence in agreement with previously reported secondary structure predictions. In gel mobility shift assays, this synthetic peptide was shown to bind in a cooperative manner the 23mer duplex oligodeoxynucleotide corresponding to the binding site of hRFX1, with a 2:1 stoichoimetry due to an inverse repeat present in the 23mer. The stoichiometry of this complex was reduced to 1:1 by decreasing the length of the DNA sequence to a 13mer oligonucleotide containing a single half-site. Surface plasmon resonance measurements were achieved using this 5′-biotylinated 13mer oligonucleotide immobilized on an avidin-coated sensor chip. Using this method an association constant (Ka = 4×105/M/s), a dissociation constant (Kd = 6×10−2/s) and an equilibrium dissociation constant (KD = 153 nM) were determined for binding of hRFX1/DBD to the double-stranded 13mer oligonucleotide. In the presence of hRFX1/DBD the melting temperature of the 13mer DNA was increased by 16°C, illustrating stabilization of the double-stranded conformation induced by the peptide

MHC-encoded HLA-DMA and-DMB molecules are atypical MHC chains that play an essential role in antigen presentation by MHC class II molecules. They resemble both MHC class I and II molecules but are not expressed at the cell surface. From the study of MHC class II regulatory mutants, it was found recently that two novel transactivators, CIITA and RFX5, are essential for the control of MHC class II gene expression. We report here that CIITA and RFX5, although operating at different levels of transcriptional control, are also both essential regulators of HLA-DMA and-DMB genes. This is true for both the constitutive and the inducible mode of DM gene expression. Indeed, both CIITA and RFX5 cDNA can correct the HLA-DMA and-DMB gene expression defect in the respective regulatory mutants. The involvement of these two transcription factors accounts for the coordinate expression of MHC class II and HLA-DM, two sets of molecules that perform quite different functions in the overall process of antigen presentation

Precise regulation of MHC class II expression plays a crucial role in the control of the immune response. The transactivator CIITA behaves as a master controller of constitutive and inducible MHC class II gene activation, but its exact mechanism of action is not known. Activation of MHC class II promoters requires binding of at least three distinct multi-protein complexes (RFX, X2BP and NF-Y). It is known that the stability of this binding results from cooperative interactions between these proteins. We show here that expression of CIITA in MHC class II- cells triggers occupation of the promoters by these complexes. This observation raised the possibility that the effect of CIITA on promoter occupation is mediated by an effect on the cooperative stabilization of the DNA-bound multi-protein complexes. We show, however, that the presence of CIITA does not affect the stability of the higher-order protein complex formed on DNA by RFX, X2BP and NF-Y. This suggests other mechanisms for CIITA-induced promoter occupancy, such as an effect on chromatin structure leading to increased accessibility of MHC class II promoters. This ability of CIITA to facilitate promoter occupation is undissociable from its transactivation potential. Finally, we conclude that this effect of CIITA is cell-type specific, since expression of CIITA is not required for normal occupation of MHC class II promoters in B lymphocytes