The Journal of Immunology, Vol 142, Issue 3 1005-1014, Copyright © 1989 by American Association of Immunologists

ARTICLES

Differences in DNA sequence specificity among MHC class II X box binding proteins

M Boothby, HC Liou and LH Glimcher
Department of Cancer Biology, Harvard School of Public Health, Boston, MA 02155.

The class II (Ia) MHC Ag are integral membrane proteins whose expression is limited to specific cell types. A pair of consensus sequences, X and Y, is found upstream from all class II genes and deletion of each of these sequences eliminates expression of transfected genes. Cells that express Ia demonstrate a coordinate response to lymphokines and other stimuli. These conserved sequences might, therefore, play a role in tissue specificity or lymphokine inducibility of Ia gene expression. The X box sequence of the murine class II A alpha gene diverges much more substantially from the X consensus than does the Y box motif of this gene. We demonstrate that this X box motif is nonetheless recognized by sequence-specific DNA- binding proteins, as is the more closely conserved Y box. Gel retardation assays and DNase I footprints were compared for a panel of Ia+ and Ia- cells as well as for cells stimulated with the Ia-inducing lymphokines IL-4 and IFN-gamma. The level, retardation pattern and region of DNA contact were comparable in all instances. Thus the availability of active DNA-binding X and Y box factors cannot alone account for the regulation of A alpha expression. To test whether the same set of proteins binds all class II MHC conserved motifs, oligonucleotide probe binding and cross-competition experiments with X box sequences from A alpha, E alpha, and E beta genes were performed. These studies demonstrated A alpha, E alpha, and E beta DNA-protein complexes with unique mobilities and specificities. In addition, all three X box oligonucleotide probes generated one faint complex with an affinity profile of E beta greater than E alpha much greater than A alpha. These three complexes comigrated and thus may represent a communal binding protein. The data are most consistent with the conclusion that multiple proteins bind class II MHC X boxes. For A alpha, the predominant complexes represent different specificities from the predominant E alpha and E beta X box binding proteins.

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