Differences in DNA sequence specificity among MHC class II X box binding proteins.
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.
Boothby
M
M
Department of Cancer Biology, Harvard School of Public Health, Boston, MA 02155.
Liou
H C
HC
Glimcher
L H
LH
D000818:Animals; D001483:Base Sequence; D001667:Binding, Competitive; D002478:Cells, Cultured; D004247:DNA / Q000302:isolation & purification* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000276:immunology; D005802:Genes, MHC Class II; D008222:Lymphokines / Q000494:pharmacology; D051379:Mice; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes
D004268:DNA-Binding Proteins; D008222:Lymphokines; D015345:Oligonucleotide Probes; D004247:DNA