IDENTIFICATION OF THE TRANSCRIPTION FACTORS NF-YA AND NF-YB AS FACTOR-A AND FACTOR-B THAT BOUND TO THE PROMOTER OF THE MAJOR HISTOCOMPATIBILITY COMPLEX CLASS-II GENE-I-A-BETA

The Y box is a conserved sequence in the promoter of major histocompat ibility complex (MHC) class II genes, which contains a CCAAT sequence (CCAAT box). Previously, we partially purified the DNA-binding protein that recognizes the Y box of the I-A beta gene and showed that it con sisted of two components (factors A and B) both of which were necessar y for optimal DNA binding. The genes for the heteromeric protein NF-Y (NF-YA and NF-YB), which binds to the I-E alpha Y box have been cloned . We subsequently isolated the genes for NF-YA and NF-YB using oligonu cleotides designed from the published sequences. NF-YA and NF-YB were tested for binding to the I-A beta and I-E alpha Y boxes. While neithe r NF-YA or NF-YB alone bound to the Y box, when the components were mi xed the complex bound to the I-A beta Y box with high affinity. Moreov er, NF-YA and NF-YB could be complemented for binding to DNA by factor B or factor A, respectively. These results suggest that the active bi nding protein is NF-YA in factor A extracts and NF-MB in factor B extr acts. Finally, antibodies against NF-YA and NF-YB were shown to induce a supershift when nuclear extracts were added to the double-stranded oligodeoxynucleotide covering the Y box of the I-A beta gene. Antisens e expression constructs of both NF-YA and NF-YB were made and their ef fect on expression from the I-A beta promoter was tested. Either antis ense construction, when transfected into cells, lowered the expression of a reporter gene linked to the I-A beta promoter. This study provid es direct evidence of the identification of NF-YA and NF-YB as the pre viously described factors A and B. Moreover, these results strongly im plicate NF-Y in the expression of the MHC class II gene I-A beta.