Identification of the transcription factors NF-YA and NF-YB as factors A and B that bound to the promoter of the major histocompatibility complex class II gene I-A β

Abstract

The Y box is a conserved sequence in the promoter of major histocompatibility 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-Aβ gene and showed that it consisted of two components (factors A and B) both of which were necessary for optimal DNA binding. The genes for the heteromeric protein NF-Y (NF-YA and NF-YB), which binds to the I-Eα Y box have been cloned. We subsequently isolated the genes for NF-YA and NF-YB using oligonucleotides designed from the published sequences. NF-YA and NF-YB were tested for binding to the I-Aβ and I-Eα Y boxes. While neither NF-YA or NF-YB alone bound to the Y box, when the components were mixed the complex bound to the I-Aβ Y box with high affinity. Moreover, 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 binding protein is NF-YA in factor A extracts and NF-YB in factor B extracts. 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-Aβ gene. Antisense expression constructs of both NF-YA and NF-YB were made and their effect on expression from the I-Aβ promoter was tested. Either antisense construction, when transfected into cells, lowered the expression of a reporter gene linked to the I-Aβ promoter. This study provides direct evidence of the identification of NF-YA and NF-YB as the previously described factors A and B. Moreover, these results strongly implicate NF-Y in the expression of the MHC class II gene I-Aβ.