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 beta.
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-A beta 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-E alpha 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-A beta and I-E alpha 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-A beta 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-A beta gene. Antisense expression constructs of both NF-YA and NF-YB were made and their effect on expression from the I-A beta promoter was tested. Either antisense construction, when transfected into cells, lowered the expression of a reporter gene linked to the I-A beta 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-A beta.
Celada
A
A
Departament de Fisiologia (Immunologia), Facultad de Biologia, Universitat de Barcelona, Spain.
McKercher
S R
SR
Maki
R A
RA
D000595:Amino Acid Sequence; D001483:Base Sequence; D022762:CCAAT-Enhancer-Binding Proteins; D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D004247:DNA; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D005802:Genes, MHC Class II; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D011485:Protein Binding
D022762:CCAAT-Enhancer-Binding Proteins; D004268:DNA-Binding Proteins; D004247:DNA; D015500:Chloramphenicol O-Acetyltransferase