Celada A, McKercher S R, Maki R A
Departament de Fisiologia (Immunologia), Facultad de Biologia, Universitat de Barcelona, Spain.
Biochem J. 1996 Aug 1;317 ( Pt 3)(Pt 3):771-7. doi: 10.1042/bj3170771.
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.
Y盒是主要组织相容性复合体（MHC）II类基因启动子中的保守序列，其中包含一个CCAAT序列（CCAAT盒）。此前，我们部分纯化了识别I-Aβ基因Y盒的DNA结合蛋白，并表明它由两个组分（因子A和B）组成，这两个组分对于最佳DNA结合都是必需的。已经克隆了与I-EαY盒结合的异源三聚体蛋白NF-Y（NF-YA和NF-YB）的基因。随后，我们使用根据已发表序列设计的寡核苷酸分离出了NF-YA和NF-YB的基因。对NF-YA和NF-YB与I-Aβ和I-EαY盒的结合进行了测试。虽然单独的NF-YA或NF-YB都不与Y盒结合，但当将这些组分混合时，复合物以高亲和力与I-AβY盒结合。此外，NF-YA和NF-YB可以分别被因子B或因子A互补以结合DNA。这些结果表明，在因子A提取物中活性结合蛋白是NF-YA，在因子B提取物中是NF-YB。最后，当将核提取物添加到覆盖I-Aβ基因Y盒的双链寡脱氧核苷酸中时，针对NF-YA和NF-YB的抗体显示出诱导超迁移。制备了NF-YA和NF-YB的反义表达构建体，并测试了它们对I-Aβ启动子表达的影响。当将任何一种反义构建体转染到细胞中时，都降低了与I-Aβ启动子相连的报告基因的表达。这项研究提供了直接证据，证明NF-YA和NF-YB就是先前描述的因子A和B。此外，这些结果强烈表明NF-Y参与了MHC II类基因I-Aβ的表达。
