A survey of 178 NF-Y binding CCAAT boxes.

The CCAAT box is one of the most common elements in eukaryotic promoters, found in the forward or reverse orientation. Among the various DNA binding proteins that interact with this sequence, only NF-Y (CBF, HAP2/3/4/5) has been shown to absolutely require all 5 nt. Analysis of a database with 178 bona fide NF-Y binding sites in 96 unrelated promoters confirms this need and points to specific additional flanking nucleotides (C, Pu, Pu on the 5'-side and C/G, A/G, G,A/C, G on the 3'-side) required for efficient binding. The frequency of CCAAT boxes appears to be relatively higher in TATA-less promoters, particularly in the reverse ATTGG orientation. In TATA-containing promoters the CCAAT box is preferentially located in the -80/-100 region (mean position -89) and is not found nearer to the Start site than -50. In TATA-less promoters it is usually closer to the +1 signal (at -66 on average) and is sometimes present in proximity to the Cap site. The consensus and location of NF-Y binding sites parallel almost perfectly a previous general statistical study on CCAAT boxes in 502 unrelated promoters. This is an indication that NF-Y is the major, if not the sole, CCAAT box recognizing protein and that it might serve different roles in TATA-containing and TATA-less promoters.     

CCAAT-box binding protein NF-Y (CBF, CP1) recognizes the minor groove and distorts DNA.

The CCAAT box is one of the most common promoter elements. The evolutionarily conserved heteromeric factor NF-Y binds this sequence with high affinity and specificity. By comparing the methylation interference patterns of different sites, performing electrophoretic mobility shift assays (EMSA) with IC-substituted oligonucleotides and competition experiments with the minor groove binding (MGB) drugs distamicin A, tallimustine and Hoechst 33258 we show that NF-Y makes key minor groove interactions. Circular permutation assays on four CCAAT boxes, MHC Class II Ea, HSP70, epsilon-globin and MSV, indicate that NF-Y is able to distort the double helix by angles of 62-82 degrees, depending on the site used, and suggest that nucleotides flanking the CCAAT pentanucleotide influence the degree of bending.     

人A4GNT基因5'调控区的结构和功能分析

为探索人α1,4-N-乙酰葡糖胺转移酶(α1,4-N-acetylglucosaminyltransferase,A4GNT)基因表达的调控机制,应用5'cDNA末端快速扩增法和引物延伸法确定了A4GNT基因的转录起始位点.在生物信息学分析的基础上,构建了系列5'缺失荧光素酶报告基因载体和定点突变载体.瞬时转染胃癌细胞MKN45和AGS.荧光素酶活性分析表明,A4GNT基因转录的核心启动子在-141bp～+116bp区域,该区域缺乏典型的TATA盒,但含有CCAAT盒、Sp1和ETS-1等转录因子潜在结合位点.突变分析显示,-136bp～-131bp的Sp1结合位点及-93bp～-89bp正向CCAAT序列对A4GNT启动子转录激活至关重要.电泳迁移率变动分析表明,这两个顺式作用元件能够与转录因子Sp1和NF-Y结合.另外,在-1464bp～-771bp区域可能含有与基因的特异性表达相关的调控元件.