Regulation of embryonic/fetal globin genes by nuclear hormone receptors: a novel perspective on hemoglobin switching.

The CCAAT box is one of the conserved motifs found in globin promoters. It binds the CP1 protein. We noticed that the CCAAT-box region of embryonic/fetal, but not adult, globin promoters also contains one or two direct repeats of a short motif analogous to DR-1 binding sites for non-steroid nuclear hormone receptors. We show that a complex previously named NF-E3 binds to these repeats. In transgenic mice, destruction of the CCAAT motif within the human epsilon-globin promoter leads to substantial reduction in epsilon expression in embryonic erythroid cells, indicating that CP1 activates epsilon expression; in contrast, destruction of the DR-1 elements yields striking epsilon expression in definitive erythropoiesis, indicating that the NF-E3 complex acts as a developmental repressor of the epsilon gene. We also show that NF-E3 is immunologically related to COUP-TF orphan nuclear receptors. One of these, COUP-TF II, is expressed in embryonic/fetal erythroid cell lines, murine yolk sac, intra-embryonic splanchnopleura and fetal liver. In addition, the structure and abundance of NF-E3/COUP-TF complexes vary during fetal liver development. These results elucidate the structure as well as the role of NF-E3 in globin gene expression and provide evidence that nuclear hormone receptors are involved in the control of globin gene switching.     

Nuclear factor I (NF I) binds to an NF I-type site but not to the CCAAT site in the human alpha-globin gene promoter.

Methylation interference and missing contact analyses demonstrate that nuclear factor I (NF I) recognizes an NF I-like site (5'-GGG(N)6GCCAG-3') within the alpha-globin promoter rather than the adjacent CCAAT box. Consistent with this, mutations within the CCAAT box do not alter significantly the affinity and specificity of the interaction whereas elimination of the 5'-GGG-3' half-site of the recognition sequence reduces the DNA binding strength of NF I by 2 orders of magnitude down to the range of unspecific interaction. On the other hand, the mutated alpha-globin promoter sequence that is no longer bound by NF I, although it retains an intact CCAAT box, interacts specifically with a protein component from nuclear extracts of HeLa cells. From these results we conclude that NF I is not the factor that interacts with the CCAAT box and that the second half of the canonical 5'-TGG(N)6GCCAA-3' NF I binding site cannot be regarded as identical with the CCAAT promoter element, as suggested previously.     

人NFBD1启动子的鉴定与初步分析

NFBD1,也称MDC1,是一个参与细胞内DNA损伤后细胞应答反应的重要分子.为了进一步深入研究其转录调控机制,本研究克隆鉴定了NFBD1的启动子.首先应用5′ RACE技术鉴定了NFBD1的转录起始位点,首次发现NFBD1至少存在3种丰度和转录起始位点不同的转录变异体.然后,通过PCR定向克隆和酶切亚克隆策略,构建了覆盖NFBD1基因5′侧翼区起始密码子ATG上游5 kb区域的一系列NFBD1启动子荧光素酶报告基因重组体.启动子活性分析表明,NFBD1启动子区域定位于主要转录起始位点区域附近1.5 kb的区域内.采用转录因子结合位点预测分析软件分析表明,NFBD1启动子缺乏TATA盒,但含有典型的CCAAT盒和GC盒以及其它潜在的转录因子结合位点,提示Sp1和NF-Y等转录因子可能参与NFBD1的转录调控     

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.     

Binding of f-PIP, a pyrrole- and imidazole-containing triamide, to the inverted CCAAT box-2 of the topoisomerase IIalpha promoter and modulation of gene expression in cells

An N-formamido pyrrole- and imidazole-containing triamide (f-PIP) has been shown by DNase I footprinting, SPR, and CD studies to bind as a stacked dimer to its cognate sequences: 5'-TACGAT-3' (5'-flank of the inverted CCAAT box-2 of the human topoisomerase IIalpha promoter) and 5'-ATCGAT-3'. A gel shift experiment provided evidence for f-PIP to inhibit protein-DNA interaction at the ICB2 site. Western blot studies showed that expression of the topoisomerase IIalpha gene in confluent NIH 3T3 cells was induced by treatment with f-PIP. The results suggested that the triamide was able to enter the nucleus, interacted with the target site within ICB2, inhibited NF-Y binding, and activated gene expression.