Activating protein factor binds in vitro to upstream control sequences in heat shock gene chromatin

DNA sequences, important for the control of Drosophila heat shock gene expression, are packaged in chromatin in a nuclease hypersensitive configuration. Recently, two protein-binding (exonuclease-resistant) sites which cover the TATA box sequence and an upstream control element were shown to occur in vivo amidst the 5' terminal hypersensitive regions of several heat shock genes. Protein-binding at the TATA box is independent of heat shock, but the binding at the upstream element is heat shock dependent, and it was proposed that a heat shock activator protein, HAP, positively regulates the genes. Here, I report the detection of HAP activity in heat shocked cell extracts by reconstituting specific binding to hsp82 gene chromatin in vitro. Inhibition of the binding by free DNA from the 5' region of heat shock genes implies a coordinate regulation of the gene family through HAP interaction with the upstream heat shock consensus sequence. Furthermore, the special ease of induction of the hsp82 gene over other heat shock genes can be explained in molecular terms by the higher affinity of HAP for the hsp82 binding site, which contains a 28 base sequence with almost perfect dyad symmetry, GAAGCCTCTAGAAG/TTTCTAGAGACTTC.     

Alternative sets of DNase I-hypersensitive sites characterize the various functional states of the chicken lysozyme gene

The structural organization of chromatin is thought to determine the state of differentiation and activity of eukaryotic genes. Local interruptions of the regular nucleosomal array, the so-called DNase-hypersensitive sites, may indicate regions of the genome which play a critical part in regulation of differential gene activity. We present here two new observations on the chromatin structure of the chicken lysozyme gene, which strongly support a regulatory function for these sites. First, different sets of DNase I-hypersensitive sites have been found upstream from the promoter, depending on whether the gene is constitutively expressed (cultured macrophages) or in the steroid hormone-controlled state (oviduct). It seems, therefore, that diverse modes of regulation of the same gene are associated with discrete patterns of DNase I hypersensitivity. Second, one of the DNase I-hypersensitive sites in the oviduct chromatin disappears and reappears on steroid hormone withdrawal and secondary induction. These reversible changes in a narrow chromatin region reflect the transition from the potentially active to the active state of the lysozyme gene.     

Methylation of a CTCF-dependent boundary controls imprinted expression of the Igf2 gene

The expression of the insulin-like growth factor 2 (Igf2) and H19 genes is imprinted. Although these neighbouring genes share an enhancer, H19 is expressed only from the maternal allele, and Igf2 only from the paternally inherited allele. A region of paternal-specific methylation upstream of H19 appears to be the site of an epigenetic mark that is required for the imprinting of these genes. A deletion within this region results in loss of imprinting of both H19 and Igf2 (ref. 5). Here we show that this methylated region contains an element that blocks enhancer activity. The activity of this element is dependent upon the vertebrate enhancer-blocking protein CTCF. Methylation of CpGs within the CTCF-binding sites eliminates binding of CTCF in vitro, and deletion of these sites results in loss of enhancer-blocking activity in vivo, thereby allowing gene expression. This CTCF-dependent enhancer-blocking element acts as an insulator. We suggest that it controls imprinting of Igf2. The activity of this insulator is restricted to the maternal allele by specific DNA methylation of the paternal allele. Our results reveal that DNA methylation can control gene expression by modulating enhancer access to the gene promoter through regulation of an enhancer boundary.     

Two tobacco DNA-binding proteins with homology to the nuclear factor CREB

The 35S promoter of the cauliflower mosaic virus (CaMV) contains a tandem repeat of the sequence TGACG in the region -83 to -63. This 21-base pair (bp) sequence, called as-1, is involved in root expression of the 35S promoter. When inserted in a promoter of a gene expressed specifically in photosynthetic tissues, as-1 confers high level expression in roots. We have described a factor, ASF-1, that binds specifically to as-1 in vitro. There is a good correlation between ASF-1 binding affinity to as-1 related sequences in vitro and the function of these sequences in vivo. These results strongly suggest that ASF-1 is responsible for the function of as-1. Here we report the isolation of tobacco complementary DNA clones encoding two TGACG-sequence-specific binding-proteins (TGA1a and TGA1b). Sequence analysis of the cDNA clones shows that both proteins contain a basic region that shows high homology to a stretch of basic amino acids in the nuclear factors CREB, GCN4, and c-Jun to a 'leucine-zipper' region. On the basis of binding specificity we propose TGA1a to be a good candidate for ASF-1.     

A hypersensitive site in hsp70 chromatin requires adjacent not internal DNA sequence

Nuclease-hypersensitive sites in chromatin exist at the 5' side of many eukaryotic genes. To gain some understanding of the molecular basis of these hypersensitive sites, we have now examined the pair of sites upstream of the Drosophila hsp70 gene in a series of plasmids that contain deletions in the hypersensitive region and have been transformed into yeast cells. Hypersensitive sites 5' to a Drosophila hsp70 gene are preserved when this gene is introduced into yeast by transformation. We find that a yeast strain containing a plasmid in which the deletion extends through the first hypersensitive site still displays the normal pair of hypersensitive sites, so DNA sequences over which the first hypersensitive site is centred are not required for hypersensitivity at this position and the site can form over a foreign DNA sequence juxtaposed against this deletion end point. Deletions progressing further into the region bracketed by the pair of 5' hypersensitive sites eliminate the first hypersensitive site and alter the downstream site. We propose that the hypersensitive sites are generated through the binding of a protein that renders flanking sequences more accessible to nucleases, perhaps by preventing normal chromatin packaging.     

Determination of spatial domains of zygotic gene expression in the Drosophila embryo by the affinity of binding sites for the bicoid morphogen

The maternal gene bicoid is a key component of the system that determines the pattern of the anterior half of Drosophila embryos. The bicoid protein forms a concentration gradient in early embryos, and is known to bind DNA. Specific binding sites are now shown to confer expression in a region of the embryo that depends on their affinity for bicoid protein: sites of high affinity allow expression further down the bicoid protein gradient than sites of low affinity.     

基于PWM扫描算法的启动子区域统计分析

为了寻找启动子区域上转录因子结合位点的分布规律,进而研究这种规律与真核基因表达调控机制之间的关系,该文从已有数据出发,运用位置权重矩阵(PWM)扫描算法对启动子区域上4种与肝脏特异表达相关的转录因子结合位点分布情况进行了初步研究,并提出了一种新的序列评分方法。通过该方法提取的统计特征,肝脏特异基因的鉴别准确率可以达93.33%。实验结果表明:肝脏特异基因的启动子区域上结合位点的分布情况与其他基因相比存在显著差异。新的序列评分方法可以更好地反映这种差异性,实现肝脏特异基因的精确鉴别。