Functional identification of the transcriptional regulatory elements within the promoter region of the human ventricular myosin alkali light chain gene.

We have identified and functionally characterized DNA sequences that regulate the expression of the human ventricular/slow twitch isoform of myosin alkali light chain (VLC1) gene. By using primer extension and S1 nuclease mapping techniques, we have shown that the VLC1 gene is transcribed from the identical site in the ventricular and slow twitch skeletal muscles. Comparison of the VLC1 sequences from +1 to -1296 in the genes for human and mouse showed that the 5'-proximal flanking region, up to about 220 nucleotides, was highly conserved (83% homology). To determine the location of sites that may be important for the function of the VLC1 promoter, a series of transient expression vectors containing progressive deletions of the VLC1 gene 5'-flanking sequence fused to the bacterial chloramphenicol acetyltransferase (CAT) gene was introduced into myogenic and nonmyogenic cells. Deletion mutagenesis of sequences between -357 and +40 revealed the presence of positive and negative activity in all the cells tested. We demonstrated that the minimal promoter sequence required to generate muscle cell-specific expression is the region between -94 to -64 upstream from the cap site and a sequence element located between -107 and -94 was found to have a positive effect in both myogenic cells and nonmyogenic cells. These two proximal regions located between -107 and -64 appear to act together to determine the cell type-specific high level expression of the VLC1 gene in muscle cells. Competition gel retardation assays revealed that the CArG sequence located between -96 and -87 interacts specifically with nuclear extracts from myogenic and nonmyogenic cells and compete for binding with the CArG sequence present in the human cardiac alpha-actin gene and with the serum response element of the c-fos gene. These results strongly suggested that similar, if not identical, the CArG box binding proteins interact with the functionally different promoter element in the VLC1, cardiac alpha-actin, and c-fos genes.     

Multimodal regulatory elements within a hormone-specific super enhancer control a heterogeneous transcriptional response

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拟南芥TCH4基因启动区转录调控元件的计算识别

TCH4基因在植物次生生长、疾病抵抗和逆境适应方面具有重要作用, 能被多种激素、环境和机械信号诱导表达。利用拟南芥TCH4的直系同源基因和芯片数据进行了启动子序列分析, 结果共识别出9个转录调控元件。它们均包含有已知元件序列, 并且在部分共表达基因和对应的直系同源基因启动子中排列顺序一致。根据已有TCH4基因启动子研究, 其中4个已被报道, 另5个为本研究新发现。根据预测结果进行知识整合, 构建了TCH4基因转录调控机制模型。     

Conserved regulatory elements in the promoter region of the N-CAM gene.

Genomic clones containing 5'-flanking sequences, the first exon, and the entire first intron from the chicken N-CAM gene were characterized by restriction mapping and DNA sequencing. A > 600-bp segment that includes the first exon is very G + C-rich and contains a large proportion of CpG dinucleotides, suggesting that it represents a CpG island. SP-1 and AP-1 consensus elements are present, but no TATA- or CCAAT-like elements were found within 300 bp upstream of the first exon. Comparison of the chicken promoter region sequence with similar regions of the human, rat, and mouse N-CAM genes revealed that some potential regulatory elements including a "purine box" seen in mouse and rat N-CAM genes, one of two homeodomain binding regions seen in mammalian N-CAM genes, and several potential SP-1 sites are not conserved within this region. In contrast, high CpG content, a homeodomain binding sequence, an SP-1 element, an octomer element, and an AP-1 element are conserved in all four genes. The first intron of the chicken gene is 38 kb, substantially smaller than the corresponding intron from mammalian N-CAM genes. Together with previous studies, this work completes the cloning of the chicken N-CAM gene, which contains at least 26 exons distributed over 85 kb.     

Evolution of regulatory elements producing a conserved gene expression pattern in Caenorhabditis

Natural selection acts at the level of function, not at the logistical level of how organisms achieve a particular function. Consequently, significant DNA sequence and regulatory differences can achieve the same function, such as a particular gene expression pattern. To investigate how regulatory features underlying a conserved function can evolve, we compared the regulation of a conserved gene expression pattern in the related species Caenorhabditis elegans and C. briggsae. We find that both C. elegans and C. briggsae express the ovo-related zinc finger gene lin-48 in the same pattern in hindgut cells. However, the regulation of this gene by the Pax-2/5/8 protein EGL-38 differs in two important ways. First, specific differences in the regulatory sequences of lin-48 result in the presence of two redundant EGL-38 response elements in C. elegans, whereas the redundancy is absent in C. briggsae. Second, there is a single egl-38 gene in C. briggsae. In contrast, the gene is duplicated in C. elegans, with only one copy retaining the ability to regulate lin-48 in vivo. These results illustrate molecular changes that can occur despite maintenance of conserved gene function in different species.     

Evolution of the proximal promoter region of the mammalian growth hormone gene.

The evolutionary relationship between the proximal growth hormone (GH) gene promoter sequences of 12 mammalian species was explored by comparison of their trinucleotide composition and by multiple sequence alignment. Both approaches yielded results that were consistent with the known fossil record-based phylogeny of the analysed sequences, suggesting that the two methods of tree reconstruction might be equally efficient and reliable. The pattern of evolution inferred for the mammalian GH gene promoters was found to vary both temporally and spatially. Thus, two distinct regions devoid of any evolutionary changes exist in primates, but only one of these 'gaps' is also observed in rodents, and neither is seen in ruminants. Furthermore, different evolutionary rates must have prevailed during different periods of evolutionary time and in different lineages, with a dramatic increase in evolutionary rate apparent in primates. Since a similar pattern of discontinuity has been previously noted for the evolution of the GH-coding regions, it may reflect the action of positive selection operating upon the GH gene as a single cohesive unit. Strong evidence for the action of gene conversion between primate GH gene promoters is provided by the fact that the human GH1 and GH2 sequences, which are thought to have diverged before the divergence of Old World monkeys from great apes, are more similar to one another than either is to the rhesus monkey GH2 promoter. Finally, it was noted that a number of nucleotide positions in the GH1 gene promoter that are polymorphic in humans appear to be highly conserved in mammals. This apparent conundrum, which could represent a caveat for the interpretation of phylogenetic footprinting studies, is potentially explicable in terms either of reduced genetic diversity in highly inbred animal species or insufficient population data from non-human species.     

Three distinct regulatory elements comprise the upstream promoter region of the nopaline synthase gene

Summary Fine deletion mutants were generated in the upstream control region of the nopaline synthase (nos) promoter to define the position and role of upstream regulatory elements. The results indicated that the 8 bp sequence (CAGAAACC) at -106/-113 and its inverted repeat (GGTTTCTG) at -140/-147 are important for promoter function. The downstream element appears more important than the upstream element since deletion of the former reduced promoter activity more significantly than deletion of the latter. Deletion of the element alone, however, did not abolish promoter function, whereas, deletion of the 10 bp potential Z-DNA-forming (Z) element located between the repeat elements nullified promoter activity. Therefore, it appears that the Z element is an essential upstream regulator and the repeated elements are upstream modulators of the nos promoter. These elements are functionally distinct since alteration of stereospecificity or insertion of short oligonucleotides between the elements did not significantly influence promoter activity. These regulatory elements were unable to function from 200 bp upstream of the CCAAT-TATA box region.