Functional analyses of promoter elements responsible for the differential expression of the human metallothionein (MT)-IG and MT-IF genes

The sequences responsible for heavy metal-inducible expression are situated within the proximal 437 and 160 base pairs (bp) of MT-IF and MT-IG 5'-flanking sequence, respectively. Only 105 bp of proximal MT-IG 5'-flanking sequence containing a TATA box, two metal responsive elements (MREs), and three GC motifs and 147 bp of proximal MT-IF 5'-flanking sequence containing a TATCA box, four MREs, and two GC motifs were required for heavy metal-inducible expression. However, the proximal 111 bp of MT-IF 5'-flanking sequences (a TATCA box, two MREs, and two GC motifs) was not responsive to heavy metals and competes less efficiently than the 105-bp MT-IG fragment in a competition transfection analysis. The MT-IF promoter fragment containing MREc and MREd is substantially stronger and a more efficient competitor than the MT-IG promoter fragment containing MREc and MREd. Furthermore, the proximal 160 bp of MT-IG 5'-flanking sequence functions as a strong metal-inducible promoter but not as a metal-inducible enhancer. Mobility shift analysis of MT-IF and MT-IG promoter subregions suggests a correlation between protein binding to MRE sequences and MT gene expression. These data illustrate that the overall structural and functional organization of the MT-IF and MT-IG promoters are very different and that the molecular mechanisms governing differential expression levels of human MT genes are quite complex.     

Communication between NF-kappa B and Sp1 controls histone acetylation within the proximal promoter of the monocyte chemoattractant protein 1 gene

The induction of the monocyte chemoattractant protein 1 gene (MCP-1) by TNF occurs through an NF-kappaB-dependent distal regulatory region and an Sp1-dependent proximal regulatory region that are separated by 2.2 kb of sequence. To investigate how these regions coordinate activation of MCP-1 in response to TNF, experiments were performed to examine the role of coactivators, changes in local chromatin structure, and the acetylation of histones at the MCP-1 regulatory regions. An E1a-sensitive coactivator was found to be required for expression. In vivo nuclease sensitivity assays identified changes in response to TNF at both the proximal and distal regions that were dependent on the p65 subunit of NF-kappaB and Sp1. Chromatin immunoprecipitations used to analyze factor assembly and histone acetylation at the distal and proximal regions showed that Sp1 binding to and histone acetylation of the proximal region was dependent on NF-kappaB p65. Conversely, Sp1 assembly at the proximal region was required for p65 binding to and acetylation of the distal region, suggesting communication between the two regions during gene activation. These data and the NF-kappaB p65-dependent histone acetylation of a middle region sequence suggest a potential order for the assembly, acetylation and accessibility of the MCP-1 regulatory regions in response to TNF.     

Building a metal-responsive promoter with synthetic regulatory elements.

A fusion gene consisting of the promoter region from the mouse metallothionein-I gene joined to the coding region of the herpes simplex virus thymidine kinase gene is efficiently regulated by zinc in a transient assay when transfected into baby hamster kidney cells. Analysis of similar plasmids in which the metallothionein-I promoter region was mutated indicated the presence of multiple metal regulatory elements (MREs) between -176 and -44 base pairs from the cap site. To further investigate the function of MREs, we inserted a synthetic DNA fragment containing the sequence of MRE-a (the element between -55 and -44 base pairs) into the nonresponsive promoter of the thymidine kinase gene in various positions and configurations. Little or no induction by zinc was observed with single insertions of the regulatory sequence, whereas many different constructions having two copies of MRE-a were inducible. The precise position of the two MREs relative to each other or to the thymidine kinase promoter elements had a relatively small effect on the efficiency of induction, but the inducibility could be further increased by the introduction of more MRE-a sequences. MRE-a can function synergistically with the thymidine kinase distal promoter elements, but in the presence of the TATA box alone it functions as a positive, zinc-dependent promoter element.     

Functional analysis of elements affecting expression of the beta-actin gene of carp.

Regulatory regions of the beta-actin gene of the common carp (Cyprinus carpio) have been examined by linking upstream, 5'-flanking sequences and regions of the first intron to a bacterial chloramphenicol acetyltransferase (CAT) reporter gene. By analysis of the mRNA products and encoded CAT activity, we have identified four putative regions that influence expression: (i) a negative regulatory region 2,300 to 1,100 base pairs (bp) ahead of the gene; (ii) a proximal promoter element, containing the highly conserved CCAAT, CC(A/T)6GG, and TATA boxes, that is within the first 204 bp upstream of the initiation site; (iii) a negative element of 426 bp in the 5' region of the first intron; and (iv) a positive 304-bp element near the end of the first intron that contains highly conserved sequences found in all characterized beta-actin genes. The positive intron element is not a classical enhancer; it is position and orientation dependent, as has been observed in other housekeeping genes in vertebrates. Depending on the elements joined together, CAT gene expression can be modulated more than 500-fold in transfected mouse cells.     

Cloning and functional characterization of 5'-upstream region of metallothionein-I gene from crucian carp (Carassius cuvieri)

Metallothioneins are low molecular weight, cysteine-rich, metal-binding proteins, which can be induced by heavy metal ions, cytokines, stress, and hormones. To investigate the roles of the main cis-acting elements involved in the inducible expression of metallothionein gene in fish, the 5'-upstream region of crucian carp (Carassius cuvieri) metallothionein-I gene had been cloned and analyzed after our previous work on metallothionein-II. In its upstream region, several putative cis-acting elements, including nine metal regulatory elements (MREs), one antioxidant response element, one E-box, and three interleukin-6 responsive elements, etc. were found. The nine metal regulatory elements are confined in less than 1000 bp from ATG start codon and organized into two clusters with different roles to the induction of the metallothionein-I expression. Deletion mutant assays demonstrated that both the distal and proximal clusters of metal regulatory elements contributed to the basal expression of the metallothionein-I, but only the proximal cluster was the chief contributor to the metal fold induction. In transient luciferase reporter assays, Zn2+ and Cd2+ served as much stronger inducers than Cu2+ to the metallothionein-I expression. H2O2 also could activate the metallothionein-I promoter about two-fold, which was mediated by the antioxidant response element (TGACAACGC, -437/-445). In conclusion, our studies demonstrate the roles of metal regulatory element and antioxidant response element in the induction of crucian carp metallothionein-I gene, and provide the regulatory mechanism for the use of fish metallothionein as a biomarker for monitoring of metal contamination in waters.     

Molecular evolution of the 5'-flanking regions of the duplicated Amy genes in Drosophila melanogaster species subgroup

The nucleotide sequences of the 5'-flanking regions of the duplicated Amy genes in eight sibling species belonging to the melanogaster species subgroup are analyzed. In Drosophila melanogaster, a region of about 450 bp immediately upstream of the translation initiation site of the two paralogous genes (the proximal and distal genes) has sequence similarities. However, we could not detect any significant sequence similarity in the region more upstream than -450. This result indicates that the coding regions of the ancestral Amy gene were duplicated together with 450 bp of the 5'-flanking region as one unit. Multiple alignment of these 450-bp sequences in the proximal and distal genes of all eight species revealed a mosaic pattern of highly conserved and divergent regions. The conserved regions included almost all the putative regulatory elements identified in previous analyses of the sequences. A phylogenetic analysis of the aligned sequences shows that these 450-bp sequences are clustered into the proximal and the distal groups. As a whole, the divergence between groups in this region is very large in contrast to that in the coding regions. Based on the divergence between groups, the 450-bp region is divided into two subregions. We found that the ratios of the divergence between groups to that within groups differ in the two subregions. From these observations, we discuss a possibility of positive selection acting on the subregion immediately upstream of the Amy coding region to cause divergence of regulatory elements of the paralogous genes.      

Structure of the Bombyx mori fibroin light-chain-encoding gene: upstream sequence elements common to the light and heavy chain.

Two overlapping genomic clones containing the fibroin light-chain (Fib-L)-encoding gene (Fib-L) were obtained from the cosmid library of the silkworm, Bombyx mori J-139, by hybridization with the Fib-L cDNA clone. Sequencing of the 14.6-kb region revealed that Fib-L was 13472 bp long containing seven exons, and that the gene contained a large first intron which occupied about 60% of the gene. Comparison of restriction patterns of the J-139 Fib-L with those of eight other B. mori breeds producing normal-level fibroin demonstrated that considerable restriction-fragment length polymorphisms were present in regions containing the first intron and the 3′-flanking sequence. However, sizes of the Fib-L mRNA and the Fib-L polypeptide were very similar among the nine breeds tested, suggesting that the exon sequences and the splice signals were all well conserved. 5′-Flanking regions of Fib-L and the fibroin heavy-chain (Fib-H)-encoding gene (Fib-H) compared in this study contained three 18-30-bp sequences of high similarity and many 8-10-bp common elements, six of which coincided with the binding sites of homeodomain proteins. Gel retardation assays with the nuclear extracts of the posterior and middle silk glands suggested that protein factors present in the posterior silk-gland nuclei could bind to a set of those common upstream elements.     

Further evidence for recombination between mouse hemoglobin beta b1 and b2 genes based on the nucleotide sequences of intron, UTR, and intergenic spacer regions

Nucleotide sequences of the intron regions and UTRs (Untranslated regions) of the hemoglobin beta adult genes, b1 and b2, and of the intergenic spacer region were determined for mouse strains representing the d, p, and w1 hemoglobin haplotypes defined by protein electrophoretic analyses. The hypothesis of recombination of the b1 and b2 genes between the d and w1 haplotypes previously reported in the cDNA nucleotide sequences was confirmed by neighbor-joining analyses of the intron regions and UTRs within the b1 and b2 genes, suggesting that all of the structures of hemoglobin beta adult genes support the hypothesis that the p haplotype was established by hybridization between d and w1 haplotype mice. The resultant recombinant of the p haplotype was found to have a d-like b1 gene and a w1-like b2 gene. In addition to the possible recombination, a break point was suggested around 2-3 kb downstream of the b1 gene within the intergenic spacer region, despite the absence of clear properties that could stimulate the recombination machinery. Some large insertions or deletions (indels) specific to the p or d haplotypes were located within the intergenic spacer region, in which the 1010-bp indel specific to the p haplotype was shared by all examined strains representing the p haplotype.     

Structural and functional characterizations of the 5'-flanking region of the mouse glucagon receptor gene: comparison with the rat gene

A putative proximal promoter was defined previously for the mouse glucagon receptor (GR) gene. In the present study, a distal promoter was characterized upstream from a novel non-coding exon revealed by the 5'-rapid amplification of cDNA ends from mouse liver tissue. The 5'-flanking region of the mouse GR gene was cloned up to 6 kb and the structural organization was compared to the 5' untranslated region of the rat gene cloned up to 7 kb. The novel exon, separated by an intron of 3.8 kb from the first coding exon, displayed a high homology (80%) with the most distal of the two untranslated exons found in the 5' region of the rat GR gene. The mouse distal promoter region, extending up to -1 kb from the novel exon, displayed 85% identity with the rat promoter. Both contain a highly GC-rich sequence with five putative binding sites for Sp1, but no consensus TATA or CAAT elements. To evaluate basal promoter activities, 5'-flanking sequences of mouse or rat GR genes were fused to a luciferase reporter gene and transiently expressed in a mouse and in a rat cell line, respectively or in rat hepatocytes. Both mouse and rat distal promoter regions directed a high level of reporter gene activity. Deletion of the Sp1 binding sites region or mutation of the second proximal Sp1 sequence markedly reduced the distal promoter activity of the reporter gene. The mouse proximal promoter activity was 2- to 3-fold less than the distal promoter, for which no functional counterpart was observed in the similar region of the rat gene.