Separated Sequence Elements Research Articles

PIF3 regulates anthocyanin biosynthesis in an HY5‐dependent manner with both factors directly binding anthocyanin biosynthetic gene promoters in Arabidopsis

Phytochromes are red/far-red light receptors that regulate various light responses by initiating the transcriptional cascades responsible for changing the expression patterns of 10-30% of the entire plant transcriptome. Several transcription factors that are thought to participate in this process have been identified, but the functional relationships among them have not yet been fully elucidated. Here we investigated the functional relationship between two such transcription factors, PIF3 and HY5, and their effects on anthocyanin biosynthesis. Our results revealed that PIF3 and HY5 do not regulate each other at either the transcriptional or the protein levels in continuous light conditions, suggesting that they are not directly linked within phytochrome-mediated signaling. We found that both PIF3 and HY5 positively regulate anthocyanin biosynthesis by activating the transcription of the same anthocyanin biosynthetic genes, but the positive effects of PIF3 required functional HY5. Chromatin immunoprecipitation analyses indicated that both PIF3 and HY5 regulate anthocyanin biosynthetic gene expression by directly binding to different regions of the gene promoters in vivo. Additional experiments revealed that PIF3 bound the promoters regardless of light and HY5. Collectively, these data show that PIF3 and HY5 regulate anthocyanin biosynthesis by simultaneously binding anthocyanin biosynthetic gene promoters at separate sequence elements.

Distinct RNA structural domains cooperate to maintain a specific cleavage site in the 3′-UTR of IGF-II mRNAs

The insulin-like growth factor II mRNAs are targets for site-specific endonucleolytic cleavage in the 3′-UTR, which results in a very stable 3′ cleavage product of 1.8 kb, consisting of 3′-UTR sequences and a poly(A) tail. The 5′ cleavage product contains the coding region and is rapidly degraded. Thus, cleavage is thought to provide an additional way to control IGF-II protein synthesis. We had established that cleavage requires two widely separated sequence elements (I and II) in the 3′-UTR that form a stable duplex of 83 nucleotides. The cleavage-site itself is located in an internal loop preceded by two stable stem-loop structures. Furthermore, in a study which was based on RNA folding algorithms, we have shown that there are specific sequence and structural requirements for the cleavage reaction. Here, the functions of the different structural domains in cleavage were assessed by deletion/mutational analyses, and biochemical structure probing assays were performed to characterize better the RNA structures formed and to verify the computer folding predictions. The data suggest that the stem-loop domain contributes to maintain a highly specific c leavage-site by preventing the formation of alternative structures in the cleavage-site domain. Involvement of the nucleotides in the cleavage-site loop itself in non-Watson-Crick interactions may be important for providing a specific recognition surface for an endoribonuclease activity.

Structural basis of DNA bending and oriented heterodimer binding by the basic leucine zipper domains of Fos and Jun.

Interactions among transcription factors that bind to separate sequence elements require bending of the intervening DNA and juxtaposition of interacting molecular surfaces in an appropriate orientation. Here, we examine the effects of single amino acid substitutions adjacent to the basic regions of Fos and Jun as well as changes in sequences flanking the AP-1 site on DNA bending. Substitution of charged amino acid residues at positions adjacent to the basic DNA-binding domains of Fos and Jun altered DNA bending. The change in DNA bending was directly proportional to the change in net charge for all heterodimeric combinations between these proteins. Fos and Jun induced distinct DNA bends at different binding sites. Exchange of a single base pair outside of the region contacted in the x-ray crystal structure altered DNA bending. Substitution of base pairs flanking the AP-1 site had converse effects on the opposite directions of DNA bending induced by homodimers and heterodimers. These results suggest that Fos and Jun induce DNA bending in part through electrostatic interactions between amino acid residues adjacent to the basic region and base pairs flanking the AP-1 site. DNA bending by Fos and Jun at inverted binding sites indicated that heterodimers bind to the AP-1 site in a preferred orientation. Mutation of a conserved arginine within the basic regions of Fos and transversion of the central C:G base pair in the AP-1 site to G:C had complementary effects on the orientation of heterodimer binding and DNA bending. The conformational variability of the Fos-Jun-AP-1 complex may contribute to its functional versatility at different promoters.

The transcriptional transactivator of simian foamy virus 1 binds to a DNA target element in the viral internal promoter.

The transcriptional transactivator (Tas) of simian foamy virus type 1 strongly augments gene expression directed by both the promoter in the viral long terminal repeat and the newly discovered internal promoter located within the env gene. A region of 121 bp, located immediately 5' to the TATA box in the internal promoter, is required for transactivation by Tas. The present study aimed to identify the precise Tas-responsive target(s) in this region and to determine the role of Tas in transcriptional regulation. By analysis of both clustered-site mutations and hybrid promoters in transient expression assays in murine and simian cells, two separate sequence elements within this 121-bp region were shown to be Tas-dependent transcriptional enhancers. These targets, each < 30 bp in length and displaying no apparent sequence homology one to the other, are designated the promoter-proximal and promoter-distal elements. By means of the gel electrophoresis mobility-shift assays, using purified glutathione S-transferase-Tas fusion protein expressed in Escherichia coli, the target proximal to the TATA box exhibited strong binding to glutathione S-transferase-Tas, whereas the distal element appears not to bind. In addition, footprint analysis revealed that 26 bp in the promoter proximal element was protected by glutathione S-transferase-Tas from DNase I. We propose a model for transactivation of the simian foamy virus type 1 internal promoter in which Tas interacts directly with the proximal target element positioned immediately 5' to the TATA box. In this model, Tas attached to this element is presumed to interact with a component(s) of the cellular RNA polymerase II initiation complex and thereby enhance transcription directed by the viral internal promoter.

Identification of a region which directs the monocytic activity of the colony-stimulating factor 1 (macrophage colony-stimulating factor) receptor promoter and binds PEBP2/CBF (AML1).

The receptor for the macrophage colony-stimulating factor (or colony-stimulating factor 1 [CSF-1]) is expressed from different promoters in monocytic cells and placental trophoblasts. We have demonstrated that the monocyte-specific expression of the CSF-1 receptor is regulated at the level of transcription by a tissue-specific promoter whose activity is stimulated by the monocyte/B-cell-specific transcription factor PU.1 (D.-E. Zhang, C.J. Hetherington, H.-M. Chen, and D.G. Tenen, Mol. Cell. Biol. 14:373-381, 1994). Here we report that the tissue specificity of this promoter is also mediated by sequences in a region II (bp -88 to -59), which lies 10 bp upstream from the PU.1-binding site. When analyzed by DNase footprinting, region II was protected preferentially in monocytic cells. Electrophoretic mobility shift assays confirmed that region II interacts specifically with nuclear proteins from monocytic cells. Two gel shift complexes (Mono A and Mono B) were formed with separate sequence elements within this region. Competition and supershift experiments indicate that Mono B contains a member of the polyomavirus enhancer-binding protein 2/core-binding factor (PEBP2/CBF) family, which includes the AML1 gene product, while Mono A is a distinct complex preferentially expressed in monocytic cells. Promoter constructs with mutations in these sequence elements were no longer expressed specifically in monocytes. Furthermore, multimerized region II sequence elements enhanced the activity of a heterologous thymidine kinase promoter in monocytic cells but not other cell types tested. These results indicate that the monocyte/B-cell-specific transcription factor PU.1 and the Mono A and Mono B protein complexes act in concert to regulate monocyte-specific transcription of the CSF-1 receptor.

Identification of a region which directs the monocytic activity of the colony-stimulating factor 1 (macrophage colony-stimulating factor) receptor promoter and binds PEBP2/CBF (AML1).

The receptor for the macrophage colony-stimulating factor (or colony-stimulating factor 1 [CSF-1]) is expressed from different promoters in monocytic cells and placental trophoblasts. We have demonstrated that the monocyte-specific expression of the CSF-1 receptor is regulated at the level of transcription by a tissue-specific promoter whose activity is stimulated by the monocyte/B-cell-specific transcription factor PU.1 (D.-E. Zhang, C.J. Hetherington, H.-M. Chen, and D.G. Tenen, Mol. Cell. Biol. 14:373-381, 1994). Here we report that the tissue specificity of this promoter is also mediated by sequences in a region II (bp -88 to -59), which lies 10 bp upstream from the PU.1-binding site. When analyzed by DNase footprinting, region II was protected preferentially in monocytic cells. Electrophoretic mobility shift assays confirmed that region II interacts specifically with nuclear proteins from monocytic cells. Two gel shift complexes (Mono A and Mono B) were formed with separate sequence elements within this region. Competition and supershift experiments indicate that Mono B contains a member of the polyomavirus enhancer-binding protein 2/core-binding factor (PEBP2/CBF) family, which includes the AML1 gene product, while Mono A is a distinct complex preferentially expressed in monocytic cells. Promoter constructs with mutations in these sequence elements were no longer expressed specifically in monocytes. Furthermore, multimerized region II sequence elements enhanced the activity of a heterologous thymidine kinase promoter in monocytic cells but not other cell types tested. These results indicate that the monocyte/B-cell-specific transcription factor PU.1 and the Mono A and Mono B protein complexes act in concert to regulate monocyte-specific transcription of the CSF-1 receptor.

RNA 3' end signals of the S.pombe ura4 gene comprise a site determining and efficiency element.

We have defined sequences in the 3' non-coding region of the Schizosaccharomyces pombe ura4 gene that are required for efficient mRNA 3' end formation. Three separate sequence elements have been identified. Two of these are site determining elements which specify alternative sites of polyadenylation [the major poly(A) site and a minor downstream poly(A) site]. The third sequence, located downstream of both poly(A) sites, functions as an efficiency element that enhances utilization of either polyadenylation site. By employing sensitive RT-PCR analysis, we demonstrate that although low levels of transcripts are detected up to the efficiency element, none is detected beyond this point. The downstream site determining element and efficiency element have both been delineated to specific 16 nt sequences which we show are together sufficient for ura4 mRNA 3' end formation. We have further characterized the interaction between these two elements and show that the efficiency element behaves in a position-independent, orientation-dependent manner, but cannot form 3' ends independently of the site determining element. Surprisingly, we find that the efficiency element can be functionally replaced by a second copy of either site determining element. We present a model for the mechanism of RNA 3' end formation of the ura4 gene and note that this bipartite structure for a poly(A) signal in S.pombe may be related to the AAUAAA and downstream GU-rich sequences of poly(A) signals in mammalian genes.

Widely separated sequence elements within cucumber mosaic virus satellites contribute to their ability to induce lethal tomato necrosis.

To determine the structural requirements for cucumber mosaic virus (CMV) satellites to elicit lethal tomato necrosis, three satellite variants D, S and Y were used in the construction and cloning of chimeric cDNAs. D and S are necrogenic and non-necrogenic 'prototype' variants, respectively, and Y possesses the 3' conserved necrosis-determining region but does not cause lethal tomato necrosis. Its 5' half harbours an insertion/deletion region that results in a molecule about 30 nucleotides longer than other variants. Tomato bio-assays were conducted with RNA transcripts of all six chimeric combinations of the 5' and 3' halves of the three satellite variants divided by a common restriction site, as well as with a mutated chimera. None of the chimeras containing the 5' half of Y induced lethal necrosis in tomato even when their 3' halves were that of the D variant with the conserved necrogenic element. Chimeras with the 3' half of Y elicited only partial or restricted necrosis which was much less severe than that induced by prototype variant D, and often was not lethal. Site-directed mutation of a single nucleotide in proximity to the necrogenic element of such a chimera containing the 3' half of Y restored much lethal necrogenicity. The results revealed the presence of structural elements in CMV satellite variant Y that modulate or even suppress the expression of the 3' conserved necrosis-determining element. They indicate that in CMV satellites widely separated sequence elements constituting a three-dimensional requirement are responsible for eliciting lethal necrosis in tomato.

Site-specific cleavage of IGF-II mRNAs requires sequence elements from two distinct regions of the IGF-II gene.

The human insulin-like growth factor II (IGF-II) gene constitutes a complex transcriptional unit that contains nine exons and four promoters. Expression of the IGF-II gene yields a family of mRNAs that all encode prepro-IGF-II. In addition, a stable 1.8 kb RNA is formed that is derived from the 3' untranslated region of exon 9. Recently, we have shown that this RNA species arises by site-specific endonucleolytic cleavage of IGF-II mRNAs and not by transcription from a separate promoter. In the present study we establish that two widely separated sequence elements of approximately 300 nucleotides, both located within exon 9, are required for this cleavage reaction. The first element encompasses about 200 nucleotides upstream and 100 nucleotides downstream of the cleavage site, while the second element is located within a region of 330 nucleotides about 2 kb upstream of the cleavage site. Interestingly, site-specific cleavage also occurred when a fragment from exon 9 of the IGF-II gene containing these two elements was inserted into the 3' untranslated part of the beta-globin gene. Apparently, the expressed hybrid beta-globin-IGF-II mRNA contains all the regulatory elements to confer site-specific endonucleolytic cleavage.

Upstream regions of the human cardiac actin gene that modulate its transcription in muscle cells: presence of an evolutionarily conserved repeated motif.

Transfection into cultured cell lines was used to investigate the transcriptional regulation of the human cardiac actin gene. We first demonstrated that in both human heart and human skeletal muscle, cardiac actin mRNAs initiate at the identical site and contain the same first exon, which is separated from the first coding exon by an intron of 700 base pairs. A region of 485 base pairs upstream from the transcription initiation site of the human cardiac actin gene directs high-level transient expression of the bacterial chloramphenicol acetyltransferase gene in differentiated myotubes of the mouse C2C12 muscle cell line, but not in mouse L fibroblast or rat PC-G2 pheochromocytoma cells. Deletion analysis of this region showed that at least two physically separated sequence elements are involved, a distal one starting between -443 and -395 and a proximal one starting between -177 and -118, and suggested that these sequences interact with positively acting transcriptional factors in muscle cells. When these two sequence elements are inserted separately upstream of a heterologous (simian virus 40) promoter, they do not affect transcription but do give a small (four- to fivefold) stimulation when tested together. Overall, these regulatory regions upstream of the cap site of the human cardiac actin gene show remarkably high sequence conservation with the equivalent regions of the mouse and chick genes. Furthermore, there is an evolutionarily conserved repeated motif that may be important in the transcriptional regulation of actin and other contractile protein genes.