Strong Negative Regulatory Element Research Articles

Influence of Cell Confluency on the Expression of the α4 Integrin Subunit of Retinal Pigment Epithelial Cells

Integrins are a family of transmembrane glycoproteins that mediate cell-cell and cell-extracellular matrix interactions. The integrin α4 subunit is widely expressed by cells from the immune system and its expression by non-hematopoietic cells is scarce. In the present study, gene and protein expression of this integrin subunit was characterized in proliferating and quiescent human RPE cells. Immunofluorescent studies confirm that the α4 subunit is expressed in vitro by RPE cells, a result that has been validated by immunofluorescence and FACS analyses. The accumulation of the α4 integrin at cell-cell junctions in post-confluent RPE cell cultures negatively correlated with the level of expression of the mRNA transcript. Accordingly, transient transfection analyses reveal that the α4 promoter activity is considerably reduced when RPE cells form a confluent monolayer. Moreover, transfection of recombinant constructs bearing 5’-deletions of the α4 promoter segment allows the localization of strong negative regulatory elements on the -76 to -300 region of the α4 gene suggesting that its expression is intimately linked to the proliferative state of primary cultured RPE cells.

The Canonical BMP Signaling Pathway Plays a Crucial Part in Stimulation of Dentin Sialophosphoprotein Expression by BMP-2

Dentin sialophosphoprotein (DSPP), a typical dentin-specific protein, is mainly expressed in the dentin extracellular matrix and plays a role in dentin mineralization. BMP-2 provides a strong signal for differentiation and mineralization of odontoblasts and osteoblasts. Previously, BMP-2 treatment is reported to stimulate Dspp expression in the MD10-F2 pre-odontoblast cells through activation of the heterotrimeric transcription factor Y (NF-Y). The canonical BMP signaling pathway is known to contribute greatly to biomineralization, however, it is not known whether it is involved in Dspp expression. Here, we investigated this question. Activation of the canonical BMP-2 signaling pathway in MDPC-23, preodontoblast cell, by overexpression of constitutively active Smad1/5 or downstream transcription factors Dlx5 and Runx2 stimulated Dspp expression. Conversely, knockdown of each element with siRNA significantly blocked the BMP-2-induced Dspp expression. To test whether these transcription factors downstream of BMP-2 are directly involved in regulating Dspp, we analyzed the mouse Dspp promoter. There are 5 well conserved homeodomain binding elements, H1 to H5, in Dspp proximal promoter regions (-791 to +54). A serial deletion of H1 and H2 greatly changed basal promoter activity and responsiveness to Dlx5 or Msx2. However, further deletions did not change the responsiveness to Dlx5 or Msx2. H1 and H2 sites can be suggested as specific response elements of Dlx5 and Msx2, respectively, based on their promoter activity modulation. Thus, the canonical BMP-2 signaling pathway plays a crucial part in the regulation of Dspp expression through the action of Smads, Dlx5, Runx2, and Msx2.

Different Configurations of Specific Thyroid Hormone Response Elements Mediate Opposite Effects of Thyroid Hormone and GC-1 on Gene Expression

T3 regulates transcription of the rat sarcoendoplasmic reticulum calcium ATPase in the heart. The T3 effect is mediated by three differently configured T3 response elements (TREs). Here we report the mutation of each individual TRE in the promoter and the contribution of each TRE on gene expression. Mutation of TRE1, a direct repeat element, exerted the strongest T3 response, compared with TRE2 and TRE3, which are inverted palindromes. The isolated TRE2 and TRE3, which showed no response (TRE2) or were weakly positive with T3 (TRE3), became strong negative regulatory elements with the T3 analog GC-1. We found that TRE1 recruits corepressor complexes containing nuclear receptor corepressor and histone deacetylase 3 in the absence of ligand, and steroid receptor coactivator-1-containing coactivator complexes with both T3 and GC-1. TRE3 bound the same corepressor complexes without ligand but showed only a weak association with steroid receptor coactivator-1 with T3 and a strong association with corepressor complexes with GC-1. Thus, GC-1 appears to control cofactor association differentially on these two sarcoendoplasmic reticulum calcium ATPase TREs, which could be the mechanism of ligand-dependent transcriptional activation and repression observed with the isolated TRE1 and TRE3 elements. Because the x-ray crystal structures of GC-1 and T3 complexed with the TR ligand binding domain are superimposable, the results imply that GC-1 and T3 induce differential effects on the receptor that are not evident in the static structures but must occur in the dynamic setting of receptor function. These results have implications for selective modulation of receptor function by agonist ligands.

Module-specific regulation of the beta-phaseolin promoter during embryogenesis.

The phas promoter displays stringent spatial regulation, being very highly expressed during embryogenesis and completely silent during all phases of vegetative development in bean, Phaseolus vulgaris. This pattern is maintained in transgenic tobacco and, as shown here, Arabidopsis. Dimethyl sulphate in vivo footprinting analyses revealed that over 20 cis-elements within the proximal 295 bp of the phas promoter are protected by factor binding in seed tissues whereas none are bound in leaves. The hypothesis that this complex profile represents a summation of several module (cotyledon, hypocotyl, and radicle)-specific factor-DNA interactions has been explored by the incorporation of site-directed substitution mutations into 10 locations within the -295phas promoter. Only 2.6% of -295phas promoter activity remained after mutation of the G-box; the CCAAAT box, the E-box and the RY elements were also found to mediate high levels of expression in embryos. Whereas the CACA element has dual positive and negative regulatory roles, the vicilin box was identified as a strong negative regulatory element. The proximal (-70 to -64) RY motif was found to bestow expression in the hypocotyl while all the RY elements contribute to expression in cotyledons but not to vascular tissue expression during embryogenesis. RY elements at positions -277 to -271, -260 to -254, and -237 to -231 were found to orchestrate radicle-specific repression. The G-box appears to be the functional abscisic acid responsive element and the E-site may be a coupling element. The results substantiate the concept that autarkical cis-element functions generate modular patterning during embryogenesis. They also reflect the existence of both redundancy and hierarchy in cis-element interactions. Importantly, the virtually identical expression patterns observed for the two distantly related plants studied argue strongly for the generality of function for the observed factor-element interactions.

Mouse connexin37: gene structure and promoter analysis

Connexin37 (Cx37) is a subunit gap junction protein which exhibits limited expression in only a few cell types, predominantly in endothelial cells and in the lung. To begin to analyze Cx37 expression, we isolated a 1.6 kb mouse Cx37 cDNA from a mouse lung cDNA library and isolated corresponding mouse genomic clones from a bacterial artificial chromosome library. Sequencing and comparison of these clones showed that the Cx37 gene contained a short first exon, an 1.0 kb single intron and a second exon containing the complete coding region and 3′-untranslated region (UTR). The 5′-UTR of the mouse cDNA showed 70% identity to that of human Cx37. Primer extension experiments performed using mouse lung RNA gave two bands of sizes consistent with the transcription start site predicted from the cDNA. Sequence analysis showed that the regions flanking exon I contained a consensus ‘TATA box’ 43 bp 5′ from the transcription start site preceded by several putative transcription factor binding sites and a 282 bp truncated L1Md interspersed element. Luciferase reporter gene transfections suggested that an area of 268 bp 5′ from the first exon acted as a basal promoter for Cx37 and that there was a strong negative regulatory element in the intron.

Mouse Connexin40: Gene Structure and Promoter Analysis

A family of related connexin genes encodes the subunit gap junction proteins that form intercellular channels in different tissues. Connexin40 (Cx40) is one of these proteins, and it exhibits limited expression only in a few cells of the cardiovascular system. To begin to analyze Cx40 expression, we isolated a 3.3-kb rat Cx40 cDNA by hybridization screening of a bacteriophage library prepared from BWEM cells and isolated corresponding mouse genomic clones from a bacterial artificial chromosome library. Restriction mapping, sequencing, and comparison to the rat cDNA showed that the mouse Cx40 gene contained a short first exon, an 11.4-kb intron, and a second exon containing the complete coding region and 3′-UTR. Exon I contained only 1 base that differed between rat and mouse. Primer extension experiments yielded a single band and confirmed the position of the transcriptional start site. We obtained 1.2 kb of sequence 5′ of the transcriptional start site and 400 bp 3′ of exon I. Exon I was closely preceded by a consensus TATA box. The flanking sequences contained a number of potential transcription factor binding sites (including AP-1, AP-2, SP1, TRE, and p53). To identify transcriptional regulatory elements in the Cx40 promoter region, a series of DNA deletion fragments flanking exon I was prepared, subcloned adjacent to a luciferase reporter gene, and used for transient transfections of BWEM, SHM, and N2A cells. The resulting luciferase activity determinations suggested that an area of 300 bp 5′ of the transcription start site acted as a basal promoter for Cx40 and that there was a strong negative regulatory element in the region from +100 to +297.

Functional Role of a Negative Regulatory Element in DevelopmentStage Specific Expression of Rat Cardiac Myosin Heavy Chain Gene. •259

The αmyosin heavy chain (αMHC) gene encodes a major cardiac contractile protein. Steadystate level of its mRNA are decreased during embryonic heart development and hypertrophy of the adult rat heart, mainly due to transcriptional regulation. While several ciselements and cognate binding factors involved in its positive regulation are described, the mechanism of its negative regulation remains unknown. We have identified a strong negative regulatory element (NRE) in the 5′ untranslated region of this gene. To study if NRE sequences play a role in the negative regulation of αMHC gene transcription in embryonic heart, we transfected the αMHC promoter/ chloramphenicol acetyl transferase (CAT) reporter constructs into primary cultures of 18 day fetal rat heart. The wild type (WT) construct included 600/420 bp sequence (in relation to transcription start site) of αMHC promoter. The deletion mutants comprised 600/63 promoter sequences and internal deletion of NRE within WT construct. The percent increase in CAT expression due to NRE deletion was compared with that obtained by direct in vivo injections of these constructs in adult heart. NRE deletion resulted in 15fold induction of CAT expression in fetal heart, but 8fold increase in adult heart. Gelshift assay with NRE sequence and nuclear extracts prepared from fetal and adult heart showed two complexes with distinct mobilities in fetal heart but one complex in adult heart. The adult heart complex corresponded to the slowermobility complex of the fetal heart. These data suggests that differential binding of nuclear factors to NRE sequence may be responsible for its stronger effect in fetal heart that may contribute to the in vivo suppression of αMHC gene transcription during embryonic heart development (Supported by a grant from Christ Hospital Med Fund).

Excitotoxic Brain Lesion Modifies Binding to a USF Binding Site Acting as a Negative Regulatory Element in theProtease Nexin-1Promoter

The expression of the serine protease inhibitorProtease nexin-1 (PN-1)is upregulated in glial cells following different types of lesion in the nervous system. A strong negative regulatory element has been shown by the missing nucleoside technique to be a CACGTG site (E-box) in the proximal part of thePN-1promoter. The factor binding to this site is specifically recognized by antibodies directed against the human upstream stimulatory factor (USF). Point mutations in the E-box binding site which abolish USF bindingin vitroincrease the transcriptional activity of thePN-1promoter. Cotransfection of aPN-1promoter/reporter construct together with an expression vector for humanUSF1confirms the negative regulatory function of this site. Finally, we show that the binding to this USF site changed after ibotenic acid-induced lesion of the caudate putamen in the rat brain.

Transcriptional regulation of the vimentin-encoding gene in mouse myeloid leukemia M1 cells

To investigate the regulatory mechanisms controlling expression of the vimentin-encoding gene ( Vim) during mouse myeloid leukemia M1 cell differentiation, mouse Vim was cloned and the transcriptional activity of its 5′ promoter region was analysed by chloramphenicol acetyltransferase (CAT) assay. Analyses of various deletion mutants revealed that a 188-bp fragment of the proximal Vim promoter ( pVim) was sufficient for effective transcription in M1 cells. This 188-bp sequence is highly conserved between mouse, hamster and human. Further deletion analyses revealed that a minimum promoter element (−44 to + 26) is essential for basic promoter function and could respond to cell differentiation. Detailed analyses of mutant and chimeric pVim constructs defined a CCAAT box at −89 to −84 to be an essential positive regulatory element. A G + C-rich element between the CCAAT and TATA boxes was found to act as a strong negative regulatory element in Vim transcription.

Endothelial-cell-specific regulation of von Willebrand factor gene expression.

In both tissue sections and cell culture, the endothelial nature of a cell is most commonly determined by demonstration of its expression of von Willebrand factor (vWf) protein and/or mRNA. Thus, the mechanism of cell-type-specific transcriptional regulation of the vWf gene is central to studying the basis of endothelial-cell-specific gene expression. In this study, deletion analyses were carried out to identify the region of the vWf gene which regulates its endothelial-cell-specific expression. A 734-bp fragment which spans the sequence from -487 to +247 relative to the transcription start site was identified as the cell-type-specific promoter. It consists of a minimal core promoter located between -90 and +22, a strong negative regulatory element located upstream of the core promoter (ca. -500 to -300), and a positive regulatory region located downstream of the core promoter in the first exon. The activity of the core promoter is not cell type specific, and the negative regulatory region is required to inhibit its activity in all cell types. The positive regulatory region relieves this inhibition only in endothelial cells and results in endothelial-cell-specific gene expression. The positive regulatory region contains sequences predicting possible SP1, GATA, and octamer binding sites. Mutations in either the SP1 or octamer sequence have no effect on transcriptional activity, while mutation in the GATA binding element totally abolishes the promoter activity. Evidence that a GATA factor is involved in this interaction is presented. Thus, the positive regulatory region with an intact GATA binding site is required to overcome the inhibitory effect of the negative regulatory element and activate vWf gene expression in an endothelial-cell-specific manner.

Endothelial-cell-specific regulation of von Willebrand factor gene expression.

In both tissue sections and cell culture, the endothelial nature of a cell is most commonly determined by demonstration of its expression of von Willebrand factor (vWf) protein and/or mRNA. Thus, the mechanism of cell-type-specific transcriptional regulation of the vWf gene is central to studying the basis of endothelial-cell-specific gene expression. In this study, deletion analyses were carried out to identify the region of the vWf gene which regulates its endothelial-cell-specific expression. A 734-bp fragment which spans the sequence from -487 to +247 relative to the transcription start site was identified as the cell-type-specific promoter. It consists of a minimal core promoter located between -90 and +22, a strong negative regulatory element located upstream of the core promoter (ca. -500 to -300), and a positive regulatory region located downstream of the core promoter in the first exon. The activity of the core promoter is not cell type specific, and the negative regulatory region is required to inhibit its activity in all cell types. The positive regulatory region relieves this inhibition only in endothelial cells and results in endothelial-cell-specific gene expression. The positive regulatory region contains sequences predicting possible SP1, GATA, and octamer binding sites. Mutations in either the SP1 or octamer sequence have no effect on transcriptional activity, while mutation in the GATA binding element totally abolishes the promoter activity. Evidence that a GATA factor is involved in this interaction is presented. Thus, the positive regulatory region with an intact GATA binding site is required to overcome the inhibitory effect of the negative regulatory element and activate vWf gene expression in an endothelial-cell-specific manner.

Organization, sequence, and expression of the murine S100 beta gene. Transcriptional regulation by cell type-specific cis-acting regulatory elements.

The organization, sequence, and transcriptional regulation of expression of the murine S100 beta gene are reported. The gene is approximately 9 kilobase pairs in length and is composed of three exons and two introns. The deduced murine S100 beta protein sequence differs from the human S100 beta protein by only 1 amino acid. The murine S100 beta gene contains a TATA box (AATAA) and a reverse CCAAT box (ATTGG) located at 30 nucleotides and 92 nucleotides upstream of the cap site, respectively. A 149-base pair DNA fragment (-157/-9) spanning the TATA box and the reverse CCAAT box functions as a promoter. The murine S100 beta promoter drives a 4-fold higher level of transcription in glial (C6) than in non-glial (3T3) cells, suggesting the existence of a potential cell type-specific regulatory element within the promoter region. The 5'-flanking region suppresses transcription from the homologous S100 beta as well as the heterologous SV40 promoters in an orientation-independent fashion. However, the 5'-flanking region exhibits cell type specificity when suppressing the S100 beta promoter-dependent transcription, indicating its involvement in the cell type-specific expression of S100 beta gene. In order to map cell type-specific regulatory elements, transcription analyses of various deletions of the 5'-region were carried out in C6 and 3T3 cells. Two cell type-specific negative regulatory elements, one active in non-glial cells and another active in glial cells, were mapped to the regions -1552/-1234 and -1234/-551, respectively. A strong negative regulatory element and a relatively weak negative element were located in the regions -551/-157 and -1669/-1552, respectively. The murine S100 beta gene is under complex transcriptional regulation involving tonic negative control exerted by combination of multiple cis-acting regulatory elements including cell type-specific elements.

Strong negative and positive regulatory elements contribute to the high-level fruit-specific expression of the tomato 2A11 gene

Fruit-specific expression of beta-glucuronidase (GUS) activity was produced in transgenic tomato plants when the GUS-coding region was flanked by 5' and 3' regions of the tomato 2A11 gene. Deletion studies on the 5' region revealed a number of strong regulatory elements involved in the proper expression of the 2A11 gene. A 4.0 kb and a 1.3 kb 5' region can confer high-level fruit-specific GUS expression, while a 1.8 kb 5' region produces no GUS activity in leaf or fruit tissue. Thus, a strong negative regulatory element is present in the region between 1324 bp and 1796 bp upstream of the 2A11 transcriptional start and a strong fruit-specific positive regulatory element is present more than 1.8 kb upstream of the transcriptional start site. The 1.8 kb promoter region can be activated by the upstream insertion of the CaMV 35S enhancer sequence, albeit not in a fruit-specific fashion. Substitution of the 3' region of the 2A11 gene with a different 3' region does not seem to affect GUS expression significantly, indicating a minor role, if any, for the 3' region in the fruit-specific expression of the 2A11 gene.

Negative regulatory element associated with potentially functional promoter and enhancer elements in the long terminal repeats of endogenous murine leukemia virus-related proviral sequences

Three series of recombinant DNA clones were constructed, with the bacterial chloramphenicol acetyltransferase (CAT) gene as a quantitative indicator, to examine the activities of promoter and enhancer sequence elements in the 5' long terminal repeat (LTR) of murine leukemia virus (MuLV)-related proviral sequences isolated from the mouse genome. Transient CAT expression was determined in mouse NIH 3T3, human HT1080, and mink CCL64 cultured cells transfected with the LTR-CAT constructs. The 700-base-pair (bp) LTRs of three polytropic MuLV-related proviral clones and the 750-bp LTRs of four modified polytropic proviral clones, in complete structures either with or without the adjacent downstream sequences, all showed very little or negligible activities for CAT expression, while ecotropic MuLV LTRs were highly active. The MuLV-related LTRs were divided into three portions and examined separately. The 3' portion of the MuLV-related LTRs that contains the CCAAC and TATAA boxes was found to be a functional promoter, being about one-half to one-third as active as the corresponding portion of ecotropic MuLV LTRs. A MboI-Bg/II fragment, representing the distinct 190- to 200-bp inserted segment in the middle, was found to be a potential enhancer, especially when examined in combination with the simian virus 40 promoter in CCL64 cells. A PstI-MboI fragment of the 5' portion, which contains the protein-binding motifs of the enhancer segment as well as the upstream LTR sequences, showed moderate enhancer activities in CCL6 cells but was virtually inactive in NIH 3T3 cells and HT1080 cells; addition of this fragment to the ecotropic LTR-CAT constructs depressed CAT expression. Further analyses using chimeric LTR constructs located the presence of a strong negative regulatory element within the region containing the 5' portion of the enhancer and the immediate upstream sequences in the MuLV-related LTRs.