Multiple Regulatory Sequences Research Articles

The hmsT 3' untranslated region mediates c-di-GMP metabolism and biofilm formation in Yersinia pestis.

Yersinia pestis, the cause of plague, forms a biofilm in the proventriculus of its flea vector to enhance transmission. Biofilm formation in Y. pestis is regulated by the intracellular levels of cyclic diguanylate (c-di-GMP). In this study, we investigated the role of the 3' untranslated region (3'UTR) in hmsT mRNA, a transcript that encodes a diguanylate cyclase that stimulates biofilm formation in Y. pestis by synthesizing the second messenger c-di-GMP. Deletion of the 3'UTR increased the half-life of hmsT mRNA, thereby upregulating c-di-GMP levels and biofilm formation. Our findings indicate that multiple regulatory sequences might be present in the hmsT 3'UTR that function together to mediate mRNA turnover. We also found that polynucleotide phosphorylase is partially responsible for hmsT 3'UTR-mediated mRNA decay. In addition, the hmsT 3'UTR strongly repressed gene expression at 37°C and 26°C, but affected gene expression only slightly at 21°C. Our findings suggest that the 3'UTR might be involved in precise and rapid regulation of hmsT expression, allowing Y. pestis to fine-tune c-di-GMP synthesis and consequently regulate biofilm production to adapt to the changing host environment.

Identification of Neural Crest and Glial Enhancers at the Mouse Sox10 Locus through Transgenesis in Zebrafish

Sox10 is a dynamically regulated transcription factor gene that is essential for the development of neural crest–derived and oligodendroglial populations. Developmental genes often require multiple regulatory sequences that integrate discrete and overlapping functions to coordinate their expression. To identify Sox10 cis-regulatory elements, we integrated multiple model systems, including cell-based screens and transposon-mediated transgensis in zebrafish, to scrutinize mammalian conserved, noncoding genomic segments at the mouse Sox10 locus. We demonstrate that eight of 11 Sox10 genomic elements direct reporter gene expression in transgenic zebrafish similar to patterns observed in transgenic mice, despite an absence of observable sequence conservation between mice and zebrafish. Multiple segments direct expression in overlapping populations of neural crest derivatives and glial cells, ranging from pan-Sox10 and pan-neural crest regulatory control to the modulation of expression in subpopulations of Sox10-expressing cells, including developing melanocytes and Schwann cells. Several sequences demonstrate overlapping spatial control, yet direct expression in incompletely overlapping developmental intervals. We were able to partially explain neural crest expression patterns by the presence of head to head SoxE family binding sites within two of the elements. Moreover, we were able to use this transcription factor binding site signature to identify the corresponding zebrafish enhancers in the absence of overall sequence homology. We demonstrate the utility of zebrafish transgenesis as a high-fidelity surrogate in the dissection of mammalian gene regulation, especially those with dynamically controlled developmental expression.

Functional Impact of Sequence Alterations Found in BRCA1 Promoter/5'UTR Region in Breast/Ovarian Cancer Families from Upper Silesia, Poland

The 5' region of BRCA1 contains multiple regulatory sequences flanking the two alternative promoters α and β and two alternative, non-coding exons, 1a and 1b. Aberrations within the 5' region BRCA1 (encompassing two alternative promoters α and β and exons 1a and 1b) may be associated with an increased risk of breast and ovarian cancer. In this study we screened 150 patients for polymorphism and mutations in this region of BRCA1. All probands came from familial breast and/or ovarian cancer that had been found to be mutation-negative in a previous search for founder mutations in BRCA1 (185delAG, C61G, 4153delA, 5382insC) or BRCA2 (6174delT, 9631delC). In our study we found several sequence alterations within the non-coding region of BRCA1 by using direct DNA sequencing and allele-specific PCR amplification. Three families with a polymorphic deletion in BRCA1 exon 1b (2223delAAAAA, Acc. U37574) were found. Moreover, two linked nucleotide substitutions (2642A>T, 2743T>C, Acc. U37574) in BRCA1 intron 1 were detected in 16 patients. In order to assess the functional significance of these two sequence variants, we constructed a reporter vector encoding firefly luciferase under the transcriptional and translational control of wild type and altered BRCA1 promoter region. The reporter assay was performed using a lung cancer cell line (NCI-H1299) and a breast cancer cell line (MCF7). We have demonstrated that the analysed sequence variants have no functional significance in our experimental system. However, we have found that the BRCA1 promoter has lower relative activity in the breast cancer cell line compared with the lung cancer cell line. Based on the results of our functional experiments we conclude that the polymorphic deletion 2223delAAAAA and two linked substitutions 2642A>T and 2743T>C do not significantly alter BRCA1 expression and are probably not disease-causing mutations.

Functional and binding studies of HS3.2 of the beta-globin locus control region

The distal locus control region (LCR) is required for high-level expression of the complex of genes (HBBC) encoding the β-like globins of mammals in erythroid cells. Several major DNase hypersensitive sites (HSs 1–5) mark the LCR. Sequence conservation and direct experimental evidence have implicated sequences within and between the HS cores in function of the LCR. In this report we confirm the mapping of a minor HS between HS3 and HS4, called HS3.2, and show that sequences including it increase the number of random integration sites at which a drug resistance gene is expressed. We also show that nuclear proteins including GATA1 and Oct1 bind specifically to sequences within HS3.2. However, the protein Pbx1, whose binding site is the best match to one highly conserved sequence, does not bind strongly. GATA1 and Oct1 also bind in the HS cores of the LCR and to promoters in HBBC. Their binding to this minor HS suggests that they may be used in assembly of a large complex containing multiple regulatory sequences.

Deletional analyses reveal an essential role for the hs3b/hs4 IgH 3' enhancer pair in an Ig-secreting but not an earlier-stage B cell line.

The Ig heavy chain (IgH) locus is controlled by multiple regulatory sequences mapping both within the IgH transcription unit (E mu) and downstream (3') of IgH coding sequences (hs3a, hs1,2, hs3b and hs4). Enhancer knockout studies in mice have implicated E mu in the control of IgH variable region gene assembly, but single-enhancer knockouts involving the 3' IgH enhancers have yet to shed light on their function. Transfection studies in mice and cell lines have suggested that the 3' enhancers behave similarly to a locus control region as first identified in the beta-globin locus. We have exploited this property to form mini-loci in a surface Ig(+) and an Ig-secreting cell line as a means for studying the functions of the 3' IgH enhancers. Importantly, this experimental system allows for the analysis of enhancer function within the context of chromatin. The mini-loci consisted of an Ig gamma 2b transcription unit linked to the four murine 3' IgH enhancers. Using targeted deletions of enhancer pairs within these mini-loci, we have discovered a critical and apparently developmentally regulated role for the hs3b/hs4 enhancer pair in IgH transgene expression.

Upregulation of amyloid precursor protein gene promoter in rat primary hippocampal neurons by phorbol ester, IL-1 and retinoic acid, but not by reactive oxygen species

The APP gene promoter has multiple regulatory sequences, some of which may contribute to the neuropathology of Alzheimer's disease (AD). In this study, we investigated the effects of phorbol ester (PMA), IL-1, retinoic acid and reactive oxygen species on APP promoter activity in primary hippocampal neurons. We transfected neurons with either of two APP promoter constructs, a −2.8 kb and a shorter −488 bp upstream fragment fused to the chloramphenicol transferase (CAT) reporter gene. We demonstrated that phorbol 12-myristate-13 acetate (PMA), retinoic acid and IL-1 all stimulated both APP promoter constructs in hippocampal neurons after 24 h treatment. PMA and IL-1 treatments led to 2-fold increases of APP promoter activity. Retinoic acid induced a 3-fold increase. In addition, the magnitude of APP promoter responses to PMA and IL-1 treatment was similar between APP −2.8 kb and −488 bp plasmid transfected neurons. This suggests that the AP-1 sequence at −350 to −344 in the APP promoter may mediate the stimulatory effects of PMA and IL-1, as previously observed in endothelial and HeLa cells. In contrast, hydrogen peroxide, which was shown to activate NF-κB in primary neurons, failed to stimulate APP promoter activity, suggesting that the regulatory elements in the APP promoter may not respond to reactive oxygen species. Overall, these data indicate that APP expression in primary neurons can be modulated by PMA, IL-1 and retinoic acid. However, the contribution of reactive oxygen to Alzheimer's disease may not be directly related to the activation of the APP gene promoter but instead to neuronal damage associated with oxidative stress. Since elevated levels of IL-1 have been observed in AD brain, IL-1 could contribute to development of Alzheimer's disease by stimulating APP synthesis in primary neurons.

A 28-bp negative element with multiple factor-binding activity controls expression of the vimentin-encoding gene

The promoter of the human vimentin-encoding gene ( VIM) contains two enhancers separated by a negative region. The distal and proximal enhancers bind the transcription factors, AP-1 and NF-KB, respectively, which contribute to serum induction of Vim synthesis. We were interested in looking for particular regulatory elements that might be responsible for tissue-specific extinction and culture-dependent activation of human VIM. We have identified a 48-bp sequence in the distal enhancer which had not been reported before. This sequence includes a negative element, NE2, which confers transcriptional repression in transfection experiments and binds at least two factors in vitro. NE2 may participate in the differentiation-stage-specific control of VIM expression which involves multiple regulatory sequences and several positive and negative trans-acting factors.

Activation of the human IGFBP-1 gene promoter by progestin and relaxin in primary culture of human endometrial stromal cells

We have studied the activity for the insulin-like growth factor binding protein-1 (IGFBP-1) gene promoter in human endometrial stromal cells by transient transfection. The promoter activity derived from p3.6CAT or p3.6Luc (3400 bp IGFBP-1 promoter 5' to the reporter gene chloramphenicol acetyltransferase or luciferase) was minimal in unstimulated cells. A time study over 13 days of culture showed that the promoter activity increased exponentially to >10 4 fold in cells treated with medroxyprogesterone acetate (MPA) and relaxin (RLX). Induction of the IGFBP-1 gene promoter activity by hormones was similar to the secretion pattern of IGFBP-1 in endometrial stromal cells. MPA alone caused a moderate induction, 3–40-fold increase over the control. Deletion analysis showed that two regions in the IGFBP-1 gene promoter were responsible for the activation of the IGFBP-1 gene. The basal promoter region, termed bpl-A (+68 bp to -1.205 kb), contains multiple sections of regulatory sequence including a cis-element CCAAT (-72 bp). A DNase I protection assay in the bp-1 A region revealed four distinct binding regions, one of which contained the CCAAT ☐ region. Another promoter region, termed bpl-B (-2.6 to -3.4kb), mediated 95% of the total promoter activity in endometrial stromal cells. The bpl-B region also contains multiple regulatory sequences. Mutation and DNase I protection assay suggest that Spl-like binding site at -2.63 kb was a regulatory site responsible for the activation of IGFBP-1 gene promoter.

Control of calcitonin/calcitonin gene-related peptide pre-mRNA processing by constitutive intron and exon elements.

The calcitonin/calcitonin gene-related peptide (CGRP) primary transcript is alternatively spliced in thyroid C cells and neurons, resulting in the tissue-specific production of calcitonin and CGRP mRNAs. Analyses of mutated calcitonin/CGRP transcription units in permanently transfected cell lines have indicated that alternative splicing is regulated by a differential capacity to utilize the calcitonin-specific splice acceptor. The analysis of an extensive series of mutations suggests that tissue-specific regulation of calcitonin mRNA production does not depend on the presence of a single, unique cis-active element but instead appears to be a consequence of suboptimal constitutive splicing signals. While only those mutations that altered constitutive splicing signals affected splice choices, the action of multiple regulatory sequences cannot be formally excluded. Further, we have identified a 13-nucleotide purine-rich element from a constitutive exon that, when placed in exon 4, entirely switches splice site usage in CGRP-producing cells. These data suggest that specific exon recruitment sequences, in combination with other constitutive elements, serve an important function in exon recognition. These results are consistent with the hypothesis that tissue-specific alternative splicing of the calcitonin/CGRP primary transcript is mediated by cell-specific differences in components of the constitutive splicing machinery.

Control of calcitonin/calcitonin gene-related peptide pre-mRNA processing by constitutive intron and exon elements.

The calcitonin/calcitonin gene-related peptide (CGRP) primary transcript is alternatively spliced in thyroid C cells and neurons, resulting in the tissue-specific production of calcitonin and CGRP mRNAs. Analyses of mutated calcitonin/CGRP transcription units in permanently transfected cell lines have indicated that alternative splicing is regulated by a differential capacity to utilize the calcitonin-specific splice acceptor. The analysis of an extensive series of mutations suggests that tissue-specific regulation of calcitonin mRNA production does not depend on the presence of a single, unique cis-active element but instead appears to be a consequence of suboptimal constitutive splicing signals. While only those mutations that altered constitutive splicing signals affected splice choices, the action of multiple regulatory sequences cannot be formally excluded. Further, we have identified a 13-nucleotide purine-rich element from a constitutive exon that, when placed in exon 4, entirely switches splice site usage in CGRP-producing cells. These data suggest that specific exon recruitment sequences, in combination with other constitutive elements, serve an important function in exon recognition. These results are consistent with the hypothesis that tissue-specific alternative splicing of the calcitonin/CGRP primary transcript is mediated by cell-specific differences in components of the constitutive splicing machinery.

Similar tissue-specific expression of the Adh genes from different Drosophila species is mediated by distinct arrangements of cis-acting sequences.

In Drosophila melanogaster transformants, the alcohol dehydrogenase (Adh) genes from D. affinidisjuncta and D. grimshawi show similar levels of expression except in the adult midgut where the D. affinidisjuncta gene is expressed about 10- to 20-fold more strongly. To study the arrangement of cis-acting sequences responsible for this regulatory difference, homologous restriction sites were used to create a series of chimeric genes that switched fragments from the 5' and 3' flanking regions of these two genes. Chimeric genes were introduced into the germ-line of D. melanogaster, and Adh gene expression was analyzed by measuring RNA levels. Various gene fragments in the promoter region and elsewhere influence expression in the adult midgut and in whole larvae and adults. Comparison of these results with earlier studies involving chimeras between the D. affinidisjuncta and D. hawaiiensis genes indicates that expression in the adult midgut is influenced by multiple regulatory sequences and that distinct arrangements of regulatory sequences can result in similar levels of expression both in the adult midgut and in the whole organism.

Regulatory sequences clustered at the 5' end of the first intron of the human thymidylate synthase gene function in cooperation with the promoter region.

A human thymidylate synthase (TS) minigene containing 5'- and 3'-flanking sequences, all the exons, and only intron 1 showed a normal frequency of stable transformation when transfected into TS-negative mutant cells, whereas minigenes in which intron 1 was replaced by intron 2 or deleted in the above construct showed only a few percent of the above frequency. Introduction of intron 1 into the above intronless or intron 2 minigene restored the transforming activities regardless of its position and orientation. Deletion analysis revealed two positive and one negative regulatory sequences in the 5' end of intron 1, each of which seemed to bind specific proteins as shown by gel shift analysis. Intron 1 also stimulated expression of a TS promoter-CAT gene construct but not that of an SV40 promoter-CAT gene construct. These results indicate that the multiple regulatory sequences clustered in intron 1 stimulate TS gene expression in concert with the 5'-flanking sequences.

Multiple promoter elements govern expression of the human ornithine decarboxylase gene in colon carcinoma cells.

Overexpression of the ornithine decarboxylase (ODC) gene may be important to the development and maintenance of colonic neoplasms, as well as tumors in general. In this study, we examined the promoter elements governing constitutive expression of the human ODC gene in HCT 116 human colon carcinoma cells and, for comparison, K562 human erythro-leukemia cells. It was determined by functional analysis that the promoter elements responsible reside within the 378 bp immediately upstream from the transcription start site. Within this sequence, there are at least three regions that modulate the efficiency of the ODC promoter cooperatively. Both DNA bandshift and footprint assays demonstrated all three regions to be rich in sites that bind to nuclear proteins isolated from HCT 116 and K562 cells; the protein binding pattern of non-transformed, diploid fibroblasts was found to be much less complex. Several of the protein binding sequences have little or no homology to common regulatory elements. We suggest that the constitutive activity of the ODC gene in HCT 116 colon carcinoma cells, and perhaps transformed cells in general, involves a complex interaction of multiple regulatory sequences and their associated nuclear proteins. Finally, the saturation of the promoter in these transformed cell lines suggests that high levels of protein binding in the ODC promoter may contribute to elevated constitutive expression of this gene.

The bx region enhancer, a distant cis-control element of the Drosophila Ubx gene and its regulation by hunchback and other segmentation genes.

The Drosophila homeotic gene Ultrabithorax (Ubx) is regulated by complex mechanisms that specify the spatial domain, the timing and the activity of the gene in individual tissues and in individual cells. In early embryonic development, Ubx expression is controlled by segmentation genes turned on earlier in the developmental hierarchy. Correct Ubx expression depends on multiple regulatory sequences located outside the basal promoter. Here we report that a 500 bp DNA fragment from the bx region of the Ubx unit, approximately 30 kb away from the promoter, contains one of the distant regulatory elements (bx region enhancer, BRE). During early embryogenesis, this enhancer element activates the Ubx promoter in parasegments (PS) 6, 8, 10, and 12 and represses it in the anterior half of the embryo. The repressor of the anterior Ubx expression is the gap gene hunchback (hb). We show that the hb protein binds to the BRE element and that such binding is essential for hb repression in vivo, hb protein also binds to DNA fragments from abx and bxd, two other regulatory regions of the Ubx gene. We conclude that hb represses Ubx expression directly by binding to BRE and probably other Ubx regulatory elements. In addition, the BRE pattern requires input from other segmentation genes, among them tailless and fushi tarazu but not Krüppel and knirps.

A nuclear protein derived from brain cells stimulates transcription of the human neurotropic virus promoter, JCVE, in vitro.

The 98-base pair enhancer/promoter sequence is critical for cell type-specific transcription of the human neurotropic viral promoter, JCVE, in glial cells. Transcriptionally active extracts were prepared from glial cells and used to identify cis- and trans-acting regulatory elements that are involved in the glial-specific activation of the JCVE promoter. Results indicate that multiple regulatory sequences within the 98-base pair repeat specifically bind to nuclear proteins present in glial cells and positively regulate viral early RNA synthesis. The central region of the repeat, designated domain-B, interacts with a 45-kDa nuclear protein present in brain cells. This brain-specific protein was purified by conventional and DNA affinity chromatography. Complementation of the highly purified protein with HeLa extract significantly increased JCVE promoter activity. Thus, association of the novel glial-origin transcription factor with its target sequence increases transcription of the JCVE promoter in a non-glial context.

Multiple polyadenylation sites downstream from the human aFGF gene encoding acidic fibroblast growth factor

We previously reported the isolation of two partial cDNA clones encoding human acidic fibroblast growth factor (aFGF). The nucleotide (nt) sequence throughout the coding region and the deduced amino acid sequence were presented [Jaye et al., Science 233 (1986) 541–545]. In this report, the isolation of additional aFGF cDNA clones and their nt sequence are presented. The human aFGF gene is shown to encode at least four functional polyadenylation sites and multiple regulatory sequences within the 3′-untranslated region. The aFGF open reading frame resides approx. 3100 bp upstream from the most frequently utilized 3′ processing and polyadenylation site. Several less abundant cDNA clones provide evidence of polyadenylation at three less distal sites, which are colinear with genomic DNA. Northern-blot analysis reveals three detectable mRNA species, whose sizes and intensities correlate with the length and relative abundance of cDNA clones representing them.