Promoter elements, regulatory elements, and chromatin structure of the yeast his3 gene.
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Base Sequence↗
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Functional dissection of the promoter of the pollen-specific gene NTP303 reveals a novel pollen-specific, and conserved cis-regulatory element.
Regulatory elements within the promoter of the pollen-specific NTP303 gene from tobacco were analysed by transient and stable expression analyses. Analysis of precisely targeted mutations showed that the NTP303 promoter is not regulated by any of the previously described pollen-specific cis-regulatory elements. However, two adjacent regions from -103 to -86 bp and from -86 to -59 bp were shown to contain sequences which positively regulated the NTP303 promoter. Both of these regions were capable of driving pollen-specific expression from a heterologous promoter, independent of orientation and in an additive manner. The boundaries of the minimal, functional NTP303 promoter were determined to lie within the region -86 to -51 bp. The sequence AAATGA localized from -94 to -89 bp was identified as a novel cis-acting element, of which the TGA triplet was shown to comprise an active part. This element was shown to be completely conserved in the similarly regulated promoter of the Bp 10 gene from Brassica napus encoding a homologue of the NTP303 gene.
Base Sequence↗
Enhancer-promoter interaction maps in pig implantation tissue identify candidate cis-regulatory elements and regulatory variants.
Due to the polygenic nature of pig reproductive traits, most variants accounting for the phenotypic variation remain poorly characterized. It is well established that trait-associated variants are often enriched in regulatory elements of relevant tissues, and coordinated endometrial-conceptus crosstalk is critical for implantation success. Accordingly, this study aimed to investigate the chromatin landscape in pig endometrium-conceptus tissues and identify regulatory variants associated with reproductive traits. Combining RNA-seq and ChIP-seq, we uncovered cis-regulatory elements whose histone modification patterns correlate with gene expression. H3K27ac-based chromatin interaction profiling further resolved 2137 cis-regulatory elements involved in enhancer-promoter (E-P) interactions. Subsequent analyses identified candidate variants within these regulatory regions that associate with reproductive traits, in which the enhancer SNP rs346038249 likely affects transcriptional activity through allele-dependent transcription factor binding. Collectively, our findings provide mechanistic insights into pig embryo implantation and nominate candidate regulatory variants that merit further functional validation for their potential role in pig genetic improvement.
Embryo implantation↗
FAIRE (Formaldehyde-Assisted Isolation of Regulatory Elements) isolates active regulatory elements from human chromatin.
DNA segments that actively regulate transcription in vivo are typically characterized by eviction of nucleosomes from chromatin and are experimentally identified by their hypersensitivity to nucleases. Here we demonstrate a simple procedure for the isolation of nucleosome-depleted DNA from human chromatin, termed FAIRE (Formaldehyde-Assisted Isolation of Regulatory Elements). To perform FAIRE, chromatin is crosslinked with formaldehyde in vivo, sheared by sonication, and phenol-chloroform extracted. The DNA recovered in the aqueous phase is fluorescently labeled and hybridized to a DNA microarray. FAIRE performed in human cells strongly enriches DNA coincident with the location of DNaseI hypersensitive sites, transcriptional start sites, and active promoters. Evidence for cell-type-specific patterns of FAIRE enrichment is also presented. FAIRE has utility as a positive selection for genomic regions associated with regulatory activity, including regions traditionally detected by nuclease hypersensitivity assays.
Cells, Cultured↗
Transactivation of the human apolipoprotein CII promoter by orphan and ligand-dependent nuclear receptors. The regulatory element CIIC is a thyroid hormone response element.
The regulatory elements CIIC (-159/-116) and CIIB (-102/-81) of the apolipoprotein CII (apoCII) promoter have distinct specificities for orphan nuclear receptors (Vorgia, P., Zannis, V. I., and Kardassis, D. (1998) J. Biol. Chem. 273, 4188-4199). In this communication we investigated the contribution of ligand-dependent and orphan nuclear receptors on the transcriptional regulation of the human apoCII gene. It was found that element CIIC in addition to ARP-1 and EAR-2 binds RXRalpha/T3Rbeta heterodimers strongly, whereas element CIIB binds hepatic nuclear factor 4 (HNF-4) exclusively. Binding is abolished by mutations that alter the HRE binding motifs. Transient cotransfection experiments showed that in the presence of T3, RXRalpha/T3Rbeta heterodimers transactivated the -205/+18 apoCII promoter 1.6- and 11-fold in HepG2 and COS-1 respectively. No transactivation was observed in the presence of 9-cis-retinoic acid. Transactivation requires the regulatory element CIIC, suggesting that this element contains a thyroid hormone response element. HNF-4 did not affect the apoCII promoter activity in HepG2 cells. However, mutations in the HNF-4 binding site on element CIIB and inhibition of HNF-4 synthesis in HepG2 cells by antisense HNF-4 constructs decreased the apoCII promoter activity to 25-40% of the control, indicating that HNF-4 is a positive regulator of the apoCII gene. ARP-1 repressed the -205/+18 but not the -104/+18 apoCII promoter activity in HepG2 cells, indicating that the repression depends on the regulatory element CIIC. In contrast, combination of ARP-1 and HNF-4 transactivated different apoCII promoter segments as well as a minimal adenovirus major late promoter driven by the regulatory element CIIB. Mutagenesis or deletion of elements CIIB or CIIC established that the observed transactivation requires DNA binding of one of the two factors and may result from HNF-4-ARP-1 interactions that elicit the transactivation functions of HNF-4. The combined data indicate that RXRalpha/T3Rbeta in the presence of T3 and HNF-4 can upregulate the apoCII promoter activity by binding to the regulatory elements CIIC and CIIB, respectively. In addition, ARP-1 can either have inhibitory or stimulatory effects on the apoCII promoter activity via different mechanisms.
Animals↗
Yin yang 1 protein negatively regulates high-density lipoprotein receptor gene transcription by disrupting binding of sterol regulatory element binding protein to the sterol regulatory element.
Because the high-density lipoprotein receptor (HDL-R) is a key element in cholesterol homeostasis and a potential therapeutic target for hypercholesterolemic drugs, an understanding of HDL-R regulation is essential. The sterol regulatory element (SRE) binding protein-1a (SREBP-1a) was shown to positively regulate HDL-R gene expression through two SREs. SREBP-1a requires the presence of a coactivator like simian-virus-40-protein-1 (Sp1) to promote maximum activation of the HDL-R promoter. Negative regulatory factors are also known to play a role in cholesterol homeostasis, and the ubiquitous Yin Yang-1 zinc finger transcription factor (YY1) has been shown to repress several sterol-responsive gene promoters. A search of the rat HDL-R promoter revealed two putative YY1 binding sites (distal, -1329 to -1321; proximal, -1211 to -1203). Upon removal of both YY1 binding sites, YY1 was unable to repress HDL-R activation under basal (unstimulated) promoter conditions. However, YY1 was still an efficient transcriptional repressor for SREBP-1a-induced activation. YY1 was able to attenuate the transcriptional synergy caused by the combined actions of SREBP-1a and Sp1. Two-hybrid studies confirmed that YY1 bound with high affinity to SREBP-1a, and mobility shift assays demonstrated that YY1 could disrupt SREBP-1a binding to both SREs. The molecular consequence of YY1 intervention seems to override any positive interactions between Sp-1 and SREBP-1a and results in the disruption of SREBP-1a binding to the SREs in the HDL-R promoter.
Animals↗
Multiple interactions of the transcription factor YY1 with human histone H4 gene regulatory elements.
Multiple regulatory elements and intricate protein-DNA interactions mediate the transcription of the human histone H4 genes in a cell growth-dependent manner. Upon analysis of the regulatory elements of the FO108 histone H4 gene, we identified several potential YY1 binding sites. In this study, we have analyzed the ability of the transcription factor YY1 to interact at these sites in vitro by using electrophoretic mobility shift assays in combination with oligonucleotide competition and antibody immunoreactivity. We show that YY1 specifically binds transcriptional regulatory elements at -340 nt (site III), -100 nt (site I) and at least two domains within the coding region of the histone H4 gene. To test if these elements were functionally responsive to YY1, we performed transient expression experiments in Drosophila S-2 cells transfected with heterologous reporter gene constructs driven by histone H4 gene segments fused to the thymidine kinase promoter. Co-expression of YY1 stimulated promoter activity of these constructs relative to the reporter construct lacking histone H4 gene fragments. Our results suggest that YY1 contributes to transcriptional regulation of the histone H4 gene through interactions at multiple regulatory elements.
Animals↗
Sp1 binds to the adhesion-molecule-on-glia regulatory element that functions as a positive transcription regulatory element in astrocytes.
We analyzed cis-acting elements regulating the expression of the gene encoding adhesion molecule on glia (AMOG) in primary cultured astrocytes from newborn rat cerebrum and cerebellum. The relative promoter activities among the series of 5' sequential deletion mutants are similar to those observed in B103 (rat neuroblastoma cell line) cells. The previously identified AMRE (AMOG regulatory element) of the GAGGCGGGG sequence functions as a positive regulatory element, not only in B103 cells, but also in astrocytes. Binding factors to the element were identified as Sp1 based on the following observations using nuclear extracts from the astrocytes and B103 cells: (1) The interaction of the factors with AMRE analyzed by DNase I footprinting and methylation interference analyses was similar to that of Sp1; (2) The binding of the factors to AMRE competed with an oligonucleotide containing the authentic Sp1 consensus sequence; (3) Sp1-specific antibody interfered with the formation of the AMRE gel retardation complexes. The functional implications of the factors in AMOG gene regulation are discussed.
Adenosine Triphosphatases↗
cAMP-dependent transcription of the human CYP21B (P-450C21) gene requires a cis-regulatory element distinct from the consensus cAMP-regulatory element.
By utilizing chimeric genes constructed from 5'-flanking sequences of the human CYP21B (P-450C21) gene and reporter genes (chloramphenicol acetyltransferase or rabbit beta-globin), a 34-nucleotide sequence has been found to be required for cAMP-dependent transcription. This sequence (-129/-96 base pairs) shows no homology to that of the consensus (CRE) cAMP-regulatory element. Gel retardation analysis shows that a protein-DNA complex is formed between this DNA sequence and nuclear proteins from mouse adrenal Y1 tumor cells or bovine adrenal cortical cells or human fetal adrenal tissue and that formation of this complex cannot be competed by DNA containing the consensus CRE sequence. Even though cAMP-enhanced accumulation of P-450C21 mRNA in primary cultures of bovine adrenocortical cells is inhibited by the protein synthesis inhibitor, cycloheximide, reporter gene transcription enhanced by the cAMP-responsive -129/-96-base pair fragment of the human CYP21B gene is not. We conclude that cAMP-dependent transcription of the human P-450C21 gene (CYP21B), an event required for maintenance of optimal steroidogenic capacity in the adrenal cortex, involves a stable transcription factor(s) distinct from the CRE-binding protein. Furthermore the cAMP-dependent cis-regulatory element of the human P-450C21 gene is distinct from those found associated with the other steroid hydroxylase genes, 17 alpha-hydroxylase cytochrome P-450, cholesterol side chain cleavage cytochrome P-450, and 11 beta-hydroxylase cytochrome P-450, suggesting that each of these genes may require its own set of specific transcription factors for cAMP-dependent regulation.
Adrenal Cortex↗
Positive regulatory elements (HF-1a and HF-1b) and a novel negative regulatory element (HF-3) mediate ventricular muscle-specific expression of myosin light-chain 2-luciferase fusion genes in transgenic mice.
The cardiac myosin light-chain 2v (MLC-2v) gene has served as a model system to identify the pathways which restrict the expression of cardiac muscle genes to particular chambers of the heart during cardiogenesis. To identify the critical cis regulatory elements which mediate ventricular chamber-specific expression of the MLC-2v gene in the in vivo context, a series of transgenic mice which harbor mutations in putative MLC-2 cis regulatory elements in a 250-bp MLC-2-luciferase fusion gene which is expressed in a ventricular chamber-specific fashion in transgenic mice were generated. These studies demonstrate that both components of HF-1 (HF-1a and HF-1b/MEF-2) are required to maintain ventricular chamber-specific expression and function as positive regulatory elements. Mutations in another conserved element (HF-2) are without statistically significant effect on ventricular chamber expression. Transgenics harboring mutations in the E-box site also displayed significant upregulation of reporter activity in the soleus, gastrocnemius, and uterus, with a borderline effect on expression in liver. Mutations in another conserved element (HF-3) result in a marked (> 75-fold) upregulation of the luciferase reporter activity in the soleus muscle of multiple independent or transgenic founders. Since the HF-3 mutations appeared to have only a marginal effect on luciferase reporter activity in liver tissue, HF-3 appears to function as a novel negative regulatory element to primarily suppress expression in muscle tissues. Thus, a combination of positive (HF-1a/HF-1b) and negative (E-box and HF-3) regulatory elements appear to be required to maintain ventricular chamber-specific expression in the in vivo context.
Animals↗
Nutritional regulation of the fatty acid synthase promoter in vivo: sterol regulatory element binding protein functions through an upstream region containing a sterol regulatory element.
The transcription of fatty acid synthase (FAS), a central enzyme in de novo lipogenesis, is dramatically induced by fasting/refeeding and insulin. We reported that upstream stimulatory factor binding to the -65 E-box is required for induction of the FAS transcription by insulin in 3T3-L1 adipocytes. On the other hand, we recently found that two upstream 5' regions are required for induction in vivo by fasting/refeeding and insulin; one at -278 to -131 albeit at a low level, and the other at -444 to -278 with an E-box at -332 where upstream stimulatory factor functions for maximal induction. Here, we generated double transgenic mice carrying the chloramphenicol acetyltransferase reporter driven by the various 5' deletions of the FAS promoter region and a truncated active form of the sterol regulatory element (SRE) binding protein (SREBP)-1a. We found that SREBP participates in the nutritional regulation of the FAS promoter and that the region between -278 and -131 bp is required for SREBP function. We demonstrate that SREBP binds the -150 canonical SRE present between -278 and -131, and SREBP can function through the -150 SRE in cultured cells. These in vivo and in vitro results indicate that SREBP is involved in the nutritional induction of the FAS promoter via the -278/-131 region and that the -150 SRE is the target sequence.
3T3 Cells↗
An octamer element functions as a regulatory element in the differentiation-responsive CD11c integrin gene promoter: OCT-2 inducibility during myelomonocytic differentiation.
The integrin CD11c/CD18 mediates leukocyte adhesion to endothelium and other cell types and is a receptor for LPS, iC3b, and fibrinogen. CD11c expression is restricted to myeloid and activated B cells, is regulated during leukocyte differentiation, and constitutes a diagnostic tool for hairy cell leukemia. Mapping of in vivo DNA-protein interactions in the CD11c proximal promoter revealed three adjacent myeloid-specific interactions, one of which lies on an octamer consensus sequence, ATTT GCAT (Oct185). Oct185 disruption increased the CD11c promoter activity while decreasing its myeloid differentiation responsiveness, indicating that Oct185 contributes to the activity of the CD11c promoter and suggesting that Oct185 is a negative regulatory element whose function changes during myeloid differentiation. Oct185 is recognized by the ubiquitous Oct-1 factor in all cell lineages and by Oct-2 in B lymphoid lineage cells. Unexpectedly, Oct-2 binding to Oct185 was induced de novo upon monocytic differentiation of U937 and HL-60 cells but not during HL-60 granulocytic differentiation, as determined by electrophoretic mobility shift assays and immunochemical studies, and Oct-2 complexes were also observed in cultured adherent monocytes. Western blotting showed that the pattern of Oct-2 isoforms in myeloid cells is similar to that seen in B cells. The Oct-2 up-regulated expression in differentiating myeloid cells and its binding to the Oct185 negative regulatory element suggests its involvement in the differentiation-regulated activity of the CD11c promoter and might represent an important parameter for the myeloid- and B cell-restricted expression of the CD11c/CD18 integrin and other molecules with similar patterns of expression.
Blotting, Western↗
Application of comparative genomics to the analysis of vertebrate regulatory elements.
Gene regulatory regions (also known as 'cis-regulatory modules') in vertebrates are poorly understood and annotated by comparison with protein-coding sequences. The short and degenerate sequences of regulatory elements and their distribution over large intergenic and intronic regions pose a major challenge to genomics scientists. Comparative genomics can be used to identify putative regulatory regions, and to analyse regulatory regions into their constituent transcription factor binding sites. There is need for high throughput assay systems to analyse the function of predicted vertebrate gene regulatory regions.
Animals↗
Identification of cis-regulatory elements in the upstream regulatory region of human papillomavirus type 59.
Human papillomavirus type 59 (HPV-59) was cloned from a vulvar intraepithelial neoplasia and the complete nucleotide sequence was determined. This virus is closely related to HPV-18 and -39 (60% homology in nucleotide sequence) and is grouped with the genital HPV types. In the present paper, we demonstrate that the HPV-59 E2 transactivator represses its E6 promoter-mediated transcription. We have also analyzed cis-regulatory elements in the upstream regulatory region (URR) of HPV-59 using chloramphenicol acetyl transferase assays as well as electrophoresis mobility shift assays (EMSA). The results allow for a subdivision of the HPV-59 URR into three regions of activity: distal (nt 7149-7493), central (nt 7493-7742), and proximal (nt 7742-7748). In particular, the 250 bp (nt 7493-7742) of the central region plays an important role as a constitutive enhancer element for the maximal transcription of the E6 promoter. Our results suggest that the transcription factors AP1, Oct1, SP1 and unidentified factors bind to the HPV-59 E6 promoter region, whereas NF1, GRE and TFIID fail to bind despite the presence of putative binding sites in the DNA sequence.
DNA-Binding Proteins↗
On the relationship between genomic regulatory element organization and gene regulatory dynamics.
In this project we study the relationship between genomic regulatory element organization and gene regulatory dynamics. This paper illustrates an approach to investigating this relationship based on the application of classical nonlinear system analysis techniques to a transcription level, statistical thermodynamical model like that used in Shea & Ackers (1985). Preliminary ideas presented at the ICMCM conference (Wolf & Eeckman, 1998) are developed in this manuscript. We show that, for prokaryotic gene circuits dominated by local promoter control, dynamical system behavior descriptors like the number and stability of equilibrium point steady states and their bifurcation potential can be largely determined from genomic organization (e.g. the number, type, and placement of regulatory protein binding sites). Concepts are illustrated on hypothetical gene regulation systems with one or two genes and varying numbers of regulatory protein binding sites (operators). Gene regulatory systems with a single gene and an arbitrary number of operator sites are shown to be globally stable, with the potential for having multiple equilibrium points and capable of bifurcating. A monomer-controlled gene regulation system with n operator sites is proven to have a maximum of 1+n/2 stable equilibria for even n, and (n+1)/2 for odd n, while a multimer-controlled, n operator site system is shown to have a maximum of 2+n/2 stable equilibria for even n, and (n+3)/2 for odd n. These results are applied to the design of a two-state switch using a gene regulation system with two operator sites. The question "what is the simplest possible gene regulation system capable of acting like a switch?" is answered. The paper ends with an analysis of a two-gene regulation system, the results of which point to the existence of a "soft-switching" mechanism that may account for the "on-off" hypothesized behavior of some gene networks.
Animals↗
GATA-3 suppresses IFN-gamma promoter activity independently of binding to cis-regulatory elements.
The regulatory mechanism by which GATA-3 suppresses IFN-gamma gene expression was investigated. A reduction of GATA-3 using RNA interference technology enhanced, whereas overexpression of GATA-3 suppressed IFN-gamma mRNA expression. IL-4 expression was reciprocally affected by GATA-3. GATA-3-mediated down-regulation of IFN-gamma was achieved through the inhibition of its promoter/enhancer activity. Two GATA elements located in the cis-regulatory elements did not contribute to the suppression of IFN-gamma promoter activity, even though they behaved as binding sites for GATA-3. The effect of GATA-3 on IFN-gamma promoter was lost upon removal of the region encompassing -257 to -172. Among several transcription factors putatively interacting with this region, Stat4, which enhanced IFN-gamma promoter activity, was down-regulated by GATA-3 at gene transcription level. Although GATA-3 has the capacity to interact with the cis-regulatory elements, it suppresses IFN-gamma gene transcription via down-regulation of Stat4.
Base Sequence↗
The hepatitis B virus PRE contains a splicing regulatory element.
The posttranscriptional regulatory element (PRE) is considered to enhance hepatitis B virus (HBV) gene expression by facilitating the nuclear export of intronless viral subgenomic RNAs. Its role in the RNA metabolism of the viral pregenomic RNA (pgRNA) is currently unknown. We identified a positively cis-acting splicing regulatory element (SRE-1) and present two lines of evidence for its functionality. Firstly, in a heterologous context SRE-1 functionally substitutes for a retroviral bidirectional exonic splicing enhancer (ESE). As expected, SRE-1 is a splicing enhancer also in its natural viral sequence context, since deletion of SRE-1 reduces splicing of pgRNA in cell culture experiments. Secondly, we show that stimulation of HBV RNA splicing by the splicing factor PSF was repressed by the PRE. Analysis of a variety of PSF mutants indicated that RNA-binding and protein-protein interaction were required to enhance splicing. In addition, we show that the PRE contributed to pgRNA stability, but has little influence on its nuclear export. Herein, we report for the first time that the PRE harbors splicing stimulating and inhibiting regulatory elements controlling processing of the viral pregenome. We discuss a model in which the regulation of pgRNA splicing depends on cellular factors interacting with the PRE.
Cell Line, Tumor↗
Polyunsaturated fatty acids decrease expression of promoters with sterol regulatory elements by decreasing levels of mature sterol regulatory element-binding protein.
Membrane physiology, plasma lipid levels, and intracellular sterol homeostasis are regulated by both fatty acids and cholesterol. Sterols regulate gene expression of key enzymes of cholesterol and fatty acid metabolism through proteolysis of the sterol regulatory element-binding protein (SREBP), which binds to sterol regulatory elements (SRE) contained in promoters of these genes. We investigated the effect of fatty acids on SRE-dependent gene expression and SREBP. Consistent results were obtained in three different cell lines (HepG2, Chinese hamster ovary, and CV-1) transfected with SRE-containing promoters linked to the luciferase expression vector. We show that micromolar concentrations of oleate and other polyunsaturated fatty acids (C18:2-C22:6) dose-dependently (0.075-0.6 mmol) decreased transcription of SRE-regulated genes by 20-75%. Few or no effects were seen with saturated free fatty acids. Fatty acid effects on SRE-dependent gene expression were independent and additive to those of exogenous sterols. Oleate decreased levels of the mature sterol regulatory element-binding proteins SREBP-1 and -2 and HMG-CoA synthase mRNA. Oleate had no effect in sterol regulation defective Chinese hamster ovary cells or in cells transfected with mutant SRE-containing promoters. We hypothesize that unsaturated fatty acids increase intracellular regulatory pools of cholesterol and thus affect mature SREBP levels and expression of SRE-dependent genes.
Animals↗