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Results for “regulatory elements”

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At least 19 recordsLinked to original sources

PubMed2026

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↗

PubMed1990

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↗

PubMed1992

A transcriptional regulatory element of the gene encoding the 67,000-M(r) form of human glutamate decarboxylase is similar to a Drosophila regulatory element.

We have isolated the 5' flanking DNA sequences of the human gene encoding the 67,000-M(r) form of glutamate decarboxylase (GAD67), the gamma-aminobutyric acid synthetic enzyme. Transcription begins at a single promoter (P1) in adult brain but at two tandem promoters, P1 and P2, in fetal brain. P1, which is 3' to P2, resembles the promoter regions of many constitutively expressed genes, whereas P2 resembles a tissue-specific promoter. P1 contains a 10-base sequence (dec-1) that closely matches the element I cis-regulatory sequence identified in the promoter region of Drosophila 3,4-dihydroxyphenylalanine decarboxylase. Gel shift and transient expression assays demonstrate that the dec-1 sequence plays a role in the transcription of the human GAD67 gene.

Animals↗

PubMed1992

Characterization of the hepatitis B virus X- and nucleocapsid gene transcriptional regulatory elements.

The regulatory DNA sequence elements that control the expression of the hepatitis B virus X- and nucleocapsid genes in the differentiated human hepatoma cell lines, Huh7, Hep3B, PLC/PRF/5, and HepG2, the dedifferentiated human hepatoma cell line, HepG2.1, and the human cervical carcinoma cell line, HeLa S3, were analyzed using transient transfection assays. In this system, the hepatitis B virus enhancer I located between coordinates 1071 (-239) and 1238 (-72) increases transcription from the X-gene promoter located between coordinates 1239 (-71) and 1376 (+67) more than 30-fold in the differentiated hepatoma and the HeLa S3 cell lines. In the dedifferentiated hepatoma cell line, HepG2.1, the enhancer I sequence increases the level of transcription from the X-gene promoter approximately 10-fold. The enhancer I subregion between coordinates 1117 (-193) and 1204 (-106) appears to be important for enhancer function only in the differentiated hepatoma cell lines, whereas the enhancer I subregion between coordinates 1222 (-88) and 1238 (-72) is required for enhancer activity in each of the cell lines examined. In all of the cell lines, the X-gene minimal promoter element was within a 138-nucleotide sequence located between coordinates 1239 (-71) and 1376 (+67). The enhancer I sequence increases transcription from the nucleocapsid promoter approximately 3- to 10-fold in the Huh7, Hep3B, PLC/PRF/5, and HeLa S3 cell lines, whereas it had little influence on the level of transcription from this promoter in HepG2 and HepG2.1 cells. The minimal nucleocapsid promoter element was within a 105 nucleotide sequence located between coordinates 1700 (-85) and 1804 (+20). This indicates that the levels of transcription from the X- and nucleocapsid gene promoters are determined in a cell-type-specific manner, in part, by the hepatitis B virus enhancer I and the corresponding minimal promoter sequence.

Base Sequence↗

PubMed1991

A binding site for the cyclic adenosine 3',5'-monophosphate-response element-binding protein as a regulatory element in the grp78 promoter.

The 78-kDa glucose-regulated protein (GRP78) is ubiquitously expressed in many cell types. Its promoter contains multiple protein-binding sites and functional elements. In this study we examined a high affinity protein-binding site spanning bp -198 to -180 of the rat grp78 promoter, using nuclear extracts from both B-lymphoid and HeLa cells. This region contains a sequence TGACGTGA which, with the exception of one base, is identical to the cAMP-response element (CRE). Site-directed mutagenesis reveals that this sequence functions as a major basal level regulatory element in hamster fibroblast cells and is also necessary to maintain high promoter activity under stress-induced conditions. By gel mobility shift analysis, we detect two specific protein complexes. The major specific complex I, while immunologically distinct from the 42-kDa CRE-binding protein (CREB), binds most strongly to the grp site, but also exhibits affinity for the CRE consensus sequence. As such, complex I may consist of other members of the CREB/activating transcription factor protein family. The minor specific complex II consists of CREB or a protein antigenically related to it. A nonspecific complex III consists of the Ku autoantigen, an abundant 70- to 80-kDa protein complex in HeLa nuclear extracts. By cotransfection experiments, we demonstrate that in F9 teratocarcinoma cells, the grp78 promoter can be transactivated by the phosphorylated CREB or when the CREB-transfected cells are treated with the calcium ionophore A23187. The differential regulation of the grp78 gene by cAMP in specific cell types and tissues is discussed.

B-Lymphocytes↗

PubMed1992

Vitamin D-responsive protein-DNA interactions at multiple promoter regulatory elements that contribute to the level of rat osteocalcin gene expression.

The observation that vitamin D-mediated enhancement of osteocalcin (OC) gene expression is dependent on and reciprocally related to the level of basal gene expression suggests that an interaction of the vitamin D responsive element (VDRE) with basal regulatory elements of the OC gene promoter contributes to both basal and vitamin D-enhanced transcription. Protein-DNA interactions at the VDRE of the rat OC gene (nucleotides -466 to -437) are reflected by direct sequence-specific and antibody-sensitive binding of the endogenous vitamin D receptor present in ROS 17/2.8 osteosarcoma nuclear protein extracts. In addition, a vitamin D-responsive increase in OC gene transcription is accompanied by enhanced non-vitamin D receptor-mediated protein-DNA interactions in the "TATA" box region (nucleotides -44 to +23), which also contains a potential glucocorticoid responsive element. Evidence for proximity of the VDRE with the basal regulatory elements is provided by two features of nuclear architecture. (i) Nuclear matrix attachment elements in the rat OC gene promoter that bind nuclear matrix proteins with sequence specificity may impose structural constraints on promoter conformation. (ii) Limited micrococcal nuclease digestion and Southern blot analysis indicate that three nucleosomes can be accommodated in the sequence spanning the OC gene VDRE, the OC/CCAAT box (nucleotides -99 to -76), and the TATA/glucocorticoid responsive element, and thereby the potential distance between the VDRE and the basal regulatory elements can be reduced. A model is presented for the contribution of both the VDRE and proximal promoter elements to the enhancement of OC gene transcription in response to vitamin D. The vitamin D receptor plus accessory proteins may function cooperatively with basal regulatory factors to modulate the extent to which the OC gene is transcribed.

Animals↗

PubMed1992

Differential trans-activation of muscle-specific regulatory elements including the mysosin light chain box by chicken MyoD, myogenin, and MRF4.

We have isolated cDNAs encoding a chicken homologue of MRF4 (cMRF4) in addition to chicken MyoD (CMD1) and myogenin (c-myogenin) described previously. In an attempt to understand the roles that cMRF4, CMD1, and c-myogenin play in chicken myogenesis, the effects of these factors on muscle-specific cis-elements identified in regulatory regions of myosin alkali light chain (MLC) genes were examined. The promoter analysis of some of MLC genes has revealed two sorts of muscle-specific positive regulatory elements to date, an enhancer located upstream of the adult type LC1 gene and a cis-element, termed an MLC box, conserved among promoters of various MLC genes. The LC1 enhancer was exclusively trans-activated by CMD1. Although c-myogenin also activated transcription driven by the LC1 promoter, it was suggested that c-myogenin requires a cis-element(s) other than the CMD1-responsive enhancer. Chicken MRF4 could not trans-activate any of the constructs containing the LC1 promoter. In contrast, the promoter of the embryonic L23 gene was trans-activated by all of the three factors. From deletion and mutation analysis, the MLC box was shown to be involved in their positive regulation. These results extend previous observations that individual myogenic regulatory factors exhibit different capabilities in transcriptional activation of muscle-specific genes by acting distinctively upon their regulatory elements.

Amino Acid Sequence↗

PubMed1991

Different combinations of regulatory elements may explain why placenta-specific expression of the glycoprotein hormone alpha-subunit gene occurs only in primates and horses.

Expression of the glycoprotein hormone alpha-subunit gene occurs in the pituitary of all mammals but in placenta of only primates and horses. In humans, two different elements, termed upstream regulatory element (URE) and cAMP response element (CRE), are required for placenta-specific expression of the alpha-subunit gene. The URE binds a protein unique to placenta whereas the CRE binds a ubiquitous protein. Comparative analysis of the promoter-regulatory region of the alpha-subunit gene from a number of mammals indicates that a functional URE has been retained and suggests the potential for placenta-specific expression. Indirect evidence also indicates that the URE-binding protein has been conserved, even in placenta from mammals that fail to express the alpha-subunit gene. Lack of expression of the alpha-subunit gene in placenta of rodents and cattle can be traced to a single nucleotide change that renders the CRE-like sequence of these genes incapable of binding the protein that confers responsiveness to cAMP. In contrast, although expression of the alpha-subunit gene occurs in horse placenta, the promoter-regulatory region lacks a functional CRE but appears to retain a functional URE. This suggests that either a different accessory element and cognate protein interacts with the horse URE to provide placenta-specific expression or that a completely different set of regulatory elements is required for placenta-specific expression in horses.

Animals↗

PubMed1989

An internal regulatory element controls troponin I gene expression.

During skeletal myogenesis, approximately 20 contractile proteins and related gene products temporally accumulate as the cells fuse to form multinucleated muscle fibers. In most instances, the contractile protein genes are regulated transcriptionally, which suggests that a common molecular mechanism may coordinate the expression of this diverse and evolutionarily unrelated gene set. Recent studies have examined the muscle-specific cis-acting elements associated with numerous contractile protein genes. All of the identified regulatory elements are positioned in the 5'-flanking regions, usually within 1,500 base pairs of the transcription start site. Surprisingly, a DNA consensus sequence that is common to each contractile protein gene has not been identified. In contrast to the results of these earlier studies, we have found that the 5'-flanking region of the quail troponin I (TnI) gene is not sufficient to permit the normal myofiber transcriptional activation of the gene. Instead, the TnI gene utilizes a unique internal regulatory element that is responsible for the correct myofiber-specific expression pattern associated with the TnI gene. This is the first example in which a contractile protein gene has been shown to rely primarily on an internal regulatory element to elicit transcriptional activation during myogenesis. The diversity of regulatory elements associated with the contractile protein genes suggests that the temporal expression of the genes may involve individual cis-trans regulatory components specific for each gene.

Animals↗

PubMed1991

Activity and enhancer binding factors for BK virus regulatory elements in differentiating embryonal carcinoma cells.

We have studied cell type specificity of expression of the human papovavirus BK regulatory elements in undifferentiated and differentiated embryonal carcinoma (EC) cells as a model system. While the activity of the regulatory elements of this virus was marginal in undifferentiated cells, differentiation by retinoic acid and DMSO resulted in a dramatic increase in the activity. To correlate in vivo activity of the regulatory elements with interaction with cellular transcription factors, we performed DNase I footprinting experiments. A GC-rich region was protected in both undifferentiated and differentiated cells. An additional four protected sites were detected in retinoic acid-differentiated cells and at least one of these additional sites was weakly protected in DMSO-differentiated cells. The sequences of the differentiated cell type-specific protected regions showed homology to a nuclear factor 1 (NF-1) binding motif and to a muscle creatine kinase gene enhancer motif. The intensity, competition, and pattern of protection of these sites were different in the two differentiated cell types, suggesting the involvement of different transcription factors regulating the activity of BKV regulatory elements in the two cell types.

BK Virus↗

PubMed1992

Transcriptional regulatory elements for basal expression of cytochrome P450IIC genes.

To analyze the transcriptional regulatory elements in rabbit cytochrome P450IIC genes, varying lengths of the 5'-flanking regions of CYP2C1 and CYP2C2 were fused to a luciferase reporter gene. Promoter activity was assayed by transfection into HepG2 cells, a hepatic cell line, and monkey kidney COS-1 cells, a nonhepatic cell line. Activity of the CYP2C1 promoter in HepG2 cells increased slightly with progressive 5' deletions of the 5'-flanking region from nucleotide -3600 to -1500 relative to the transcription start site. Additional deletions to -900, -358, and -116 each reduced activity by about 50%, and deletion of the sequence from -116 to -67 reduced activity by a factor of 12. Activity of the CYP2C2 promoter increased about 3-fold with progressive 5' deletions of sequence from nucleotide -3500 to -410. In contrast, deletions of sequences from -251 to -193 and from -133 to -64 reduced promoter activity by factors of 2 and 8, respectively. In COS-1 cells, the maximum activities of the CYP2C1 and CYP2C2 promoters normalized to a Rous sarcoma viral promoter were about 10-20% of that in the HepG2 cells. The changes in activity between different constructions in COS-1 cells largely paralleled those in the HepG2 cells except for deletions of the sequences -133 to -64 and -116 to -67 for CYP2C1 and CYP2C2, respectively, which produced the largest reduction of promoter activity in HepG2 cells but had little effect in COS-1 cells. These results show that HepG2-specific regulatory elements are present in the regions between -120 and -65 in both genes. Nuclear proteins from HepG2 cells, but not from COS-1 cells, bound to sequences within these regions, and the binding was inhibited by an oligonucleotide containing a sequence conserved in rabbit P450IIC genes which has been designated the HepG2-specific P450 2C factor-1 (HPF1) motif. Mutation of this sequence eliminated the binding of nuclear proteins and reduced transcriptional activity 25-fold. The HPF1 binding sequence is conserved in CYP2A, CYP2C, and CYP2D genes and resembles the binding motif for hepatic nuclear factor-4. These results demonstrate that CYP2C1 and CYP2C2 contain several potential regulatory elements for basal expression, including one HepG2-specific sequence that may be important for liver expression.

Animals↗

PubMed1992

Identification of cis-regulatory elements in the myelin proteolipid protein (PLP) gene.

Regulatory elements of the proteolipid protein (PLP) gene were identified physically by footprinting and gel mobility shift assays and functionally by transfecting glial cell lines with PLP-chloramphenicol acetyltransferase chimeric genes. In both human and rat glial cells, only several hundred base pairs of upstream sequence were sufficient for high level activity of the human PLP promoter. This region contains five sites that contact nuclear proteins in vitro. More distal recognition sites may exist, as regions upstream of -524 displayed silencing activity indicative of a negative regulatory element. A series of site directed mutations revealed one essential positive element (ATGGA at -118) which is found in other genes encoding myelin proteins. Our combined biochemical and functional analyses indicate that the key cis sites for maximal tissue-specific expression of PLP in cultured glial cells are clustered near the promoter. Within this cluster are several conserved motifs that may coordinate the regulation of myelin-specific genes.

Animals↗

PubMed1991

Regulation of the human ApoA-II gene by the synergistic action of factors binding to the proximal and distal regulatory elements.

We have recently shown that the human apoA-II promoter contains a set of 11 distal regulatory elements between nucleotides -903 and -255 and three proximal regulatory elements between nucleotides -126 and -33 that are essential for hepatic and intestinal transcription of the apoA-II gene (Chambaz, J., Cardot, P., Pastier, D., Zannis, V. I., and Cladaras, C. (1991) J. Biol. Chem. 266, 11676-11685). Deletion or nucleotide substitution analysis has shown that alterations in elements L (nucleotides -803 to -773) and K (nucleotides -760 to -743) reduced hepatic transcription to 25 and 20% and intestinal transcription to 8 and 4% of control, respectively, as measured by chloramphenicol acetyltransferase assays, indicating that these elements play an important regulatory role. Nucleotide substitutions in element AB (nucleotides -65 to -33) reduced hepatic and intestinal transcription to 60 and 36% of control, respectively. The factors that recognize regulatory regions L, K, and AB were analyzed by DNA binding gel electrophoretic and competition assays. This analysis has shown that elements AB, K, and L bind with different affinities to a newly characterized heat-stable factor, CIIIB1, which is a transcriptional activator of the human apoC-III gene (Ogami, K., Kardassis, D., Cladaras, C., and Zannis, V. I. (1991) J. Biol. Chem. 266, 9640-9646). In addition, elements AB and K bind a heat-labile activity, designated AIIAB1, and element L binds to several CCAAT box binding activities. Mutations in domain L that prevented the binding of CCAAT box binding activities reduced both hepatic and intestinal transcription to 30% of control, indicating the importance of these factors in transcription. Simultaneous nucleotide substitutions that prevented the binding of CIIIB1 activity in elements AB, K, and L reduced hepatic and intestinal transcription to 7 and 6% of control, respectively, suggesting that the synergistic interaction of CIIIB1 (bound to the proximal and distal regulatory elements) with CCAAT box proteins (bound to element L) can modulate the level of transcription of the human apoA-II gene.

Animals↗

PubMed1988

Identification of negative and positive regulatory elements associated with a class I major histocompatibility complex gene.

Regulatory DNA sequence elements were functionally identified in the 5'-flanking region of a gene, PD1, which encodes a porcine classical transplantation antigen. Both a positive regulatory element and a novel negative regulatory DNA element were mapped within 1.1 kilobases upstream of exon 1. The negative regulatory element reduced the activity of both the homologous PD1 promoter and a heterologous simian virus 40 promoter. In vivo competition experiments indicated that the functions of the PD1 positive and negative regulatory elements are mediated by distinct cellular trans-acting factors. The PD1 positive regulatory element interacted with cellular factors in common with those binding to the simian virus 40 enhancer. Finally, the negative regulatory element required the presence of a positive regulatory element to function. This interaction between positive and negative regulatory elements represents a novel mechanism for regulating gene expression.

Amino Acid Sequence↗

PubMed1990

Regulatory elements controlling expression of the Drosophila homeotic gene fork head.

The region-specific homeotic gene fork head (fkh) is expressed and required in a variety of tissues of the developing Drosophila embryo. In order to identify the cis regulatory elements directing the complex spatio-temporal expression pattern of fkh, we have studied the subpatterns directed by defined fragments of fkh genomic DNA. These experiments enabled us to distinguish separate regulatory elements specific for the different expression domains of fkh. In addition, our analysis revealed several unexpected features such as the redundancy of regulatory elements and the overlap of regulatory elements with the transcribed regions of other genes. Moreover, the separation of normally contiguous elements effecting expression in the posterior terminal fkh domain appears to lead to novel expression domains which do not correspond to known developmental units in the embryo.

Alleles↗

PubMed1990

Identification of cis-acting repressive sequences within the negative regulatory element of human immunodeficiency virus type 1.

The negative regulatory element of human immunodeficiency virus type 1 is a 260-nucleotide-long sequence that decreases the rate of RNA transcription initiation specified by the long terminal repeat. This region has the potential to bind several cellular transcription factors. Here it is shown that sequences which recognize the NFAT-1 and USF cellular transcription factors contribute to this negative regulatory effect. The sequences within the negative regulatory element which resemble the AP-1 site and the URS do not negatively regulate human immunodeficiency virus long terminal repeat transcription initiation.

Base Sequence↗

PubMed2026

Genetic Deletion of Cis-Regulatory Elements to Dissect the Function of the Non-coding Genome in human Preimplantation Models.

Cis-regulatory elements coordinate gene expression in a spatially and temporally controlled manner and contribute to the establishment of distinct cellular states during development. A substantial proportion of transcriptionally active cis-regulatory elements in primate embryos originated from ancient retroviral integrations into the germline. These endogenous retroviruses, also known as long terminal repeat retrotransposons, retain intrinsic regulatory activity and are often species-specific, making them strong candidates for regulating species-divergent aspects of embryonic development. Ethical and legal restrictions on human embryo research have historically limited direct investigation of gene regulation during human embryogenesis. Human naive pluripotent stem cells and three-dimensional stem cell-based blastocyst models provide alternative systems for studying early developmental processes. This protocol describes the CRISPR-Cas9-mediated deletion of endogenous retrovirus-derived cis-regulatory elements in human naive pluripotent stem cells. Preassembled Cas9 and single-guide RNA ribonucleoprotein complexes are delivered by nucleofection, followed by single-cell cloning, PCR-based genotyping, Sanger sequencing, expansion, cryopreservation, and genomic stability assessment of the edited lines. The resulting wild-type, heterozygous, and homozygous or hemizygous deletion clones provide a platform for investigating the contribution of individual endogenous retrovirus-derived elements to gene regulation in human preimplantation models. This method enables direct functional interrogation of species-specific non-coding regulatory sequences and supports the study of transcriptional mechanisms involved in early human development.

Humans↗

PubMed1989

Rat ornithine decarboxylase gene. Nucleotide sequence, potential regulatory elements, and comparison to the mouse gene.

Expression of ornithine decarboxylase is regulated by a variety of hormonal and other stimuli in rat cells and tissues. To study this phenomenon at the molecular level, we isolated and sequenced a cDNA-encoding rat ornithine decarboxylase and deduced its amino acid sequence. The cDNA clone was used to isolate a clone from a rat genomic library which contained the sequence of the entire rat ornithine decarboxylase gene. The gene comprised 12 exons and 11 introns and spanned 7.7 kilobases. Two polyadenylation signals (AATAAA) were located 310 and 697 base pairs 3' to the translational termination codon and were responsible for the occurrence of two hybridizing mRNA species in Northern blots of rat cells and tissues. S1 nuclease mapping suggested that there were multiple transcriptional start sites; the major one appeared to be located 2269 base pairs of genomic sequence 5' to the ATG translational initiation site, representing 274 bases of mRNA. Several potential regulatory elements were identified in the 5'-promoter regions or in the first intron: a TATA box, GC boxes, AP-1 and AP-2 binding sites, a cAMP-responsive element, a glucocorticoid regulatory element, and RNA polymerase III promoter sequences. The 5'-noncoding region of the mRNA was extremely rich in G + C; secondary structure predictions suggested that almost this entire region could form stable secondary structures, with an overall free energy of formation (delta G) of -114 kcal/mol. The potential regulatory elements identified in both the promoter region of the gene and the 5'-untranslated region of the mRNA may be involved in the complex regulation of rat ornithine decarboxylase expression.

Amino Acid Sequence↗