scholarly journals The transcriptional tissue specificity of the human proα1(I) collagen gene is determined by a negative cis-regulatory element in the promoter

1992 ◽  
Vol 286 (1) ◽  
pp. 179-185 ◽  
Author(s):  
C P Simkevich ◽  
J P Thompson ◽  
H Poppleton ◽  
R Raghow
Keyword(s):  
Tissue Specificity ◽  
Regulatory Element ◽  
Mesenchymal Cell ◽  
Cell Types ◽  
Regulatory Elements ◽  
Negative Influence ◽  
Collagen Gene ◽  
Specific Expression ◽  
Cis Acting ◽  
First Intron

The transcriptional activity of plasmid pCOL-KT, in which human pro alpha 1 (I) collagen gene upstream sequences up to -804 and most of the first intron (+474 to +1440) drive expression of the chloramphenicol acetyltransferase (CAT) gene [Thompson, Simkevich, Holness, Kang & Raghow (1991) J. Biol. Chem. 266, 2549-2556], was tested in a number of mesenchymal and non-mesenchymal cells. We observed that pCOL-KT was readily expressed in fibroblasts of human (IMR-90 and HFL-1), murine (NIH 3T3) and avian (SL-29) origin and in a human rhabdomyosarcoma cell line (A204), but failed to be expressed in human erythroleukaemia (K562) and rat pheochromocytoma (PC12) cells, indicating that the regulatory elements required for appropriate tissue-specific expression of the human pro alpha 1 (I) collagen gene were present in pCOL-KT. To delineate the nature of cis-acting sequences which determine the tissue specificity of pro alpha 1 (I) collagen gene expression, functional consequences of deletions in the promoter and first intron of pCOL-KT were tested in various cell types by transient expression assays. Cis elements in the promoter-proximal and intronic sequences displayed either a positive or a negative influence depending on the cell type. Thus deletion of fragments using EcoRV (nt -625 to -442 deleted), XbaI (-804 to -331) or SstII (+670 to +1440) resulted in 2-10-fold decreased expression in A204 and HFL-1 cells. The negative influences of deletions in the promoter-proximal sequences was apparently considerably relieved by deleting sequences in the first intron, and the constructs containing the EcoRV/SstII or XbaI/SstII double deletions were expressed to a much greater extent than either of the single deletion constructs. In contrast, the XbaI* deletion (nt -804 to -609), either alone or in combination with the intronic deletion, resulted in very high expression in all cells regardless of their collagen phenotype; the XbaI*/(-SstII) construct, which contained the intronic SstII fragment (+670 to +1440) in the reverse orientation, was not expressed in either mesenchymal or nonmesenchymal cells. Based on these results, we conclude that orientation-dependent interactions between negatively acting 5′-upstream sequences and the first intron determine the mesenchymal cell specificity of human pro alpha 1 (I) collagen gene transcription.

scholarly journals Separable cis-regulatory elements that contribute to tissue- and site-specific alpha 2(XI) collagen gene expression in the embryonic mouse cartilage.

1996 ◽  
Vol 134 (6) ◽  
pp. 1573-1582 ◽  
Author(s):  
N Tsumaki ◽  
T Kimura ◽  
Y Matsui ◽  
K Nakata ◽  
T Ochi
Keyword(s):  
Promoter Region ◽  
Regulatory Elements ◽  
Collagen Gene ◽  
Lacz Expression ◽  
Specific Expression ◽  
Site Specific ◽  
Cis Acting ◽  
First Intron ◽  
Vertebral Bodies ◽  
Type Xi Collagen

Type XI collagen is a structural component of the cartilage extracellular matrix and plays an important role in skeletal morphogenesis. As a step toward defining the molecular mechanisms responsible for the regulation of type XI collagen expression, we characterized the promoter region of the mouse alpha 2(XI) collagen gene (Coll1a2). We also generated transgenic mice harboring various fragments of the promoter and the first intron of Coll1a2 linked to the Escherichia coli beta-galactosidase gene to identify the cis-acting elements responsible for tissue- and site-specific expression during development. Cloning and sequence analysis of the 5' flanking region of Coll1a2 showed that the putative 3' end of the retinoid X receptor beta gene was located 742 bp upstream of the Coll1a2 start site. This suggested that the promoter region of Coll1a2 was localized within this 742-bp sequence, which contained multiple consensus regulatory elements. Examination of the transgenic mice revealed that the longest DNA construct (containing the entire promoter and first intron sequences) directed lacZ expression in the notochord as well as in the primordial cartilage throughout the body, with the pattern of expression mimicking that of endogenous Coll1a2 transcripts. On the other hand, deletion of the upstream approximately 290 bp resulted in the elimination of lacZ expression in the primordial cartilage of the carpals, tarsals, and vertebral bodies, whereas lacZ expression in the notochord and in the other primordial cartilage elsewhere was not affected. Deletion of the first intron sequence also resulted in the loss of lacZ expression in the primordial cartilage of the carpals, tarsals, and vertebral bodies, as well as in the notochord. These results demonstrate that the upstream 742-bp and first intron segments of the mouse Coll1a2 gene contain the necessary information to confer high level tissue-specific expression in mouse embryos. In addition, our observations suggest the presence of site-specific cis-acting elements that control Coll11a2 gene expression in different cartilaginous components of the skeleton.

scholarly journals An internal regulatory element controls troponin I gene expression.

1989 ◽  
Vol 9 (4) ◽  
pp. 1397-1405 ◽  
Author(s):  
K E Yutzey ◽  
R L Kline ◽  
S F Konieczny
Keyword(s):  
Transcriptional Activation ◽  
Troponin I ◽  
Protein Gene ◽  
Regulatory Element ◽  
Consensus Sequence ◽  
Regulatory Elements ◽  
Contractile Protein ◽  
Specific Expression ◽  
Cis Acting ◽  
Specific Expression Pattern

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.

An internal regulatory element controls troponin I gene expression

1989 ◽  
Vol 9 (4) ◽  
pp. 1397-1405
Author(s):  
K E Yutzey ◽  
R L Kline ◽  
S F Konieczny
Keyword(s):  
Transcriptional Activation ◽  
Troponin I ◽  
Protein Gene ◽  
Regulatory Element ◽  
Consensus Sequence ◽  
Regulatory Elements ◽  
Contractile Protein ◽  
Specific Expression ◽  
Cis Acting ◽  
Specific Expression Pattern

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.

Cooperativity of sequence elements mediates tissue specificity of the rat insulin II gene

1990 ◽  
Vol 10 (4) ◽  
pp. 1784-1788
Author(s):  
Y P Hwung ◽  
Y Z Gu ◽  
M J Tsai
Keyword(s):  
Beta Cell ◽  
Tissue Specificity ◽  
Promoter Element ◽  
Heterologous Promoter ◽  
Specific Expression ◽  
Tissue Specific ◽  
Cis Acting ◽  
Insulin Promoter ◽  
Sequence Elements ◽  
Flanking Region

The 5'-flanking region of the rat insulin II gene (-448 to +50) is sufficient for tissue-specific expression. To further determine the tissue-specific cis-acting element(s), important sequences defined by linker-scanning mutagenesis were placed upstream of a heterologous promoter and transfected into insulin-producing and -nonproducing cells. Rat insulin promoter element 3 (RIPE3), which spans from -125 to -86, was shown to confer beta-cell-specific expression in either orientation. However, two subregions of RIPE3, RIPE3a and RIPE3b (defined by linker-scanning mutations), displayed only marginal activities. These results suggest that the two subregions cooperate to confer tissue specificity, presumably via their cognate binding factors.

Identification of upstream and intragenic regulatory elements that confer cell-type-restricted and differentiation-specific expression on the muscle creatine kinase gene

1988 ◽  
Vol 8 (7) ◽  
pp. 2896-2909 ◽  
Author(s):  
E A Sternberg ◽  
G Spizz ◽  
W M Perry ◽  
D Vizard ◽  
T Weil ◽  
...  
Keyword(s):  
Transcription Initiation ◽  
Regulatory Element ◽  
Simian Virus 40 ◽  
Muscle Cells ◽  
Regulatory Elements ◽  
Initiation Site ◽  
Simian Virus ◽  
Cell Type ◽  
Specific Expression ◽  
Developing Muscle

Terminal differentiation of skeletal myoblasts is accompanied by induction of a series of tissue-specific gene products, which includes the muscle isoenzyme of creatine kinase (MCK). To begin to define the sequences and signals involved in MCK regulation in developing muscle cells, the mouse MCK gene has been isolated. Sequence analysis of 4,147 bases of DNA surrounding the transcription initiation site revealed several interesting structural features, some of which are common to other muscle-specific genes and to cellular and viral enhancers. To test for sequences required for regulated expression, a region upstream of the MCK gene from -4800 to +1 base pairs, relative to the transcription initiation site, was linked to the coding sequences of the bacterial chloramphenicol acetyltransferase (CAT) gene. Introduction of this MCK-CAT fusion gene into C2 muscle cells resulted in high-level expression of CAT activity in differentiated myotubes and no detectable expression in proliferating undifferentiated myoblasts or in nonmyogenic cell lines. Deletion mutagenesis of sequences between -4800 and the transcription start site showed that the region between -1351 and -1050 was sufficient to confer cell type-specific and developmentally regulated expression on the MCK promoter. This upstream regulatory element functioned independently of position, orientation, or distance from the promoter and therefore exhibited the properties of a classical enhancer. This upstream enhancer also was able to confer muscle-specific regulation on the simian virus 40 promoter, although it exhibited a 3- to 5-fold preference for its own promoter. In contrast to the cell type- and differentiation-specific expression of the upstream enhancer, the MCK promoter was able to function in myoblasts and myotubes and in nonmyogenic cell lines when combined with the simian virus 40 enhancer. An additional positive regulatory element was identified within the first intron of the MCK gene. Like the upstream enhancer, this intragenic element functioned independently of position, orientation, and distance with respect to the MCK promoter and was active in differentiated myotubes but not in myoblasts. These results demonstrate that expression of the MCK gene in developing muscle cells is controlled by complex interactions among multiple upstream and intragenic regulatory elements that are functional only in the appropriate cellular context.

scholarly journals Promoter analysis of the DHCR24 (3β-hydroxysterol Δ24-reductase) gene: characterization of SREBP (sterol-regulatoryelement-binding protein)-mediated activation

2012 ◽  
Vol 33 (1) ◽  
Author(s):  
Lidia A. Daimiel ◽  
María E. Fernández-Suárez ◽  
Sara Rodríguez-Acebes ◽  
Lorena Crespo ◽  
Miguel A. Lasunción ◽  
...  
Keyword(s):  
Cellular Response ◽  
Binding Protein ◽  
Regulatory Element ◽  
Cholesterol Biosynthesis ◽  
Regulatory Elements ◽  
Proximal Promoter ◽  
Luciferase Reporter ◽  
Mobility Shift ◽  
Gene Characterization ◽  
Cis Acting

DHCR24 (3β-hydroxysterol Δ24-reductase) catalyses the reduction of the C-24 double bond of sterol intermediates during cholesterol biosynthesis. DHCR24 has also been involved in cell growth, senescence and cellular response to oncogenic and oxidative stress. Despite its important roles, little is known about the transcriptional mechanisms controlling DHCR24 gene expression. We analysed the proximal promoter region and the cholesterol-mediated regulation of DHCR24. A putative SRE (sterol-regulatory element) at −98/−90 bp of the transcription start site was identified. Other putative regulatory elements commonly found in SREBP (SRE-binding protein)-targeted genes were also identified. Sterol responsiveness was analysed by luciferase reporter assays of approximately 1 kb 5′-flanking region of the human DHCR24 gene in HepG2 and SK-N-MC cells. EMSAs (electrophoretic mobility-shift assays) and ChIP (chromatin immunoprecipitation) assays demonstrated cholesterol-dependent recruitment and binding of SREBPs to the putative SRE. Given the presence of several CACCC-boxes in the DHCR24 proximal promoter, we assessed the role of KLF5 (Krüppel-like factor 5) in androgen-regulated DHCR24 expression. DHT (dihydrotestosterone) increased DHCR24 expression synergistically with lovastatin. However, DHT was unable to activate the DHCR24 proximal promoter, whereas KLF5 did, indicating that this mechanism is not involved in the androgen-induced stimulation of DHCR24 expression. The results of the present study allow the elucidation of the mechanism of regulation of the DHCR24 gene by cholesterol availability and identification of other putative cis-acting elements which may be relevant for the regulation of DHCR24 expression.

Distinct regulation of the interleukin-1 and interleukin-6 response elements of the rat haptoglobin gene in rat and human hepatoma cells

1990 ◽  
Vol 10 (11) ◽  
pp. 5967-5976
Author(s):  
H Baumann ◽  
K K Morella ◽  
G P Jahreis ◽  
S Marinković
Keyword(s):  
Interleukin 1 ◽  
Gene Promoter ◽  
Cell Types ◽  
Regulatory Elements ◽  
Hepatoma Cells ◽  
Expression Vectors ◽  
Human Hepatoma ◽  
Human Hepatoma Cells ◽  
Cis Acting ◽  
Chloramphenicol Acetyltransferase Gene

The transcription rate of the haptoglobin (Hp) gene is stimulated by interleukin-1 (IL-1), IL-6, and dexamethasone in rat hepatoma (H-35) cells. To identify the cis-acting regulatory elements responsive to these hormones, various lengths of 5' Hp gene-flanking regions, including the promoter, were inserted into chloramphenicol acetyltransferase gene expression vectors and transiently introduced into H-35 cells. The first 4 kb of 5' region mediated a severalfold increase in expression after treatment with IL-6 and dexamethasone. No response to IL-1 was detectable. When, however, upstream sequences were deleted to position -165 relative to the transcription start site, a significant stimulation by IL-1 was gained without appreciably affecting the IL-6 response. With the apparent removal of an inhibitory sequence, the promoter-proximal 165-bp region also displayed a severalfold enhanced response to the combination of dexamethasone, IL-1, and IL-6. The sequence from -165 to -147, termed the A-element, was found to be crucial for all hormone regulatory functions. Two copies of the A-element linked to a heterologous promoter responded to the three hormones, but to a lesser degree than in the Hp gene promoter context. The regulatory elements of the rat Hp gene were similarly active in human hepatoma cells. Optimal regulation by IL-6 in HepG2 cells was, however, independent of the A-element. The A-element functioned in these cells exclusively as an IL-1 response sequence. The results suggest that genomic sequences upstream of the rat Hp gene suppress the regulation by specific cytokines more prominently in transient expression assays than in the normal chromosomal context. Moreover, the functional comparison indicated that specific regulatory regions of the rat Hp gene do not function identically in different hepatic cell types.

Expression of the K-fgf proto-oncogene is controlled by 3' regulatory elements which are specific for embryonal carcinoma cells

1990 ◽  
Vol 10 (6) ◽  
pp. 2475-2484
Author(s):  
A M Curatola ◽  
C Basilico
Keyword(s):  
Dna Sequences ◽  
Embryonal Carcinoma ◽  
Protein S ◽  
Cell Types ◽  
Regulatory Elements ◽  
Developmentally Regulated ◽  
Cis Acting ◽  
Dna Elements ◽  
Ec Cells ◽  
Cat Expression

Expression of the K-fgf/hst proto-oncogene appears to be restricted to cells in the early stages of development, such as embryonal carcinoma (EC) cells. When EC cells are induced to differentiate, K-fgf expression is drastically repressed. To identify cis-acting DNA elements responsible for this type of regulation, we constructed a plasmid in which cat gene expression was driven by about 1 kilobase of upstream K-fgf human DNA sequences, including the putative promoter, and transfected it into undifferentiated F9 EC cells or HeLa cells as prototypes of cells which express or do not express, respectively, the K-fgf proto-oncogene. This plasmid was essentially inactive in both cell types, and the addition of more than 8 kilobases of DNA sequences upstream of the K-fgf promoter did not lead to any increase in chloramphenicol acetyltransferase (CAT) expression. On the other hand, when we inserted in this plasmid DNA sequences which are 3' of the human K-fgf coding sequences, we could detect a significant stimulation of CAT activity. Analysis of these sequences led to the identification of enhancerlike DNA elements which are part of the 3' noncoding region of K-fgf exon 3 and promote CAT expression only in undifferentiated mouse F9 or human NT2/D1 EC cells, but not in HeLa, 3T3, or differentiated F9 cells, therefore mimicking the physiological expression of the K-fgf proto-oncogene. Similar elements are also present in the 3' region of the murine K-fgf proto-oncogene, in a region showing high homology to the human K-fgf sequences. These regulatory elements can promote CAT expression from heterologous promoters in an EC-specific manner, suggesting that they interact with a specific cellular transacting protein(s) whose expression is developmentally regulated.

Developmental expression of the maternal protein XDCoH, the dimerization cofactor of the homeoprotein LFB1 (HNF1)

Development ◽  
1995 ◽  
Vol 121 (4) ◽  
pp. 1217-1226
Author(s):  
E. Pogge yon Strandmann ◽  
G.U. Ryffel
Keyword(s):  
Transcription Factors ◽  
Anterior Part ◽  
Regulatory Element ◽  
Cdna Probe ◽  
Cell Types ◽  
Developmental Expression ◽  
Variant Form ◽  
Cis Acting ◽  
Liver And Kidney

The tissue-specific transcription factors LFB1 (HNF1) and LFB3 (vHNF1) mainly expressed in liver, kidney and intestine are homeoproteins that interact with the regulatory element HP1. The HP1 sequence constitutes one of the most important cis-acting elements in liver-specifically expressed genes, while its function in other cell types containing LFB1 and LFB3 is not fully understood. In mammals, LFB1 activity is modulated by DCoH, a cofactor that stimulates the LFB1 transactivation significantly. Using the rat cDNA probe, we cloned the corresponding Xenopus sequence XDCoH, encoding a 104 amino acid protein, that is 85% identical to the rat protein. XDCoH enhances the LFB1-dependent transactivation potential in transfection experiments and interacts in vitro directly with LFB1 and its variant form LFB3. The protein is detectable in liver and kidney extracts of adult frogs and in small amounts also in lung and stomach, organs expressing LFB1 and/or LFB3 protein as well. To investigate the possible involvement of XDCoH in Xenopus development, we analyzed its temporal and spatial expression pattern during early embryogenesis. XDCoH is a maternal factor, although LFB1 is absent in the egg. In early cleavage stages, the protein is detectable in the cytoplasm of each blastomere and enters the nuclei of the cells as early as the zygotic transcription in the Xenopus embryo starts. The amount of XDCoH increases dramatically following neurulation, when the formation of liver, pronephros and other organs takes place. Whole-mount immunostaining demonstrates that, in the developing larvae, XDCoH is localized in the nuclei of the hepatocytes, the gut cells and the pronephric cells, tissues of mesodermal and endodermal origin known to contain LFB1 and LFB3. Surprisingly it is also present in the pigmented epithelium surrounding the eye of the embryo, which is derived from the anterior part of the ectodermal neural plates and lacks LFB1. The tissue distribution of XDCoH during embryogenesis suggests that XDCoH is involved in determination and differentiation of various unrelated cell types. It seems likely that XDCoH interaction is not only essential for the function of LFB1 and LFB3 but also for certain other transcription factors.

scholarly journals DNA Methylation Contributes to the Differential Expression Levels of Mecp2 in Male Mice Neurons and Astrocytes

2019 ◽  
Vol 20 (8) ◽  
pp. 1845 ◽  
Author(s):  
Vichithra R.B. Liyanage ◽  
Carl O. Olson ◽  
Robby M. Zachariah ◽  
James R. Davie ◽  
Mojgan Rastegar
Keyword(s):  
Dna Methylation ◽  
Current Knowledge ◽  
Regulatory Element ◽  
Regulatory Elements ◽  
Autism Spectrum ◽  
Brain Cells ◽  
Specific Expression ◽  
Expression Levels ◽  
Mecp2 Duplication Syndrome ◽  
Cell Type Specific Expression

Methyl CpG binding protein-2 (MeCP2) isoforms (E1 and E2) are important epigenetic regulators in brain cells. Accordingly, MeCP2 loss- or gain-of-function mutation causes neurodevelopmental disorders, including Rett syndrome (RTT), MECP2 duplication syndrome (MDS), and autism spectrum disorders (ASD). Within different types of brain cells, highest MeCP2 levels are detected in neurons and the lowest in astrocytes. However, our current knowledge of Mecp2/MeCP2 regulatory mechanisms remains largely elusive. It appears that there is a sex-dependent effect in X-linked MeCP2-associated disorders, as RTT primarily affects females, whereas MDS is found almost exclusively in males. This suggests that Mecp2 expression levels in brain cells might be sex-dependent. Here, we investigated the sex- and cell type-specific expression of Mecp2 isoforms in male and female primary neurons and astrocytes isolated from the murine forebrain. Previously, we reported that DNA methylation of six Mecp2 regulatory elements correlated with Mecp2 levels in the brain. We now show that in male brain cells, DNA methylation is significantly correlated with the transcript expression of these two isoforms. We show that both Mecp2 isoforms are highly expressed in male neurons compared to male astrocytes, with Mecp2e1 expressed at higher levels than Mecp2e2. Our data indicate that higher DNA methylation at the Mecp2 regulatory element(s) is associated with lower levels of Mecp2 isoforms in male astrocytes compared to male neurons.

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