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 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 H19 and Igf2 monoallelic expression is regulated in two distinct ways by a shared cis acting regulatory region upstream of H19

2000 ◽  
Vol 14 (10) ◽  
pp. 1186-1195 ◽  
Author(s):  
Madhulika Srivastava ◽  
Sandra Hsieh ◽  
Alexander Grinberg ◽  
Lisa Williams-Simons ◽  
Sing-Ping Huang ◽  
...  
Keyword(s):  
Promoter Region ◽  
Regulatory Region ◽  
Regulatory Elements ◽  
Upstream Region ◽  
Monoallelic Expression ◽  
Direct Role ◽  
Specific Expression ◽  
Cis Acting ◽  
Allele Specific ◽  
Mammalian System

H19 and Igf2 are expressed in a monoallelic fashion from the maternal and paternal chromosomes, respectively. A region upstream of H19 has been shown to regulate such imprinted expression of both genes in cis. We have taken advantage of aloxP/cre recombinase-based strategy to delete this region in mice in a conditional manner to determine the temporal requirement of the upstream region in initiating and maintaining the imprinted expression of H19 and Igf2. Analysis of allele-specific expression of H19 and Igf2 and DNA methylation at the H19 promoter demonstrates that this region controls the monoallelic expression of the two genes in different ways, suggesting that it harbors two functionally distinct regulatory elements. Continued presence of the region is required to silence maternal Igf2 in accordance with its proposed role as an insulator. However, it does not have a direct role in keeping the paternal H19 promoter silenced. Instead, on the paternal chromosome, the upstream element mediates epigenetic modifications of the H19 promoter region during development, leading to transcriptional silencing of H19. Thereafter, its presence is redundant for preventing transcription. Presently, this temporal requirement of the silencing element appears to be a unique cisactivity in the mammalian system. However, it is likely that othercis-acting elements, positive and negative, have the ability to effect stable changes in the chromatin structure and are not constantly required to give signals to the transcriptional machinery.

Factors Involved in the Regulation of Type I Collagen Gene Expression: Implication in Fibrosis

2002 ◽  
Vol 227 (5) ◽  
pp. 301-314 ◽  
Author(s):  
Asish K. Ghosh
Keyword(s):  
Gene Expression ◽  
Collagen Synthesis ◽  
Molecular Mechanisms ◽  
Type I Collagen ◽  
Regulatory Elements ◽  
Transcriptional Level ◽  
Type I ◽  
Collagen Gene ◽  
Cis Acting ◽  
Collagen Gene Expression

Type I collagen, the major component of extracellular matrix in skin and other tissues, is a heterotrimer of two α1 and one α2 collagen polypeptides. The synthesis of both chains is highly regulated by different cytokines at the transcriptional level. Excessive synthesis and deposition of collagen in the dermal region causes thick and hard skin, a clinical manifestation of scleroderma. To better understand the causes of scleroderma or other tissue fibrosis, it is very Important to investigate the molecular mechanisms that cause upregulation of the Type I collagen synthesis in these tissues. Several cis-acting regulatory elements and trans-acting protein factors, which are involved in basal as well as cytokine-modulated Type I collagen gene expression, have been identified and characterized. Hypertranscription of Type I collagen in scleroderma skin fibroblasts may be due to abnormal activities of different positive or negative transcription factors In response to different abnormally induced signaling pathways. In this review, I discuss the present day understanding about the involvement of different factors in the regulation of basal as well as cytokine-modulated Type I collagen gene expression and its implication in scleroderma research.

scholarly journals Regulation of the human cardiac/slow-twitch troponin C gene by multiple, cooperative, cell-type-specific, and MyoD-responsive elements.

1993 ◽  
Vol 13 (11) ◽  
pp. 6752-6765 ◽  
Author(s):  
T H Christensen ◽  
H Prentice ◽  
R Gahlmann ◽  
L Kedes
Keyword(s):  
Skeletal Muscle ◽  
Heart Muscle ◽  
Regulatory Elements ◽  
Troponin C ◽  
Cell Type ◽  
Promoter Elements ◽  
Specific Expression ◽  
First Intron ◽  
Cell Type Specific ◽  
Slow Twitch

The cardiac troponin C (cTnC) gene produces identical transcripts in slow-twitch skeletal muscle and in heart muscle (R. Gahlmann, R. Wade, P. Gunning, and L. Kedes, J. Mol. Biol. 201:379-391, 1988). A separate gene encodes the fast-twitch skeletal muscle troponin C and is not expressed in heart muscle. We have used transient transfection to characterize the regulatory elements responsible for skeletal and cardiac cell-type-specific expression of the human cTnC (HcTnC) gene. At least four separate elements cooperate to confer tissue-specific expression of this gene in differentiated myotubes; a basal promoter (between -61 and -13) augments transcription 9-fold, upstream major regulatory sequences (between -68 and -142 and between -1319 and -4500) augment transcription as much as 39-fold, and at least two enhancer-like elements in the first intron (between +58 and +1028 and between +1029 and +1523) independently augment transcription 4- to 5-fold. These enhancers in the first intron increase myotube-specific chloramphenicol acetyltransferase activity when linked to their own promoter elements or to the heterologous simian virus 40 promoter, and the effects are multiplicative rather than additive. Each of the major myotube regulatory regions is capable of responding directly or indirectly to the myogenic determination factor, MyoD.A MyoD expression vector in 10T1/2 cells induced constructs carrying either the upstream HcTnC promoter elements or the first intron of the gene 300- to 500-fold. Expression was inhibited by cotransfection with Id, a negative regulator of basic helix-loop-helix transcription factors. The basal promoter contains five tandem TGGGC repeats that interact with Sp1 or an Sp1-like factor in nuclear extracts. Mutational analysis of this element demonstrated that two of the five repeat sequences were sufficient to support basal level muscle cell-specific transcription. Whereas the basal promoter is also critical for expression in cardiac myocytes, the elements upstream of -67 appear to play little or no role. Major augmentation of expression in cardiomyocytes is also provided by sequences in the first intron, but these are upstream (between +58 and +1028). The downstream segment of the first intron has no enhancer activity in cardiomyocytes. A specific DNA-protein complex is formed by this C2 cell enhancer with extracts from C2 cells but not cardiomyocytes. These observations suggest that tissue-specific expression of the HcTnC gene is cooperatively regulated by the complex interactions of multiple regulatory elements and that different elements are used to regulate expression in myogenic and cardiac cells.

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.

scholarly journals Site-specific methylation of the rat prolactin and growth hormone promoters correlates with gene expression.

1996 ◽  
Vol 16 (7) ◽  
pp. 3245-3254 ◽  
Author(s):  
V Ngô ◽  
D Gourdji ◽  
J N Laverrière
Keyword(s):  
Gene Expression ◽  
Growth Hormone ◽  
Cell Lines ◽  
Anterior Pituitary ◽  
Regulatory Elements ◽  
Specific Expression ◽  
Site Specific ◽  
Cpg Dinucleotides ◽  
Cpg Sites

The methylation patterns of the rat prolactin (rPRL) (positions -440 to -20) and growth hormone (rGH) (positions -360 to -110) promoters were analyzed by bisulfite genomic sequencing. Two normal tissues, the anterior pituitary and the liver, and three rat pituitary GH3 cell lines that differ considerably in their abilities to express both genes were tested. High levels of rPRL gene expression were correlated with hypomethylation of the CpG dinucleotides located at positions -277 and -97, near or within positive cis-acting regulatory elements. For the nine CpG sites analyzed in the rGH promoter, an overall hypomethylation-expression coupling was also observed for the anterior pituitary, the liver, and two of the cell lines. The effect of DNA methylation was tested by measuring the transient expression of the chloramphenicol acetyltransferase reporter gene driven by a regionally methylated rPRL promoter. CpG methylation resulted in a decrease in the activity of the rPRL promoter which was proportional to the number of modified CpG sites. The extent of the inhibition was also found to be dependent on the position of methylated sites. Taken together, these data suggest that site-specific methylation may modulate the action of transcription factors that dictate the tissue-specific expression of the rPRL and rGH genes in vivo.

Promoter assay using transgenic mice reveals the presence of cis-acting elements governing tissue- and site-specific expression of Col11a2

1996 ◽  
Vol 15 (3) ◽  
pp. 194-195
Author(s):  
N. Tsumaki ◽  
M. Sugimoto ◽  
Y. Matsui ◽  
K. Nakata ◽  
T. Ochi ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Specific Expression ◽  
Promoter Assay ◽  
Site Specific ◽  
Cis Acting

Regulation of the human cardiac/slow-twitch troponin C gene by multiple, cooperative, cell-type-specific, and MyoD-responsive elements

1993 ◽  
Vol 13 (11) ◽  
pp. 6752-6765
Author(s):  
T H Christensen ◽  
H Prentice ◽  
R Gahlmann ◽  
L Kedes
Keyword(s):  
Skeletal Muscle ◽  
Heart Muscle ◽  
Regulatory Elements ◽  
Troponin C ◽  
Cell Type ◽  
Promoter Elements ◽  
Specific Expression ◽  
First Intron ◽  
Cell Type Specific ◽  
Slow Twitch

The cardiac troponin C (cTnC) gene produces identical transcripts in slow-twitch skeletal muscle and in heart muscle (R. Gahlmann, R. Wade, P. Gunning, and L. Kedes, J. Mol. Biol. 201:379-391, 1988). A separate gene encodes the fast-twitch skeletal muscle troponin C and is not expressed in heart muscle. We have used transient transfection to characterize the regulatory elements responsible for skeletal and cardiac cell-type-specific expression of the human cTnC (HcTnC) gene. At least four separate elements cooperate to confer tissue-specific expression of this gene in differentiated myotubes; a basal promoter (between -61 and -13) augments transcription 9-fold, upstream major regulatory sequences (between -68 and -142 and between -1319 and -4500) augment transcription as much as 39-fold, and at least two enhancer-like elements in the first intron (between +58 and +1028 and between +1029 and +1523) independently augment transcription 4- to 5-fold. These enhancers in the first intron increase myotube-specific chloramphenicol acetyltransferase activity when linked to their own promoter elements or to the heterologous simian virus 40 promoter, and the effects are multiplicative rather than additive. Each of the major myotube regulatory regions is capable of responding directly or indirectly to the myogenic determination factor, MyoD.A MyoD expression vector in 10T1/2 cells induced constructs carrying either the upstream HcTnC promoter elements or the first intron of the gene 300- to 500-fold. Expression was inhibited by cotransfection with Id, a negative regulator of basic helix-loop-helix transcription factors. The basal promoter contains five tandem TGGGC repeats that interact with Sp1 or an Sp1-like factor in nuclear extracts. Mutational analysis of this element demonstrated that two of the five repeat sequences were sufficient to support basal level muscle cell-specific transcription. Whereas the basal promoter is also critical for expression in cardiac myocytes, the elements upstream of -67 appear to play little or no role. Major augmentation of expression in cardiomyocytes is also provided by sequences in the first intron, but these are upstream (between +58 and +1028). The downstream segment of the first intron has no enhancer activity in cardiomyocytes. A specific DNA-protein complex is formed by this C2 cell enhancer with extracts from C2 cells but not cardiomyocytes. These observations suggest that tissue-specific expression of the HcTnC gene is cooperatively regulated by the complex interactions of multiple regulatory elements and that different elements are used to regulate expression in myogenic and cardiac cells.

Tissue-specific and hormonally regulated expression of a rat alpha 2u globulin gene in transgenic mice

1987 ◽  
Vol 7 (10) ◽  
pp. 3749-3758
Author(s):  
V da C Soares ◽  
R M Gubits ◽  
P Feigelson ◽  
F Costantini
Keyword(s):  
Transgenic Mice ◽  
Gene Family ◽  
Hormonal Regulation ◽  
Germ Line ◽  
Regulatory Elements ◽  
Preputial Gland ◽  
Specific Expression ◽  
Tissue Specific ◽  
Cis Acting ◽  
Globulin Gene

To investigate the tissue-specific and hormonal regulation of the rat alpha 2u globulin gene family, we introduced one cloned member of the gene family into the mouse germ line and studied its expression in the resulting transgenic mice. Alpha 2u globulingene 207 was microinjected on a 7-kilobase DNA fragment, and four transgenic lines were analyzed. The transgene was expressed at very high levels, specifically in the liver and the preputial gland of adult male mice. The expression in male liver was first detected at puberty, and no expression was detected in female transgenic mice. This pattern of expression is similar to the expression of endogenous alpha 2u globulin genes in the rat but differs from the expression of the homologous mouse major urinary protein (MUP) gene family in that MUPs are synthesized in female liver and not in the male preputial gland. We conclude that these differences between rat alpha 2u globulin and mouse MUP gene expression are due to evolutionary differences in cis-acting regulatory elements. The expression of the alpha 2u globulin transgene in the liver was abolished by castration and fully restored after testosterone replacement. The expression could also be induced in the livers of female mice by treatment with either testosterone or dexamethasone, following ovariectomy and adrenalectomy. Therefore, the cis-acting elements responsible for regulation by these two hormones, as well as those responsible for tissue-specific expression, are closely linked to the alpha 2u globulin gene.

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