Multiple negative elements in a gene that codes for an extracellular matrix protein, collagen X, restrict expression to hypertrophic chondrocytes.

During skeletal development, chondrocytes go through several stages of differentiation. The last stage, chondrocyte hypertrophy, occurs in areas of endochondral ossification. Mature hypertrophic chondrocytes differ from immature chondrocytes in that they become postmitotic, increase their cellular volume up to eightfold, and synthesize a unique set of matrix molecules. One such molecule is a short collagenous protein, collagen X. Previous studies have shown that collagen X is not expressed by other cell types and that its specific expression in hypertrophic chondrocytes is controlled by transcriptional mechanisms. To define these mechanisms, plasmid constructs containing the chicken collagen X gene promoter and 5' flanking regions fused to a reporter gene (chloramphenicol acetyl transferase, CAT) were transfected into primary cultures of collagen X-expressing and nonexpressing cells. A construct containing a short (558 bp) promoter exhibited high levels of CAT activity in all cell types (fibroblasts, immature, and hypertrophic chondrocytes). Adding a 4.2-kb fragment of 5' flanking DNA to this construct resulted in a dramatic reduction of CAT activity in fibroblasts and immature chondrocytes, but had no effect in hypertrophic chondrocytes. Addition of three subfragments of the 4.2-kb fragment to the initial construct, either individually or in various combinations, showed that all subfragments reduced CAT activity somewhat in non-collagen X-expressing cells, and that their effects were additive. Unrelated DNA had no effect on CAT activity. The results suggest that multiple, diffuse upstream negative regulatory elements act in an additive manner to restrict transcription of the collagen X gene to hypertrophic chondrocytes.

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Molecular Structure and Tissue Distribution of Matrilin-3, a Filament-forming Extracellular Matrix Protein Expressed during Skeletal Development

Journal of Biological Chemistry ◽

10.1074/jbc.275.6.3999 ◽

2000 ◽

Vol 275 (6) ◽

pp. 3999-4006 ◽

Cited By ~ 92

Author(s):

Andreas R. Klatt ◽

D. Patric Nitsche ◽

Birgit Kobbe ◽

Matthias Mörgelin ◽

Mats Paulsson ◽

...

Keyword(s):

Molecular Structure ◽

Extracellular Matrix ◽

Tissue Distribution ◽

Matrix Protein ◽

Skeletal Development ◽

Extracellular Matrix Protein

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

Biochemical Journal ◽

10.1042/bj2860179 ◽

1992 ◽

Vol 286 (1) ◽

pp. 179-185 ◽

Cited By ~ 24

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.

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Cdc42 and formin activity control non-muscle myosin dynamics during Drosophila heart morphogenesis

The Journal of Cell Biology ◽

10.1083/jcb.201405075 ◽

2014 ◽

Vol 206 (7) ◽

pp. 909-922 ◽

Cited By ~ 20

Author(s):

Georg Vogler ◽

Jiandong Liu ◽

Timothy W. Iafe ◽

Ede Migh ◽

József Mihály ◽

...

Keyword(s):

Cell Fate ◽

Matrix Protein ◽

Small Gtpase ◽

Extracellular Matrix Protein ◽

Specific Expression ◽

Heart Morphogenesis ◽

Lumen Formation ◽

Spatiotemporal Regulation ◽

Genetic Mechanisms ◽

Heart Formation

During heart formation, a network of transcription factors and signaling pathways guide cardiac cell fate and differentiation, but the genetic mechanisms orchestrating heart assembly and lumen formation remain unclear. Here, we show that the small GTPase Cdc42 is essential for Drosophila melanogaster heart morphogenesis and lumen formation. Cdc42 genetically interacts with the cardiogenic transcription factor tinman; with dDAAM which belongs to the family of actin organizing formins; and with zipper, which encodes nonmuscle myosin II. Zipper is required for heart lumen formation, and its spatiotemporal activity at the prospective luminal surface is controlled by Cdc42. Heart-specific expression of activated Cdc42, or the regulatory formins dDAAM and Diaphanous caused mislocalization of Zipper and induced ectopic heart lumina, as characterized by luminal markers such as the extracellular matrix protein Slit. Placement of Slit at the lumen surface depends on Cdc42 and formin function. Thus, Cdc42 and formins play pivotal roles in heart lumen formation through the spatiotemporal regulation of the actomyosin network.

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

Molecular and Cellular Biology ◽

10.1128/mcb.10.11.5967-5976.1990 ◽

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.

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E-box- and MEF-2-independent muscle-specific expression, positive autoregulation, and cross-activation of the chicken MyoD (CMD1) promoter reveal an indirect regulatory pathway

Molecular and Cellular Biology ◽

10.1128/mcb.14.8.5474-5486.1994 ◽

1994 ◽

Vol 14 (8) ◽

pp. 5474-5486

Author(s):

C A Dechesne ◽

Q Wei ◽

J Eldridge ◽

L Gannoun-Zaki ◽

P Millasseau ◽

...

Keyword(s):

Binding Site ◽

Binding Sites ◽

Primary Cultures ◽

Regulatory Elements ◽

The Other ◽

Indirect Pathway ◽

Specific Expression ◽

Fibroblast Cultures ◽

E Box ◽

Myogenic Factors

Members of the MyoD family of gene-regulatory proteins (MyoD, myogenin, myf5, and MRF4) have all been shown not only to regulate the transcription of numerous muscle-specific genes but also to positively autoregulate and cross activate each other's transcription. In the case of muscle-specific genes, this transcriptional regulation can often be correlated with the presence of a DNA consensus in the regulatory region CANNTG, known as an E box. Little is known about the regulatory interactions of the myogenic factors themselves; however, these interactions are thought to be important for the activation and maintenance of the muscle phenotype. We have identified the minimal region in the chicken MyoD (CMD1) promoter necessary for muscle-specific transcription in primary cultures of embryonic chicken skeletal muscle. The CMD1 promoter is silent in primary chick fibroblast cultures and in muscle cell cultures treated with the thymidine analog bromodeoxyuridine. However, CMD1 and chicken myogenin, as well as, to a lesser degree, chicken Myf5 and MRF4, expressed in trans can activate transcription from the minimal CMD1 promoter in these primary fibroblast cultures. Here we show that the CMD1 promoter contains numerous E-box binding sites for CMD1 and the other myogenic factors, as well as a MEF-2 binding site. Surprisingly, neither muscle-specific and the other myogenic factors, as well as a MEF-2 binding site. Surprisingly, neither muscle-specific expression, autoregulation, or cross activation depends upon the presence of of these E-box or MEF-2 binding sites in the CMD1 promoter. These results demonstrate that the autoregulation and cross activation of the chicken MyoD promoter through the putative direct binding of the myogenic basic helix-loop-helix regulatory factors is mediated through an indirect pathway that involves unidentified regulatory elements and/or ancillary factors.

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Glioblastoma invasion factor ODZ1 is induced by microenvironmental signals through activation of a Stat3-dependent transcriptional pathway

Scientific Reports ◽

10.1038/s41598-021-95753-6 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Veronica Vidal ◽

Olga Gutierrez ◽

Ana Talamillo ◽

Carlos Velasquez ◽

Jose L. Fernandez-Luna

Keyword(s):

Extracellular Matrix ◽

Matrix Protein ◽

Transmembrane Protein ◽

Gene Promoter ◽

Dominant Negative ◽

Surrounding Tissue ◽

Extracellular Matrix Protein ◽

Luciferase Reporter ◽

Dominant Negative Variant ◽

Transcriptional Pathway

AbstractWe have previously shown that the transmembrane protein ODZ1 serves for glioblastoma (GBM) cells to invade the surrounding tissue through activation of RhoA/ROCK pathway. However, the transcriptional machinery used by GBM cells to regulate the expression of ODZ1 is unknown. Here we show that interaction with tumor microenvironment elements, mainly activated monocytes through IL-6 secretion, and the extracellular matrix protein fibronectin, induces the Stat3 transcriptional pathway and upregulates ODZ1 which results in GBM cell migration. This signaling route is abrogated by blocking the IL-6 receptor, inhibiting Jak kinases or knocking down Stat3. Furthermore, we have identified a Stat3 responsive element in the ODZ1 gene promoter, about 1 kb from the transcription start site. Luciferase-reporter assays confirmed that the promoter responds to the presence of monocytic cells and this activation is greatly reduced when the Stat3 site is mutated or following treatment with a neutralizing anti-IL-6 receptor antibody or transfecting GBM cells with a dominant negative variant of Stat3. Overall, we show that monocyte-secreted IL-6 and the extracellular matrix protein fibronectin activate the axis Stat3-ODZ1 and promote migration of GBM cells. This is the first described transcriptional mechanism used by tumor cells to promote the expression of the invasion factor ODZ1.

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Cell-Selective Regulation of CFTR Gene Expression: Relevance to Gene Editing Therapeutics

Genes ◽

10.3390/genes10030235 ◽

2019 ◽

Vol 10 (3) ◽

pp. 235 ◽

Cited By ~ 6

Author(s):

Hannah Swahn ◽

Ann Harris

Keyword(s):

Gene Expression ◽

Cystic Fibrosis ◽

Gene Editing ◽

Genetic Diseases ◽

3D Structure ◽

Three Dimensional ◽

Cell Types ◽

Regulatory Elements ◽

Cftr Gene ◽

Specific Expression

The cystic fibrosis transmembrane conductance regulator (CFTR) gene is an attractive target for gene editing approaches, which may yield novel therapeutic approaches for genetic diseases such as cystic fibrosis (CF). However, for gene editing to be effective, aspects of the three-dimensional (3D) structure and cis-regulatory elements governing the dynamic expression of CFTR need to be considered. In this review, we focus on the higher order chromatin organization required for normal CFTR locus function, together with the complex mechanisms controlling expression of the gene in different cell types impaired by CF pathology. Across all cells, the CFTR locus is organized into an invariant topologically associated domain (TAD) established by the architectural proteins CCCTC-binding factor (CTCF) and cohesin complex. Additional insulator elements within the TAD also recruit these factors. Although the CFTR promoter is required for basal levels of expression, cis-regulatory elements (CREs) in intergenic and intronic regions are crucial for cell-specific and temporal coordination of CFTR transcription. These CREs are recruited to the promoter through chromatin looping mechanisms and enhance cell-type-specific expression. These features of the CFTR locus should be considered when designing gene-editing approaches, since failure to recognize their importance may disrupt gene expression and reduce the efficacy of therapies.

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A combination of closely associated positive and negative cis-acting promoter elements regulates transcription of the skeletal alpha-actin gene.

Molecular and Cellular Biology ◽

10.1128/mcb.10.2.528 ◽

1990 ◽

Vol 10 (2) ◽

pp. 528-538 ◽

Cited By ~ 69

Author(s):

K L Chow ◽

R J Schwartz

Keyword(s):

Transcriptional Activity ◽

Primary Cultures ◽

Gene Promoter ◽

Regulatory Sequence ◽

Actin Gene ◽

Cell Type ◽

Specific Expression ◽

Cis Acting ◽

Cell Type Specific Expression ◽

Alpha Actin

The chicken skeletal alpha-actin gene promoter region provides at least a 75-fold-greater transcriptional activity in muscle cells than in fibroblasts. The cis-acting sequences required for cell type-restricted expression within this 200-base-pair (bp) region were elucidated by chloramphenicol acetyltransferase assays of site-directed Bg/II linker-scanning mutations transiently transfected into primary cultures. Four positive cis-acting elements were identified and are required for efficient transcriptional activity in myogenic cells. These elements, conserved across vertebrate evolution, include the ATAAAA box (-24 bp), paired CCAAT-box-associated repeats (CBARs; at -83 bp and -127 bp), and the upstream T+A-rich regulatory sequence (at -176 bp). Basal transcriptional activity in fibroblasts was not as dependent on the upstream CBAR or regions of the upstream T+A-rich regulatory sequence. Transfection experiments provided evidence that positive regulatory factors required for alpha-actin expression in fibroblasts are limiting. In addition, negative cis-acting elements were detected and found closely associated with the G+C-rich sequences that surround the paired CBARs. Negative elements may have a role in restricting developmentally timed expression in myoblasts and appear to inhibit promoter activity in nonmyogenic cells. Cell type-specific expression of the skeletal alpha-actin gene promoter is regulated by combinatorial and possibly competitive interactions between multiple positive and negative cis-acting elements.

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Adeno-Associated Virus Technologies and Methods for Targeted Neuronal Manipulation

10.1101/759936 ◽

2019 ◽

Author(s):

Leila Haery ◽

Benjamin E. Deverman ◽

Katherine Matho ◽

Ali Cetin ◽

Kenton Woodard ◽

...

Keyword(s):

Viral Vectors ◽

Cell Types ◽

Regulatory Elements ◽

Specific Cell ◽

Specific Expression ◽

Adeno Associated Virus ◽

Cell Type Specific Expression ◽

Cell Type Specific ◽

And Function ◽

Novel Applications

AbstractCell-type-specific expression of molecular tools and sensors is critical to construct circuit diagrams and to investigate the activity and function of neurons within the nervous system. Strategies for targeted manipulation include combinations of classical genetic tools such as Cre/loxP and Flp/FRT, use of cis-regulatory elements, targeted knock-in transgenic mice, and gene delivery by AAV and other viral vectors. The combination of these complex technologies with the goal of precise neuronal targeting is a challenge in the lab. This report will discuss the theoretical and practical aspects of combining current technologies and establish best practices for achieving targeted manipulation of specific cell types. Novel applications and tools, as well as areas for development, will be envisioned and discussed.

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Ksp-cadherin gene promoter. II. Kidney-specific activity in transgenic mice

AJP Renal Physiology ◽

10.1152/ajprenal.1999.277.4.f599 ◽

1999 ◽

Vol 277 (4) ◽

pp. F599-F610 ◽

Cited By ~ 22

Author(s):

Peter Igarashi ◽

Cooduvalli S. Shashikant ◽

R. Brent Thomson ◽

Dilys A. Whyte ◽

Shuxian Liu-Chen ◽

...

Keyword(s):

Epithelial Cells ◽

Transgenic Mice ◽

Collecting Duct ◽

Gene Promoter ◽

Regulatory Elements ◽

Developmental Expression ◽

Tubular Epithelial Cells ◽

Specific Expression ◽

Tissue Specific

Kidney-specific cadherin (Ksp-cadherin, cadherin 16) is a tissue-specific member of the cadherin superfamily that is expressed exclusively in the basolateral membrane of tubular epithelial cells in the kidney. To determine the basis for tissue-specific expression of Ksp-cadherin in vivo, we evaluated the activity of the promoter in transgenic mice. Transgenic mice containing 3.3 kb of the mouse Ksp-cadherin promoter and an Escherichia coli lacZ reporter gene were generated by pronuclear microinjection. Assays of β-galactosidase enzyme activity showed that the transgene was expressed exclusively in the kidney in both adult and developing mice. Within the kidney, the transgene was expressed in a subset of renal tubular epithelial cells that endogenously expressed Ksp-cadherin and that were identified as collecting ducts by colabeling with Dolichos biflorus agglutinin. In the developing metanephros, expression of the transgene in the branching ureteric bud correlated with the developmental expression of Ksp-cadherin. Identical patterns of expression were observed in multiple founder mice, indicating that kidney specificity was independent of transgene integration site. However, heterocellular expression was observed consistent with repeat-induced gene silencing. We conclude that the Ksp-cadherin gene promoter directs kidney-specific expression in vivo. Regulatory elements that are sufficient to recapitulate the tissue- and differentiation-specific expression of Ksp-cadherin in the renal collecting duct are located within 3.3 kb upstream to the transcriptional start site.

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