scholarly journals Control of differentiation-induced calbindin-D9k gene expression in Caco-2 cells by cdx-2 and HNF-1α

2004 ◽  
Vol 287 (5) ◽  
pp. G943-G953 ◽  
Author(s):  
Liyong Wang ◽  
Anna Klopot ◽  
Jean-Noel Freund ◽  
Lauren N. Dowling ◽  
Stephen D. Krasinski ◽  
...  
Keyword(s):  
Gene Expression ◽  
Calcium Absorption ◽  
Hepatocyte Nuclear Factor ◽  
Promoter Sequences ◽  
Physiological Importance ◽  
Calbindin D9k ◽  
Enhancer Binding Protein ◽  
Promoter Construct ◽  
Calbindin D

Calbindin D9k (CaBP) is critical for intestinal calcium absorption; its in vivo expression is restricted to differentiated enterocytes of the small intestine. Our goal was to identify factors controlling the transcriptional regulation of this gene in the human intestine. Both the natural gene and a 4600-bp promoter construct were strongly regulated by differentiation (>100-fold) but not by treatment with 1,25(OH)2 vitamin D (<2-fold) in the Caco-2 clone TC7. Deletion-mutation studies revealed that conserved promoter sequences for cdx-2 (at −3158 bp) and hepatocyte nuclear factor (HNF)-1 (at −3131 and at −98 bp) combined to control CaBP expression during differentiation. Other putative response elements were not important for CaBP regulation in TC7 cells (CCAAT enhancer binding protein, pancreatic duodenal homebox-1 (pdx-1), a proximal cdx-2 element). Mutation of the distal HNF-1 site had the greatest impact on CaBP gene expression through disruption of HNF-1α binding; both basal and differentiation-mediated CaBP expression was reduced by 80%. In contrast, mutation of the distal cdx-2 element reduced only basal CaBP expression. Whereas a 60% reduction of CaBP mRNA in the duodenum of HNF-1α null mice confirmed the physiological importance of HNF-1α for CaBP gene regulation, additional studies showed that maximal CaBP expression requires the presence of both HNF-1α and cdx-2. Our data suggest that cdx-2 is a permissive factor that influences basal CaBP expression in enterocytes and that HNF-1α modulates CaBP gene expression during cellular differentiation.

In vivo regulation of β-MHC gene in rodent heart: role of T3 and evidence for an upstream enhancer

1999 ◽  
Vol 276 (4) ◽  
pp. C883-C891 ◽  
Author(s):  
Carola E. Wright ◽  
F. Haddad ◽  
A. X. Qin ◽  
P. W. Bodell ◽  
K. M. Baldwin
Keyword(s):  
Gene Expression ◽  
Thyroid Hormone ◽  
Reporter Gene ◽  
Normal Control ◽  
Transcriptional Activity ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Promoter Construct ◽  
Hormone Responsiveness

Cardiac β-myosin heavy chain (β-MHC) gene expression is mainly regulated through transcriptional processes. Although these results are based primarily on in vitro cell culture models, relatively little information is available concerning the interaction of key regulatory factors thought to modulate MHC expression in the intact rodent heart. Using a direct gene transfer approach, we studied the in vivo transcriptional activity of different-length β-MHC promoter fragments in normal control and in altered thyroid states. The test β-MHC promoter was fused to a firefly luciferase reporter gene, whereas the control α-MHC promoter was fused to the Renilla luciferase reporter gene and was used to account for variations in transfection efficiency. Absolute reporter gene activities showed that β- and α-MHC genes were individually and reciprocally regulated by thyroid hormone. The β-to-α ratios of reporter gene expression demonstrated an almost threefold larger β-MHC gene expression in the longest than in the shorter promoter fragments in normal control animals, implying the existence of an upstream enhancer. A mutation in the putative thyroid response element of the −408-bp β-MHC promoter construct caused transcriptional activity to drop to null. When studied in the −3,500-bp β-MHC promoter, construct activity was reduced (∼100-fold) while thyroid hormone responsiveness was retained. These findings suggest that, even though the bulk of the thyroid hormone responsiveness of the gene is contained within the first 215 bp of the β-MHC promoter sequence, the exact mechanism of triiodothyronine (T3) action remains to be elucidated.

scholarly journals Stable integration of an optimized inducible promoter system enables spatiotemporal control of gene expression throughout avian development

Biology Open ◽  
2020 ◽  
Vol 9 (10) ◽  
pp. bio055343 ◽  
Author(s):  
Daniel Chu ◽  
An Nguyen ◽  
Spenser S. Smith ◽  
Zuzana Vavrušová ◽  
Richard A. Schneider
Keyword(s):  
Gene Expression ◽  
Embryonic Development ◽  
Inducible Promoter ◽  
In Ovo ◽  
Control Of Gene Expression ◽  
Promoter Construct ◽  
Avian Development ◽  
Spatiotemporal Control ◽  
Promoter System

ABSTRACTPrecisely altering gene expression is critical for understanding molecular processes of embryogenesis. Although some tools exist for transgene misexpression in developing chick embryos, we have refined and advanced them by simplifying and optimizing constructs for spatiotemporal control. To maintain expression over the entire course of embryonic development we use an enhanced piggyBac transposon system that efficiently integrates sequences into the host genome. We also incorporate a DNA targeting sequence to direct plasmid translocation into the nucleus and a D4Z4 insulator sequence to prevent epigenetic silencing. We designed these constructs to minimize their size and maximize cellular uptake, and to simplify usage by placing all of the integrating sequences on a single plasmid. Following electroporation of stage HH8.5 embryos, our tetracycline-inducible promoter construct produces robust transgene expression in the presence of doxycycline at any point during embryonic development in ovo or in culture. Moreover, expression levels can be modulated by titrating doxycycline concentrations and spatial control can be achieved using beads or gels. Thus, we have generated a novel, sensitive, tunable, and stable inducible-promoter system for high-resolution gene manipulation in vivo.

255 THE ESTROGENIC EVALUATION OF PARABENS USING RAT UTERINE CALBINDIN-D9k

2010 ◽  
Vol 22 (1) ◽  
pp. 285
Author(s):  
T. T. B. Vo ◽  
E. B. Jeung
Keyword(s):  
Gene Expression ◽  
Adverse Effects ◽  
Treated Group ◽  
Environmental Chemicals ◽  
Female Rats ◽  
Protein Levels ◽  
Calbindin D9k ◽  
The Difference

In the current study, calbindin-D9k (CaBP-9k), a potent biomarker for screening estrogen-like environmental chemicals in vivo and in vitro, was adopted to examine the potential estrogen-like property of the following parabens: propyl-, isopropyl-, butyl-, and isobutyl-paraben. Immature female rats were administered for 3 days from postnatal day 14 to 16 with 17?-ethinylestradiol (EE, 1 mg/kg of body weight (BW) per day) or parabens (62.5, 250, and 1000 mg/kg of BW per day). In uterotrophic assays, significantly increased uterus weights were detected in the EE-treated group and in the groups treated with the greatest dose of isopropyl-, butyl- and isobutyl-paraben. In addition, these parabens induced uterine CaBP-9k mRNA and protein levels, whereas co-treatment of parabens and fulvestrant (Faslodex, formerly known as ICI 182, 780), a pure estrogen receptor (ER) antagonist, completely reversed the paraben-induced gene expression and increased uterine weights. To investigate the ER-mediated mechanism(s) by which parabens exert their effects, the expression level of ERα and progesterone receptor (PR) was analyzed. Exposure to EE or parabens caused a dramatic decrease in expression of both ER? mRNA and protein levels, whereas co-treatment with fulvestrant reversed these effects. These data showed the difference of CaBP-9k and ER? expression, suggesting that CaBP-9k might not express via ER? pathway. In the effect of parabens on CaBP-9k expression through PR mediation, a significantly increased expression of uterine PR gene, a well-known ER regulating gene, at both transcriptional and translational levels was indicated in the greatest dose of isopropyl- and butyl-paraben. These parabens induced PR gene expression that was completely blocked by fulvestrant. This result indicates that CaBP-9k expression might involve PR mediates in the estrogenic effect of paraben in immature rat uteri. Taken together, parabens exhibited an estrogen-like property in vivo, which might be mediated by a PR and/or ER? signaling pathway. In addition, our results expanded the current understanding of the potential adverse effects of parabens associated with their estrogen-like activities. Further investigation is needed to elucidate in greater detail the adverse effects of parabens in humans and wildlife.

scholarly journals Factors involved in the duodenal expression of the human calbindin-D9k gene

1999 ◽  
Vol 341 (3) ◽  
pp. 491-500 ◽  
Author(s):  
Natalie F. BARLEY ◽  
S. Radhika PRATHALINGAM ◽  
Pang ZHI ◽  
Stephen LEGON ◽  
Alison HOWARD ◽  
...  
Keyword(s):  
Vitamin D ◽  
Transcription Factors ◽  
Regional Differences ◽  
Calcium Absorption ◽  
Receptor Protein ◽  
Distal Intestine ◽  
Calbindin D9k ◽  
Enhancer Binding Protein ◽  
Potential Binding ◽  
Mcf 7

Calbindin-D9k is expressed in the cytoplasm of intestinal cells, where it is critical for dietary calcium absorption. Two striking aspects of the expression of this gene are its vitamin-D dependency and regional differences in expression, with high levels only in duodenum. We report studies of the human calbindin-D9k promoter. Differences between the reported sequences of the human calbindin-D9k promoter were first clarified before undertaking a functional analysis of this sequence. Studies of the rat gene have indicated that several transcription factors, including the caudal-related homeobox factor (CDX-2), hepatic nuclear factor-4 and CCAAT-enhancer-binding protein α (C/EBPα), could interact with elements in the promoter. Although these elements are conserved in the human gene, we show here that their intestinal distribution makes them unlikely to be critical positive factors. The calbindin-D9k gene contains multiple potential binding sites for homeobox transcription factors; one of these, known as IPF-1 or PDX-1, co-localizes in the intestine with calbindin-D9k. We show in gel-shift assays that the sequence within a putative vitamin-D-response element in the human calbindin-D9k promoter can bind expressed IPF-1/PDX-1 protein, although we cannot confirm binding of the vitamin-D-receptor protein. CDX-2 binds to the region around the TATA box, as in the rat gene, and may act as a negative factor in the distal intestine. Transfection studies in Caco-2 and MCF-7 cells with heterologous reporter vectors containing up to 1303 bp of the gene showed that this functioned as a weak promoter and indicated the presence of suppressor sequences, but did not show vitamin-D responsiveness. This indicates that other elements are also needed for the control of human calbindin-D9k expression.

scholarly journals TonEBP regulates the hyperosmotic expression of aquaporin 1 and 5 in the intervertebral disc

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
J. W. Snuggs ◽  
S. Tessier ◽  
R. A. B. Bunning ◽  
I. M. Shapiro ◽  
M. V. Risbud ◽  
...  
Keyword(s):  
Gene Expression ◽  
Intervertebral Disc ◽  
Nucleus Pulposus ◽  
Central Region ◽  
Aquaporin 1 ◽  
Enhancer Binding Protein ◽  
Ivd Degeneration ◽  
Hyperosmotic Environment

AbstractThe central region of the intervertebral disc (IVD) is rich in proteoglycans, leading to a hyperosmotic environment, which fluctuates with daily loading. The cells of the nucleus pulposus (NP cells) have adapted to this environment via the function of tonicity enhancer binding protein (TonEBP), and NP cells have been shown to express several water channels known as aquaporins (AQP). We have previously shown that AQP1 and 5 decrease during IVD degeneration. Here, the regulation of AQP1 and 5 by hyperosmotic conditions and the role of TonEBP in this regulation was investigated. AQP1 and 5 gene expression was upregulated by hyperosmotic conditions mimicking the osmolality of the healthy IVD, which was abrogated by TonEBP knockdown. Furthermore, AQP1 and 5 immunopositivity was significantly reduced in TonEBPΔ/Δ E17.5 mice when compared with wildtype controls, indicating in vivo expression of AQP1 and 5 is controlled at least in part by TonEBP. This hyperosmotic regulation of AQP1 and 5 could help to explain the decreased AQP1 and 5 expression during degeneration, when the osmolality of the NP decreases. Together this data suggests that TonEBP-regulated osmo-adaptation may be disrupted during IVD degeneration when the expression of both AQPs is reduced.

scholarly journals Normative models of enhancer function

2020 ◽  
Author(s):  
Rok Grah ◽  
Benjamin Zoller ◽  
Gašper Tkačik
Keyword(s):  
Gene Expression ◽  
High Specificity ◽  
Equilibrium Models ◽  
Gene Expression Noise ◽  
Promoter Sequences ◽  
Physical Measurements ◽  
Enhancer Function ◽  
Non Equilibrium

In prokaryotes, thermodynamic models of gene regulation provide a highly quantitative mapping from promoter sequences to gene expression levels that is compatible with in vivo and in vitro bio-physical measurements. Such concordance has not been achieved for models of enhancer function in eukaryotes. In equilibrium models, it is difficult to reconcile the reported short transcription factor (TF) residence times on the DNA with the high specificity of regulation. In non-equilibrium models, progress is difficult due to an explosion in the number of parameters. Here, we navigate this complexity by looking for minimal non-equilibrium enhancer models that yield desired regulatory phenotypes: low TF residence time, high specificity and tunable cooperativity. We find that a single extra parameter, interpretable as the “linking rate” by which bound TFs interact with Mediator components, enables our models to escape equilibrium bounds and access optimal regulatory phenotypes, while remaining consistent with the reported phenomenology and simple enough to be inferred from upcoming experiments. We further find that high specificity in non-equilibrium models is in a tradeoff with gene expression noise, predicting bursty dynamics — an experimentally-observed hallmark of eukaryotic transcription. By drastically reducing the vast parameter space to a much smaller subspace that optimally realizes biological function prior to inference from data, our normative approach holds promise for mathematical models in systems biology.

scholarly journals Quadruplex structures of muscle gene promoter sequences enhance in vivo MyoD-dependent gene expression

2010 ◽  
Vol 38 (7) ◽  
pp. 2369-2377 ◽  
Author(s):  
Jeny Shklover ◽  
Pnina Weisman-Shomer ◽  
Anat Yafe ◽  
Michael Fry
Keyword(s):  
Gene Expression ◽  
Gene Promoter ◽  
Promoter Sequences ◽  
Muscle Gene

Selective stimulation of G-6-Pase catalytic subunit but not G-6-Ptransporter gene expression by glucagon in vivo and cAMP in situ

2004 ◽  
Vol 286 (5) ◽  
pp. E795-E808 ◽  
Author(s):  
Lauri A. Hornbuckle ◽  
Carrie A. Everett ◽  
Cyrus C. Martin ◽  
Stephanie S. Gustavson ◽  
Christina A. Svitek ◽  
...  
Keyword(s):  
Gene Expression ◽  
Hepg2 Cells ◽  
Binding Protein ◽  
Catalytic Subunit ◽  
Conscious Dogs ◽  
Transporter Gene ◽  
Camp Response Element ◽  
Subunit Mrna ◽  
Enhancer Binding Protein

We recently compared the regulation of glucose-6-phosphatase (G-6-Pase) catalytic subunit and glucose 6-phosphate (G-6- P) transporter gene expression by insulin in conscious dogs in vivo (Hornbuckle LA, Edgerton DS, Ayala JE, Svitek CA, Neal DW, Cardin S, Cherrington AD, and O'Brien RM. Am J Physiol Endocrinol Metab 281: E713–E725, 2001). In pancreatic-clamped, euglycemic conscious dogs, a 5-h period of hypoinsulinemia led to a marked increase in hepatic G-6-Pase catalytic subunit mRNA; however, G-6- P transporter mRNA was unchanged. Here, we demonstrate, again using pancreatic-clamped, conscious dogs, that glucagon is a candidate for the factor responsible for this selective induction. Thus glucagon stimulated G-6-Pase catalytic subunit but not G-6- P transporter gene expression in vivo. Furthermore, cAMP stimulated endogenous G-6-Pase catalytic subunit gene expression in HepG2 cells but had no effect on G-6- P transporter gene expression. The cAMP response element (CRE) that mediates this induction was identified through transient transfection of HepG2 cells with G-6-Pase catalytic subunit-chloramphenicol acetyltransferase fusion genes. Gel retardation assays demonstrate that this CRE binds several transcription factors including CRE-binding protein and CCAAT enhancer-binding protein.

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