Molecular mechanism of rat NHE3 gene promoter regulation by sodium butyrate

2007 ◽  
Vol 293 (1) ◽  
pp. C64-C74 ◽  
Author(s):  
Pawel R. Kiela ◽  
Nesrin Kuscuoglu ◽  
Anna J. Midura ◽  
Monica T. Midura-Kiela ◽  
Claire B. Larmonier ◽  
...  
Keyword(s):  
Gene Transcription ◽  
Sodium Butyrate ◽  
Gene Promoter ◽  
Mobility Shift ◽  
Upstream Site ◽  
Potent Inducer ◽  
Maximal Induction ◽  
Gel Mobility Shift

Sodium butyrate (NaB) stimulates sodium and water absorption by inducing colonic Na+/H+ exchange. NaB induces Na+/H+ exchanger (NHE)3 activity and protein and mRNA expression both in vivo and in vitro. Our previously published observations indicated that this induction is Ser/Thr kinase dependent and that NaB-responsive elements were localized within −320/−34 bp of the rat NHE3 promoter. Here we further delineate the mechanism of NaB-mediated NHE3 gene transcription. Transient and stable transfection of Caco-2 cells with NHE3 gene reporter constructs identified Sp binding site SpB at position −58/−55 nt as critical for NaB-mediated induction. Gel mobility shift (GMSA) and DNA affinity precipitation assays indicated NaB-induced binding of Sp3 and decreased binding of Sp1 to SpB element. While no changes in expression of Sp1 or Sp3 were noted, NaB induced phosphorylation of Sp1 and acetylation of Sp3. Sp3 was a more potent inducer of NHE3 gene transcription, which suggested that change in balance, favoring binding of Sp3 to the SpB site, would result in significant increase in NHE3 promoter activity. Small interfering RNA studies in Caco-2 cells and data from NaB-treated SL2 cells used as a reconstitution model confirmed this hypothesis. In addition to the SpB site, which played a permissive role, an upstream novel butyrate response element located at −196/−175 nt was necessary for maximal induction. GMSA identified a protein-DNA complex with a −196/−175 nt probe; this interaction was not affected by NaB treatment, thus suggesting that in response to NaB Sp3 binding to site SpB precedes and results in recruitment of the putative factor to this upstream site.

Transcription from a murine T-cell receptor V beta promoter depends on a conserved decamer motif similar to the cyclic AMP response element

1989 ◽  
Vol 9 (11) ◽  
pp. 4835-4845
Author(s):  
S J Anderson ◽  
S Miyake ◽  
D Y Loh
Keyword(s):  
Cyclic Amp ◽  
Regulatory Region ◽  
Cell Receptor ◽  
Transcription Activator ◽  
Mobility Shift ◽  
Response Element ◽  
Cyclic Amp Response Element ◽  
Gel Mobility Shift

We identified a regulatory region of the murine V beta promoter by both in vivo and in vitro analyses. The results of transient transfection assays indicated that the dominant transcription-activating element within the V beta 8.3 promoter is the palindromic motif identified previously as the conserved V beta decamer. Elimination of this element, by linear deletion or specific mutation, reduced transcriptional activity from this promoter by 10-fold. DNase I footprinting, gel mobility shift, and methylation interference assays confirmed that the palindrome acts as the binding site of a specific nuclear factor. In particular, the V beta promoter motif functioned in vitro as a high-affinity site for a previously characterized transcription activator, ATF. A consensus cyclic AMP response element (CRE) but not a consensus AP-1 site, can substitute for the decamer in vivo. These data suggest that cyclic AMP response element-binding protein (ATF/CREB) or related proteins activate V beta transcription.

scholarly journals Identification of an Adeno-Associated Virus Rep Protein Binding Site in the Adenovirus E2a Promoter

2005 ◽  
Vol 79 (1) ◽  
pp. 28-38 ◽  
Author(s):  
John M. Casper ◽  
Jennifer M. Timpe ◽  
John David Dignam ◽  
James P. Trempe
Keyword(s):  
Gene Expression ◽  
Random Sequence ◽  
Gene Promoter ◽  
Helper Virus ◽  
Host Cells ◽  
Mobility Shift ◽  
Adeno Associated Virus ◽  
Rep Protein

ABSTRACT Adeno-associated virus (AAV) and other parvoviruses inhibit proliferation of nonpermissive cells. The mechanism of this inhibition is not thoroughly understood. To learn how AAV interacts with host cells, we investigated AAV's interaction with adenovirus (Ad), AAV's most efficient helper virus. Coinfection with Ad and AAV results in an AAV-mediated inhibition of Ad5 gene expression and replication. The AAV replication proteins (Rep) activate and repress gene expression from AAV and heterologous transcription promoters. To investigate the role of Rep proteins in the suppression of Ad propagation, we performed chromatin immunoprecipitation analyses that demonstrated in vivo AAV Rep protein interaction with the Ad E2a gene promoter. In vitro binding of purified AAV Rep68 protein to the Ad E2a promoter was characterized by electrophoretic mobility shift assays (Kd = 200 ± 25 nM). A 38 bp, Rep68-protected region (5′-TAAGAGTCAGCGCGCAGTATTTACTGAAGAGAGCCT-3′) was identified by DNase I footprint analysis. The 38-bp protected region contains the weak E2a TATA box, sequence elements that resemble the Rep binding sites identified by random sequence oligonucleotide selection, and the transcription start site. These results suggest that Rep binding to the E2a promoter contributes to the inhibition of E2a gene expression from the Ad E2a promoter and may affect Ad replication.

scholarly journals In vitro transcription from the human A gamma-globin gene promoter

Blood ◽  
1993 ◽  
Vol 82 (4) ◽  
pp. 1344-1350 ◽  
Author(s):  
M Castle ◽  
D O'Neill ◽  
A Bank
Keyword(s):  
Gene Transcription ◽  
Globin Gene ◽  
Gene Promoter ◽  
K562 Cells ◽  
In Vitro Transcription ◽  
Systematic Analysis ◽  
Gamma Globin ◽  
Octamer Motif

Abstract We report enhanced transcription from the human A gamma-globin gene promoter in nuclear extracts (NE) of erythroleukemia (K562) cells compared with that in HeLa NE. We do not observe differences in transcription levels in the two extracts with nonglobin promoter templates. Our findings, indicating preferential recognition of the globin gene promoter by nuclear factors in K562 cells, are consistent with results of studies previously reported by ourselves and others. A novel finding described here is that the addition of a double-stranded octamer motif oligonucleotide to K562 NE increases the level of transcription from the A gamma-globin gene promoter, suggesting a potential role for an octamer motif-binding factor in the repression of A gamma-globin gene transcription. A cosmid construct containing extensive human gamma- and beta-globin gene promoter and structural sequences as well as upstream control sequences also exhibits higher levels of globin gene transcription in K562 NE than in HeLa NE. Our demonstration of the feasibility of efficient, globin promoter-specific in vitro transcription of this complex template offers a novel approach for the systematic analysis of the effects of putative regulatory factors on globin gene expression in vitro in the context of a genetic environment approximating that found in vivo.

Regulation of the rat NHE3 gene promoter by sodium butyrate

2001 ◽  
Vol 281 (4) ◽  
pp. G947-G956 ◽  
Author(s):  
Pawel R. Kiela ◽  
Eric R. Hines ◽  
James F. Collins ◽  
Fayez K. Ghishan
Keyword(s):  
Gene Transcription ◽  
Transcriptional Activation ◽  
Hdac Inhibitor ◽  
Promoter Activity ◽  
Actinomycin D ◽  
Trichostatin A ◽  
Gene Promoter ◽  
Short Chain Fatty Acids

Short-chain fatty acids, and especially butyrate (NaB), stimulate sodium and water absorption by inducing colonic Na+/H+ exchange (NHE). NaB induces NHE3 activity and protein and mRNA expression both in vivo and in vitro. NaB, as a histone deacetylase (HDAC) inhibitor, regulates gene transcription. We therefore studied whether NaB regulates transcription of the rat NHE3 promoter in transiently transfected Caco-2 cells. NaB (5 mM) strongly stimulated reporter gene activity, and this stimulation was prevented with actinomycin D, indicating transcriptional activation. NaB effects on the NHE3 promoter depended on the activity of Ser/Thr kinases, in particular, protein kinase A (PKA). However, PKA stimulation alone did not have an effect on promoter activity, and it did not act synergistically with NaB. Another HDAC inhibitor, Trichostatin A (TSA), stimulated NHE3 promoter in a Ser/Thr kinase-independent fashion. The putative NaB-responsive elements were localized within −320/−34 bp of the NHE3 promoter. These findings suggest that PKA mediates NaB effects on NHE3 gene transcription and that the mechanism of NaB action is different from that of TSA.

scholarly journals Transcription from a murine T-cell receptor V beta promoter depends on a conserved decamer motif similar to the cyclic AMP response element.

1989 ◽  
Vol 9 (11) ◽  
pp. 4835-4845 ◽  
Author(s):  
S J Anderson ◽  
S Miyake ◽  
D Y Loh
Keyword(s):  
Cyclic Amp ◽  
Regulatory Region ◽  
Cell Receptor ◽  
Transcription Activator ◽  
Mobility Shift ◽  
Response Element ◽  
Cyclic Amp Response Element ◽  
Gel Mobility Shift

We identified a regulatory region of the murine V beta promoter by both in vivo and in vitro analyses. The results of transient transfection assays indicated that the dominant transcription-activating element within the V beta 8.3 promoter is the palindromic motif identified previously as the conserved V beta decamer. Elimination of this element, by linear deletion or specific mutation, reduced transcriptional activity from this promoter by 10-fold. DNase I footprinting, gel mobility shift, and methylation interference assays confirmed that the palindrome acts as the binding site of a specific nuclear factor. In particular, the V beta promoter motif functioned in vitro as a high-affinity site for a previously characterized transcription activator, ATF. A consensus cyclic AMP response element (CRE) but not a consensus AP-1 site, can substitute for the decamer in vivo. These data suggest that cyclic AMP response element-binding protein (ATF/CREB) or related proteins activate V beta transcription.

scholarly journals Regulation of the Human Secretin Gene Is Controlled by the Combined Effects of CpG Methylation, Sp1/Sp3 Ratio, and the E-Box Element

2004 ◽  
Vol 18 (7) ◽  
pp. 1740-1755 ◽  
Author(s):  
Leo Tsz-On Lee ◽  
Kian-Cheng Tan-Un ◽  
Ronald Ting-Kai Pang ◽  
David Tai-Wai Lam ◽  
Billy Kwok-Chong Chow
Keyword(s):  
Gene Expression ◽  
Hepg2 Cells ◽  
Cpg Island ◽  
Methylation Status ◽  
Gene Promoter ◽  
Mobility Shift ◽  
Specific Pcr ◽  
E Box

Abstract To unravel the mechanisms that regulate the human secretin gene expression, in this study, we have used secretin-expressing (HuTu-80 cells, human duodenal adenocarcinoma) and non-secretin-expressing [PANC-1 (human pancreatic ductile carcinoma) and HepG2 (human hepatocellular carcinoma) cells] cell models for in vitro and in vivo analyses. By transient transfection assays, within the promoter region (−11 to −341 from ATG, relative to the ATG initiation codon), we have initially identified several functional motifs including an E-box and 2 GC-boxes. Results from gel mobility shift and chromatin immunoprecipitation assays confirmed further that NeuroD, E2A, Sp1, and Sp3 bind to these E- and GC-boxes in HuTu-80 cells in vitro and in vivo, whereas only high levels of Sp3 is observed to bind the promoter in HepG2 cells. In addition, overexpression of Sp3 resulted in a dose-dependent repression of the Sp1-mediated transactivation. Collectively, these data suggest that the Sp1/Sp3 ratio is instrumental to controlling secretin gene expression in secretin-producing and non-secretin-producing cells. The functions of GC-box and Sp proteins prompted us to investigate the possible involvement of DNA methylation in regulating this gene. Consistent with this idea, we found a putative CpG island (−336 to 262 from ATG) that overlaps with the human secretin gene promoter. By methylation-specific PCR, all the CpG dinucleo-tides (26 of them) within the CpG island in HuTu-80 cells are unmethylated, whereas all these sites are methylated in PANC-1 and HepG2 cells. The expressions of secretin in PANC-1 and HepG2 cells were subsequently found to be significantly activated by a demethylation agent, 5′-Aza-2′ deoxycytidine. Taken together, our data indicate that the human secretin gene is controlled by the in vivo Sp1/Sp3 ratio and the methylation status of the promoter.

scholarly journals Mechanism of action of a repressor of dioxin-dependent induction of Cyp1a1 gene transcription.

1992 ◽  
Vol 12 (5) ◽  
pp. 2115-2123 ◽  
Author(s):  
A J Watson ◽  
K I Weir-Brown ◽  
R M Bannister ◽  
F F Chu ◽  
S Reisz-Porszasz ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Transcriptional Activation ◽  
High Salt ◽  
In Vitro Assays ◽  
Ah Receptor ◽  
Mobility Shift ◽  
Nuclear Extracts ◽  
Gel Mobility Shift

A dominant mutant of Hepa-1 cells, c31, expresses a repressor that prevents 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-dependent stimulation of Cyp1a1 transcription. The repressor acts via the xenobiotic-responsive elements (XREs), which are the DNA-binding sites for the aryl hydrocarbon (Ah) receptor-TCDD complex during transcriptional activation of the gene. High-salt nuclear extracts prepared from c31 cells grown with TCDD contained normal levels of the Ah receptor which bound the XRE with normal affinity, as judged by in vitro gel mobility shift assays. Furthermore, extracts prepared from these cells, grown either with or without TCDD, contained no novel XRE-binding proteins compared with extracts from wild-type Hepa-1 cells. However, in vivo genomic footprinting demonstrated that TCDD treatment leads to binding of the Ah receptor to the XREs in Hepa-1 but not mutant cells. This finding suggests that the repressor associates with the Ah receptor to prevent its binding to the XREs and that high-salt treatment either causes dissociation of the receptor/repressor complex or fails to extract the repressor from nuclei. The results underscore the importance of using both in vivo and in vitro assays for analyzing DNA-protein interactions.

scholarly journals Regulatory Interactions among Adhesin Gene Systems of Uropathogenic Escherichia coli

2007 ◽  
Vol 76 (2) ◽  
pp. 771-780 ◽  
Author(s):  
Stina Lindberg ◽  
Yan Xia ◽  
Berit Sondén ◽  
Mikael Göransson ◽  
Jörg Hacker ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Regulatory Protein ◽  
Mobility Shift ◽  
E Coli ◽  
Fimbrial Adhesins ◽  
Gel Mobility Shift

ABSTRACT Uropathogenic Escherichia coli strain J96 carries multiple determinants for fimbrial adhesins. The regulatory protein PapB of P fimbriae has previously been implicated in potential coregulatory events. The focB gene of the F1C fimbria determinant is highly homologous to papB; the translated sequences share 81% identity. In this study we investigated the role of PapB and FocB in regulation of the F1C fimbriae. By using gel mobility shift assays, we showed that FocB binds to sequences in both the pap and foc operons in a somewhat different manner than PapB. The results of both in vitro cross-linking and in vivo oligomerization tests indicated that FocB could function in an oligomeric fashion. Furthermore, our results suggest that PapB and FocB can form heterodimers and that these complexes can repress expression of the foc operon. The effect of FocB on expression of type 1 fimbriae was also tested. Taken together, the results that we present expand our knowledge about a regulatory network for different adhesin gene systems in uropathogenic E. coli and suggest a hierarchy for expression of the fimbrial adhesins.

scholarly journals hDREF Regulates Cell Proliferation and Expression of Ribosomal Protein Genes

2007 ◽  
Vol 27 (6) ◽  
pp. 2003-2013 ◽  
Author(s):  
Daisuke Yamashita ◽  
Yukako Sano ◽  
Yuka Adachi ◽  
Yuma Okamoto ◽  
Hirotaka Osada ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Proliferation ◽  
Ribosomal Proteins ◽  
Human Fibroblasts ◽  
Gene Promoter ◽  
Ribosomal Protein Genes ◽  
Mobility Shift ◽  
Positive Elements

ABSTRACT Although ribosomal proteins (RPs) are essential cellular constituents in all living organisms, mechanisms underlying regulation of their gene expression in mammals remain unclear. We have established that 22 out of 79 human RP genes contain sequences similar to the human DREF (DNA replication-related element-binding factor; hDREF) binding sequence (hDRE) within 200-bp regions upstream of their transcriptional start sites. Electrophoretic gel mobility shift assays and chromatin immunoprecipitation analysis indicated that hDREF binds to hDRE-like sequences in the RP genes both in vitro and in vivo. In addition, transient luciferase assays revealed that hDRE-like sequences act as positive elements for RP gene transcription and cotransfection of an hDREF-expressing plasmid was found to stimulate RP gene promoter activity. Like that of hDREF, expression of RP genes is increased during the late G1 to S phases, and depletion of hDREF using short hairpin RNA-mediated knockdown decreased RP gene expression and cell proliferation in normal human fibroblasts. Knockdown of the RPS6 gene also resulted in impairment of cell proliferation. These data suggest that hDREF is an important transcription factor for cell proliferation which plays roles in cell cycle-dependent regulation of a number of RP genes.

scholarly journals A soluble transcription factor, Oct-1, is also found in the insoluble nuclear matrix and possesses silencing activity in its alanine-rich domain.

1996 ◽  
Vol 16 (8) ◽  
pp. 4366-4377 ◽  
Author(s):  
M K Kim ◽  
L A Lesoon-Wood ◽  
B D Weintraub ◽  
J H Chung
Keyword(s):  
Nuclear Matrix ◽  
Transcription Initiation ◽  
Initiation Site ◽  
Homeodomain Protein ◽  
Mobility Shift ◽  
Rich Domain ◽  
Gel Mobility Shift ◽  
Silencer Element

Expression of the human thyrotropin beta (hTSHbeta) gene is restricted to thyrotrophs, at least in part, by silencing. Using transient-transfection assays, we have localized a silencer element to a region between -128 and -480 bp upstream of the transcription initiation site. The silencing activity was overcome in a thyrotroph-specific manner by an unknown enhancer located in the sequences at -approximately 10000 to -1200 bp. The ubiquitous POU homeodomain protein Oct-1 recognized the A/T-rich silencer element at multiple sites in gel mobility shift assays and in vitro footprinting analyses. The silencing activity of Oct-1 was localized in its C-terminal alanine-rich domain, suggesting that Oct-1 plays a role in silencing of the hTSHbeta promoter. Further, a significant fraction of Oct-1 was shown to be associated with the nuclear matrix, and the hTSHbeta silencer region was tethered to a nuclear matrix of human cells in vivo, suggesting a possible role of the Oct-1-hTSHbeta silencer region interaction in chromatin organization.

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