The DAF-3 Smad binds DNA and represses gene expression in the Caenorhabditis elegans pharynx

Development ◽  
1999 ◽  
Vol 126 (1) ◽  
pp. 97-107 ◽  
Author(s):  
J.D. Thatcher ◽  
C. Haun ◽  
P.G. Okkema
Keyword(s):  
Gene Expression ◽  
Caenorhabditis Elegans ◽  
Recognition Sequence ◽  
Mobility Shift ◽  
Enhancer Activity ◽  
Gene Target ◽  
Gfp Reporter ◽  
Pharyngeal Muscles ◽  
Gel Mobility Shift ◽  
Dauer Larvae

Gene expression in the pharyngeal muscles of Caenorhabditis elegans is controlled in part by organ-specific signals, which in the myo-2 gene target a short DNA sequence termed the C subelement. To identify genes contributing to these signals, we performed a yeast one-hybrid screen for cDNAs encoding factors that bind the C subelement. One clone recovered was from daf-3, which encodes a Smad most closely related to vertebrate Smad4. We demonstrated that DAF-3 binds C subelement DNA directly and specifically using gel mobility shift and DNase1 protection assays. Mutation of any base in the sequence GTCTG interfered with binding in the gel mobility shift assay, demonstrating that this pentanucleotide is a core recognition sequence for DAF-3 binding. daf-3 is known to promote formation of dauer larvae and this activity is negatively regulated by TGFbeta-like signaling. To determine how daf-3 affects C subelement enhancer activity in vivo, we examined expression a gfp reporter controlled by a concatenated C subelement oligonucleotide in daf-3 mutants and other mutants affecting the TGFbeta-like signaling pathway controlling dauer formation. Our results demonstrate that wild-type daf-3 can repress C subelement enhancer activity during larval development and, like its dauer-promoting activity, daf-3's repressor activity is negatively regulated by TGFbeta-like signaling. We have examined expression of this gfp reporter in dauer larvae and have observed no daf-3-dependent repression of C activity. These results suggest daf-3 directly regulates pharyngeal gene expression during non-dauer development.

A novel cis-acting DNA element required for a high level of inducible expression of the rat P-450c gene

1990 ◽  
Vol 10 (4) ◽  
pp. 1470-1475
Author(s):  
A Yanagida ◽  
K Sogawa ◽  
K I Yasumoto ◽  
Y Fujii-Kuriyama
Keyword(s):  
Regulatory Element ◽  
Consensus Sequence ◽  
Inducible Expression ◽  
Recognition Sequence ◽  
Mobility Shift ◽  
Enhancer Activity ◽  
Cis Acting ◽  
Tata Sequence ◽  
Gel Mobility Shift ◽  
High Level

A novel cis-acting regulatory element (designated BTE for basic transcription element) was found in the region proximal to the TATA sequence of the P-450c gene by the use of deletion mutations. This DNA element is considered to be involved in the basic transcription of the gene and does not show distinct enhancer activity in itself. Together with the XRE sequence (A. Fujisawa-Sehara, K. Sogawa, M. Yamane, and Y. Fujii-Kuriyama, Nucleic Acids Res. 15:4179-4191, 1987), however, this sequence is required for a high inducible expression of the P-450c gene in response to xenobiotic inducers. The BTE sequence contained the GC box consensus sequence and half of the NF-1-binding consensus or CAT box sequence, but their synthetic oligonucleotides, used as competitors in the gel mobility shift assays, did not compete with the BTE sequence for the binding protein, suggesting that the BTE sequence functions as a different recognition sequence from that for Sp1 or NF-1. Analogous sequences to BTE are found in the region proximal to the TATA sequence of other genes, especially other P-450 genes with different modes of regulation, suggesting that the BTE sequence plays a common regulatory role in basic transcription of genes including a group of the P-450 superfamily. The ubiquitous distribution of nuclear factor(s) binding to this element supports this suggestion.

scholarly journals A novel cis-acting DNA element required for a high level of inducible expression of the rat P-450c gene.

1990 ◽  
Vol 10 (4) ◽  
pp. 1470-1475 ◽  
Author(s):  
A Yanagida ◽  
K Sogawa ◽  
K I Yasumoto ◽  
Y Fujii-Kuriyama
Keyword(s):  
Regulatory Element ◽  
Consensus Sequence ◽  
Inducible Expression ◽  
Recognition Sequence ◽  
Mobility Shift ◽  
Enhancer Activity ◽  
Cis Acting ◽  
Tata Sequence ◽  
Gel Mobility Shift ◽  
High Level

A novel cis-acting regulatory element (designated BTE for basic transcription element) was found in the region proximal to the TATA sequence of the P-450c gene by the use of deletion mutations. This DNA element is considered to be involved in the basic transcription of the gene and does not show distinct enhancer activity in itself. Together with the XRE sequence (A. Fujisawa-Sehara, K. Sogawa, M. Yamane, and Y. Fujii-Kuriyama, Nucleic Acids Res. 15:4179-4191, 1987), however, this sequence is required for a high inducible expression of the P-450c gene in response to xenobiotic inducers. The BTE sequence contained the GC box consensus sequence and half of the NF-1-binding consensus or CAT box sequence, but their synthetic oligonucleotides, used as competitors in the gel mobility shift assays, did not compete with the BTE sequence for the binding protein, suggesting that the BTE sequence functions as a different recognition sequence from that for Sp1 or NF-1. Analogous sequences to BTE are found in the region proximal to the TATA sequence of other genes, especially other P-450 genes with different modes of regulation, suggesting that the BTE sequence plays a common regulatory role in basic transcription of genes including a group of the P-450 superfamily. The ubiquitous distribution of nuclear factor(s) binding to this element supports this suggestion.

The orphan nuclear receptor NGFI-B regulates expression of the gene encoding steroid 21-hydroxylase

1993 ◽  
Vol 13 (2) ◽  
pp. 861-868
Author(s):  
T E Wilson ◽  
A R Mouw ◽  
C A Weaver ◽  
J Milbrandt ◽  
K L Parker
Keyword(s):  
Nuclear Receptor ◽  
Transcriptional Activation ◽  
Core Promoter ◽  
Orphan Nuclear Receptor ◽  
Recognition Sequence ◽  
Mobility Shift ◽  
Adrenocortical Cells ◽  
Gene Encoding ◽  
Adult Rat ◽  
Gel Mobility Shift

As part of its trophic action to maintain the steroidogenic capacity of adrenocortical cells, corticotropin (ACTH) increases the transcription of the cytochrome P-450 steroid hydroxylase genes, including the gene encoding steroid 21-hydroxylase (21-OHase). We previously identified several promoter elements that regulate 21-OHase gene expression in mouse Y1 adrenocortical tumor cells. One of these elements, located at nucleotide -65, closely resembles the recognition sequence of the orphan nuclear receptor NGFI-B, suggesting that NGFI-B regulates this essential steroidogenic enzyme. To explore this possibility, we first used in situ hybridization to demonstrate high levels of NGFI-B transcripts in the adrenal cortex of the adult rat. In cultured mouse Y1 adrenocortical cells, treatment with ACTH, the major regulator of 21-OHase transcription, rapidly increased NGFI-B expression. Gel mobility shift and DNase I footprinting experiments showed that recombinantly expressed NGFI-B interacts specifically with the 21-OHase -65 element and identified one complex formed by Y1 extracts and the 21-OHase -65 element that contains NGFI-B. Expression of NGFI-B significantly augmented the activity of the intact 21-OHase promoter, while mutations of the -65 element that abolish NGFI-B binding markedly diminished NGFI-B-mediated transcriptional activation. Specific mutations of NGFI-B shown previously to impair either DNA binding or transcriptional activation diminished the effect of NGFI-B coexpression on 21-OHase expression. Finally, an oligonucleotide containing the NGFI-B response element conferred ACTH response to a core promoter from the prolactin gene, showing that this element is sufficient for ACTH induction. Collectively, these results identify a cellular promoter element that is regulated by NGFI-B and implicate NGFI-B in the transcriptional induction of 21-OHase by ACTH.

NG-monomethyl-l-arginine inhibits erythropoietin gene expression by stimulating GATA-2

Blood ◽  
2000 ◽  
Vol 96 (5) ◽  
pp. 1716-1722 ◽  
Author(s):  
Takahisa Tarumoto ◽  
Shigehiko Imagawa ◽  
Ken Ohmine ◽  
Tadashi Nagai ◽  
Masato Higuchi ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
Messenger Rna ◽  
Promoter Activity ◽  
Binding Activity ◽  
Guanosine Monophosphate ◽  
Mobility Shift ◽  
Enhancer Activity ◽  
Gata Transcription Factor ◽  
Hep3b Cells

Abstract NG-monomethyl-l-arginine (L-NMMA) has been reported to be elevated in uremic patients. Based on the hypothesis that the pathogenesis of the anemia of renal disease might be due to the perturbation of transcription factors of the erythropoietin (Epo) gene by L-NMMA, the present study was designed to investigate the effect of L-NMMA on Epo gene expression through the GATA transcription factor. L-NMMA caused decreased levels of NO, cyclic guanosine monophosphate (cGMP), and Epo protein in Hep3B cells. L-NAME (analogue of L-NMMA) also inhibited Epo production in anemic mice. Transfection of the Epo promoter-luciferase gene into Hep3B cells revealed that L-NMMA inhibited the Epo promoter activity. However, L-NMMA did not inhibit the Epo promoter activity when mutated Epo promoter (GATA to TATA) was transfected, and L-NMMA did not affect the enhancer activity. Electrophoretic mobility shift assays demonstrated the stimulation of GATA binding activity by L-NMMA. However, L-NMMA had no effect on the binding activity of hepatic nuclear factor-4, COUP-TF1, hypoxia-inducing factor-1, or NF-κB. Furthermore, cGMP inhibited the L-NMMA–induced GATA binding activity. L-NMMA also increased GATA-2 messenger RNA expression. These results demonstrate that L-NMMA suppresses Epo gene expression by up-regulation of the GATA transcription factor and support the hypothesis that L-NMMA is one of the candidate substances that underlie the pathogenesis of renal anemia.

scholarly journals Regulation of d-Xylose Metabolism in Caulobacter crescentus by a LacI-Type Repressor

2007 ◽  
Vol 189 (24) ◽  
pp. 8828-8834 ◽  
Author(s):  
Craig Stephens ◽  
Beat Christen ◽  
Kelly Watanabe ◽  
Thomas Fuchs ◽  
Urs Jenal
Keyword(s):  
Gene Expression ◽  
Energy Source ◽  
Regulatory Mechanism ◽  
Caulobacter Crescentus ◽  
Heterologous Gene Expression ◽  
Xylose Metabolism ◽  
Mobility Shift ◽  
Inducible Promoters ◽  
Gel Mobility Shift ◽  
Operator Binding

ABSTRACT In the oligotrophic freshwater bacterium Caulobacter crescentus, d-xylose induces expression of over 50 genes, including the xyl operon, which encodes key enzymes for xylose metabolism. The promoter (P xylX ) controlling expression of the xyl operon is widely used as a tool for inducible heterologous gene expression in C. crescentus. We show here that P xylX and at least one other promoter in the xylose regulon (P xylE ) are controlled by the CC3065 (xylR) gene product, a LacI-type repressor. Electrophoretic gel mobility shift assays showed that operator binding by XylR is greatly reduced in the presence of d-xylose. The data support the hypothesis that there is a simple regulatory mechanism in which XylR obstructs xylose-inducible promoters in the absence of the sugar; the repressor is induced to release DNA upon binding d-xylose, thereby freeing the promoter for productive interaction with RNA polymerase. XylR also has an effect on glucose metabolism, as xylR mutants exhibit reduced expression of the Entner-Doudoroff operon and their ability to utilize glucose as a sole carbon and energy source is compromised.

scholarly journals A complex array of positive and negative elements regulates the chicken alpha A-crystallin gene: involvement of Pax-6, USF, CREB and/or CREM, and AP-1 proteins.

1994 ◽  
Vol 14 (11) ◽  
pp. 7363-7376 ◽  
Author(s):  
A Cvekl ◽  
C M Sax ◽  
E H Bresnick ◽  
J Piatigorsky
Keyword(s):  
Gene Expression ◽  
Promoter Activity ◽  
Cellular Differentiation ◽  
Ocular Lens ◽  
Mobility Shift ◽  
Specific Expression ◽  
Negative Element ◽  
Cis Acting ◽  
Direct Data ◽  
Gel Mobility Shift

The abundance of crystallins (> 80% of the soluble protein) in the ocular lens provides advantageous markers for selective gene expression during cellular differentiation. Here we show by functional and protein-DNA binding experiments that the chicken alpha A-crystallin gene is regulated by at least five control elements located at sites A (-148 to -139), B (-138 to -132), C (-128 to -101), D (-102 to -93), and E (-56 to -41). Factors interacting with these sites were characterized immunologically and by gel mobility shift experiments. The results are interpreted with the following model. Site A binds USF and is part of a composite element with site B. Site B binds CREB and/or CREM to enhance expression in the lens and binds an AP-1 complex including CREB, Fra2 and/or JunD which interacts with USF on site A to repress expression in fibroblasts. Sites C and E (which is conserved across species) bind Pax-6 in the lens to stimulate alpha A-crystallin promoter activity. These experiments provide the first direct data that Pax-6 contributes to the lens-specific expression of a crystallin gene. Site D (-104 to -93) binds USF and is a negative element. Thus, the data indicate that USF, CREB and/or CREM (or AP-1 factors), and Pax-6 bind a complex array of positive and negative cis-acting elements of the chicken alpha A-crystallin gene to control high expression in the lens and repression in fibroblasts.

Functional analysis of the murine T-cell receptor beta enhancer and characteristics of its DNA-binding proteins

1990 ◽  
Vol 10 (10) ◽  
pp. 5027-5035
Author(s):  
J Takeda ◽  
A Cheng ◽  
F Mauxion ◽  
C A Nelson ◽  
R D Newberry ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Receptor ◽  
Cell Receptor ◽  
Lymphoid Cells ◽  
Mobility Shift ◽  
Enhancer Activity ◽  
Mobility Shift Assay ◽  
Gel Mobility Shift ◽  
Beta Enhancer

The minimal T-cell receptor (TCR) beta-chain (TCR beta) enhancer has been identified by transfection into lymphoid cells. The minimal enhancer was active in T cells and in some B-lineage cells. When a larger fragment containing the minimal enhancer was used, its activity was apparent only in T cells. Studies with phytohemagglutinin and 4 beta-phorbol-12,13-dibutyrate revealed that the enhancer activity was increased by these agents. By a combination of DNase I footprinting, gel mobility shift assay, and methylation interference analysis, seven different motifs were identified within the minimal enhancer. Furthermore, competition experiments showed that some of these elements bound identical or similar factors that are known to bind to the TCR V beta promoter decamer or to the immunoglobulin enhancer kappa E2 or muEBP-E motif. These shared motifs may be important in the differential gene activity among the different lymphoid subsets.

scholarly journals Activation of mouse Pi-class glutathione S-transferase gene by Nrf2(NF-E2-related factor 2) and androgen

2002 ◽  
Vol 364 (2) ◽  
pp. 563-570 ◽  
Author(s):  
Hiromi IKEDA ◽  
Mohamed S. SERRIA ◽  
Ikuko KAKIZAKI ◽  
Ichiro HATAYAMA ◽  
Kimihiko SATOH ◽  
...  
Keyword(s):  
Androgen Receptor ◽  
Receptor Binding ◽  
Binding Sites ◽  
Related Factor ◽  
Glutathione S Transferase ◽  
Mobility Shift ◽  
Enhancer Activity ◽  
Drug Induction ◽  
Mobility Shift Assay ◽  
Gel Mobility Shift

The Pi-class glutathione S-transferases (GSTs) play pivotal roles in the detoxification of xenobiotics, carcinogenesis and drug resistance. The mechanisms of regulation of these genes during drug induction and carcinogenesis are yet to be elucidated. Recently, Nrf2 (NF-E2-related factor 2; a bZip-type transcription factor) knockout mice were shown to display impaired induction of Pi-class GST genes by drugs. It is known that the mouse Pi-class GST gene GST-P1 is expressed predominantly in the male liver, and is regulated by androgen. To determine whether Nrf2 and the androgen receptor regulate GST-P1 directly, we analysed the molecular mechanism of activation of this gene by these factors. The promoter of the GST-P1 gene was activated markedly by Nrf2 in transient transfection analyses. Gel mobility shift assay and footprinting analyses revealed three Nrf2 binding sites: one at the proximal and two at distal elements, located at positions −59, −915 and −937 from the cap site. The fifth intron of the GST-P1 gene contains the androgen-responsive region. Multiple androgen receptor binding sites are clustered within a 500bp region of this intron. The whole fragment contains a minimum of seven androgen receptor binding sites, which collectively display strong androgen-dependent enhancer activity. However, on division into small fragments containing two or three elements each, individual enhancer activities were dramatically decreased. This suggests that multiple elements work synergistically as a strong androgen-responsive enhancer. Our findings indicate that Nrf2 and the androgen receptor directly bind to and activate the mouse GST-P1 gene.

scholarly journals An estrogen-inducible protein binds specifically to a sequence in the 3' untranslated region of estrogen-stabilized vitellogenin mRNA.

1994 ◽  
Vol 14 (5) ◽  
pp. 3130-3138 ◽  
Author(s):  
R E Dodson ◽  
D J Shapiro
Keyword(s):  
Binding Site ◽  
Untranslated Region ◽  
Specific Binding ◽  
High Specificity ◽  
Proteinase K ◽  
Recognition Sequence ◽  
Mobility Shift ◽  
Inducible Protein ◽  
Gel Mobility Shift ◽  
Vitellogenin Mrna

The 3' untranslated region (3'-UTR) has been implicated in the estrogen stabilization of hepatic Xenopus laevis vitellogenin mRNA. We used RNA gel mobility shift assays to demonstrate that Xenopus liver contains a factor which binds with very high specificity to a segment of the 3'-UTR of vitellogenin B1 and B2 mRNAs. We detected a single high-affinity binding site in the vitellogenin mRNA 3'-UTR and localized the binding site to a 27-nucleotide region. Since binding was abolished by proteinase K digestion, at least a component of the factor is a protein. Following estrogen administration, binding was induced approximately four- to fivefold in extracts from liver polysomes. The hepatic vitellogenin mRNA-binding protein was found in both polysomes and cytosol. Since the protein was also estrogen inducible in cytosol, this represents a genuine induction, not simply recruitment of the cytosolic protein into polysomes. UV cross-linking studies with the 27-nucleotide recognition sequence revealed bands corresponding to bound proteins with apparent molecular weights of 71,000 and 141,000. This appears to be the first example of steroid hormone-inducible proteins binding to an mRNA 3'-UTR. Its induction by estrogen and its sequence-specific binding to a region of vitellogenin mRNA important in estrogen-mediated stabilization suggest that the protein may play a role in the regulation of mRNA stability.

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