scholarly journals Temporal Regulation of Enhancer Function in Intestinal Epithelium

2006 ◽  
Vol 281 (43) ◽  
pp. 32263-32271 ◽  
Author(s):  
Elizabeth A. Maier ◽  
Mary R. Dusing ◽  
Dan A. Wiginton
Keyword(s):  
Expression Patterns ◽  
Regulatory Region ◽  
Specific Gene ◽  
Temporal Region ◽  
Mobility Shift ◽  
Temporal Regulation ◽  
Gata Factors ◽  
Dnase I Footprinting ◽  
Duodenal Epithelium ◽  
Electrophoretic Mobility Shift Assays

An intestine-specific gene regulatory region was previously identified near the second exon of the human adenosine deaminase (ADA) gene. In mammalian intestine, ADA is expressed at high levels only along the villi of the duodenal epithelium, principally if not exclusively in enterocytes. Within the ADA intestinal regulatory region, a potent duodenum-specific enhancer was identified that controls this pattern of expression. This enhancer has been shown to rely on PDX-1, GATA factors, and Cdx factors for its function. Upstream of the enhancer, a separate temporal regulatory region was identified that has no independent enhancer capability but controls the timing of enhancer activation. DNase I footprinting and electrophoretic mobility shift assays were used to begin to characterize temporal region function at the molecular level. In this manner, binding sites for the Onecut (OC) family of factors, YY1, and NFI family members were identified. Identification of the OC site was especially interesting, because almost nothing is known about the function of OC factors in intestine. In transgenic mice, mutation of the OC site to ablate binding resulted in a delay of 2–3 weeks in enhancer activation in the developing postnatal intestine, a result very similar to that observed previously when the entire temporal region was deleted. In mammals, the OC family is comprised of OC-1/HNF-6, OC-2, and OC-3. An examination of intestinal expression patterns showed that all three OC factors are expressed at detectable levels in adult mouse duodenum, with OC-2 predominant. In postnatal day 2 mice only OC-2 and OC-3 were detected in intestine, with OC-2 again predominant.

scholarly journals Regulation ofperRExpression by Iron and PerR in Campylobacter jejuni

2011 ◽  
Vol 193 (22) ◽  
pp. 6171-6178 ◽  
Author(s):  
Minkyeong Kim ◽  
Sunyoung Hwang ◽  
Sangryeol Ryu ◽  
Byeonghwa Jeon
Keyword(s):  
Oxidative Stress ◽  
Campylobacter Jejuni ◽  
Regulatory Region ◽  
Transcriptional Level ◽  
Mobility Shift ◽  
Content Type ◽  
Dnase I Footprinting ◽  
Transcriptional Changes ◽  
Electrophoretic Mobility Shift Assays ◽  
Binding Sequence

Campylobacter jejuniis a leading food-borne pathogen causing gastroenteritis in humans. Although OxyR is a widespread oxidative stress regulator in many Gram-negative bacteria,C. jejunilacks OxyR and instead possesses the metalloregulator PerR. Despite the important role played by PerR in oxidative stress defense, little is known about the factors influencingperRexpression inC. jejuni. In this study, aperRpromoter-lacZfusion assay demonstrated that iron significantly reduced the level ofperRtranscription, whereas other metal ions, such as copper, cobalt, manganese, and zinc, did not affectperRtranscription. Notably, aperRmutation substantially increased the level ofperRtranscription and intranscomplementation restored the transcriptional changes, suggestingperRis transcriptionally autoregulated inC. jejuni. In theperRmutant, iron did not repressperRtranscription, indicating the iron dependence ofperRexpression results fromperRautoregulation. Electrophoretic mobility shift assays showed that PerR binds to theperRpromoter, and DNase I footprinting assays identified a PerR binding site overlapping the −35 region of the twoperRpromoters, further supportingperRautoregulation at the transcriptional level. Alignment of the PerR binding sequence in theperRpromoter with the regulatory region of other PerR regulon genes ofC. jejunirevealed a 16-bp consensus PerR binding sequence, which shares high similarities to theBacillus subtilisPerR box. The results of this study demonstrated that PerR directly interacts with theperRpromoter and regulatesperRtranscription and thatperRautoregulation is responsible for the repression ofperRtranscription by iron inC. jejuni.

scholarly journals SlyA and H-NS Regulate Transcription of the Escherichia coli K5 Capsule Gene Cluster, and Expression of slyA in Escherichia coli Is Temperature-dependent, Positively Autoregulated, and Independent of H-NS

2007 ◽  
Vol 282 (46) ◽  
pp. 33326-33335 ◽  
Author(s):  
David Corbett ◽  
Hayley J. Bennett ◽  
Hamdia Askar ◽  
Jeffrey Green ◽  
Ian S. Roberts
Keyword(s):  
Escherichia Coli ◽  
Gene Cluster ◽  
Regulation Of Transcription ◽  
Temperature Dependent ◽  
Mobility Shift ◽  
E Coli ◽  
Dnase I Footprinting ◽  
Nucleoprotein Complex ◽  
Electrophoretic Mobility Shift Assays

In this paper, we present the first evidence of a role for the transcriptional regulator SlyA in the regulation of transcription of the Escherichia coli K5 capsule gene cluster and demonstrate, using a combination of reporter gene fusions, DNase I footprinting, and electrophoretic mobility shift assays, the dependence of transcription on the functional interplay between H-NS and SlyA. Both SlyA and H-NS bind to multiple overlapping sites within the promoter in vitro, but their binding is not mutually exclusive, resulting in a remodeled nucleoprotein complex. In addition, we show that expression of the E. coli slyA gene is temperature-regulated, positively autoregulated, and independent of H-NS.

SoxS regulates the expression of the Salmonella enterica serovar Typhimurium ompW gene

Microbiology ◽  
2009 ◽  
Vol 155 (8) ◽  
pp. 2490-2497 ◽  
Author(s):  
F. Gil ◽  
I. Hernández-Lucas ◽  
R. Polanco ◽  
N. Pacheco ◽  
B. Collao ◽  
...  
Keyword(s):  
Salmonella Enterica ◽  
Salmonella Enterica Serovar Typhimurium ◽  
Regulatory Region ◽  
Transcription Start ◽  
Mobility Shift ◽  
Serovar Typhimurium ◽  
A Minor ◽  
Electrophoretic Mobility Shift Assays ◽  
Putative Binding Site ◽  
Transcriptional Fusions

OmpW of Salmonella enterica serovar Typhimurium has been described as a minor porin involved in osmoregulation, and is also affected by environmental conditions. Biochemical and genetic evidence from our laboratory indicates that OmpW is involved in efflux of and resistance towards paraquat (PQ), and its expression has been shown to be activated in response to oxidative stress. In this study we have explored ompW expression in response to PQ. Primer extension and transcriptional fusions showed that its expression was induced in the presence of PQ. In silico analyses suggested a putative binding site for the SoxS transcriptional factor at the ompW regulatory region. Electrophoretic mobility shift assays (EMSAs) and footprinting experiments showed that SoxS binds at a region that starts close to −54 and ends at about −197 upstream of the transcription start site. Transcriptional fusions support the relevance of this region in ompW activation. The SoxS site is in the forward orientation and its location suggests that the ompW gene has a class I SoxS-dependent promoter.

scholarly journals The ArgP Protein Stimulates the Klebsiella pneumoniae gdhA Promoter in a Lysine-Sensitive Manner

2008 ◽  
Vol 190 (12) ◽  
pp. 4351-4359 ◽  
Author(s):  
Thomas J. Goss
Keyword(s):  
Klebsiella Pneumoniae ◽  
Upstream Region ◽  
Mobility Shift ◽  
Base Pairs ◽  
Dnase I Footprinting ◽  
Sensitive Factor ◽  
Electrophoretic Mobility Shift Assays ◽  
Knockout Mutation

ABSTRACT The lysine-sensitive factor that binds to the upstream region of the Klebsiella pneumoniae gdhA promoter and stimulates gdhA transcription during growth in minimal medium has been proposed to be the K. pneumoniae ArgP protein (M. R. Nandineni, R. S. Laishram, and J. Gowrishankar, J. Bacteriol. 186:6391-6399, 2004). A knockout mutation of the K. pneumoniae argP gene was generated and used to assess the roles of exogenous lysine and argP in the regulation of the gdhA promoter. Disruption of argP reduced the strength and the lysine-dependent regulation of the gdhA promoter. Electrophoretic mobility shift assays using crude extracts prepared from wild-type and argP-defective strains indicted the presence of an argP-dependent factor whose ability to bind the gdhA promoter was lysine sensitive. DNase I footprinting studies using purified K. pneumoniae ArgP protein indicated that ArgP bound the region that lies approximately 50 to 100 base pairs upstream of the gdhA transcription start site in a manner that was sensitive to the presence of lysine. Substitutions within the region bound by ArgP affected the binding of ArgP to the gdhA promoter region in vitro and the argP-dependent stimulation of the gdhA promoter in vivo. These observations suggest that elevated intracellular levels of lysine reduce the affinity of ArgP for its binding site at the gdhA promoter, preventing ArgP from binding to and stimulating transcription from the promoter in vivo.

scholarly journals Gata4 Regulates Testis Expression of Dmrt1

2004 ◽  
Vol 24 (1) ◽  
pp. 377-388 ◽  
Author(s):  
Ning Lei ◽  
Leslie L. Heckert
Keyword(s):  
Transient Transfection ◽  
Mobility Shift ◽  
Specific Expression ◽  
Dnase I Footprinting ◽  
Unknown Factor ◽  
Related Transcription Factor ◽  
Important Regulator ◽  
Functional Relevance ◽  
Electrophoretic Mobility Shift Assays

ABSTRACT The doublesex and mab-3 related transcription factor 1 (Dmrt1) is a putative transcriptional regulator that is expressed exclusively in the gonads and is required for postnatal testis differentiation. Here we describe the transcriptional mechanisms regulating testis-specific expression of the Dmrt1 gene. Transient-transfection analysis identified a region of the promoter between kb −3.2 and −2.8 that is important for Sertoli cell-specific expression. DNase I footprinting revealed four sites of DNA-protein interaction within this region, three of which were prominent in primary Sertoli cells. Analysis of these sites, using electrophoretic mobility shift assays, revealed that Gata4 and another unknown factor bound within these regions. Further transient-transfection assays of various mutant promoters established the functional relevance of the Gata4-response and unknown factor-response elements, while studies of Dmrt1 expression in 13.5 days postcoitum Fog2 null gonads supported the in vivo importance of Gata4's regulation. As a whole, these studies identify Gata4 as an important regulator in the Dmrt1 transcriptional machinery that is responsible for robust expression of Dmrt1 in the testis.

scholarly journals Transcriptional Activation of Multiple Operons Involved inpara-Nitrophenol Degradation by Pseudomonas sp. Strain WBC-3

2014 ◽  
Vol 81 (1) ◽  
pp. 220-230 ◽  
Author(s):  
Wen-Mao Zhang ◽  
Jun-Jie Zhang ◽  
Xuan Jiang ◽  
Hongjun Chao ◽  
Ning-Yi Zhou
Keyword(s):  
Transcriptional Activation ◽  
Transcriptional Regulator ◽  
Pseudomonas Sp ◽  
Mobility Shift ◽  
Content Type ◽  
High Affinity ◽  
Dnase I Footprinting ◽  
Nitrophenol Degradation ◽  
Transcriptional Start Sites ◽  
Electrophoretic Mobility Shift Assays

ABSTRACTPseudomonassp. strain WBC-3 utilizespara-nitrophenol (PNP) as a sole carbon and energy source. The genes involved in PNP degradation are organized in the following three operons:pnpA,pnpB, andpnpCDEFG. How the expression of the genes is regulated is unknown. In this study, an LysR-type transcriptional regulator (LTTR) is identified to activate the expression of the genes in response to the specific inducer PNP. While the LTTR coding genepnpRwas found to be not physically linked to any of the three catabolic operons, it was shown to be essential for the growth of strain WBC-3 on PNP. Furthermore, PnpR positively regulated its own expression, which is different from the function of classical LTTRs. A regulatory binding site (RBS) with a 17-bp imperfect palindromic sequence (GTT-N11-AAC) was identified in allpnpA,pnpB,pnpC, andpnpRpromoters. Through electrophoretic mobility shift assays and mutagenic analyses, this motif was proven to be necessary for PnpR binding. This consensus motif is centered at positions approximately −55 bp relative to the four transcriptional start sites (TSSs). RBS integrity was required for both high-affinity PnpR binding and transcriptional activation ofpnpA,pnpB, andpnpR. However, this integrity was essential only for high-affinity PnpR binding to the promoter ofpnpCDEFGand not for its activation. Intriguingly, unlike other LTTRs studied, no changes in lengths of the PnpR binding regions of thepnpAandpnpBpromoters were observed after the addition of the inducer PNP in DNase I footprinting.

Regulation of the Bacillus subtilis mannitol utilization genes: promoter structure and transcriptional activation by the wild-type regulator (MtlR) and its mutants

Microbiology ◽  
2014 ◽  
Vol 160 (1) ◽  
pp. 91-101 ◽  
Author(s):  
Kambiz Morabbi Heravi ◽  
Josef Altenbuchner
Keyword(s):  
Bacillus Subtilis ◽  
Transcriptional Activation ◽  
Transcription Initiation ◽  
Phosphotransferase System ◽  
Mobility Shift ◽  
Dnase I Footprinting ◽  
Proposed Model ◽  
Rna Polymerase Holoenzyme ◽  
Electrophoretic Mobility Shift Assays

Expression of mannitol utilization genes in Bacillus subtilis is directed by P mtlA , the promoter of the mtlAFD operon, and P mtlR , the promoter of the MtlR activator. MtlR contains phosphoenolpyruvate-dependent phosphotransferase system (PTS) regulation domains, called PRDs. The activity of PRD-containing MtlR is mainly regulated by the phosphorylation/dephosphorylation of its PRDII and EIIBGat-like domains. Replacing histidine 342 and cysteine 419 residues, which are the targets of phosphorylation in these two domains, by aspartate and alanine provided MtlR-H342D C419A, which permanently activates P mtlA in vivo. In the mtlR-H342D C419A mutant, P mtlA was active, even when the mtlAFD operon was deleted from the genome. The mtlR-H342D C419A allele was expressed in an Escherichia coli strain lacking enzyme I of the PTS. Electrophoretic mobility shift assays using purified MtlR-H342D C419A showed an interaction between the MtlR double-mutant and the Cy5-labelled P mtlA and P mtlR DNA fragments. These investigations indicate that the activated MtlR functions regardless of the presence of the mannitol-specific transporter (MtlA). This is in contrast to the proposed model in which the sequestration of MtlR by the MtlA transporter is necessary for the activity of MtlR. Additionally, DNase I footprinting, construction of P mtlA -P licB hybrid promoters, as well as increasing the distance between the MtlR operator and the −35 box of P mtlA revealed that the activated MtlR molecules and RNA polymerase holoenzyme likely form a class II type activation complex at P mtlA and P mtlR during transcription initiation.

scholarly journals Transcriptional regulation of the rat fatty acid synthase gene: identification and functional analysis of positive and negative effectors of basal transcription

1996 ◽  
Vol 317 (1) ◽  
pp. 257-265 ◽  
Author(s):  
Babak OSKOUIAN ◽  
Vangipuram S. RANGAN ◽  
Stuart SMITH
Keyword(s):  
Fatty Acid ◽  
Electrophoretic Mobility ◽  
Fatty Acid Synthase ◽  
Regulatory Element ◽  
Dnase I ◽  
Mobility Shift ◽  
Negative Regulatory Element ◽  
Dnase I Footprinting ◽  
Ccaat Box ◽  
Electrophoretic Mobility Shift Assays

The gene for fatty acid synthase (FAS), which contains both GC-rich sequences and a TATA box in its promoter region, is expressed in a tissue-specific manner in response to developmental, nutritional and hormonal signals. Here we report the identification of sequence elements in the 5´-flanking region responsible for modulation of basal promoter activity. Transient transfection of H4IIE hepatoma cells and 3T3-30A5 preadipocytes with plasmids containing the chloroamphenicol acetyltransferase gene driven by FAS promoter sequences of different lengths revealed that two regions between nucleotides -249 and -30 contain elements capable of enhancing transcription. One of these positive regulatory elements was localized to nucleotides -241/-236 using DNase I footprinting, electrophoretic mobility-shift assays and mutagenesis. The sequence element is a typical GC box and the nuclear protein binding to this region appears immunochemically indistinguishable from Sp1. The second positive regulatory element, an inverted CCAAT box, was localized to nucleotides -98/-92 by electrophoretic mobility-shift assays and mutagenesis. A putative negative regulatory element, initially identified by reporter gene transfection experiments, was localized between nucleotides -319 and -301 by DNase I footprinting, electrophoretic mobility-shift assays and deletion mutagenesis; this region consists of 78% G residues. In conclusion, initiation of FAS transcription from a single start site is enhanced by the presence of an adjacent TATA motif, an inverted CCAAT box and an upstream binding site for the transcription factor Sp1; further modulation of transcription is achieved through complex interactions between these promoter elements and an upstream negative regulatory element.

scholarly journals VraR Binding to the Promoter Region of agr Inhibits Its Function in Vancomycin-Intermediate Staphylococcus aureus (VISA) and Heterogeneous VISA

2017 ◽  
Vol 61 (5) ◽  
Author(s):  
Yuanyuan Dai ◽  
Wenjiao Chang ◽  
Changcheng Zhao ◽  
Jing Peng ◽  
Liangfei Xu ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Promoter Region ◽  
Reference Strain ◽  
Vancomycin Resistance ◽  
Mobility Shift ◽  
Content Type ◽  
Promoter Sequences ◽  
Dnase I Footprinting ◽  
Resistant Strains ◽  
Electrophoretic Mobility Shift Assays

ABSTRACT Acquisition of vancomycin resistance in Staphylococcus aureus is often accompanied by a reduction in virulence, but the mechanisms underlying this change remain unclear. The present study was undertaken to investigate this process in a clinical heterogeneous vancomycin-intermediate S. aureus (hVISA) strain, 10827; an hVISA reference strain, Mu3; and a VISA reference strain, Mu50, along with their respective series of vancomycin-induced resistant strains. In these strains, increasing MICs of vancomycin were associated with increased expression of the vancomycin resistance-associated regulator gene (vraR) and decreased expression of virulence genes (hla, hlb, and coa) and virulence-regulated genes (RNAIII, agrA, and saeR). These results suggested that VraR might have a direct or indirect effect on virulence in S. aureus. In electrophoretic mobility shift assays, VraR did not bind to promoter sequences of hla, hlb, and coa genes, but it did bind to the agr promoter region. In DNase I footprinting assays, VraR protected a 15-nucleotide (nt) sequence in the intergenic region between the agr P2 and P3 promoters. These results indicated that when S. aureus is subject to induction by vancomycin, expression of vraR is upregulated, and VraR binding inhibits the function of the Agr quorum-sensing system, causing reductions in the virulence of VISA/hVISA strains. Our results suggested that VraR in S. aureus is involved not only in the regulation of vancomycin resistance but also in the regulation of virulence.

scholarly journals Variants in a cis-regulatory element of TBX1 in conotruncal heart defect patients impair GATA6-mediated transactivation

2021 ◽  
Vol 16 (1) ◽  
Author(s):  
Xuechao Jiang ◽  
Tingting Li ◽  
Sijie Liu ◽  
Qihua Fu ◽  
Fen Li ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Regulatory Element ◽  
Expression Patterns ◽  
Human Embryos ◽  
Mobility Shift ◽  
T Box ◽  
Conotruncal Heart Defect ◽  
Velo Cardio Facial Syndrome ◽  
Electrophoretic Mobility Shift Assays

Abstract Background TBX1 (T-box transcription factor 1) is a major candidate gene that likely contributes to the etiology of velo-cardio-facial syndrome/DiGeorge syndrome (VCFS/DGS). Although the haploinsufficiency of TBX1 in both mice and humans results in congenital cardiac malformations, little has been elucidated about its upstream regulation. We aimed to explore the transcriptional regulation and dysregulation of TBX1. Methods Different TBX1 promoter reporters were constructed. Luciferase assays and electrophoretic mobility shift assays (EMSAs) were used to identify a cis-regulatory element within the TBX1 promoter region and its trans-acting factor. The expression of proteins was identified by immunohistochemistry and immunofluorescence. Variants in the cis-regulatory element were screened in conotruncal defect (CTD) patients. In vitro functional assays were performed to show the effects of the variants found in CTD patients on the transactivation of TBX1. Results We identified a cis-regulatory element within intron 1 of TBX1 that was found to be responsive to GATA6 (GATA binding protein 6), a transcription factor crucial for cardiogenesis. The expression patterns of GATA6 and TBX1 overlapped in the pharyngeal arches of human embryos. Transfection experiments and EMSA indicated that GATA6 could activate the transcription of TBX1 by directly binding with its GATA cis-regulatory element in vitro. Furthermore, sequencing analyses of 195 sporadic CTD patients without the 22q11.2 deletion or duplication identified 3 variants (NC_000022.11:g.19756832C > G, NC_000022.11:g.19756845C > T, and NC_000022.11:g. 19756902G > T) in the non-coding cis-regulatory element of TBX1. Luciferase assays showed that all 3 variants led to reduced transcription of TBX1 when incubated with GATA6. Conclusions Our findings showed that TBX1 might be a direct transcriptional target of GATA6, and variants in the non-coding cis-regulatory element of TBX1 disrupted GATA6-mediated transactivation.

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