scholarly journals Angiotensin II-Dependent Transcriptional Activation of Human Steroidogenic Acute Regulatory Protein Gene by a 25-kDa cAMP-Responsive Element Modulator Protein Isoform and Yin Yang 1

Endocrinology ◽  
2012 ◽  
Vol 153 (3) ◽  
pp. 1256-1268 ◽  
Author(s):  
Renate K. Meier ◽  
Barbara J. Clark
Keyword(s):  
Rna Polymerase Ii ◽  
Reporter Gene ◽  
Transcriptional Activation ◽  
Regulatory Protein ◽  
Gene Activity ◽  
Proximal Promoter ◽  
Ang Ii ◽  
Reporter Gene Activity ◽  
Steroidogenic Acute Regulatory ◽  
Responsive Element

Transcriptional activation of the steroidogenic acute regulatory protein (STAR) gene is a critical component in the angiotensin II (Ang II)-dependent increase in aldosterone biosynthesis in the adrenal gland. The purpose of this study was to define the molecular mechanisms that mediate the Ang II-dependent increase in STARD1 gene (STAR) expression in H295R human adrenocortical cells. Mutational analysis of the STAR proximal promoter revealed that a nonconsensus cAMP-responsive element located at −78 bp relative to the transcription start site (−78CRE) is required for the Ang II-stimulated STAR reporter gene activity. DNA immunoaffinity chromatography identified a 25-kDa cAMP-responsive element modulator isoform and Yin Yang 1 (YY1) as −78CRE DNA-binding proteins, and Ang II treatment of H295R cells increased expression of that 25-kDa CREM isoform. Small interfering RNA silencing of CREM and YY1 attenuated the Ang II-dependent increases in STAR reporter gene activity and STAR mRNA levels. Conversely, overexpression of CREM and YY1 in COS-1 cells resulted in transactivation of STAR reporter gene activity. Chromatin immunoprecipitation analysis demonstrated recruitment of CREM and YY1 to the STAR promoter along with increased association of the coactivator cAMP response element-binding protein-binding protein (CBP) and increased phosphorylated RNA polymerase II after Ang II treatment. Together our data reveal that the Ang II-stimulated increase in STAR expression in H295R cells requires 25 kDa CREM and YY1. The recruitment of these transcription factors to the STAR proximal promoter results in association of CBP and activation of RNA polymerase II leading to increased STAR transcription.

scholarly journals Cyclic Adenosine 3′,5′-Monophosphate (cAMP) Enhances cAMP-Responsive Element Binding (CREB) Protein Phosphorylation and Phospho-CREB Interaction with the Mouse Steroidogenic Acute Regulatory Protein Gene Promoter

Endocrinology ◽  
2005 ◽  
Vol 146 (3) ◽  
pp. 1348-1356 ◽  
Author(s):  
Brian F. Clem ◽  
Elizabeth A. Hudson ◽  
Barbara J. Clark
Keyword(s):  
Binding Protein ◽  
Regulatory Protein ◽  
Proximal Promoter ◽  
Steroidogenic Acute Regulatory Protein ◽  
Gene Promoters ◽  
Cis Acting ◽  
Steroidogenic Acute Regulatory ◽  
Responsive Element ◽  
Phosphorylated Creb ◽  
Camp Responsive Element Binding

Steroidogenic acute regulatory protein (StAR) transcription is regulated through cAMP-protein kinase A-dependent mechanisms that involve multiple transcription factors including the cAMP-responsive element binding protein (CREB) family members. Classically, binding of phosphorylated CREB to cis-acting cAMP-responsive elements (5′-TGACGTCA-3′) within target gene promoters leads to recruitment of the coactivator CREB binding protein (CBP). Herein we examined the extent of CREB family member phosphorylation on protein-DNA interactions and CBP recruitment with the StAR promoter. Immunoblot analysis revealed that CREB, cAMP-responsive element modulator (CREM), and activating transcription factor (ATF)-1 are expressed in MA-10 mouse Leydig tumor cells, yet only CREB and ATF-1 are phosphorylated. (Bu)2cAMP treatment of MA-10 cells increased CREB phosphorylation approximately 2.3-fold within 30 min but did not change total nuclear CREB expression levels. Using DNA-affinity chromatography, we now show that CREB and ATF-1, but not CREM, interact with the StAR promoter, and this interaction is dependent on the activator protein-1 (AP-1) cis-acting element within the cAMP-responsive region. In addition, (Bu)2cAMP-treatment increased phosphorylated CREB (P-CREB) association with the StAR promoter but did not influence total CREB interaction. In vivo chromatin immunoprecipitation assays demonstrated CREB binding to the StAR proximal promoter is independent of (Bu)2cAMP-treatment, confirming our in vitro analysis. However, (Bu)2cAMP-treatment increased P-CREB and CBP interaction with the StAR promoter, demonstrating for the first time the physical role of P-CREB:DNA interactions in CBP recruitment to the StAR proximal promoter.

scholarly journals Functional Analysis of Multiple Transcription Factor Sites in a Regulatory Element of Human ε-Globin Gene

2004 ◽  
Vol 36 (10) ◽  
pp. 673-680
Author(s):  
Chun-Hui Hou ◽  
Jian Huang ◽  
Ruo-Lan Qian
Keyword(s):  
Reporter Gene ◽  
Regulatory Element ◽  
Globin Gene ◽  
K562 Cells ◽  
Regulatory Elements ◽  
Gene Activity ◽  
Proximal Promoter ◽  
Mobility Shift ◽  
Sv40 Promoter ◽  
Reporter Gene Activity

Abstract The developmental control of the human ε-globin gene expression is mediated by transcriptional regulatory elements in the 5′ flanking DNA of this gene. A previously identified negative regulatory element (–3028 to –2902 bp, termed ε-NRAII) was analyzed and one putative NF-κB site and two GATA sites locate at –3004 bp, –2975 bp and –2948 bp were characterized. Electrophoresis mobility shift assay (EMSA) showed that the putative NF-κB site was specifically bound by nuclear proteins of K562 cells. Data obtained from transient transfection showed that the expression of reporter gene could be upregulated about 50% or 100% respectively when ε-NRAII was inserted upstream of the SV40 promoter or ε-globin gene proximal promoter (−177 bp to +1 bp), suggesting that ε-NRAII might not be a classic silencer. Mutation in the putative NF-κB site or in the GATA site (at –2975 bp) slightly reduced the expression of reporter gene driven by SV40 promoter or ε-globin gene proximal promoter. However, the mutation of GATA site at –2948 bp remarkably reduced the reporter gene activity driven by SV40 promoter, but not by ε-globin gene proximal promoter. Further mutation analysis showed that the negative effect of mutation in GATA site at –2948 bp on SV40 promoter was not affected by the mutation of the putative NF-κB site, whereas it could be abolished by the mutation of GATA site at –2975 bp. Furthermore, the mutation of both GATA sites could synergistically reduce the reporter gene activity driven by ε-globin gene proximal promoter. Those results suggested that ε-NRAII might function differently on the SV40 promoter and ε-globin gene proximal promoter.

scholarly journals Role of Intramitochondrial Arachidonic Acid and Acyl-CoA Synthetase 4 in Angiotensin II-Regulated Aldosterone Synthesis in NCI-H295R Adrenocortical Cell Line

Endocrinology ◽  
2012 ◽  
Vol 153 (7) ◽  
pp. 3284-3294 ◽  
Author(s):  
Pablo G. Mele ◽  
Alejandra Duarte ◽  
Cristina Paz ◽  
Alessandro Capponi ◽  
Ernesto J. Podestá
Keyword(s):  
Arachidonic Acid ◽  
Angiotensin Ii ◽  
Regulatory Protein ◽  
Steroidogenic Acute Regulatory Protein ◽  
Ang Ii ◽  
Steroid Production ◽  
Glomerulosa Cells ◽  
Steroidogenic Acute Regulatory ◽  
Export System

Although the role of arachidonic acid (AA) in angiotensin II (ANG II)- and potassium-stimulated steroid production in zona glomerulosa cells is well documented, the mechanism responsible for AA release is not fully described. In this study we evaluated the mechanism involved in the release of intramitochondrial AA and its role in the regulation of aldosterone synthesis by ANG II in glomerulosa cells. We show that ANG II and potassium induce the expression of acyl-coenzyme A (CoA) thioesterase 2 and acyl-CoA synthetase 4, two enzymes involved in intramitochondrial AA generation/export system well characterized in other steroidogenic systems. We demonstrate that mitochondrial ATP is required for AA generation/export system, steroid production, and steroidogenic acute regulatory protein induction. We also demonstrate the role of protein tyrosine phosphatases regulating acyl-CoA synthetase 4 and steroidogenic acute regulatory protein induction, and hence ANG II-stimulated aldosterone synthesis.

Isotopic Assays for Reporter Gene Activity

2001 ◽  
Vol 63 (1) ◽  
Author(s):  
Robert E. Kingston ◽  
Jen Sheen ◽  
David Moore
Keyword(s):  
Reporter Gene ◽  
Gene Activity ◽  
Reporter Gene Activity

scholarly journals Alternative promoters and repetitive DNA elements define the species-dependent tissue-specific expression of the FMO1 genes of human and mouse

2007 ◽  
Vol 406 (3) ◽  
pp. 491-499 ◽  
Author(s):  
Elizabeth A. Shephard ◽  
Pritpal Chandan ◽  
Milena Stevanovic-Walker ◽  
Mina Edwards ◽  
Ian R. Phillips
Keyword(s):  
Reporter Gene ◽  
Adult Mouse ◽  
Direct Repeat ◽  
Mouse Gene ◽  
Gene Activity ◽  
Upstream Sequence ◽  
Alternative Promoters ◽  
Specific Expression ◽  
Tissue Specific ◽  
Reporter Gene Activity

In humans, expression of the FMO1 (flavin-containing mono-oxygenase 1) gene is silenced postnatally in liver, but not kidney. In adult mouse, however, the gene is active in both tissues. We investigated the basis of this species-dependent tissue-specific transcription of FMO1. Our results indicate the use of three alternative promoters. Transcription of the gene in fetal human and adult mouse liver is exclusively from the P0 promoter, whereas in extra-hepatic tissues of both species, P1 and P2 are active. Reporter gene assays showed that the proximal P0 promoters of human (hFMO1) and mouse (mFmo1) genes are equally effective. However, sequences upstream (−2955 to −506) of the proximal P0 of mFmo1 increased reporter gene activity 3-fold, whereas hFMO1 upstream sequences (−3027 to −541) decreased reporter gene activity by 75%. Replacement of the upstream sequence of human P0 with the upstream sequence of mouse P0 increased activity of the human proximal P0 8-fold. Species-specific repetitive elements are present immediately upstream of the proximal P0 promoters. The human gene contains five LINE (long-interspersed nuclear element)-1-like elements, whereas the mouse gene contains a poly A region, an 80-bp direct repeat, an LTR (long terminal repeat), a SINE (short-interspersed nuclear element) and a poly T tract. The rat and rabbit FMO1 genes, which are expressed in adult liver, lack some (rat) or all (rabbit) of the elements upstream of mouse P0. Thus silencing of FMO1 in adult human liver is due apparently to the presence upstream of the proximal P0 of L1 (LINE-1) elements rather than the absence of retrotransposons similar to those found in the mouse gene.

scholarly journals Human Serum Induces Streptococcal C5a Peptidase Expression

2009 ◽  
Vol 77 (9) ◽  
pp. 3817-3825 ◽  
Author(s):  
Ute Gleich-Theurer ◽  
Simone Aymanns ◽  
Gregor Haas ◽  
Stefanie Mauerer ◽  
Julia Vogt ◽  
...  
Keyword(s):  
Human Serum ◽  
Reporter Gene ◽  
Bovine Serum ◽  
Calf Serum ◽  
Gene Activity ◽  
Pcr Analysis ◽  
Reporter Gene Activity ◽  
Reverse Transcription Pcr ◽  
Bovine Strain ◽  
Human Infections

ABSTRACTStreptococcus agalactiaeis a major pathogen in humans and animals. Virulence factors are often associated with mobile genetic elements, and their expression can be modulated by host factors.S. agalactiaeharbors the genes for C5a peptidase (scpB) and Lmb on a composite transposon structure which is absent in many bovine isolates. To investigate whether these genes participate in the adaptation to human hosts, we determined the influence of human and bovine serum on the promoter activity ofscpBandlmbby using fluorescence-activated cell sorter analysis. Culture in the presence of 1 to 50% human serum resulted in a dose-dependent induction of reporter gene activity forscpBbut notlmb. Reporter gene activity was, however, unchanged following growth in fetal calf serum. Interestingly, a bovine strain did not display any induction ofscpBby either bovine or human serum. Reverse transcription-PCR analysis was used to confirm differential induction ofscpBinS. agalactiaeand showed a similar induction of theStreptococcus pyogenesC5a peptidase genescpAby human but not bovine serum. The specific induction of the streptococcal C5a peptidase by human serum corresponds to the absence ofscpBin many bovineS. agalactiaeisolates and underlines the importance of this virulence factor for human infections.

scholarly journals A simple protocol for preparation of a liposomal vesicle with encapsulated plasmid DNA that mediate high accumulation and reporter gene activity in tumor tissue

2011 ◽  
Vol 1 (1) ◽  
pp. 49-56 ◽  
Author(s):  
Torben Gjetting ◽  
Thomas Lars Andresen ◽  
Camilla Laulund Christensen ◽  
Frederik Cramer ◽  
Thomas Tuxen Poulsen ◽  
...  
Keyword(s):  
Reporter Gene ◽  
Plasmid Dna ◽  
Tumor Tissue ◽  
Gene Activity ◽  
High Accumulation ◽  
Reporter Gene Activity ◽  
Simple Protocol
Sign in / Sign up

Export Citation Format

Share Document