Reconstitution of the vitamin D-responsive osteocalcin transcription unit in Saccharomyces cerevisiae

1989 ◽  
Vol 9 (8) ◽  
pp. 3517-3523
Author(s):  
D P McDonnell ◽  
J W Pike ◽  
D J Drutz ◽  
T R Butt ◽  
B W O'Malley
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Vitamin D ◽  
Transcription Factors ◽  
Promoter Activity ◽  
Transcription Unit ◽  
Proximal Promoter ◽  
Regulatory Sequences ◽  
Cis Acting ◽  
Osteocalcin Gene ◽  
Definition Of

The human osteocalcin gene is regulated in mammalian osteoblasts by 1,25(OH)2D3-dependent and -independent mechanisms. The sequences responsible for this activity have been mapped to within the -1339 region of the gene. We show here that this enhancer region functions analogously in Saccharomyces cerevisiae cells engineered to produce active 1,25(OH)2D3 receptor. When fused to the proximal promoter elements of the yeast iso-1-cytochrome c gene, the enhancer demonstrated substantial promoter activity. This activity was elevated further by 1,25(OH)2D3 when the reporter constructs were assayed in cells containing the 1,25(OH)2D3 receptor. This system affords a model for 1,25(OH)2D3 action and represents a simple assay system that will enable definition of the important cis-acting regulatory sequences within the osteocalcin gene and identification of their cognate transcription factors.

scholarly journals Reconstitution of the vitamin D-responsive osteocalcin transcription unit in Saccharomyces cerevisiae.

1989 ◽  
Vol 9 (8) ◽  
pp. 3517-3523 ◽  
Author(s):  
D P McDonnell ◽  
J W Pike ◽  
D J Drutz ◽  
T R Butt ◽  
B W O'Malley
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Vitamin D ◽  
Transcription Factors ◽  
Promoter Activity ◽  
Transcription Unit ◽  
Proximal Promoter ◽  
Regulatory Sequences ◽  
Cis Acting ◽  
Osteocalcin Gene ◽  
Definition Of

The human osteocalcin gene is regulated in mammalian osteoblasts by 1,25(OH)2D3-dependent and -independent mechanisms. The sequences responsible for this activity have been mapped to within the -1339 region of the gene. We show here that this enhancer region functions analogously in Saccharomyces cerevisiae cells engineered to produce active 1,25(OH)2D3 receptor. When fused to the proximal promoter elements of the yeast iso-1-cytochrome c gene, the enhancer demonstrated substantial promoter activity. This activity was elevated further by 1,25(OH)2D3 when the reporter constructs were assayed in cells containing the 1,25(OH)2D3 receptor. This system affords a model for 1,25(OH)2D3 action and represents a simple assay system that will enable definition of the important cis-acting regulatory sequences within the osteocalcin gene and identification of their cognate transcription factors.

Regulation of human coagulation factor X gene expression by GATA-4 and the Sp family of transcription factors

Blood ◽  
2001 ◽  
Vol 97 (4) ◽  
pp. 946-951 ◽  
Author(s):  
Hsiao-Ling Hung ◽  
Eleanor S. Pollak ◽  
Rama D. Kudaravalli ◽  
Valder Arruda ◽  
Kirk Chu ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Protein Binding ◽  
Promoter Activity ◽  
Critical Role ◽  
Coagulation Factor ◽  
Proximal Promoter ◽  
Coagulation Cascade ◽  
Clotting Factor ◽  
Factor X ◽  
Specific Expression

Abstract Serine protease factor Xa plays a critical role in the coagulation cascade. Zymogen factor X is synthesized and modified in the liver. To understand the mechanisms governing the liver-specific expression of factor X, the proximal promoter of human factor X was previously characterized. Two crucial cis elements at −73 and −128 and their cognate binding proteins, HNF-4 and NF-Y, respectively, were identified. In this report, studies are extended to 3 additionalcis elements within the factor X promoter. Using gel mobility shift assays, the liver-enriched protein GATA-4 was identified as the protein binding to the GATA element at −96. GATA-4 transactivates the factor X promoter 28-fold in transient transfection experiments. It was also determined that the Sp family of transcription factors binds 2 DNase I–footprinted sites at −165 and −195. Disruption of Sp protein binding at either site reduces the promoter activity by half. Simultaneous disruption of both sites reduces the promoter activity 8-fold. This is the first report indicating the involvement of GATA-4 in the regulation of clotting factor expression. These observations provide novel insight into mechanisms by which the vitamin K–dependent coagulation factors are regulated.

scholarly journals Genomic organization and regulation of the human orexin (hypocretin) receptor 2 gene: identification of alternative promoters

2010 ◽  
Vol 427 (3) ◽  
pp. 377-390 ◽  
Author(s):  
Jing Chen ◽  
Harpal S. Randeva
Keyword(s):  
Transcription Factors ◽  
Protein Kinase ◽  
Promoter Activity ◽  
Camp Response Element Binding ◽  
Proximal Promoter ◽  
Activator Protein 1 ◽  
Alternative Promoters ◽  
Specific Expression ◽  
Reverse Transcription Pcr ◽  
Distal Promoter

Orexins (hypocretins), acting via their receptors, are involved in the control of feeding behaviour, sleep, arousal and energy homoeostasis. However, regulation of the human orexin receptor 2 (hOX2R) gene remains unknown. We have identified four transcripts arising from alternative splicing from three exons. These exon 1 variants were designated exons 1A, 1B and 1C on the basis of their 5′–3′ order. RT (reverse transcription)–PCR demonstrates the differential expression in various human tissues. The alternative 5′-UTRs (untranslated regions) possessed by these isoforms have different translational efficiencies, which regulate the level of protein expression. In the present study, we have demonstrated that the hOX2R gene is regulated by two promoters and the novel transcripts are regulated by the distal promoter located upstream of exon 1A. We have demonstrated that the AP-1 (activator protein 1) motif is critical for sustaining the basal activity of distal promoter. Analysis of the proximal promoter revealed the region regulating promoter activity contained putative binding elements including those for CREB (cAMP-response-element-binding protein), GATA-2 and Oct-1. Using the chromatin immunoprecipitation assay, we demonstrated that CREB, GATA-2 and Oct-1 transcription factors bind to these critical regulatory promoter elements. Mutational studies suggested that these motifs functioned independently, but have a compound effect regulating hOX2R gene transcription. Furthermore, proximal promoter activity is enhanced by both PKA (protein kinase A) and PKC (protein kinase C) pathway activation, via binding of CREB and GATA-2 transcription factors. In conclusion, we have demonstrated that expression of hOX2R is regulated by a complex involving a proximal PKA/PKC-regulated promoter and a distal promoter regulating tissue-specific expression of alternative transcripts which in turn post-transcriptionally regulate receptor levels.

Positive cis-acting regulatory sequences mediate proper control of POL1 transcription in Saccharomyces cerevisiae

1992 ◽  
Vol 21 (3) ◽  
pp. 183-189 ◽  
Author(s):  
Antonella Pizzagalli ◽  
Simonetta Piatti ◽  
Daniele Derossi ◽  
Irene Gander ◽  
Paolo Plevani ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Regulatory Sequences ◽  
Cis Acting

scholarly journals GATA augments GNRH-mediated increases in Adcyap1 gene expression in pituitary gonadotrope cells

2013 ◽  
Vol 51 (3) ◽  
pp. 313-324 ◽  
Author(s):  
Robin L Thomas ◽  
Natalie M Crawford ◽  
Constance M Grafer ◽  
Weiming Zheng ◽  
Lisa M Halvorson
Keyword(s):  
Gene Expression ◽  
Transcription Factors ◽  
Promoter Activity ◽  
Gene Promoter ◽  
Proximal Promoter ◽  
Mrna Levels ◽  
Paracrine Factor ◽  
Mobility Shift ◽  
Subunit Expression ◽  
Mobility Shift Assay

Pituitary adenylate cyclase-activating polypeptide 1 (PACAP or ADCYAP1) regulates gonadotropin biosynthesis and secretion, both alone and in conjunction with GNRH. Initially identified as a hypothalamic-releasing factor, ADCYAP1 subsequently has been identified in pituitary gonadotropes, suggesting it may act as an autocrine–paracrine factor in this tissue. GNRH has been shown to increase pituitaryAdcyap1gene expression through the interaction of CREB and jun/fos with CRE/AP1cis-elements in the proximal promoter. In these studies, we were interested in identifying additional transcription factors and cognatecis-elements which regulateAdcyap1gene promoter activity and chose to focus on the GATA family of transcription factors known to be critical for both pituitary cell differentiation and gonadotropin subunit expression. By transient transfection and electrophoretic mobility shift assay analysis, we demonstrate that GATA2 and GATA4 stimulateAdcyap1promoter activity via a GATAcis-element located at position −191 in the ratAdcyap1gene promoter. Furthermore, we show that addition of GATA2 or GATA4 significantly augments GNRH-mediated stimulation ofAdcyap1gene promoter activity in the gonadotrope LβT2 cell line. Conversely, blunting GATA expression with specific siRNA inhibits the ability of GNRH to stimulate ADCYAP1 mRNA levels in these cells. These data demonstrate a complex interaction between GNRH and GATA on ADCYAP1 expression, providing important new insights into the regulation of gonadotrope function.

Differential configurations involving binding of USF transcription factors and Twist1 regulate Alx3 promoter activity in mesenchymal and pancreatic cells

2013 ◽  
Vol 450 (1) ◽  
pp. 199-208 ◽  
Author(s):  
Patricia García-Sanz ◽  
Antonio Fernández-Pérez ◽  
Mario Vallejo
Keyword(s):  
Gene Expression ◽  
Transcription Factors ◽  
Promoter Activity ◽  
Cell Types ◽  
Regulatory Elements ◽  
Proximal Promoter ◽  
Homeodomain Protein ◽  
Glucose Homoeostasis ◽  
Pancreatic Cells ◽  
Primary Mouse

During embryonic development, the aristaless-type homeodomain protein Alx3 is expressed in the forehead mesenchyme and contributes to the regulation of craniofacial development. In the adult, Alx3 is expressed in pancreatic islets where it participates in the control of glucose homoeostasis. In the present study, we investigated the transcriptional regulation of Alx3 gene expression in these two cell types. We found that the Alx3 promoter contains two E-box regulatory elements, named EB1 and EB2, that provide binding sites for the basic helix–loop–helix transcription factors Twist1, E47, USF (upstream stimulatory factor) 1 and USF2. In primary mouse embryonic mesenchymal cells isolated from the forehead, EB2 is bound by Twist1, whereas EB1 is bound by USF1 and USF2. Integrity of both EB1 and EB2 is required for Twist1-mediated transactivation of the Alx3 promoter, even though Twist1 does not bind to EB1, indicating that binding of USF1 and USF2 to this element is required for Twist1-dependent Alx3 promoter activity. In contrast, in pancreatic islet insulin-producing cells, the integrity of EB2 is not required for proximal promoter activity. The results of the present study indicate that USF1 and USF2 are important regulatory factors for Alx3 gene expression in different cell types, whereas Twist1 contributes to transcriptional transactivation in mesenchymal, but not in pancreatic, cells.

scholarly journals SMAD3 augments FoxO3-induced MuRF-1 promoter activity in a DNA-binding-dependent manner

2014 ◽  
Vol 307 (3) ◽  
pp. C278-C287 ◽  
Author(s):  
Lance M. Bollinger ◽  
Carol A. Witczak ◽  
Joseph A. Houmard ◽  
Jeffrey J. Brault
Keyword(s):  
Transcription Factors ◽  
Dna Binding ◽  
Protein Degradation ◽  
Protein Content ◽  
Promoter Activity ◽  
Proximal Promoter ◽  
Skeletal Muscle Atrophy ◽  
Dependent Manner ◽  
Direct Role ◽  
Human Embryonic Kidney Cells

Muscle-specific RING finger-1 (MuRF-1), a ubiquitin ligase and key regulator of proteasome-dependent protein degradation, is highly expressed during skeletal muscle atrophy. The transcription factor forkhead box O3 (FoxO3) induces MuRF-1 expression, but the direct role of other major atrophy-related transcription factors, such as SMAD3, is largely unknown. The goal of this study was to determine whether SMAD3 individually regulates, or with FoxO3 coordinately regulates, MuRF-1 expression. In cultured myotubes or human embryonic kidney cells, MuRF-1 mRNA content and promoter activity were increased by FoxO3 but not by SMAD3 overexpression. However, FoxO3 and SMAD3 coexpression synergistically increased MuRF-1 mRNA and promoter activity. Mutation of the SMAD-binding element (SBE) in the proximal MuRF-1 promoter or overexpression of a SMAD3 DNA-binding mutant attenuated FoxO3-dependent MuRF-1 promoter activation, showing that SMAD binding to DNA is required for optimal activation of FoxO3-induced transcription of MuRF-1. Using chromatin immunoprecipitation, SMAD3 DNA binding increased FoxO3 abundance and SBE mutation reduced FoxO3 abundance on the MuRF-1 promoter. Furthermore, SMAD3 overexpression dose-dependently increased FoxO3 protein content, and coexpression of FoxO3 and SMAD3 synergistically increased FoxO-dependent gene transcription [assessed with a FoxO response element (FRE)-driven reporter]. Collectively, these results show that SMAD3 regulates transcription of MuRF-1 by increasing FoxO3 binding at a conserved FRE-SBE motif within the proximal promoter region, and by increasing FoxO3 protein content and transcriptional activity. These data are the first to indicate that two major transcription factors regulating protein degradation, FoxO3 and SMAD3, converge to coordinately and directly regulate transcription of MuRF-1.

scholarly journals Graded PU.1 Levels Regulate Granulocyte Vs. Macrophage Genes Via Multiple Enhancer Elements

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 403-403
Author(s):  
Vitek Pospisil ◽  
Pavle Krsmanovic ◽  
Jan Valecka ◽  
Kamila Chramostová ◽  
Vojtech Kulvait ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Cell Fate ◽  
Binding Sites ◽  
Target Genes ◽  
Conflicts Of Interest ◽  
Dna Looping ◽  
Proximal Promoter ◽  
Regulatory Sequences ◽  
3D Interaction ◽  
Chromosome Conformation

Abstract PU.1 is a transcription factor absolutely required for normal hematopoiesis. Cumulating evidence indicates that precise levels of PU.1 expression are critical for differentiation to distinct blood lineages, and if perturbed, even modest decreases in PU.1 can lead to leukemogenesis. In contrast to extensive knowledge of regulation of PU.1 gene itself, the mechanism of how target genes senses different PU.1 levels remain largely unknown. To address this, we used PU.1-/- mouse myeloid progenitors encoding inducible PU.1 transgene (PU.1ER, PUER, Walsh 2002) that allows tight control of PU.1 activity. Interestingly, intermediate PU.1 activity induced differentiation of PUER progenitors into granulocyte like cells, while high PU.1 produced macrophages, supporting the model that different PU.1 expression is not a consequence but a driver of cell fate choice. Global expression analysis using 4 different levels of PU.1 at 8 time points (2-96 hrs) revealed that granulocyte specific genes were activated exclusively by intermediate PU.1 levels in 3 distinct modes: 1. not expressed in progenitors while strongly induced at intermediate PU.1 (e.g. Gelatinase B (Mmp9) and Neutrophil collagenase (NC) 2. moderately expressed in progenitors while strongly activated at intermediate PU.1 and repressed at high PU.1 (e.g. Myeloperaxidase (Mpo) 3. highly expressed in unstimulated progenitors with expression maintained at intermediate PU.1 but strongly repressed at high PU.1 (e.g. Neutrophil elastase (NE), Proteinase 3 (primary granule proteins), Cebpe and Gfi1 (Growth factor independent1) Majority of macrophage genes (incl. CD14, Csf1R, Egr2) were regulated as early PU.1 target genes; being gradually activated by high PU.1 activity within 8hrs. However, most granulocyte genes (NE, Mmp9, Mpo, NC but not Cebpe and GFI1) were late activated PU.1 targets (48 and 96hrs) indicating that these genes are coregulated by additional factor(s), likely an early PU.1 target. Next we analyzed the regulatory sequences (+-50kb) of two genes activated exclusively by intermediate PU.1, Mpo and Mmp9, using own and public ChIP(seq) data of transcription factors (TFs) (PU.1, GFI.1), DNAseI hypersensitive sites, histone modifications (H3K4Me, H3K27Ac, H3K9Ac) and expression of enhancer specific bidirectional ncRNAs (eRNA) (CAGE). 14 Mpo and 16 Mmp9 putative enhancers, selected by above mentioned criteria, were cloned into luciferase vector containing their proximal promoter (PP) and were tested for functional activity in response to PU.1 levels. Interestingly, the PU.1 binding motifs within these regions have a low to intermediate affinity (log of score, Jaspar) and are often present in multiples and/or enriched for binding sites of other lineage determining transcription factors. Although PU.1 bound to all of these DNA regions resembling superenhancer, just a small fraction of PU.1 binding was functionally responsive. Specifically, we identified novel enhancer elements at -3.4 kb and -15kb of MPO which were activated by intermediate (but not high) PU.1 levels. Interestingly, activity of -3.4 kb enhancer required presence of PP, while the -15kb element required presence of both PP and the -3.4kb element. Similar phenomenon was observed at -5kb and +4.6kb (intronic) MMP9 enhancers. Collectively, these observations suggest that a cooperative assembly of several cell type-specific enhancers is required for optimal Mpo and Mmp9 activation. This model is supported by our Chromosome conformation capture (3C) data identifying 3D interaction of these enhancer elements at intermediate PU.1 levels suggesting that PU.1 binding mediates DNA looping that allows enhancer cooperation. In addition, activity of these enhancers at intermediate PU.1 levels was associated with expression of bidirectional noncoding enhancer RNAs, confirming functionality of these elements. In conclusion, our data support the model that PU.1 at intermediate concentration binds to low and intermediate affinity binding sites in several enhancers of granulocyte genes, causing their successive looping and interaction with proximal promoter that leads to transcription activation. The role of cooperating TFs, mechanisms of how granulocyte genes are switched off at high PU.1 concentration and deregulation of these mechanisms in AML are being further studied. Grants 16-05649S P305/12/1033 16-31586A 16-27790A 16-31586A UNCE 204021 PRVOUK P24 Disclosures No relevant conflicts of interest to declare.

scholarly journals SPT13 (GAL11) of Saccharomyces cerevisiae negatively regulates activity of the MCM1 transcription factor in Ty1 elements.

1993 ◽  
Vol 13 (1) ◽  
pp. 63-71 ◽  
Author(s):  
G Yu ◽  
J S Fassler
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Transcription Factor ◽  
Binding Site ◽  
Heterologous Gene Expression ◽  
Transcription Unit ◽  
Negative Control ◽  
Negative Regulation ◽  
Regulatory Sequences ◽  
Protein Levels ◽  
Previous Proposal

The Ty transposable elements of Saccharomyces cerevisiae consist of a single large transcription unit whose expression is controlled by a combination of upstream and downstream regulatory sequences. Errede (B. Errede, Mol. Cell. Biol. 13:57-62, 1993) has shown that among the downstream control sequences is a binding site for the transcription factor, MCM1. A small restriction fragment containing the Ty1 MCM1-binding site exhibits very weak activation of heterologous gene expression. The absence of SPT13 (GAL11) causes a dramatic increase in activity directed by these sequences. This effect is mediated through the MCM1-binding site itself. MCM1 mRNA and protein levels, as well as its affinity for its binding site, are unchanged in the absence of SPT13. Our results suggest that SPT13 has a role in the negative control of MCM1 activity that is likely to be posttranslational. A role for SPT13 in the negative regulation of the activity of the Ty1 MCM1-binding site is consistent with our previous proposal that spt13-mediated suppression of Ty insertion mutations could be attributed to the loss of negative regulation of genes adjacent to Ty elements.

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