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.

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.

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 A specific distal promoter controls γ-glutamyl transpeptidase gene expression in undifferentiated rat transformed liver cells

1997 ◽  
Vol 326 (2) ◽  
pp. 311-320 ◽  
Author(s):  
Seiji NOMURA ◽  
Olivier LAHUNA ◽  
Takanobu SUZUKI ◽  
Arthur BROUILLET ◽  
Marie Noêle CHOBERT ◽  
...  
Keyword(s):  
Hepatoma Cells ◽  
Liver Cells ◽  
Proximal Region ◽  
Activator Protein 1 ◽  
Alternate Splicing ◽  
Specific Expression ◽  
Cdna Sequences ◽  
Cis Element ◽  
Distal Promoter ◽  
Glutamyl Transpeptidase

In rat undifferentiated hepatoma cells, the γ-glutamyl transpeptidase (GGT) gene is transcribed into a 2.3 and a 2.6 kb mRNA which, in contrast with other rat GGT transcripts, are not detected in more differentiated liver cells or adult tissues. Analysis of the cDNA sequences obtained from H5 hepatoma cells reveals that these two transcripts differ from other GGT mRNAs by a 312-nt unique untranslated leader sequence; this sequence maps on the gene in a single exon 10 kb upstream from the GGT promoter IV transcription start site. We established that the 2.6 kb mRNA V-1 and the 2.3 kb GGT mRNA V-2 derive, by alternate splicing, from a primary transcript initiated on a distal promoter on the rat GGT gene. This gene appears to be transcribed from five promoters, and the specific expression of this new distal promoter in undifferentiated hepatoma cells requires binding of activator protein-1 and hepatic nuclear factor 3 specific transcription factors to a composite cis-element in the proximal region of the promoter. The distal GGT promoter, specifically expressed in undifferentiated liver cells, might reflect the expression of that gene in liver precursor cells before they differentiate in the hepatocytic or biliary lineage.

scholarly journals POU domain transcription factor brain 4 confers pancreatic alpha-cell-specific expression of the proglucagon gene through interaction with a novel proximal promoter G1 element.

1997 ◽  
Vol 17 (12) ◽  
pp. 7186-7194 ◽  
Author(s):  
M A Hussain ◽  
J Lee ◽  
C P Miller ◽  
J F Habener
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
Transcription Factors ◽  
Alpha Cell ◽  
Proximal Promoter ◽  
Major Constituent ◽  
Alpha Cells ◽  
Specific Expression ◽  
Pou Domain ◽  
Pancreatic Alpha Cell

The proglucagon gene is expressed in a highly restricted tissue-specific manner in the alpha cells of the pancreatic islet, the hypothalamus, and the small and large intestines. Proglucagon is processed to glucagon and glucagon-like peptides GLP-1 and -2. Glucagon is expressed in alpha cells and regulates glucose homeostasis. GLP-1 is implicated in the control of insulin secretion, food intake, and satiety signaling, and GLP-2 is implicated in regulating small-bowel growth. Cell-specific expression of the proglucagon gene is mediated by proteins that interact with the proximal G1 promoter element which contains several AT-rich domains with binding sites for homeodomain transcription factors. In an attempt to identify major homeodomain proteins involved in pancreatic alpha-cell-specific proglucagon expression, we found that the POU domain transcription factor brain 4 is abundantly expressed in proglucagon-producing islet cell lines and rat pancreatic islets. In the latter, brain 4 and glucagon immunoreactivity colocalize in the outer mantle of islets. Electrophoretic mobility shift assays with specific antisera identify brain 4 as a major constituent of nuclear proteins of glucagon-producing cells that bind to the G1 element of the proglucagon gene proximal promoter. Transcriptional transactivation experiments reveal that brain 4 is a major regulator of proglucagon gene expression by its interaction with the G1 element. The finding that a neuronal transcription factor is involved in glucagon gene transcription may explain the presence of proglucagon in certain areas of the brain as well as in pancreatic alpha cells. Further, this finding supports the idea that the neuronal properties of endodermis-derived endocrine pancreatic cells may find their basis in regulation of gene expression by neuronal transcription factors.

scholarly journals Genetic Variation in the Proximal Promoter of ABC and SLC Superfamilies: Liver and Kidney Specific Expression and Promoter Activity Predict Variation

PLoS ONE ◽  
2009 ◽  
Vol 4 (9) ◽  
pp. e6942 ◽  
Author(s):  
Stephanie E. Hesselson ◽  
Pär Matsson ◽  
James E. Shima ◽  
Hisayo Fukushima ◽  
Sook Wah Yee ◽  
...  
Keyword(s):  
Genetic Variation ◽  
Promoter Activity ◽  
Proximal Promoter ◽  
Specific Expression ◽  
Liver And Kidney

scholarly journals Regulation of human prohormone convertase 2 promoter activity by the transcription factor EGR-1

1997 ◽  
Vol 328 (1) ◽  
pp. 69-74 ◽  
Author(s):  
Erik JANSEN ◽  
A. Y. Torik AYOUBI ◽  
M. P. Sandra MEULEMANS ◽  
Wim J. M. VAN DE VEN
Keyword(s):  
Transcription Factor ◽  
Promoter Region ◽  
Promoter Activity ◽  
Secretory Pathway ◽  
Tata Box ◽  
Proximal Promoter ◽  
Prohormone Convertase ◽  
Specific Expression ◽  
Proximal Promoter Region ◽  
Prohormone Convertase 2

Prohormone convertases are involved in the tissue-specific endoproteolytic processing of prohormones and neuropeptide precursors within the secretory pathway. In the present study, we have isolated genomic clones comprising the 5ʹ-terminal region of the human prohormone convertase 2 (PC2) gene and established characteristics of the PC2 promoter region. The proximal promoter region is very G+C-rich and does not contain a canonical TATA box or a CAAT box. Transient expression assays with a set of human PC2 gene fragments containing progressive 5ʹ deletions demonstrate that the proximal promoter region is capable of directing high levels of neuroendocrine-specific expression of reporter gene constructs. In addition, we show that the transcription factor EGR-1 interacts with two distinct elements within the proximal human PC2 promoter region. Transfection experiments also demonstrate that EGR-1 is able to enhance PC2 promoter activity.

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 Regulation of the gene promoter for extracellular signal-regulated protein kinase 2 by transcription factors NF-Y and Sp3

2000 ◽  
Vol 347 (1) ◽  
pp. 155-161 ◽  
Author(s):  
Naoaki SUGIURA ◽  
Kunio TAKISHIMA
Keyword(s):  
Transcription Factors ◽  
Protein Kinase ◽  
Promoter Activity ◽  
Dominant Negative ◽  
Mitogen Activated Protein Kinase ◽  
Flanking Sequence ◽  
Mobility Shift ◽  
Extracellular Signal ◽  
Promoter Deletion Analysis ◽  
Ccaat Box

We have previously shown that the maximal promoter activity of the gene for extracellular signal-regulated protein kinase 2 (ERK2; also known as p42 mitogen-activated protein kinase) resides in the 371 bp 5ʹ-flanking sequence. In the present study we defined roles for a CCAAT box and two adjacent GC boxes in the activity of this promoter. Deletion analysis and DNase I footprinting of this 371 bp region indicated that the CCAAT box at -64 and GC boxes at -86 and -39 are crucial for promoter activity. Electrophoretic mobility-shift assays showed that transcription factor NF-Y/CBF binds to the CCAAT box. Sp1 and Sp3, members of the Sp family of transcription factors, bind to the GC boxes of the ERK2 promoter. The binding of Sp3 was predominant over that of Sp1. Disruption by mutation of any of the CCAAT box and GC boxes similarly decreased promoter activity. These three cis elements exhibited a moderate synergy in promoter function. The transactivating role of NF-Y was corroborated by the finding that a dominant-negative form of NF-YA diminished the promoter activity. These results provide clues for refining our understanding of not only the regulation of expression of the gene for ERK2 but also mechanisms by which NF-Y and Sp1/Sp3 regulate transcription.

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