scholarly journals Transcriptional Regulation of the Human Growth Hormone Receptor (hGHR) Gene V2 Promoter by Transcriptional Activators and Repressor

2009 ◽  
Vol 23 (3) ◽  
pp. 373-387 ◽  
Author(s):  
Yuhong Wei ◽  
Svetlana Puzhko ◽  
Martin Wabitsch ◽  
Cynthia Gates Goodyer
Keyword(s):  
Mammalian Cells ◽  
Growth Hormone Receptor ◽  
Gene Promoter ◽  
Human Growth ◽  
Housekeeping Gene ◽  
Site Directed Mutagenesis ◽  
Proximal Promoter ◽  
Luciferase Reporter ◽  
Homologous Protein ◽  
As Stress

Abstract The V2 transcript is the major ubiquitously expressed human GH receptor (hGHR) mRNA in all tissues examined to date. In a previous investigation, we defined the V2 promoter as TATA-less and exhibiting many characteristics of a housekeeping gene promoter. We also demonstrated that its basal activity is determined by several different cis-regulatory regions within both the promoter and the V2 exon. In the present study, we used luciferase-reporter, site-directed mutagenesis, gel shift, chromatin immunoprecipitation, and quantitative RT-PCR assays to investigate the ability of certain transcription factors to regulate hGHR V2 transcription through these regions in mammalian cells, including human adipocytes. Ets1 was found to transactivate the V2 proximal promoter through specific Ets sites. Two CCAAT/enhancer-binding protein (C/EBP) family members [C/EBP-homologous protein (CHOP) and C/EBPβ] enhanced V2 transcription via different pathways: indirectly, by association with a V2 exon region (CHOP), and directly, using a V2 proximal promoter noncanonical binding site (C/EBPβ). The Notch signaling mediator, Hes1, potently suppressed V2 promoter activity through interaction with two Hes sites within the V2 exon. We propose that these transcriptional factors regulate hGHR V2 expression by acting as downstream nuclear effectors, linking specific signaling cascades (e.g. MAPK and Notch) triggered by different growth factor-, development-, and nutrition- as well as stress-related stimuli. Our data also suggest that these factors are likely to be important in the differentiation-induced increase in V2 mRNA expression in adipocytes, with Ets1 and CHOP functioning at the preadipocyte stage to prepare the cells for differentiation and increasing C/EBPs and decreasing Hes1 levels contributing during adipocyte maturation.

scholarly journals Stimulated initiation of mitogen-activated protein kinase phosphatase-1 (MKP-1) gene transcription involves the synergistic action of multiple cis-acting elements in the proximal promoter

2004 ◽  
Vol 378 (2) ◽  
pp. 473-484 ◽  
Author(s):  
Stephan RYSER ◽  
Abbas MASSIHA ◽  
Isabelle PIUZ ◽  
Werner SCHLEGEL
Keyword(s):  
Gene Transcription ◽  
Transcription Initiation ◽  
Site Directed Mutagenesis ◽  
Mitogen Activated Protein Kinase ◽  
Proximal Promoter ◽  
In Vivo Studies ◽  
Luciferase Reporter ◽  
Basal Transcription ◽  
Band Shift ◽  
Mitogen Activated Protein

Mitogen-activated protein kinases (MAPKs) are inactivated by a dual specificity phosphatase, MAPK phosphatase-1 (MKP-1). MKP-1 is transcribed as an immediate early response gene (IEG) following various stimuli. In the pituitary cell line GH4C1, MKP-1 gene transcription is strongly induced by thyrotropin-releasing hormone (TRH) as well as by epidermal growth factor (EGF) as a consequence of activated MAPK/extracellular-signal-regulated kinase (ERK) signalling. Intriguingly, reporter gene analysis with the MKP-1 promoter showed strong basal transcription, but only limited induction by TRH and EGF. Site-directed mutagenesis of the reporter construct combined with band-shift and in vivo studies revealed that part of the constitutive activity of the MKP-1 promoter resides in two GC boxes bound by Sp1 and Sp3 transcription factors in the minimal promoter. Basal transcription of transiently transfected luciferase reporter can be initiated by either of the two GC boxes or also by either of the two cAMP/Ca2+ responsive elements or by the E-box present in the proximal promoter. On the other hand, when analysed by stable transfection, the five responsive elements are acting in synergy to transactivate the MKP-1 proximal promoter. We show in this study that the MKP-1 promoter can function as a constitutive promoter or as a rapid and transient sensor for the activation state of MAPKs/ERKs. This dual mode of transcription initiation may have different consequences for the control of a block to elongation situated in the first exon of the MKP-1 gene, as described previously [Ryser, Tortola, van Haasteren, Muda, Li and Schlegel (2001) J. Biol. Chem. 276, 33319–33327].

A 3-Bp Deletion Between Transcription Factor Binding Motifs In the HBS1L-MYB Intergenic Region on Chromosome 6q23 Is Associated with HbF Expression

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1013-1013
Author(s):  
John J. Farrell ◽  
Richard M. Sherva ◽  
Zhi-yi Chen ◽  
Luo Hong-yuan ◽  
Banjamin F. Chu ◽  
...  
Keyword(s):  
Hong Kong ◽  
Transcription Factors ◽  
Dna Sequences ◽  
Globin Gene ◽  
Gene Promoter ◽  
Proximal Promoter ◽  
Luciferase Reporter ◽  
Deletion Polymorphism ◽  
Functional Motif ◽  
A Genome

Abstract Abstract 1013 More than 3% of Chinese in Hong Kong are heterozygous carriers of β-thalassemia. Homozygotes or compound heterozygotes for β-thalassemia are usually severely ill and require monthly transfusions. Increased production of fetal hemoglobin (HbF) can modulate the disease severity by compensating for the shortfall of HbA caused by the β-thalassemia mutations. HbF level in adults varies and is regulated as a multigenic trait. Three major HbF quantitative trait loci (QTL) have been identified: the C/T SNP also known as the Xmn I site at the Gγ-globin gene promoter, the BCL11A polymorphism on chromosome 2p16, and the HBS1L-MYB intergenic polymorphism (HMIP) on chromosome 6q23. The functional motif for each of these 3 QTLs responsible for their effects upon HbF is not known. We undertook a genome-wide association study (GWAS), using Illumina Human 610-Quad BeadChip array, on 619 Chinese β-thalassemia heterozygotes from Hong Kong. In this population, the variance in HbF due to HMIP is 13.5%, significantly higher than that due to BCL11A polymorphism (6.4%). We used 1,000 Genomes Project data, SNP imputation, comparisons of association results across populations, predicted binding of transcription factors, and phylogenetic conservation to identify the functional variant in HMIP. Based on these lines of evidence, a hitherto unreported association between HbF expression and a 3-bp deletion on chromosome 6q23 was found. In 335 Chinese β-thalassemia heterozygotes, the 3-bp deletion polymorphism is in complete linkage disequilibrium with rs9399137, the SNP found in multiple GWAS to be most significantly associated with HbF (P=1.4E-24 in the Chinese cohort GWAS). Flanking this deletion are conserved binding sites for TAL1/SCL1, E47, GATA, and RUNX1/AML1, which are essential erythropoiesis-related transcription factors. The 3-bp deletion changes the normal DNA binding configuration of these transcription factors and spatial configuration for DNA-protein binding and/or protein-protein interactions. Furthermore, this 3-bp deletion polymorphism resides within a likely erythroid distal regulatory region manifested by DNase I hypersensitivity and GATA-1 binding (Wahlberg et al, Blood 114:1254, 2009). We hypothesized that a 61-bp fragment of DNA that encompasses the site of the 3-bp deletion polymorphism might have enhancer-like activity. When ligated to the Gγ-globin gene 1.4 kb proximal promoter linked to a luciferase reporter gene, the 61-bp fragment of DNA enhances the Gγ-globin gene promoter activity by more than 3-fold after transient transfection into K562 cells. A 58-bp fragment of DNA that includes the 3-bp deletion has 60% more enhancer-like activity than the 61-bp fragment without the deletion. These findings suggest that this 3-bp deletion polymorphism is most likely the functional motif accounting for HMIP modulation of HbF. Further studies are needed to identify target genes for this enhancer-like activity mediated by the DNA sequences encompassing the 3-bp deletion polymorphism in HMIP. These studies also suggest that this experimental approach could be used to identify functional motifs in other genotype-phenotype association studies. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Expression of the Hepatic Specific V1 Messenger Ribonucleic Acid of the Human Growth Hormone Receptor Gene Is Regulated by Hepatic Nuclear Factor (HNF)-4α2 and HNF-4α8

2008 ◽  
Vol 22 (2) ◽  
pp. 485-500 ◽  
Author(s):  
Cynthia Cynthia ◽  
Zakaria Rhani ◽  
Hong Zheng
Keyword(s):  
Growth Hormone Receptor ◽  
Receptor Gene ◽  
Regulatory Region ◽  
Human Growth ◽  
Regulatory Elements ◽  
Proximal Promoter ◽  
Target Cells ◽  
Nuclear Factor ◽  
Messenger Ribonucleic Acid ◽  
Specific Regulation

Abstract Human (h) GH plays an essential role in growth and metabolism, and its effectiveness is modulated by the availability of its specific receptor [hGH receptor (hGHR)] on target cells. The hGHR gene has a complex 5′-regulatory region containing multiple first exons. Seven are clustered within two small regions: V2,V3,V9 (module A) and V1,V4,V7,V8 (module B). Module A-derived mRNAs are ubiquitously expressed whereas those from module B are only found in postnatal liver, suggesting developmental- and liver-specific regulation of module B hGHR gene expression. To characterize the elements regulating module B activity, we studied a 1.8-kb promoter of the highest expressing exon in liver, V1. This promoter was repressed in transfection assays; however, either 5′- or 3′-deletions relieved this, suggesting the presence of multiple negative regulatory elements. Six putative hepatic nuclear factor 4 (HNF-4) response elements were identified. We determined that HNF-4α is developmentally regulated in the human liver: HNF-4α2 and HNF-4α8 are expressed in fetal hepatocytes but only HNF-4α2 is expressed in postnatal liver. Transient transfection assays demonstrated that HNF-4α2 and HNF-4α8 have a similar dual effect on V1 transcription: activation via site 1 in the proximal promoter and repression through site 6, approximately 1.7 kb upstream. EMSA/electrophoretic mobility supershift assays and chromatin immunoprecipitation analyses confirmed these two sites are bound by HNF-4α. Based on these data, we speculate there are multiple regions working together to repress the expression of V1 hGHR transcripts in tissues other than the normal postnatal liver, and that HNF-4α is a good candidate for regulating V1 hGHR expression in the human hepatocyte.

scholarly journals Gamma-Globin Gene Promoter Elements Required for Interaction With Globin Enhancers

Blood ◽  
1998 ◽  
Vol 91 (1) ◽  
pp. 309-318 ◽  
Author(s):  
Scott D. Langdon ◽  
Russel E. Kaufman
Keyword(s):  
Binding Sites ◽  
Globin Gene ◽  
Gene Promoter ◽  
Regulatory Elements ◽  
Site Directed Mutagenesis ◽  
Luciferase Reporter ◽  
Promoter Strength ◽  
Gene Promoters ◽  
Wild Type ◽  
Luciferase Reporter Gene

Abstract Normal expression of the human β-globin domain genes is dependent on at least three types of regulatory elements located within the β-globin domain: the locus control region (LCR), globin enhancer elements (3′β and 3′Aγ), and the individual globin gene promoter and upstream regions. It has been postulated that regulation occurs through physical interactions between factors bound to these elements, which are located at considerable distances from each other. To identify the elements required for promoter-enhancer interactions from a distance, we have investigated the expression of the wild-type, truncated, and mutated γ-globin promoters linked to the 5′HS2 enhancer. We show that in K562 cells, 5′HS2 increases activity approximately 20-fold from both a wild-type and truncated (-135 → +25) γ promoter and that truncation or site-directed mutagenesis of the tandem CCAAT boxes eliminated the enhancement by 5′HS2. Mutation of the γ-globin gene promoter GATA-1 binding sites did not decrease either promoter strength or enhancement of activity by 5′HS2. To determine if enhanced expression of γ-globin gene promoters carrying mutations associated with hereditary persistence of fetal hemoglobin (HPFH) was due to greater interactions with enhancers, we linked these HPFH γ-globin gene promoters to 5′HS2 and demonstrated a twofold to threefold higher expression than the corresponding wild-type promoter plus enhancer in MEL cells. Addition of the Aγ-globin gene 3′ enhancer to a plasmid containing the γ-globin gene promoter and 5′HS2 did not further enhance promoter strength. Furthermore, we have demonstrated that the previously identified core 5′HS2 enhancer (46-bp tandem AP-1/NF-E2 sites) increased expression only when located 5′, but not 3′, to the γ-globin-luciferase reporter gene, suggesting that its enhancer effect is not by DNA looping. Our results suggest that CCAAT boxes, but not GATA or CACCC binding sites, are required for interaction between the γ-globin promoter and the LCR/5′HS2 and that regulatory elements in addition to the core enhancer may be required for the enhancer to act from a distance.

scholarly journals Regulation of Human Growth Hormone Receptor Expression by MicroRNAs

2014 ◽  
Vol 28 (9) ◽  
pp. 1448-1459 ◽  
Author(s):  
Samar Elzein ◽  
Cynthia Gates Goodyer
Keyword(s):  
Gene Expression ◽  
Growth Hormone Receptor ◽  
Human Growth ◽  
Receptor Expression ◽  
Hek293 Cells ◽  
Luciferase Reporter ◽  
Target Cells ◽  
Protein Levels ◽  
Coding Regions ◽  
Hormone Receptor Expression

Human GH binds to its receptor (GHR) on target cells and activates multiple intracellular pathways, leading to changes in gene expression, differentiation, and metabolism. GHR deficiency is associated with growth and metabolic disorders whereas increased GHR expression has been reported in certain cancers, suggesting that the GHR gene requires tight controls. Several regulatory mechanisms have been found within its 5′-untranslated region (UTR) promoter and coding regions. However, the 3′-UTR has not been previously examined. MicroRNAs (miRNAs) are small (19–22 nucleotides) noncoding RNAs that downregulate gene expression mainly through targeting the 3′-UTR of mRNAs and enhancing their degradation or inhibiting translation. In the present study, we investigated whether miRNAs regulate GHR expression. To define putative miRNA binding sites in the GHR 3′-UTR, we used multiple in silico prediction tools, analyzed conservation across species and the presence of parallel sites in GH/IGF axis-related genes, and searched for reports linking miRNAs to GHR-related physiological or pathophysiological activities. To test prioritized sites, we cotransfected a wild-type GHR 3′-UTR luciferase reporter vector as well as miRNA binding site mutants into HEK293 cells with miRNA mimics. Furthermore, we tested whether the miRNAs altered endogenous GHR mRNA and protein levels in HEK293 cells and in 2 cancer cell lines (MCF7 and LNCaP). Our experiments have identified miRNA (miR)-129–5p, miR-142–3p, miR-202, and miR-16 as potent inhibitors of human GHR expression in normal (HEK293) and cancer (MCF7 and LNCaP) cells. This study paves the way for the development of miRNA inhibitors as therapeutic agents in GH/GHR-related pathophysiologies, including cancer.

scholarly journals Gamma-Globin Gene Promoter Elements Required for Interaction With Globin Enhancers

Blood ◽  
1998 ◽  
Vol 91 (1) ◽  
pp. 309-318
Author(s):  
Scott D. Langdon ◽  
Russel E. Kaufman
Keyword(s):  
Binding Sites ◽  
Globin Gene ◽  
Gene Promoter ◽  
Regulatory Elements ◽  
Site Directed Mutagenesis ◽  
Luciferase Reporter ◽  
Promoter Strength ◽  
Gene Promoters ◽  
Wild Type ◽  
Luciferase Reporter Gene

Normal expression of the human β-globin domain genes is dependent on at least three types of regulatory elements located within the β-globin domain: the locus control region (LCR), globin enhancer elements (3′β and 3′Aγ), and the individual globin gene promoter and upstream regions. It has been postulated that regulation occurs through physical interactions between factors bound to these elements, which are located at considerable distances from each other. To identify the elements required for promoter-enhancer interactions from a distance, we have investigated the expression of the wild-type, truncated, and mutated γ-globin promoters linked to the 5′HS2 enhancer. We show that in K562 cells, 5′HS2 increases activity approximately 20-fold from both a wild-type and truncated (-135 → +25) γ promoter and that truncation or site-directed mutagenesis of the tandem CCAAT boxes eliminated the enhancement by 5′HS2. Mutation of the γ-globin gene promoter GATA-1 binding sites did not decrease either promoter strength or enhancement of activity by 5′HS2. To determine if enhanced expression of γ-globin gene promoters carrying mutations associated with hereditary persistence of fetal hemoglobin (HPFH) was due to greater interactions with enhancers, we linked these HPFH γ-globin gene promoters to 5′HS2 and demonstrated a twofold to threefold higher expression than the corresponding wild-type promoter plus enhancer in MEL cells. Addition of the Aγ-globin gene 3′ enhancer to a plasmid containing the γ-globin gene promoter and 5′HS2 did not further enhance promoter strength. Furthermore, we have demonstrated that the previously identified core 5′HS2 enhancer (46-bp tandem AP-1/NF-E2 sites) increased expression only when located 5′, but not 3′, to the γ-globin-luciferase reporter gene, suggesting that its enhancer effect is not by DNA looping. Our results suggest that CCAAT boxes, but not GATA or CACCC binding sites, are required for interaction between the γ-globin promoter and the LCR/5′HS2 and that regulatory elements in addition to the core enhancer may be required for the enhancer to act from a distance.

scholarly journals Slc7a11 Modulated by POU2F1 is Involved in Pigmentation in Rabbit

2019 ◽  
Vol 20 (10) ◽  
pp. 2493 ◽  
Author(s):  
Yang Chen ◽  
Shuaishuai Hu ◽  
Lin Mu ◽  
Bohao Zhao ◽  
Manman Wang ◽  
...  
Keyword(s):  
Skin Color ◽  
Site Directed Mutagenesis ◽  
Molecular Regulation ◽  
Luciferase Reporter ◽  
Directed Mutagenesis ◽  
Regulation Mechanism ◽  
Solute Carrier Family ◽  
Depth Analysis ◽  
Solute Carrier ◽  
Fur Color

Solute carrier family 7 member 11 (Slc7a11) is a cystine/glutamate xCT transporter that controls the production of pheomelanin pigment to change fur and skin color in animals. Previous studies have found that skin expression levels of Slc7a11 varied significantly with fur color in Rex rabbits. However, the molecular regulation mechanism of Slc7a11 in pigmentation is unknown. Here, rabbit melanocytes were first isolated and identified. The distribution and expression pattern of Slc7a11 was confirmed in skin from rabbits with different fur colors. Slc7a11 affected the expression of pigmentation related genes and thus affected melanogenesis. Meanwhile, Slc7a11 decreased melanocyte apoptosis, but inhibition of Slc7a11 enhanced apoptosis. Furthermore, the POU2F1 protein was found to bind to the −713 to −703 bp region of Slc7a11 promoter to inhibit its activity in a dual-luciferase reporter and site-directed mutagenesis assay. This study reveals the function of the Slc7a11 in melanogenesis and provides in-depth analysis of the mechanism of fur pigmentation.

scholarly journals Silencing of the Gene for the α-Subunit of Human Chorionic Gonadotropin by the Embryonic Transcription Factor Oct-3/4

1997 ◽  
Vol 11 (11) ◽  
pp. 1651-1658 ◽  
Author(s):  
Limin Liu ◽  
Douglas Leaman ◽  
Michel Villalta ◽  
R. Michael Roberts
Keyword(s):  
Transcription Factor ◽  
Binding Site ◽  
Gene Promoter ◽  
Site Directed Mutagenesis ◽  
Mobility Shift ◽  
Α Subunit ◽  
Choriocarcinoma Cells ◽  
Human Choriocarcinoma Cells ◽  
Electrophoretic Mobility Shift Assays ◽  
Jar Cells

Abstract CG is required for maintenance of the corpus luteum during pregnancy in higher primates. As CG is a heterodimeric molecule, some form of coordinated control must be maintained over the transcription of its two subunit genes. We recently found that expression of human CG β-subunit (hCGβ) in JAr human choriocarcinoma cells was almost completely silenced by the embryonic transcription factor Oct-3/4, which bound to a unique ACAATAATCA octameric sequence in the hCGβ gene promoter. Here we report that Oct-3/4 is also a potent inhibitor of hCG α-subunit (hCGα) expression in JAr cells. Oct-3/4 reduced human GH reporter expression from the −170 hCGα promoter in either the presence or absence of cAMP by about 70% in transient cotransfection assays, but had no effect on expression from either the −148 hCGα or the −99 hCGα promoter. Unexpectedly, no Oct-3/4-binding site was identified within the −170 to −148 region of the hCGα promoter, although one was found around position −115 by both methylation interference footprinting and electrophoretic mobility shift assays. Site-directed mutagenesis of this binding site destroyed the affinity of the promoter for Oct-3/4, but did not affect repression of the promoter. Therefore, inhibition of hCGα gene transcription by Oct-3/4 appears not to involve direct binding of this factor to the site responsible for silencing. When stably transfected into JAr cells, Oct-3/4 reduced the amounts of both endogenous hCGα mRNA and protein by 70–80%. Oct-3/4 is therefore capable of silencing both hCGα and hCGβ gene expression. We suggest that as the trophoblast begins to form, reduction of Oct-3/4 expression permits the coordinated onset of transcription from the hCGα and hCGβ genes.

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