The functions and mechanisms of sequence differences of DGAT1 gene on milk fat synthesis between dairy cow and buffalo

2020 ◽  
Vol 87 (2) ◽  
pp. 170-174
Author(s):  
Dinesh Bhattarai ◽  
Rahim Dad ◽  
Tesfay Worku ◽  
Sutong Xu ◽  
Farman Ullah ◽  
...  
Keyword(s):  
Dairy Cows ◽  
Milk Fat ◽  
Promoter Region ◽  
Promoter Activity ◽  
Core Promoter ◽  
293T Cell ◽  
Buffalo Milk ◽  
Luciferase Reporter ◽  
Plasmid Vectors ◽  
Candidate Mutation

AbstractIn this research communication we describe the DGAT1 sequence and promoter region in dairy cows and buffalo and compare the activities of DGAT1 between the two species in order to increase knowledge of the cause of milk fat variation. pGL-3 basic vectors were used to construct the reporter gene. Based on the predicted promoter region, 4 truncated plasmid vectors were constructed in cow-DGAT1 and 3 plasmid vectors in buffalo-DGAT1. Each reporter plasmid was transfected into the bovine mammary epithelial cell (BMEC), 293T cell, and CHO cells to analyze the activity using Dual-Luciferase Reporter Assay System. The results show that the region between −93 to −556 bp was essential for cow promoter activity while −84 to −590 bp was essential for buffalo promoter activity revealing these regions contain core promoter. The buffalo has higher promoter activity than cow yet it was not statistically significant. Comparison of candidate mutation K232A between cow and buffalo population revealed the presence of both the allelic population in dairy cows (lysine and alanine) however, only K (lysine) allelic amino acid was found in buffalo population. The absence of the alanine allelic population from buffalo explains the higher fat content of buffalo milk.

scholarly journals Functional analysis of the promoter region of the human phosphotyrosine phosphatase activator gene: Yin Yang 1 is essential for core promoter activity

1999 ◽  
Vol 344 (3) ◽  
pp. 755-763 ◽  
Author(s):  
Veerle JANSSENS ◽  
Christine VAN HOOF ◽  
Ivo DE BAERE ◽  
Wilfried MERLEVEDE ◽  
Jozef GORIS
Keyword(s):  
Binding Sites ◽  
Promoter Region ◽  
Promoter Activity ◽  
Cpg Island ◽  
Luciferase Reporter ◽  
Binding Motif ◽  
Phosphotyrosine Phosphatase ◽  
Yin Yang 1 ◽  
Yin Yang

The phosphotyrosine phosphatase activator (PTPA) has been isolated as an in vitro regulator of protein phosphatase 2A. Human PTPA is encoded by a single gene, the structure and chromosomal localization of which have been determined in our previous work. Here we describe the further isolation, sequencing and functional characterization of the PTPA promoter region. In agreement with its ubiquitous expression, the PTPA promoter displays several characteristics of housekeeping genes: it lacks both a TATA-box and a CAAT-box, it is very GC-rich and it contains an unmethylated CpG island surrounding the transcription initiation site. Transient transfection experiments in different cell types with several truncated chimaeric luciferase reporter gene plasmids revealed the importance of the region between positions -67 and -39 for basal promoter activity. This region coincides remarkably well with the determined CpG island. Further analysis of this region demonstrated the presence of a Yin Yang 1 (YY1) binding motif at positions -52 to -44. Binding of YY1 to this sequence is demonstrated in bandshift and DNase I footprinting experiments. Another YY1 binding motif is found in the 5ʹ untranslated region, at positions +27 to +35. Mutations in either of these sites, abolishing YY1 binding in vitro, have differential effects on promoter activity. Point mutations in both sites completely abolish promoter activity. Moreover, induction of promoter activity by co-transfection with a YY1 expression plasmid is fully dependent upon the presence of both intact YY1 binding sites. Thus YY1 apparently mediates basal transcription of the human PTPA gene through two binding sites within its proximal promoter.

Selective Sp1 Binding Is Critical for Maximal Activity of the Human c-kit Promoter

Blood ◽  
1998 ◽  
Vol 92 (11) ◽  
pp. 4138-4149
Author(s):  
Gyeong H. Park ◽  
Howard K. Plummer ◽  
Geoffrey W. Krystal
Keyword(s):  
Binding Site ◽  
Transcription Initiation ◽  
Promoter Activity ◽  
Core Promoter ◽  
Maximal Activity ◽  
Site Directed Mutagenesis ◽  
Luciferase Reporter ◽  
Rnase Protection ◽  
Kit Gene ◽  
Core Promoter Activity

The receptor tyrosine kinase c-kit is necessary for normal hematopoiesis, the development of germ cells and melanocytes, and the pathogenesis of certain hematologic and nonhematologic malignancies. To better understand the regulation of the c-kit gene, a detailed analysis of the core promoter was performed. Rapid amplification of cDNA ends (RACE) and RNase protection methods showed two major transcriptional initiation sites. Luciferase reporter assays using 5′ promoter deletion-reporter constructs containing up to 3 kb of 5′ sequence were performed in hematopoietic and small-cell lung cancer cell lines which either did or did not express the endogenous c-kit gene. This analysis showed the region 83 to 124 bp upstream of the 5′ transcription initiation site was crucial for maximal core promoter activity. Sequence analysis showed several potential Sp1 binding sites within this highly GC-rich region. Gel shift and DNase footprinting showed that Sp1 selectively bound to a single site within this region. Supershift studies using an anti-Sp1 antibody confirmed specific Sp1 binding. Site-directed mutagenesis of the −93/−84 Sp1 binding site reduced promoter-reporter activity to basal levels in c-kit–expressing cells. Cotransfection into DrosophilaSL2 cells of a c-kit promoter-reporter construct with an Sp1 expression vector showed an Sp1 dose-dependent enhancement of expression that was markedly attenuated by mutation of the −93/−84 site. These results indicate that despite the fact that the human c-kit promoter contains multiple potential Sp1 sites, Sp1 binding is a selective process that is essential for core promoter activity.

Characterization of the rat intestinal Fc receptor (FcRn) promoter: transcriptional regulation of FcRn gene by the Sp family of transcription factors

2004 ◽  
Vol 286 (6) ◽  
pp. G922-G931 ◽  
Author(s):  
Lingling Jiang ◽  
Jiafang Wang ◽  
R. Sergio Solorzano-Vargas ◽  
Hugh V. Tsai ◽  
Edgar M Gutierrez ◽  
...  
Keyword(s):  
Transcriptional Regulation ◽  
Cell Lines ◽  
Promoter Region ◽  
Core Promoter ◽  
Dnase I ◽  
Fc Receptor ◽  
Minimal Promoter ◽  
Luciferase Reporter ◽  
Core Promoter Region

The regulatory elements that control the transcriptional regulation of the intestinal Fc receptor ( FcRn) have not been elucidated. The objective of this study was to characterize the core promoter region of the rat FcRn gene. Chimeric clones that contained various regions of the promoter located upstream of the luciferase reporter were transiently transfected into either IEC-6 or Caco-2 cell lines and nuclear extracts were used to perform DNase I footprint and DNA binding assays (EMSA). Transfection of chimeric upstream nested deletions-luciferase reporter clones into either of these cell lines supported robust reporter activity and identified the location of the minimal promoter at −157/+135. DNase I footprint analysis revealed two complexes located within the gene's core promoter region, and site-directed mutagenesis identified two regions that were critical to maintain basal expression. EMSA identified the presence of five Sp elements within the immediate promoter region that are capable of binding members of the Sp family of proteins. Among the five Sp elements, one element appears to not bind Sp1, Sp2, or Sp3 while influencing the interaction of Sp proteins with an adjacent Sp site. Overexpression of either Sp1 or Sp3 augments activity of the minimal promoter in Sp-deficient Drosophila SL2 cells. In summary, we report on the characterization of the rat FcRn minimal promoter, including the characterization of five Sp elements within this region that interact with members of the Sp family of transcriptional factors and drive promoter activity in intestinal cell lines.

scholarly journals The NRF-1/α-PAL transcription factor regulates human E2F6 promoter activity

2004 ◽  
Vol 383 (3) ◽  
pp. 529-536 ◽  
Author(s):  
Zoulika KHERROUCHE ◽  
Yvan DE LAUNOIT ◽  
Didier MONTE
Keyword(s):  
Promoter Region ◽  
Promoter Activity ◽  
Core Promoter ◽  
Dominant Negative ◽  
Site Directed Mutagenesis ◽  
Nuclear Respiratory Factor ◽  
Nuclear Respiratory Factor 1 ◽  
Dnase I Footprinting ◽  
Dominant Negative Form

E2F6 is widely expressed in human tissues and cell lines. Recent studies have demonstrated its involvement in developmental patterning and in the regulation of various genes implicated in chromatin remodelling. Despite a growing number of studies, nothing is really known concerning the E2F6 expression regulation. To understand how cells control E2F6 expression, we analysed the activity of the previously cloned promoter region of the human E2F6 gene. DNase I footprinting, gel electrophoreticmobility shift, transient transfection and site-directed mutagenesis experiments allowed the identification of two functional NRF-1/α-PAL (nuclear respiratory factor-1/α-palindrome-binding protein)-binding sites within the human E2F6 core promoter region, which are conserved in the mouse and rat E2F6 promoter region. Moreover, ChIP (chromatin immunoprecipitation) analysis demonstrated that overexpressed NRF-1/α-PAL is associated in vivo with the E2F6 promoter. Furthermore, overexpression of full-length NRF-1/α-PAL enhanced E2F6 promoter activity, whereas expression of its dominant-negative form reduced the promoter activity. Our results indicate that NRF-1/α-PAL is implicated in the regulation of basal E2F6 gene expression.

Abstract 551: Deficiency of Hepatic Nuclear Factor 4 Alpha Results in Impaired Metabolism of Endogenous Methylarginines and Beta-aminoisobutiric Acid - A Novel Mechanism of Cardiovascular Complications in Patients With Type 2 Diabetes?

2015 ◽  
Vol 35 (suppl_1) ◽  
Author(s):  
Dmitrii V Burdin ◽  
Alexey A Kolobov ◽  
Anton V Demyanov ◽  
Alexey A Soshnev ◽  
Chad N Broker ◽  
...  
Keyword(s):  
Lipid Metabolism ◽  
Binding Site ◽  
Promoter Region ◽  
Core Promoter ◽  
Luciferase Reporter ◽  
Diabetic Patients ◽  
Mrna Levels ◽  
Nuclear Factor ◽  
Wild Type

Introduction: Alanine-glyoxylate aminotransferase 2 (AGXT2) is the only known enzyme capable of degradation of all three endogenous methylarginines, which serve as markers and potentially mediators of cardiovascular disease. Recent studies also suggest that AGXT2 and its alternative substrate beta-aminoisobutyric acid (BAIB) play important role in lipid metabolism. The predicted core promoter region of mammalian AGXT2 promoter contains a highly conserved putative binding site for hepatic nuclear factor 4 alpha (HNF4A). Patients with severe deficiency in HNF4a develop maturity onset diabetes of young 1. Furthermore, polymorphisms of HNF4A are associated with increased risk of diabetes type 2. The aim of this study was to test the hypothesis that HNF4A is a major regulator of AGXT2 expression and activity. Methods and results: We demonstrated direct binding of HNF4A to the Agxt2 promoter region in hepatic cell line Hepa 1-6 using chromatin immunoprecipitation assays. Then we showed that mutations of the predicted HNF4A binding site in the Agxt2 core promoter result in up to 80% decrease in the promoter activity as assessed by luciferase reporter assays (p<0.001). We used siRNA-mediated knockdown of HNF4A to determine whether this factor is required for basal Agxt2 expression in Hepa 1-6 cells. Knockdown of HNF4A led to almost 50% reduction in Agxt2 mRNA levels compared to controls (p<0.01). We took advantage of the previously characterized inducible liver-specific Hnf4a knockout (KO) mice to determine whether HNF4A regulates Agxt2 expression in vivo and showed a 90% (p<0.001) decrease in liver Agxt2 expression and a 85% (p<0.01) decrease in liver AGXT2 activity towards methylarginines in Hnf4a KO mice compared with the wild-type littermates. Finaly, on a functional level, Hnf4a KO mice had significant amounts of BAIB present in plasma, whereas BAIB was not detectable in the plasma of the wild-type littermates. Conclusions: In our study we identified HNF4A as the major regulator of Agxt2 gene expression. This finding suggests that diabetic patients with HNF4A deficiency might have a unique mechanism for development of cardiovascular complication via AGXT2-dependent impairment of lipid metabolism and methylarginines-mediated vascular dysfunction.

Genomic structure and promoter characterization of the human Sprouty4 gene, a novel regulator of lung morphogenesis

2004 ◽  
Vol 287 (1) ◽  
pp. L52-L59 ◽  
Author(s):  
Wei Ding ◽  
Saverio Bellusci ◽  
Wei Shi ◽  
David Warburton
Keyword(s):  
Promoter Activity ◽  
Molecular Mechanisms ◽  
Core Promoter ◽  
Genomic Structure ◽  
Inducible Promoter ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Significant Similarity ◽  
The Core ◽  
Flanking Region

The expression of Sprouty4 ( Spry4), an intracellular FGF receptor antagonist, shows a temporally and spatially restricted pattern in embryonic lung and is induced by ERK signaling. To clarify the molecular mechanisms regulating Spry4 transcription, the genomic structure of the human Sprouty4 ( hSpry4) gene was first determined by using the GenomeWalker kit. The hSpry4 gene spans > 14 kb and is organized in three exons and two introns. Multiple transcription start sites were subsequently mapped by 5′-rapid amplification of cDNA ends. Analysis of up to 4 kb of sequence in the 5′-flanking region of the gene showed the presence of multiple potential transcription factor binding sites but no TATA or CAAT boxes. Transient transfection using luciferase reporter gene constructs with progressive deletions of the hSpry4 5′-flanking region revealed that the core promoter activity is located within the proximal 0.4-kb region, whereas the minimal ERK-inducible promoter activity is between −69 and −31. Homology analysis further showed that the core promoter region of the hSpry4 gene exhibits significant similarity to the 5′-flanking region of the mouse gene.

scholarly journals Identification and analysis of the promoter region of the human methionine sulphoxide reductase A gene

2005 ◽  
Vol 393 (1) ◽  
pp. 321-329 ◽  
Author(s):  
Antonella De Luca ◽  
Paolo Sacchetta ◽  
Carmine Di Ilio ◽  
Bartolo Favaloro
Keyword(s):  
Cell Lines ◽  
Transcription Initiation ◽  
Promoter Region ◽  
Promoter Activity ◽  
Regulatory Region ◽  
Initiation Site ◽  
Luciferase Reporter ◽  
Mcf7 Cells ◽  
Luciferase Reporter Gene ◽  
Hek 293

MsrA (methionine sulphoxide reductase A) is an antioxidant repair enzyme that reduces oxidized methionine to methionine. Moreover, the oxidation of methionine residues in proteins is considered to be an important consequence of oxidative damage to cells. To understand mechanisms of human msrA gene expression and regulation, we cloned and characterized the 5′ promoter region of the human msrA gene. Using 5′-RACE (rapid amplification of cDNA ends) analysis of purified mRNA from human cells, we located the transcription initiation site 59 nt upstream of the reference MsrA mRNA sequence, GenBank® accession number BC 054033. The 1.3 kb of sequence located upstream of the first exon of msrA gene was placed upstream of the luciferase reporter gene in a pGL3-Basic vector and transfected into different cell lines. Sequentially smaller fragments of the msrA promoter region were generated by PCR, and expression levels were monitored from these constructs within HEK-293 and MCF7 human cell lines. Analysis of deletion constructs revealed differences in promoter activity in these cell lines. In HEK-293 cells, the promoter activity was constant from the minimal promoter region to the longest fragment obtained. On the other hand, in MCF7 cells we detected a down-regulation in the longest fragment. Mutation of a putative negative regulatory region that is located between −209 and −212 bp (the CCAA box) restored promoter activity in MCF7 cells. The location of the msrA promoter will facilitate analysis of the transcriptional regulation of this gene in a variety of pathological contexts.

The g.763G>C SNP of the bovine FASN gene affects its promoter activity via Sp-mediated regulation: implications for the bovine lactating mammary gland

2008 ◽  
Vol 34 (2) ◽  
pp. 144-148 ◽  
Author(s):  
Laura Ordovás ◽  
Rosa Roy ◽  
Sandra Pampín ◽  
Pilar Zaragoza ◽  
Rosario Osta ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Mammary Gland ◽  
Milk Fat ◽  
Promoter Activity ◽  
Transcription Factor Binding ◽  
Fat Content ◽  
Luciferase Reporter ◽  
Binding Ability ◽  
Factor Binding ◽  
Lactating Mammary Gland

Fatty acid synthase (FASN) is an enzyme that catalyzes de novo synthesis of fatty acids in cells. The bovine FASN gene maps to BTA 19, where several quantitative trait loci for fat-related traits have been described. Our group recently reported the identification of a single nucleotide polymorphism (SNP), g.763G>C, in the bovine FASN 5′ flanking region that was significantly associated with milk fat content in dairy cattle. The g.763G>C SNP was part of a GC-rich region that may constitute a cis element for members of the Sp transcription factor family. Thus the SNP could alter the transcription factor binding ability of the FASN promoter and consequently affect the promoter activity of the gene. However, the functional consequences of the SNP on FASN gene expression are unknown. The present study was therefore directed at elucidating the underlying molecular mechanism that could explain the association of the SNP with milk fat content. Three cellular lines (3T3L1, HepG2, and MCF-7) were used to test the promoter and the transcription factor binding activities by luciferase reporter assays and electrophoretic mobility shift assays, respectively. Band shift assays were also carried out with nuclear extracts from lactating mammary gland (LMG) to further investigate the role of the SNP in this tissue. Our results demonstrate that the SNP alters the bovine FASN promoter activity in vitro and the Sp1/Sp3 binding ability of the sequence. In bovine LMG, the specific binding of Sp3 may account for the association with milk fat content.

scholarly journals Gfi1 Is Repressed by p53 and Inhibits DNA Damage-Induced Apoptosis

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1228-1228
Author(s):  
Pei Du ◽  
Fangqiang Tang ◽  
Yaling Qiu ◽  
Fan Dong
Keyword(s):  
Dna Damage ◽  
Promoter Region ◽  
Core Promoter ◽  
Critical Role ◽  
Hematopoietic Cells ◽  
Luciferase Reporter ◽  
Mrna Levels ◽  
Core Promoter Region ◽  
Promoter Fragment ◽  
Induced Apoptosis

Abstract Abstract 1228 Gfi1 is a transcriptional repressor that plays a critical role in hematopoiesis. Gfi1 has also been implicated in lymphomagenesis when aberrantly activated as a result of deregulated expression. It is still poorly understood how Gfi1 expression is regulated and how it acts in the hematopoietic system. We show here that Gfi1 transcription was repressed by the tumor suppressor p53 in hematopoietic cells. Treatment of cells with doxorubicin (Doxo), which induces topoisomerase II-mediated DNA double strand breaks (DSB), led to a steady increase in p53 protein level, which was accompanied by a gradual decline in Gfi1 expression at both protein and mRNA levels. Knockdown of p53 resulted in increased Gfi1 expression and also abolished Doxo-induced Gfi1 downregulation. In contrast, Gfi1 expression was reduced and its downregulation in response to DNA damage was restored in p53-deficient cells transfected with the p53/estrogen receptor ligand binding domain fusion protein (p53ERTAM) in the presence of 4-hydroxytamoxifen (4-OHT). In luciferase reporter assays, Doxo treatment inhibited the activity of a ∼2.4-kb Gfi1 promoter fragment in p53+/+ cells, but not in p53−/− cells. Consistent with this, the wild type p53, but not a DNA binding-defective p53 mutant, repressed the Gfi1 promoter fragment. Chromatin immunoprecipitation (ChIP) assays demonstrated that p53 bound to the proximal region of the Gfi1 promoter. Detailed mapping of the Gfi1 promoter indicated that the core promoter region of Gfi1 spanning −33 to +6 bp is sufficient for p53-mediated repression. This core promoter region contains a putative p53 repressive response element (RRE) and, when the RRE was mutated, p53 failed to bind to and repress the Gfi1 promoter. Significantly, apoptosis induction by Doxo treatment was inhibited upon Gfi1 overexpression, but augmented following Gfi1 knockdown. Together, these data establish for the first time that Gfi1 is repressed by p53 and indicate that Gfi1 acts to protect hematopoietic cells from DNA damage-induced apoptosis. Our findings have important implications for understanding the roles of Gfi-1 in normal hematopoiesis and lymphomagenesis. Disclosures: No relevant conflicts of interest to declare.

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