scholarly journals HDAC6/FOXP3/HNF4α axis promotes bile acids induced gastric intestinal metaplasia

2021 ◽  
Author(s):  
Na Wang ◽  
Siran Wu ◽  
Luyao Zhang ◽  
Min Chen ◽  
Jiaoxia Zeng ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Intestinal Metaplasia ◽  
Bile Acids ◽  
Epigenetic Modification ◽  
Bile Reflux ◽  
Precancerous Lesion ◽  
Ectopic Expression ◽  
Histone Deacetylation ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene

Abstract Background: Gastric intestinal metaplasia (IM) is an important precancerous lesion. Our previous study has shown that ectopic expression of HDAC6 promotes the activation of intestinal markers in bile acids (BA) induced gastric IM cells; however, the mechanism underlying how HDAC6-mediated epigenetic modifications regulate intestinal markers is not clear.Methods: RNA-sequencing (RNA-seq) was used to detect the molecular changes in GES-1 cells after HDAC6 overexpression. The potential binding sites of FOXP3 with the promoter region of HNF4α were verified by ChIP and luciferase reporter gene assays. The ChIP assay was also used to detect the histone deacetylation. The levels of mucin in gastric or intestinal mucosa were detected by AB-PAS staining. Transgenic mice were used to explore the pro-metaplastic function of DCA and HNF4α in vivo.Results: Deoxycholic acid (DCA) upregulated HDAC6 in gastric cells, which further inhibited the transcription of FOXP3. Then, FOXP3 transcriptionally inhibited HNF4α, which further inhibits the expression of downstream intestinal markers. These molecules have been shown to be clinically relevant, as FOXP3 levels were negatively correlated with HDAC6 and HNF4α in IM tissues. Transgenic mice experiments confirmed that HNF4α overexpression combined with DCA induced gastric mucosa to secrete intestinal mucus and caused an abnormal mucosal structure. Conclusions: Our findings suggest that HDAC6 reduces FOXP3 through epigenetic modification, thus forming HDAC6/FOXP3/HNF4α axis to promote gastric IM. Inhibition of HDAC6 may be a potential approach to prevent gastric IM in patients with bile reflux.

scholarly journals HDAC6/FOXP3/HNF4α Axis Promotes Bile Acids Induced Gastric Intestinal Metaplasia

2021 ◽  
Author(s):  
Na Wang ◽  
Siran Wu ◽  
Luyao Zhang ◽  
Min Chen ◽  
Jiaoxia Zeng ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Intestinal Metaplasia ◽  
Bile Acids ◽  
Epigenetic Modification ◽  
Bile Reflux ◽  
Precancerous Lesion ◽  
Ectopic Expression ◽  
Histone Deacetylation ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene

Abstract Background: Gastric intestinal metaplasia (IM) is an important precancerous lesion. Our previous study has shown that ectopic expression of HDAC6 promotes the activation of intestinal markers in bile acids (BA) induced gastric IM cells; however, the mechanism underlying how HDAC6-mediated epigenetic modifications regulate intestinal markers is not clear.Methods: RNA-sequencing (RNA-seq) was used to detect the molecular changes in GES-1 cells after HDAC6 overexpression. The potential binding sites of FOXP3 with the promoter region of HNF4α were verified by ChIP and luciferase reporter gene assays. The ChIP assay was also used to detect the histone deacetylation. The levels of mucin in gastric or intestinal mucosa were detected by AB-PAS staining. Transgenic mice were used to explore the pro-metaplastic function of DCA and HNF4α in vivo.Results: Deoxycholic acid (DCA) upregulated HDAC6 in gastric cells, which further inhibited the transcription of FOXP3. Then, FOXP3 transcriptionally inhibited HNF4α, which further inhibits the expression of downstream intestinal markers. These molecules have been shown to be clinically relevant, as FOXP3 levels were negatively correlated with HDAC6 and HNF4α in IM tissues. Transgenic mice experiments confirmed that HNF4α overexpression combined with DCA induced gastric mucosa to secrete intestinal mucus and caused an abnormal mucosal structure. Conclusions: Our findings suggest that HDAC6 reduces FOXP3 through epigenetic modification, thus forming HDAC6/FOXP3/HNF4α axis to promote gastric IM. Inhibition of HDAC6 may be a potential approach to prevent gastric IM in patients with bile reflux.

scholarly journals HDAC6/HNF4α loop mediated by miR-1 promotes bile acids-induced gastric intestinal metaplasia

2020 ◽  
Vol 24 (1) ◽  
pp. 103-116
Author(s):  
Na Wang ◽  
Min Chen ◽  
Zhen Ni ◽  
Ting Li ◽  
Jiaoxia Zeng ◽  
...  
Keyword(s):  
Mouse Model ◽  
Intestinal Metaplasia ◽  
Bile Acids ◽  
Transgenic Mouse ◽  
Cell Model ◽  
Critical Role ◽  
Precancerous Lesion ◽  
Transgenic Mouse Model ◽  
Luciferase Reporter ◽  
Gastric Intestinal Metaplasia

Abstract Background Gastric intestinal metaplasia (IM) is considered a precancerous lesion, and bile acids (BA) play a critical role in the induction of IM. Ectopic expression of HNF4α was observed in a BA-induced IM cell model. However, the mechanisms underlying the upregulation of the protein in IM cells remains to be elucidated. Methods The effects of HNF4α on gastric mucosal cells in vivo were identified by a transgenic mouse model and RNA-seq was used to screen downstream targets of deoxycholic acid (DCA). The expression of pivotal molecules and miR-1 was detected by immunohistochemistry and in situ hybridization in normal, gastritis and IM tissue slides or microarrays. The transcriptional regulation of HDAC6 was investigated by chromatin immunoprecipitation (ChIP) and luciferase reporter assays. Results The transgenic mouse model validated that HNF4α stimulated the HDAC6 expression and mucin secretion in gastric mucosa. Increased HDAC6 and HNF4α expression was also detected in the gastric IM cell model and patient specimens. HNF4α could bind to and activate HDAC6 promoter. In turn, HDAC6 enhanced the HNF4α protein level in GES-1 cells. Furthermore, miR-1 suppressed the expression of downstream intestinal markers by targeting HDAC6 and HNF4α. Conclusions Our findings show that the HDAC6/HNF4α loop regulated by miR-1 plays a critical role in gastric IM. Blocking the activation of this loop could be a potential approach to preventing BA-induced gastric IM or even gastric cancer (GC).

In vitro and in vivo analysis of murine lipoprotein lipase gene promoter: tissue-specific expression

1995 ◽  
Vol 268 (2) ◽  
pp. E213-E218 ◽  
Author(s):  
J. M. Gimble ◽  
X. Hua ◽  
F. Wanker ◽  
C. Morgan ◽  
C. Robinson ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Lipoprotein Lipase ◽  
Reporter Gene ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Specific Expression ◽  
Tissue Specific ◽  
Brown Adipose ◽  
Octamer Motif

Lipoprotein lipase, an enzyme of central importance to lipid metabolism, is most abundant in adipose tissues, cardiac and skeletal muscle, and portions of the brain. The current work examined the murine lipoprotein lipase promoter using transient transfection, gel-retention analyses, and transgenic mice. Maximum expression of the luciferase reporter gene in transfected cells was observed with -101 bp of the promoter. Nuclear extracts from tissues expressing lipoprotein lipase contained DNA binding proteins that recognize the CCAAT box (-64 bp) and an octamer motif (-46 bp); this combination of factors was absent in nonexpressing tissues. Transgenic mice from three of five founders prepared with -1,824-bp promoter constructs expressed the luciferase reporter gene at highest levels in brown adipose tissue and brain. These findings suggest that the -1,824-bp promoter region contains sequence elements responsible for the tissue-specific transcription of lipoprotein lipase in vivo.

Krüppel-like factor 4 represses transcription of the survivin gene in esophageal cancer cell lines

2009 ◽  
Vol 390 (5/6) ◽  
Author(s):  
Guo Zhang ◽  
Hongxia Zhu ◽  
Yihua Wang ◽  
Shangbin Yang ◽  
Mei Liu ◽  
...  
Keyword(s):  
Cancer Cells ◽  
Binding Sites ◽  
Ectopic Expression ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Mobility Shift ◽  
Aberrant Expression ◽  
Survivin Gene ◽  
Survivin Promoter

Abstract Aberrant expression of survivin has been shown to be regulated at the transcription level in cancer cells. In this study, we demonstrate that there are six putative binding sites of Krüppel-like factor 4 (KLF4) within the 2000-bp region upstream of the transcription start site of the human survivin gene. Luciferase reporter gene assays revealed that survivin promoter activity is repressed upon overexpression of KLF4 in EC9706 cells. A chromatin immunoprecipitation assay indicated that KLF4 indeed binds the survivin promoter in vivo. It specifically binds the site located at position -40 among the six binding sites as determined by electrophoretic mobility shift assay. Ectopic expression of KLF4 decreases the mRNA and protein levels of survivin. Furthermore, overexpression of survivin partially reverses KLF4-induced cell apoptosis. These results indicate that KLF4 is a transcriptional repressor of the human survivin gene in esophageal squamous cancer cells.

scholarly journals Endothelial-specific gene expression directed by the tie gene promoter in vivo

Blood ◽  
1995 ◽  
Vol 86 (5) ◽  
pp. 1828-1835 ◽  
Author(s):  
J Korhonen ◽  
I Lahtinen ◽  
M Halmekyto ◽  
L Alhonen ◽  
J Janne ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Transgenic Mice ◽  
Reporter Gene ◽  
Promoter Activity ◽  
Cultured Cells ◽  
Luciferase Reporter ◽  
Specific Gene ◽  
Luciferase Reporter Gene ◽  
Human And Mouse

The tie gene encodes a receptor tyrosine kinase that is expressed in the endothelium of blood vessels, particularly during embryonic development and angiogenesis in adults. We have cloned and characterized the mouse tie gene and isolated the human and mouse tie promoters. The promoter activities of human and mouse tie were analyzed using luciferase reporter gene constructs in transfected cell lines and beta-galactosidase constructs in transgenic mice. In transfection assays of cultured cells, both human and mouse promoter DNA fragments showed activity that was not restricted to endothelial cells. In contrast, in transgenic mice both promoters directed expression of the reporter gene to endothelial cells undergoing vasculogenesis and angiogenesis. In adult mice, tie promoter activity in lung and many vessels of the kidney was as high as in the vessels of the corresponding embryonic tissues, whereas in the heart, brain and liver, tie promoter activity was downregulated and restricted to coronaries, cusps, capillaries, and arteries. Our results show that the endothelial cell-type specificity of the tie promoter in vivo can be transferred to heterologous genes by using relatively short promoter fragments. The tie promoter, thus, has useful properties for potential gene therapy.

422 GENERATION OF REPORTER TRANSGENIC MICE FOR THE CHEMOKINE CXCL2 USING TWO DIFFERENT DNA CONCENTRATIONS

2010 ◽  
Vol 22 (1) ◽  
pp. 368
Author(s):  
M. Crispo ◽  
M. Cárdenas-Rodriguez ◽  
G. Schlapp ◽  
G. Fernández ◽  
M. Rumbo
Keyword(s):  
Immune Response ◽  
Transgenic Mice ◽  
Innate Immune Response ◽  
Gene Promoter ◽  
Innate Immune ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Chi Square ◽  
Dna Concentration ◽  
Chi Square Test

Transgenic mice have important implications in biomedicine, and are widely employed to understand gene functions and their regulation. The improvement of transgenic efficiency is relevant because of the low rate of success for this technology. CXCL2 is a chemokine secreted by macrophages and epithelial cells under proinflammatory stimulus of the innate immune response such as bacterial endotoxins. The main effect of CXCL2 is the recruitment of neutrophils to the site of production to fight infections. The objective of this study was to evaluate the effect of 2 DNA concentrations in the efficiency of the transgenesis process. To this aim we used a luciferase reporter under the control of CXCL2 promoter for the generation of a transgenic line to report activation of innate immune response. A total of 1727 1-cell embryos were divided into 2 experimental groups to be microinjected with 0.5 or 1.0 ng μL-1 of DNA in 25 sessions. Three-week-old B6SJL F1 females (n = 131) were superovulated with 5 IU of eCG i.p. (Novormon, Syntex, Buenos Aires, Argentina) and 5 IU of hCG i.p. (Ovusyn, Syntex) 46 h later, and mated with B6SJL F1 stud males. At the moment of hCG treatment, foster females were mated with vasectomized males to induce pseudogestation. Donor females were euthanized by cervical dislocation 20 h after hCG treatment, and embryos were recovered from the ampulla, denuded in 300 μg mL-1 hyaluronidase (Sigma, St. Louis, MO, USA) and incubated at 37°C with 5% CO2, in drops of M16 media (Sigma) under mineral oil, until microinjection. DNA construction consisted of the luciferase reporter gene under the control of the murine CXCL2 gene promoter. Embryos were microinjected into 1 pronucleus under an inverted microscope (Nikon, NY, USA) using glass microtools and mechanic micromanipulators (Eppendorf, Hamburg, Germany). Intact/injected embryos were assessed 30 min after microinjection. Fifteen to 20 embryos per foster female were transferred in both oviducts. Birth rate, survival of pups at Day 7 after birth, number of transgenic pups assessed by standard PCR, and overall transgenic efficiency was registered for each group. Data were analyzed by Yates-corrected chi-square test. No statistical differences were founded except for a higher number of pups alive/embryo transferred in the lower DNA concentration, suggesting the advantage of using 0.5 ng μL-1 v. 1.0 ng μL-1. Table 1.Effect of DNA concentration in the generation of CXCL2-luc transgenic mice

scholarly journals Insulin activates the rat sterol-regulatory-element-binding protein 1c (SREBP-1c) promoter through the combinatorial actions of SREBP, LXR, Sp-1 and NF-Y cis-acting elements

2004 ◽  
Vol 385 (1) ◽  
pp. 207-216 ◽  
Author(s):  
Lauren M. CAGEN ◽  
Xiong DENG ◽  
Henry G. WILCOX ◽  
Edwards A. PARK ◽  
Rajendra RAGHOW ◽  
...  
Keyword(s):  
Binding Protein ◽  
Regulatory Element ◽  
Ectopic Expression ◽  
Rat Hepatocytes ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Cis Acting ◽  
Element Binding Protein ◽  
Sterol Regulatory Element

The enhanced synthesis of fatty acids in the liver and adipose tissue in response to insulin is critically dependent on the transcription factor SREBP-1c (sterol-regulatory-element-binding protein 1c). Insulin increases the expression of the SREBP-1c gene in intact liver and in hepatocytes cultured in vitro. To learn the mechanism of this stimulation, we analysed the activation of the rat SREBP-1c promoter and its truncated or mutated congeners driving a luciferase reporter gene in transiently transfected rat hepatocytes. The rat SREBP-1c promoter contains binding sites for LXR (liver X receptor), Sp1, NF-Y (nuclear factor-Y) and SREBP itself. We have found that each of these sites is required for the full stimulatory response of the SREBP-1c promoter to insulin. Mutation of either the putative LXREs (LXR response elements) or the SRE (sterol response element) in the proximal SREBP-1c promoter reduced the stimulatory effect of insulin by about 50%. Insulin and the LXR agonist TO901317 increased the association of SREBP-1 with the SREBP-1c promoter. Ectopic expression of LXRα or SREBP-1c increased activity of the SREBP-1c promoter, and this effect is further enhanced by insulin. The Sp1 and NF-Y sites adjacent to the SRE are also required for full activation of the SREBP-1c promoter by insulin. We propose that the combined actions of the SRE, LXREs, Sp1 and NF-Y elements constitute an insulin-responsive cis-acting unit of the SREBP-1c gene in the liver.

Bmi1- Is a SALL4 Target Gene in Hematopoeitic and Leukemic Cells.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1411-1411
Author(s):  
Yupo Ma ◽  
Jianchang Yang ◽  
Li Chai ◽  
Fang Liu ◽  
Hesham M. Amin
Keyword(s):  
Stem Cell ◽  
Transgenic Mice ◽  
Cell Line ◽  
Leukemic Cells ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Gene Target ◽  
Gene Assay ◽  
Human Acute Myeloid Leukemia ◽  
Stem Cell Pluripotency

Abstract Bmi-1 and SALL4 are putative oncogenes modulating stem cell pluripotency and playing a role in leukemogenesis. Previously, we report that SALL4, a zinc finger transcription factor, is constitutively expressed in human acute myeloid leukemia (AML) and induces AML in transgenic mice. Here we demonstrate that transcription from the Bmi-1 promoter is strikingly activated by SALL4. The SALL4 responsiveness of the Bmi-1 promoter exhibits dose-dependent activation in a luciferase reporter gene assay. We mapped the N-terminal domain of SALL4 responsible for activation of the Bmi-1 promoter. Chromatin immunoprecipitation from a myeloid stem cell line, 32D demonstrated that SALL4 bound directly to the Bmi-1 promoter in a region involving SALL4 stimulation. The over-expression of SALL4 in the 32D cell line up-regulated Bmi-1expression and was associated with increased proliferation and inhibition of apoptosis. Deletion of one copy of SALL4 by the gene target in the mouse marrow significantly reduced Bmi-1 expression. SALL4 up-regulated Bmi-1 expression in SALL4 transgenic mice and the up-regulated levels of Bmi-1 in SALL4 transgenic mice correlated to that of disease progression from normal, to preleukemic and leukemic stages. Similar to Bmi-1, SALL4 was expressed highly in hematopoietic cells (HSCs) and was down-regulated as differentiation proceeds. These findings, when taken together, reveal a novel link between SALL4 and Bmi-1 in regulating self-renewal of normal and leukemic stem cells.

scholarly journals Disruption of zinc transporter ZnT3 transcriptional activity and synaptic vesicular zinc in the brain of Huntington’s disease transgenic mouse

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Li Niu ◽  
Li Li ◽  
Shiming Yang ◽  
Weixi Wang ◽  
Cuifang Ye ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Huntington's Disease ◽  
Huntington’S Disease ◽  
Transcriptional Activity ◽  
Zinc Transporter ◽  
Zinc Homeostasis ◽  
Luciferase Reporter ◽  
Synaptic Dysfunction ◽  
Mutant Huntingtin ◽  
Luciferase Reporter Gene

Abstract Background Huntington’s disease (HD) is a neurodegenerative disease that involves a complex combination of psychiatric, cognitive and motor impairments. Synaptic dysfunction has been implicated in HD pathogenesis. However, the mechanisms have not been clearly delineated. Synaptic vesicular zinc is closely linked to modulating synaptic transmission and maintaining cognitive ability. It is significant to assess zinc homeostasis for further revealing the pathogenesis of synaptic dysfunction and cognitive impairment in HD. Results Histochemical staining by autometallography indicated that synaptic vesicular zinc was decreased in the hippocampus, cortex and striatum of N171-82Q HD transgenic mice. Analyses by immunohistochemistry, Western blot and RT-PCR found that the expression of zinc transporter 3 (ZnT3) required for transport of zinc into synaptic vesicles was obviously reduced in these three brain regions of the HD mice aged from 14 to 20 weeks  and  BHK  cells  expressing  mutant  huntingtin. Significantly, dual-luciferase reporter gene and chromatin immunoprecipitation assays demonstrated that transcription factor Sp1 could activate ZnT3 transcription via its binding to the GC boxes in ZnT3 promoter. Moreover, mutant huntingtin was found to inhibit the binding of Sp1 to the promoter of ZnT3 and down-regulate ZnT3 expression, and the decline in ZnT3 expression could be ameliorated through overexpression of Sp1. Conclusions This is first study to reveal a significant loss of synaptic vesicular zinc and a decline in ZnT3 transcriptional activity in the HD transgenic mice. Our work sheds a novel mechanistic insight into pathogenesis of HD that mutant huntingtin down-regulates expression of ZnT3 through inhibiting binding of Sp1 to the promoter of ZnT3 gene, causing disruption of synaptic vesicular zinc homeostasis. Disrupted vesicular zinc ultimately leads to early synaptic dysfunction and cognitive deficits in HD. It is also suggested that maintaining normal synaptic vesicular zinc concentration is a potential therapeutic strategy for HD.

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