scholarly journals Functional mapping of the mouse hairless gene promoter region

2018 ◽  
Author(s):  
Eric G. Folco ◽  
Stefan Nonchev

AbstractThe mouse hairless gene (Hr) encodes a protein of 127 kDa, acting as corepressor of nuclear hormone receptors. The Hairless protein (HR) is involved in the control of the cellular transition to the first hair cycle in adult Mammals. In its absence hair follicles disintegrate leading to a complete and irreversible hair loss with formation of cutaneous cysts. The hairless phenotype is therefore linked to defective proliferation and migration of the hair follicle stem cells apparently unable to respond to various signalling molecules. The Hr gene is expressed at high levels in skin and brain, and hairless transcripts were detected in gonads, thymus and colon. Although the patterns of Hr expression appear to be spatially and temporally regulated, very little is known about the molecular basis of the transcriptional control underlying Hr gene function. In this work we determine the precise transcriptional initiation start site of the mouse Hr gene and identify a new 1,1 kb cis-control element (RE1) that encompasses the promoter region and is able to drive luciferase reporter expression in skin and brain derived cell lines. We performed a deletion analysis and explored functionally regulatory motifs within this fragment to show that the role of this upstream regulatory region is linked to the presence of TRE and VDRE binding sites. We find that a TRE situated at –300 bp from the cap site is essential for gene expression in both skin NIH 3T3 and GHFT1 cells, while a VDRE positioned 94 bp upstream of the TRE modulates reporter expression specifically in skin derived cell lines. In addition, we define a novel cis-regulatory motif UE60, situated at the 5’-end of RE1 and likely to interact with both TRE and VDRE. Our data complete previous results on the possible existence of an autoregulatory pathway, implicated in Hr gene regulation. Taken together these findings reveal a complex molecular network that potentially links several signalling pathways in hair follicle formation. We discuss the organisation of the regulatory modules in the mouse Hr gene upstream DNA sequences in the light of the high homology of this region in mouse, rat and human.

2020 ◽  
Author(s):  
Xicen Zhang ◽  
Mei Ding ◽  
Yi Liu ◽  
Yongping Liu ◽  
Jiaxin Xing ◽  
...  

Abstract Background: In previous studies, we researched the association of the DRD2 gene promoter region SNP loci rs7116768, rs1047479195, rs1799732, rs1799978 and schizophrenia using Sanger sequencing. rs7116768 and rs1799978 were found to be slightly associated with schizophrenia. This study investigated the effects of haplotypes consisted of the four SNPs on protein expression level in vitro and identified the functional sequence in the 5’ regulatory region of DRD2 gene which has a potential link with schizophrenia.Methods: Recombinant plasmids with haplotypes, SNPs and 13 recombinant vectors containing deletion fragments from the DRD2 gene 5' regulatory region were transfected into HEK293 and SK-N-SH cell lines. Relative luciferase activity of the haplotypes, SNPs and different sequences was compared using a dual luciferase reporter assay system.Results: Haplotype H4(G-C-InsC-G) could significantly increase the gene expression in SK-N-SH cell lines. Allele C of rs7116768, allele A of rs1047479195 and allele del C of rs1799732 could up-regulate the gene expression. There were 5~7 functional regions in the promoter region of DRD2 gene that could affect the level of gene expression.Conclusion: We cannot rule out the possibility that different haplotypes may influence DRD2 gene expression in vivo. We observed that allele C of rs7116768, allele A of rs1047479195 and allele del C of rs1799732 could up-regulate gene expression. The truncation results confirmed the existence of functional regions in the promoter region of DRD2 gene that could affect the level of gene expression.


2005 ◽  
Vol 393 (1) ◽  
pp. 321-329 ◽  
Author(s):  
Antonella De Luca ◽  
Paolo Sacchetta ◽  
Carmine Di Ilio ◽  
Bartolo Favaloro

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.


2021 ◽  
Vol 12 ◽  
Author(s):  
Fangzheng Shang ◽  
Yu Wang ◽  
Rong Ma ◽  
Zhengyang Di ◽  
Zhihong Wu ◽  
...  

BackgroundInner Mongolian cashmere goats have hair of excellent quality and high economic value, and the skin hair follicle traits of cashmere goats have a direct and important effect on cashmere yield and quality. Circular RNA has been studied in a variety of tissues and cells.ResultIn this study, high-throughput sequencing was used to obtain the expression profiles of circular RNA (circRNA) in the hair follicles of Inner Mongolian cashmere goats at different embryonic stages (45, 55, 65, and 75 days). A total of 21,784 circRNAs were identified. At the same time, the differentially expressed circRNA in the six comparison groups formed in the four stages were: d75vsd45, 59 upregulated and 33 downregulated DE circRNAs; d75vsd55, 61 upregulated and 102 downregulated DE circRNAs; d75vsd65, 32 upregulated and 33 downregulated DE circRNAs; d65vsd55, 67 upregulated and 169 downregulated DE circRNAs; d65vsd45, 96 upregulated and 63 downregulated DE circRNAs; and d55vsd45, 76 upregulated and 42 downregulated DE circRNAs. Six DE circRNA were randomly selected to verify the reliability of the sequencing results by quantitative RT-PCR. Subsequently, the circRNA corresponding host genes were analyzed by the Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway. The results showed that the biological processes related to hair follicle growth and development enriched by GO mainly included hair follicle morphogenesis and cell development, and the signaling pathways related to hair follicle development included the Notch signaling pathway and NF-κB signaling pathway. We combined the DE circRNA of d75vsd45 with miRNA and mRNA databases (unpublished) to construct the regulatory network of circRNA–miRNA–mRNA, and formed a total of 102 pairs of circRNA–miRNA and 126 pairs of miRNA–mRNA interactions. The binding relationship of circRNA3236–chi-miR-27b-3p and circRNA3236–chi-miR-16b-3p was further verified by dual-luciferase reporter assays, and the results showed that circRNA3236 and chi-miR-27b-3p, and circRNA3236 and chi-miR-16b-3p have a targeted binding relationship.ConclusionTo summarize, we established the expression profiling of circRNA in the fetal skin hair follicles of cashmere goats, and found that the host gene of circRNA may be involved in the development of hair follicles of cashmere goats. The regulatory network of circRNA–miRNA–mRNA was constructed and preliminarily verified using DE circRNAs.


2004 ◽  
Vol 286 (6) ◽  
pp. G922-G931 ◽  
Author(s):  
Lingling Jiang ◽  
Jiafang Wang ◽  
R. Sergio Solorzano-Vargas ◽  
Hugh V. Tsai ◽  
Edgar M Gutierrez ◽  
...  

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.


2020 ◽  
Author(s):  
Fangzheng Shang ◽  
Yu Wang ◽  
Rong Ma ◽  
Zhengyang Di ◽  
Zhihong Wu ◽  
...  

Abstract Background:Inner Mongolian cashmere goats have hair of excellent quality and high economic value, and the skin hair follicle traits of cashmere goats have a direct and important effect on cashmere yield and quality.Circular RNA has been studied in a variety of tissues and cells. Result:In this study, high-throughput sequencing was used to obtain the expression profiles of circRNA in the hair follicles of Inner Mongolia cashmere goats at different embryonic stages (45, 55 , 65 and 75 days). A total of 21784 circRNAs were identified. At the same time, the differentially expression circRNA in the six control groups formed in the four stages were: d75vsd45, circRNA up-regulated by 59 and down-regulated by 33; d75vsd55, circRNA up-regulated by 61 and down-regulated by 102; d75vsd65, circRNA up-regulated by 32 and down-regulated by 33; d65vsd55, circRNA up-regulated by 67 and down-regulated by 169; d65vsd45, circRNA up-regulated by 96 and down-regulated by 63; and d55vsd45, circRNA up-regulated by 76 and down-regulated by 42. Six DE circRNA were randomly selected to verify the reliability of the sequencing results by qRT-PCR. Subsequently,the circRNA corresponding host genes were analyzed by GO and the KEGG pathaway. The results showed that the biological processes related to hair follicle growth and development enriched by GO mainly included hair follicle morphogenesis, hair follicle maturation and cell development, and the signal pathways related to hair follicle development included the Notch signaling pathway and NF-kappa B signaling pathway.We combined the DE circRNA of d75vsd45 with miRNA and mRNA databases(unpublished) to construct the co-expression network of circRNA-miRNA-mRNA, and formed a total of 102 pairs of circRNA-miRNA and 126 pairs of miRNA-mRNA interactions. The binding relationship of circRNA3236-chi-miR-27b-3p and circRNA3236-chi-miR-16b-3p was further verified by dual-luciferase reporter assays,and the results showed that circRNA3236 and chi-miR-27b-3p,circRNA3236 and chi-miR-16b-3p have a targeted binding relationship.Conclusion:To summarize, we established the expression profile of circRNA in the fetal skin hair follicles of cashmere goats, and found that the host gene of circRNA may be involved in the development of hair follicles of cashmere goats. The co-expression network of circRNA-miRNA-mRNA that DE circRNA was constructed and preliminary verification was carried out.


2019 ◽  
Vol 71 (4) ◽  
pp. 1527-1539 ◽  
Author(s):  
Jun Yao ◽  
Zedan Shen ◽  
Yanli Zhang ◽  
Xia Wu ◽  
Jianhui Wang ◽  
...  

Abstract Plasma membrane proton pumps play a crucial role in maintaining ionic homeostasis in salt-resistant Populus euphratica under saline conditions. High levels of NaCl (200 mM) induced PeHA1 expression in P. euphratica roots and leaves. We isolated a 2022 bp promoter fragment upstream of the translational start of PeHA1 from P. euphratica. The promoter–reporter construct PeHA1-pro::GUS was transferred to tobacco plants, demonstrating that β-glucuronidase activities increased in root, leaf, and stem tissues under salt stress. DNA affinity purification sequencing revealed that PeWRKY1 protein targeted the PeHA1 gene. We assessed the salt-induced transcriptional response of PeWRKY1 and its interaction with PeHA1 in P. euphratica. PeWRKY1 binding to the PeHA1 W-box in the promoter region was verified by a yeast one-hybrid assay, EMSA, luciferase reporter assay, and virus-induced gene silencing. Transgenic tobacco plants overexpressing PeWRKY1 had improved expression of NtHA4, which has a cis-acting W-box in the regulatory region, and improved H+ pumping activity in both in vivo and in vitro assays. We conclude that salt stress up-regulated PeHA1 transcription due to the binding of PeWRKY1 to the W-box in the promoter region of PeHA1. Thus, we conclude that enhanced H+ pumping activity enabled salt-stressed plants to retain Na+ homeostasis.


2004 ◽  
Vol 186 (3) ◽  
pp. 661-671 ◽  
Author(s):  
Deborah R. Yoder ◽  
Lee Kroos

ABSTRACT Myxococcus xanthus utilizes extracellular signals during development to coordinate cell movement, differentiation, and changes in gene expression. One of these signals, the C signal, regulates the expression of many genes, including Ω4400, a gene identified by an insertion of Tn5 lac into the chromosome. Expression of Tn5 lac Ω4400 is reduced in csgA mutant cells, which fail to perform C signaling, and the promoter region has several sequences similar to sequences found in the regulatory regions of other C-signal-dependent genes. One such gene, Ω4403, depends absolutely on the C signal for expression, and its promoter region has been characterized previously by mutational analysis. To determine if the similar sequences within the Ω4400 and Ω4403 regulatory regions function in the same way, deletion analysis and site-directed mutagenesis of the Ω4400 promoter region were performed. A 7-bp sequence centered at −49 bp, termed a C box, is identical in the Ω4400 and Ω4403 promoter regions, yet mutations in the individual base pairs affected expression from the two promoters very differently. Also, a single-base-pair change within a similar 5-bp element, which is centered at −61 bp in both promoter regions, had very different effects on the activities of the two promoters. Further mutational analysis showed that two regions are important for Ω4400 expression; one region, from −63 to −31 bp, is required for Ω4400 expression, and the other, from −86 to −81 bp, exerts a two- to fourfold effect on expression and is at least partially responsible for the C signal dependence of the Ω4400 promoter. Mutations in sigD and sigE, which are genes that encode σ factors, abolished and reduced Ω4400 expression, respectively. Expression of Ω4400 in actB or actC mutants correlated well with the altered levels of C signal produced in these mutants. Our results provide the first detailed analysis of an M. xanthus regulatory region that depends partially on C signaling for expression and indicate that similar DNA sequences in the Ω4400 and Ω4403 promoter regions function differently.


2019 ◽  
Vol 2019 ◽  
pp. 1-11
Author(s):  
Seung-Hun Kim ◽  
Kwang-Hwan Choi ◽  
Dong-Kyung Lee ◽  
Mingyun Lee ◽  
Jae Yeon Hwang ◽  
...  

OCT4 plays pivotal roles in maintaining pluripotency during early mammalian embryonic development and in embryonic stem cells. It is essential to establish a reporter system based on the OCT4 promoter region to study pluripotency. However, there is still a lack of information about the porcine OCT4 upstream reporter system. To improve our understanding of the porcine OCT4 regulatory region, we identified conserved regions in the porcine OCT4 promoter upstream region by sequence-based comparative analysis using various mammalian genome sequences. The similarity of nucleotide sequences in the 5′ upstream region was low among mammalian species. However, the OCT4 promoter and four regulatory regions, including distal and proximal enhancer elements, had high similarity. Next, a functional analysis of the porcine OCT4 promoter region was conducted. Luciferase reporter assay results indicated that the porcine OCT4 distal enhancer and proximal enhancer were highly activated in mouse embryonic stem cells and embryonic carcinoma cells, respectively. A comparison analysis of naïve and primed state marker gene expression in a dual-reporter assay showed that the expression levels of naïve and primed markers differed in fluorescence signal between high-expressing cells and low-expressing cells. Similar to OCT4 upstream-based reporter systems derived from other species, the porcine OCT4 upstream region-based reporter constructs showed exclusive expression patterns depending on the state of pluripotency. This work provides basic information about the porcine OCT4 upstream region and various porcine OCT4 fluorescence reporter constructs, which can be applied to study species-specific pluripotency in early embryo development and the establishment of embryonic stem cells in pigs.


2020 ◽  
Author(s):  
Jun-feng Huo ◽  
Xiao-bing Chen

Abstract Background: Abnormal DNA methylation in the promoter region of vascular endothelial growth factor (VEGF) has been observed in multiple types of cancer. Increasing evidence shows that miR-148a-3p is involved in the regulation of methylation. However, it is unclear how miR-148a-3p regulates VEGF methylation.Methods: Methylation-specific polymerase chain reaction (MSP) and bisulfate-sequencing PCR (BSP) were performed to detect the methylation level of the VEGF promoter region in glioma tissues and cell lines. Then, we used RT-qPCR to detect the expression of miR-148a-3p and VEGF in glioma tissues and cell lines. Human glioma U87 cells were transfected with miR-148a-3p mimic or a pcDNA3.1 overexpression vector of VEGF, and then, the proliferation, invasion and apoptosis of U87 cells were examined with cell counting kit-8 (CCK-8), Transwell assay and Flow cytometry, respectively. We next examined the relationship between DNA methyltransferase 1 (DNMT1) and miR-148a-3p with online prediction and luciferase reporter gene experiments. Furthermore, we also used Western blotting to detect the protein levels of VEGF, DNMT1, PI3K, Akt and eNOS.Results: Both miR-148a-3p and VEGF were significantly up-regulated, and the promoter methylation of VEGF was hypomethylation in glioma. Overexpression miR-148a-3p or VEGF promoted the proliferation and invasion of U87 cells and inhibited the apoptosis. Silencing VEGF inhibited U87 cells proliferation and invasion and induced the apoptosis. Furthermore, miR-148a-3p regulated DNMT1 at the post-transcriptional. Overexpression of DNMT1 mediated an increase in the methylation level of VEGF, which reduced the expression of VEGF in U87 cells. The opposite was exact when DNMT1 was silenced. We also observed that silencing VEGF inhibited the activation of the PI3K/Akt/eNOS pathway and induced U87 cell apoptosis.Conclusion: These results indicated that miR-148a-3p down-regulates VEGF methylation by targeting DNMT1 and promotes the proliferation, invasion of the glioma cell through the PI3K/Akt/eNOS pathway.


2001 ◽  
Vol 21 (21) ◽  
pp. 7256-7267 ◽  
Author(s):  
Amar Abderrahmani ◽  
Myriam Steinmann ◽  
Valérie Plaisance ◽  
Guy Niederhauser ◽  
Jacques-Antoine Haefliger ◽  
...  

ABSTRACT Islet-brain 1 (IB1) is the human and rat homologue of JIP-1, a scaffold protein interacting with the c-Jun amino-terminal kinase (JNK). IB1 expression is mostly restricted to the endocrine pancreas and to the central nervous system. Herein, we explored the transcriptional mechanism responsible for this preferential islet and neuronal expression of IB1. A 731-bp fragment of the 5′ regulatory region of the human MAPK8IP1 gene was isolated from a human BAC library and cloned upstream of a luciferase reporter gene. This construct drove high transcriptional activity in both insulin-secreting and neuron-like cells but not in unrelated cell lines. Sequence analysis of this promoter region revealed the presence of a neuron-restrictive silencer element (NRSE) known to bind repressor zinc finger protein REST. This factor is not expressed in insulin-secreting and neuron-like cells. By mobility shift assay, we confirmed that REST binds to the NRSE present in the IB1 promoter. Once transiently transfected in β-cell lines, the expression vector encoding REST repressed IB1 transcriptional activity. The introduction of a mutated NRSE in the 5′ regulating region of the IB1 gene abolished the repression activity driven by REST in insulin-secreting β cells and relieved the low transcriptional activity of IB1 observed in unrelated cells. Moreover, transfection in non-β and nonneuronal cell lines of an expression vector encoding REST lacking its transcriptional repression domain relieved IB1 promoter activity. Last, the REST-mediated repression of IB1 could be abolished by trichostatin A, indicating that deacetylase activity is required to allow REST repression. Taken together, these data establish a critical role for REST in the control of the tissue-specific expression of the humanIB1 gene.


Sign in / Sign up

Export Citation Format

Share Document