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Nanog, a homeodomain protein, has been investigated in humans and mice using embryonic stem cells (ESCs). Because of the limited availability of ESCs, few studies have reported the function and role of Nanog in porcine ESCs. Therefore, in this study, we investigated the location of the porcine Nanog chromosome and its basal promoter activity, which might have potential applications in development of ESCs specific marker as well as understanding its operating systems in the porcine.
Bovine embryonic stem cells have potential for use in research, such as transgenic cattle generation and the study of developmental gene regulation. The Nanog may play a critical role in maintenance of the undifferentiated state of embryonic stem cells in the bovine, as in murine and human. Nevertheless, efforts to study the bovine Nanog for pluripotency-maintaining factors have been insufficient. In this study, in order to understand the mechanisms of transcriptional regulation of the bovine Nanog, the 5'-flanking region of the Nanog was isolated from ear cells of Hanwoo. Results of transient transfection using a luciferase reporter gene under the control of serially deleted 5'-flanking sequences revealed that the -134 to -19 region contained the positive regulatory sequences for the transcription of the bovine Nanog. Results from mutagenesis studies demonstrated that the Sp1-binding site that is located in the proximal promoter region plays an important role in transcriptional activity of the bovine Nanog promoter. The electrophoretic mobility shift assay with the Sp1 specific antibody confirmed the specific binding of Sp1 transcription factor to this site. In addition, significant inhibition of Nanog promoter activity by the Sp1 mutant was observed in murine embryonic stem cells. Furthermore, chromatin-immunoprecipitation assay with the Sp1 specific antibody confirmed the specific binding of Sp1 transcription factor to this site. These results suggest that Sp1 is an essential regulatory factor for bovine Nanog transcriptional activity.
Adiponectin, a cytokine hormone secreted exclusively by adipose tissue, has key roles in energy homeostasis, and in glucose and lipid metabolism. To understand the regulatory expression of pig adiponectin, the 5'-flanking region of the adiponectin gene was isolated from a pig BAC library. 5'-RACE analysis revealed that there were three transcriptional start sites, including one that is novel. The luciferase reporter assay detected a positive cis-acting element for efficient expression of the adiponectin gene at the region spanned by nucleotides -1150 to -1130 with serially deleted 5'-flanking sequences as its promoters. Analysis of oligonucleotide competition by the electrophoretic mobility-shift assay revealed the presence of a cAMP-responsive element (CRE) (nucleotides -1150 to -1130) for the cAMP-responsive element binding protein (CREB), which has not been reported in human or mouse adiponectin genes. These results indicated that CREB is an essential regulatory factor for the transcriptional activity of pig adiponectin.
Pyridoxal kinase is a key enzyme for the biosynthesis of pyridoxal 5'-phosphate. Pyridoxal 5'-phosphate is the catalytically active form of vitamin B6, and acts as a cofactor in >140 different enzyme reactions. It is still unknown how the kinase synthesis is regulated in the cells, and nothing has been reported about the gene promoter. In the present study, based on the bioinformatics analysis of the 5'-flanking region of the human PDXK gene, we cloned the promoter region by PCR. Through the construction of a series of luciferase expression vectors containing the human PDXK promoter region, we characterized the promoter in terms of its structure and function. The transcription start site is at 198bp upstream of the ATG translation initiation site. An important regulatory region is located at -665/-433bp upstream of the transcription start site. The promoter lacks the canonical TATA box, but contains three GC-boxes and one E-box. A deletion and mutation experiment revealed that the transcription factor Sp1 binding site C (-553/-543) is critical in maintaining the robust promoter activity. Knockdown of Sp1 by RNA interference and chromatin immunoprecipitation analysis further proved that the Sp1 is involved in the regulation of the PDXK gene expression.
The Unfolded Protein Response pathway is a conserved signaling mechanism having important roles in cellular physiology and is perturbed accompanying disease. We previously identified the novel UPR target gene CHAC1, a direct target of ATF4, downstream of PERK-EIF2A and activated by the UPR pathway. CHAC1 enzyme directs catalysis of γ-linked glutamate bonds within specific molecular targets. CHAC1 is the first enzyme characterized that can catalyze intracellular glutathione degradation in eukaryotes, having implications for regulation of oxidative stress. DDIT3 (CHOP) is a terminal UPR transcription factor, regulated by ATF4 and an output promoting cell death signaling. Herein we examine the relationship of CHOP controlling CHAC1 transcription in humans and mice. We note parallel induction of CHOP and CHAC1 in human cells after agonist induced UPR. Expanding upon previous reports, we define transcriptional induction of CHAC1 in humans and mice driven by ATF4 through a synergistic relationship with conserved ATF/CRE and CARE DNA sequences of the CHAC1 promoter. Using this system, we also tested effects of CHOP on CHAC1 transcription, and binding at the CHAC1 ATF/CRE using IM-EMSA. These data indicate a novel inhibitory effect of CHOP on CHAC1 transcription, which was ablated in the absence of the ATF/CRE control element. While direct binding of ATF4 to CHAC1 promoter sequences was confirmed, binding of CHOP to the CHAC1 ATF/CRE was not evident at baseline or after UPR induction. These data reveal CHAC1 as a novel CHOP inhibited target gene, acting through an upstream ATF/CRE motif via an indirect mechanism.
Synthesis of 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) is catalysed by the enzyme 25-hydroxyvitamin D(3)-1alpha-hydroxylase (CYP27B1). Regulation of CYP27B1 gene expression is poorly understood, particularly in non-renal tissues including bone where 1,25(OH)(2)D(3) is hypothesised to serve autocrine/paracrine roles. Transient transfection of ROS 17/2.8 osteoblast-like cells with reporter gene constructs containing deletions of the 5'-flanking region of the human CYP27B1 gene revealed a proximal promoter, enhancer region and strong upstream repressive region. Putative CCAAT and GC boxes, as well as Ets protein binding sites were shown to contribute to promoter and enhancer activities respectively in common with kidney and prostate cells. Inhibition of basal expression was largely attributed to a palindrome 5'-GTCTCAGAC-3' (-1015/-1007bp) that contains two putative canonical Smad binding elements. We conclude that repression of CYP27B1 gene expression may be a common event but the novel inhibitory elements we have identified may be unique to osteoblasts.
The Na, K-ATPase is formed by two major subunits (alpha and beta) encoded by a gene family of at least four alpha and three beta isoforms. These genes show distinctive expression patterns involving complex tissue-specific and developmental regulation, although the control mechanisms are not well understood. Here we study the role of chromatin structure in the tissue-specific expression of rat Na, K-ATPase beta2 isoform, which is mainly found in the central nervous system. We have examined the presence and characteristics of nuclease hypersensitive sites and the cytosine methylation patterns in the 5'-flanking region of the beta2 isoform gene from various nuclear preparations. Our results show that in this 5'-flanking region there is only one nuclease hypersensitive site. It is located upstream of the transcription initiation site and shows tissue-specific characteristics. Digestion with deoxyribonuclease I (DNase I), S1 nuclease and micrococcal nuclease yield patterns consistent with a triple-helix structure present only in the active state of the promoter. We also demonstrate that the 5'-flanking region of the beta2 gene co-localizes with a CpG island free of methylation in every tissue tested. The results presented here support a role for specific chromatin remodeling events in the regulation of the Na, K-ATPase beta2 gene expression. They also provide the basis for future studies of the transcription factors involved in the regulation of this gene.
Oestrogens influence the pathology and development of hormone-sensitive breast cancers. Tissue factor pathway inhibitor (TFPI) has been shown to be associated with breast cancer pathogenesis. Recently, we found TFPI mRNA levels to be significantly reduced by oestrogens in a breast cancer cell line (MCF7), a process mediated through the oestrogen receptor alpha (ERα). The aim of the present study was to investigate the mechanism(s) by which oestrogens may regulate TFPI at the transcriptional level. The TFPI 5'-flanking region contains three oestrogen response element (ERE) half-sites at positions -845, -769 and -50. Constructs containing the wild type or mutated ERE half-sites of the TFPI 5'-flanking region were generated in a luciferase reporter gene vector and transiently co-transfected with an ERα expression vector into HEK293 cells and subsequently treated with oestrogens. We found that luciferase activity was significantly downregulated after oestrogen stimulation in cells transfected with the wild type construct, an effect that was abolished by mutating either ERE half-sites. Electrophoretic mobility shift assay suggested direct and specific interaction of ERα with the ERE half-sites in the TFPI 5'-flanking region. Chromatin immunoprecipitation showed that ERα was recruited to the region -899 to -578 of the TFPI 5'-flanking region in vivo, where the ERE half-sites -845 and -769 are located. Our results indicate that ERα can interact with all three ERE half-sites in the TFPI 5'-flanking region and thus participate in the repression of oestrogen mediated TFPI transcription in breast cancer cells.
The human SLC52A1 gene encodes the riboflavin transporter-1 (RFVT-1), a plasma membrane protein that transports vitamin B2 (riboflavin, RF) into cells, and thus, plays a role in controlling cellular homeostasis of RF in those tissues that express the carrier protein (e.g. placenta and intestine). Currently, there is nothing known about transcriptional regulation of the SLC52A1 gene, therefore, we aimed to clone and characterize its 5'-flanking region. Using rapid amplification of the cDNA ends (5'-RACE), we identified one transcription start site (TSS). A 579 bp segment of the 5'-flanking region of this gene was cloned which exhibited robust promoter activity upon transfection in human intestinal epithelial cells. Deletion analysis revealed that the core promoter activity to be embedded in a region between -234 and -23 that lacked TATA element, was GC-rich, and harbored several putative cis-regulatory sites including KLFs, AP-2, EGRF and Sp-1. Mutating each of these sites led to a significant decrease in promoter activity (which was highest for the Sp-1 site), suggesting their possible involvement in regulating SLC52A1 transcription. Focusing on the Sp-1 site, EMSA, super-shift and ChIP analysis was performed that established the interaction of the Sp-1 transcription factor with the SLC52A1 promoter; also, co-transfection of the minimal SLC52A1 promoter with an Sp-1 containing vector in Drosophila SL-2 cells led to significant promoter activation. These results are the first to reveal the identity of the minimal SLC52A1 promoter and to establish an important role for Sp-1 in its activity.
The genetic determination of eggshell coloration has not been determined in birds. Here we report that the blue eggshell is caused by an EAV-HP insertion that promotes the expression of SLCO1B3 gene in the uterus (shell gland) of the oviduct in chicken. In this study, the genetic map location of the blue eggshell gene was refined by linkage analysis in an F(2) chicken population, and four candidate genes within the refined interval were subsequently tested for their expression levels in the shell gland of the uterus from blue-shelled and non-blue-shelled hens. SLCO1B3 gene was found to be the only one expressed in the uterus of blue-shelled hens but not in that of non-blue-shelled hens. Results from a pyrosequencing analysis showed that only the allele of SLCO1B3 from blue-shelled chickens was expressed in the uterus of heterozygous hens (O*LC/O*N). SLCO1B3 gene belongs to the organic anion transporting polypeptide (OATP) family; and the OATPs, functioning as membrane transporters, have been reported for the transportation of amphipathic organic compounds, including bile salt in mammals. We subsequently resequenced the whole genomic region of SLCO1B3 and discovered an EAV-HP insertion in the 5' flanking region of SLCO1B3. The EAV-HP insertion was found closely associated with blue eggshell phenotype following complete Mendelian segregation. In situ hybridization also demonstrated that the blue eggshell is associated with ectopic expression of SLCO1B3 in shell glands of uterus. Our finding strongly suggests that the EAV-HP insertion is the causative mutation for the blue eggshell phenotype. The insertion was also found in another Chinese blue-shelled breed and an American blue-shelled breed. In addition, we found that the insertion site in the blue-shelled chickens from Araucana is different from that in Chinese breeds, which implied independent integration events in the blue-shelled chickens from the two continents, providing a parallel evolutionary example at the molecular level.
The Breast cancer 1, early onset gene (BRCA1) is known to be significantly associated with human familial breast cancer and is identified to play an important role in canine mammary tumors. Here, genetic variations in the coding region and DNA methylation in the 5' flanking region of BRCA1 in canine mammary tumor samples, 15 each of benign and malignant against 10 normal canine mammary tissue samples, were analyzed using the direct sequencing method. The results indicated two point mutations each in the coding region of canine BRCA1 in one benign mammary tumor sample (4702G >T and 4765G >T) and in one malignant canine mammary tumor sample (3619A >G and 4006G >A). No mutations were detected in the normal canine mammary tissue samples. The 4702G >T mutation was found to terminate further translation. The physical effect of the 4765G >T mutation was found to be the repalacement of the glutamate residue with glutamine. The physical effect of the 3619A >G mutation was found to be the replacement of the threonine residue with alanine, and that of mutation 4006G >A was the replacement of the valine residue with isoleucine in the BRCA1 protein. Bisulfite sequencing detected methylated CpG sites in one canine malignant mammary tumor sample. In conclusion, the present study elucidated the mutational status of the BRCA1 coding region and methylation status of the 5' flanking region of BRCA1 in canine mammary tumors.
The insulin-like growth factor 1 has an important role in reproduction, foetal development and growth. It regulates the secretion of gonadotrophin releasing hormone, stimulates ovarian function and steroidogenesis. The present study was conducted to characterise the 5' flanking region of goat IGF 1 gene, ascertain ovarian expression of the IGF1 gene, detect SNPs and assess the association with prolificacy in the two indigenous goat breeds of South India viz., low prolific Attappady Black and high prolific Malabari. The 5' flanking region of IGF1 gene was PCR amplified, cloned and sequenced from both breeds. Genotyping was performed in 277 goats from the two genetic groups using the PCR-Single Strand Conformational Polymorphism (SSCP) and the expression of the IGF1 gene in the ovary was analysed by quantitative real time PCR. The 5' flanking region of the IGF1 gene was 601 bp long and located at 450 bp upstream of the start codon. Sequence exhibited 97-99% similarity with that of the sheep, cattle and sika deer IGF1 genes. Three genotypes, PP, PQ and QR were observed at this locus with the frequency of 0.62, 0.30 and 0.08, respectively. Sequencing of the representative PCR products from each genotype revealed two SNPs, g.224A>G and g.227C>T. The population was found to be in Hardy-Weinberg disequilibrium at both loci. Statistical results indicated that these loci were associated with litter size (P ≤ 0.05). However, no significant difference was found in the expression of the IGF1 gene in the ovaries of the two goat breeds. These results suggest the significant influence of the IGF1 gene on prolificacy in goats and identified SNPs would benefit the selection of prolific animals in future breeding programs.
Belted cattle have a circular belt of unpigmented hair and skin around their midsection. The belt is inherited as a monogenic autosomal dominant trait. We mapped the causative variant to a 37 kb segment on bovine chromosome 3. Whole genome sequence data of 2 belted and 130 control cattle yielded only one private genetic variant in the critical interval in the two belted animals. The belt-associated variant was a copy number variant (CNV) involving the quadruplication of a 6 kb non-coding sequence located approximately 16 kb upstream of the TWIST2 gene. Increased copy numbers at this CNV were strongly associated with the belt phenotype in a cohort of 333 cases and 1322 controls. We hypothesized that the CNV causes aberrant expression of TWIST2 during neural crest development, which might negatively affect melanoblasts. Functional studies showed that ectopic expression of bovine TWIST2 in neural crest in transgenic zebrafish led to a decrease in melanocyte numbers. Our results thus implicate an unsuspected involvement of TWIST2 in regulating pigmentation and reveal a non-coding CNV underlying a captivating Mendelian character.
It has been reported that binding of STAT3 protein to the 5'-flanking region of the relaxin gene may result in downregulation of the relaxin expression. There is a Guanine(G)-rich segment located in about 3.8 Kb upstream of the relaxin gene and very close to the STAT3's binding site. In our study, NMR spectroscopy revealed the formation of G-quadruplex by this G-rich strand, and the result was confirmed by ESI mass spectrometry and CD spectroscopy. The theoretical structure of RLX G-quadruplex was constructed and refined by molecular modeling. When this relaxin G-quadruplex was stabilized by berberine(ΔTm = 10°C), a natural alkaloid from a Chinese herb, the gene expression could be up-regulated in a dose-dependent manner which was proved by luciferase assay. This result is different from the general G-quadruplex function that inhibiting the telomere replication or down-regulating many oncogenes expression. Therefore, our study reported a novel G-quadruplex in the relaxin gene and complemented the regulation mechanism about gene expression by G-quadruplexes.
Serine-threonine protein phosphatase 2A (PP2A) is a trimeric holoenzyme that plays an integral role in the regulation of cell growth, differentiation, and apoptosis. The substrate specificity and (sub)cellular localization of the PP2A holoenzymes are highly regulated by interaction with a family of regulatory B subunits (PP2A-Bs). The regulatory subunit PP2A-B/PR55δ (PP2A-Bδ) is involving in the dephosphorylation of PP2A substrates and is crucial for controlling entry into and exit from mitosis. The molecular mechanisms involved in the regulation of expression of PP2A-Bδ gene (PPP2R2D) remain largely unknown. To explore genetic variations in the 5'-flanking region of PPP2R2D gene as well as their frequent haplotypes in the Han Chinese population and determine whether such variations have an impact on transcriptional activity, DNA samples were collected from 70 healthy Chinese donors and sequenced for identifying genetic variants in the 5'-flanking region of PPP2R2D. Four genetic variants were identified in the 1836 bp 5'-flanking region of PPP2R2D. Linkage disequilibrium (LD) patterns and haplotype profiles were constructed for the genetic variants. Using serially truncated human PPP2R2D promoter luciferase constructs, we found that a 601 bp (-540 nt to +61 nt) fragment constitutes the core promoter region. The subcloning of individual 5'-flanking fragment revealed the existence of three haplotypes in the distal promoter of PPP2R2D. The luciferase reporter assay showed that different haplotypes exhibited distinct promoter activities. The EMSA revealed that the -462 G>A variant influences DNA-protein interactions involving the nuclear factor 1 (NF1). In vitro reporter gene assay indicated that cotransfection of NF1/B expression plasmid could positively regulate the activity of PPP2R2D proximal promoter. Introduction of exogenous NF1/B expression plasmid further confirmed that the NF1 involves in the regulation of PPP2R2D gene expression. Our findings suggest that functional genetic variants and their haplotypes in the 5'-flanking region of PPP2R2D are critical for transcriptional regulation of PP2A-Bδ.
Sex-determining region Y is a crucial gene that initiates male sex determination in mammals. Mutations of the Sp1-binding site in the 5' flanking region of SRY are associated with clinical male-to-female sex reversal syndrome, although such occurrences are rare and, until now, have not been reported in animal models. In this study, we mutated Sp1-binding sites in the 5' flanking region of the rabbit SRY gene using the CRISPR/Cas9 system. As expected, the SRY-Sp1 knockout rabbits had female external and internal genitalia and exhibited normal female copulatory behaviors, but they were infertile, and the adults displayed reduced follicles. Interestingly, we successfully obtained offspring from sex-reversed SRY-Sp1 knockout rabbits using embryo transfer. In summary, our study demonstrates that Sp1 is a major regulator in SRY gene transcription, and mutations of the Sp1 binding sites (Sp1-B and Sp1-C) in the 5' flanking region of SRY induce sex reversal in rabbits, which can be used as targets for clinical research of male-to-female sex reversal syndrome. Additionally, we provide the first evidence that sex reversal syndrome patients have the potential to become pregnant with the use of embryo transfer.
17Beta hydroxysteroid dehydrogenase type 7 (HSD17B7) was described to possess dual functionality in steroidogenesis as well as in postsqualene cholesterol biosynthesis in vitro. In order to gain insight into the transcriptional regulation, and thereby into in vivo functionality of HSD17B7, we analyzed and compared the 5' flanking regions of the corresponding human and murine genes. For this task we used bioinformatic and experimental approaches. The identified proximal promoter regions of both human and murine HSD17B7 genes contain multiple transcription factor binding sites and show strong similarity to cholesterogenic genes, especially to other postsqualene genes, but not to other steroidogenic genes. In liver cell lines, the transcriptional activity is dependent on the level of cholesterol, but not estradiol. The results of our study lead us to the conclusion that HSD17B7 is involved in postsqualene cholesterol biosynthesis in both human and mice.
Vanadate, a protein tyrosine phosphatase inhibitor which elicits insulin-like effects, has previously been shown to inhibit expression of the insulin receptor gene at the transcriptional level in rat hepatoma cells. In an attempt to identify the DNA sequence and transcription factors potentially involved in this effect, a fragment of the proximal 5'flanking region of the IR gene (-1143/-252 upstream the ATG codon) has been cloned and functionally characterized. RNase protection allowed the identification of several transcription start sites in the conserved region of the gene, among which two major sites at -455 and -396. Upon fusion to the luciferase gene and transient transfection into hepatoma cells, the -1143/-252 fragment showed promoter activity. This was unaffected by deletion of the -1143/-761 sequence, but markedly decreased (90%) by additional deletion of the -760/-465 sequence. Treatment of hepatoma cells with vanadate led to a dose-dependent decrease in promoter activity of the 1143/-252, -760/-252 and -464/-252 constructs (change relative to untreated cells, 40, 55 and 23% at 125 μM, and 70, 85 and 62% at 250 μM, respectively). These data suggest that although the entire DNA sequence upstream the transcription start sites is probably involved in vanadate-induced inhibition, the short sequence downstream of position -464 and is sufficient for inhibition. Potential targets of vanadate are the transcription factors FoxO1 and HMGA1, two downstream targets of the insulin signaling pathway which have been shown to mediate the inhibitory effect of insulin on IR gene expression.
Farnesyl diphosphate synthetase (FDPS) is a key enzyme in the isoprenoid pathway responsible for cholesterol biosynthesis, post-translational protein modifications and synthesis of steroid hormones, whose expression is regulated by phorbol esters and polyunsaturated fatty acids. Genomic comparison of the 5' upstream sequence of the FDPS genes identifies conserved binding sites for NF-Y, SP1, SRE3, and YY1 regulatory elements in rat, mouse, dog and chimpanzee. Two additional specific consensus sequences, upstream of the core promoter that had not been analysed previously, are shared only by human and chimpanzee genomes. The work presented here aimed at characterizing these genomic sequence elements in the human FDPS promoter region and their contribution to gene expression. We have characterized functionally the minimal basal promoter of the human FDPS gene by means of deletion mutants and we have identified two cis-acting elements which modulate the FDPS gene expression and are recognized by Pax5 and OCT-1 transcription factors.
Inflammation induced by H.pylori colonization in the stomach is related to the development of gastric cancer and the genetic variations of the genes involved in the immune responses modify the host response to the infection. The aim of this study was to evaluate whether polymorphisms in the toll-like receptor 4 (TLR4) gene, a key regulator of both innate and adaptive immunity, were related to the susceptibility to gastric cancer in a Chinese population.
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