Zinc-sensitive genes as potential new target genes of the metal transcription factor-1 (MTF-1)

2005 ◽  
Vol 83 (2) ◽  
pp. 221-229 ◽  
Author(s):  
Birgit Kindermann ◽  
Frank Döring ◽  
Jan Budczies ◽  
Hannelore Daniel
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
Steady State ◽  
Cell Line ◽  
Target Genes ◽  
Complement Factor ◽  
Mrna Levels ◽  
Control Of Gene Expression ◽  
Conditional Expression ◽  
Ht 29

Zinc is an essential trace element that serves as a structural constituent of a large number of transcription factors, which explains its pivotal role in the control of gene expression. Previous studies investigating the effect of zinc deficiency and zinc supplementation on gene expression in the human adenocarcinoma cell line HT-29 led to the identification of a considerable number of genes responding to alterations in cellular zinc status with changes in steady state mRNA levels. For 9 of 20 genes from these previous screenings that were studied in more detail, mRNA steady state levels responded to both high and low media zinc concentrations. As they are primarily zinc-dependent, we assessed whether these genes are controlled by the zinc-finger metal transcription factor MTF-1. To test this hypothesis we generated a doxycyline-inducible Tet-On HT-29 cell line overexpressing MTF-1. Using this conditional expression system, we present evidence that Kruppel-like factor 4 (klf4), hepatitis A virus cellular receptor 1 (hhav), and complement factor B (cfbp) are 3 potential new target genes of MTF-1. To support this, we used in silico analysis to screen for metal-responsive elements (MREs) within promotors of zinc-sensitive genes. We conclude that zinc responsiveness of klf4, hhav, and cfbp in HT-29 cells is mediated at least in part by MTF-1.Key words: zinc-sensitive genes, target genes, MTF-1, HT-29 cells, metal-response element.

scholarly journals A CRISPR/Cas9 genome editing pipeline in the EndoC-βH1 cell line to study genes implicated in beta cell function

2019 ◽  
Vol 4 ◽  
pp. 150 ◽  
Author(s):  
Antje K. Grotz ◽  
Fernando Abaitua ◽  
Elena Navarro-Guerrero ◽  
Benoit Hastoy ◽  
Daniel Ebner ◽  
...  
Keyword(s):  
Gene Expression ◽  
Beta Cell ◽  
Er Stress ◽  
Cell Line ◽  
Cell Lines ◽  
Target Genes ◽  
Gene Knockout ◽  
Mrna Levels ◽  
Beta Cell Line ◽  
Human Beta Cell

Type 2 diabetes (T2D) is a global pandemic with a strong genetic component, but most causal genes influencing the disease risk remain unknown. It is clear, however, that the pancreatic beta cell is central to T2D pathogenesis. In vitro gene-knockout (KO) models to study T2D risk genes have so far focused on rodent beta cells. However, there are important structural and functional differences between rodent and human beta cell lines. With that in mind, we have developed a robust pipeline to create a stable CRISPR/Cas9 KO in an authentic human beta cell line (EndoC-βH1). The KO pipeline consists of a dual lentiviral sgRNA strategy and we targeted three genes (INS, IDE, PAM) as a proof of concept. We achieved a significant reduction in mRNA levels and complete protein depletion of all target genes. Using this dual sgRNA strategy, up to 94 kb DNA were cut out of the target genes and the editing efficiency of each sgRNA exceeded >87.5%. Sequencing of off-targets showed no unspecific editing. Most importantly, the pipeline did not affect the glucose-responsive insulin secretion of the cells. Interestingly, comparison of KO cell lines for NEUROD1 and SLC30A8 with siRNA-mediated knockdown (KD) approaches demonstrate phenotypic differences. NEUROD1-KO cells were not viable and displayed elevated markers for ER stress and apoptosis. NEUROD1-KD, however, only had a modest elevation, by 34%, in the pro-apoptotic transcription factor CHOP and a gene expression profile indicative of chronic ER stress without evidence of elevated cell death. On the other hand, SLC30A8-KO cells demonstrated no reduction in KATP channel gene expression in contrast to siRNA silencing. Overall, this strategy to efficiently create stable KO in the human beta cell line EndoC-βH1 will allow for a better understanding of genes involved in beta cell dysfunction, their underlying functional mechanisms and T2D pathogenesis.

Activation of NF-kappa B and elevation of MnSOD gene expression by thiol reducing agents in lung adenocarcinoma (A549) cells

1995 ◽  
Vol 269 (5) ◽  
pp. L588-L602 ◽  
Author(s):  
K. C. Das ◽  
Y. Lewis-Molock ◽  
C. W. White
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
Transcription Factors ◽  
Steady State ◽  
A549 Cells ◽  
Reducing Agents ◽  
Mrna Levels ◽  
Nf Kappa B ◽  
Oxygen Intermediates ◽  
Mnsod Gene

The effect of reducing agents, including N-acetylcysteine (NAC), dithiothreitol (DTT), and 2-mercaptoethanol (2-ME) on nuclear transcription factor-kappa B (NF-kappa B) activation and manganese superoxide dismutase (MnSOD) expression was investigated in a pulmonary adenocarcinoma (A549) cell line. NAC, DTT, and 2-ME each activated the transcription factor NF-kappa B and increased steady-state levels of MnSOD mRNA and enzyme activity in these cells. In addition, NAC, DTT, and 2-ME increased chloramphenicol acetyltransferase (CAT) activity in cells transfected with a construct containing the CAT gene under the control of the rat MnSOD promoter. SOD and catalase (500 U/ml) plus ethanol (1 mM) did not inhibit activation of NF-kappa B or elevation of steady-state MnSOD mRNA levels by NAC, DTT, or 2-ME. Controls in which comparable amounts of O2-. to those produced by thiols were generated by hypoxanthine and xanthine oxidase, or in which H2O2 was added directly, had neither activated NF-kappa B nor elevated MnSOD mRNA. This shows that reactive oxygen intermediates, which may be formed during autooxidation, may not contribute to activation of NF-kappa B. Because the MnSOD promoter also contains potential binding sites for other transcription factors, such as promoter-selective transcription factor-1 (SP-1), activator protein-1 (AP-1), AP-2, adenosine 3',5'-cyclic monophosphate-regulator element binding factor (CREB), and transcription factor IID complex (TFIID), the effect of thiols on their activation also were evaluated. In contrast to findings with NF-kappa B, there was only minor activation of AP-1 by thiols, and none of the other transcription factors were activated by thiols. AP-1 activation was inhibited by catalase (500 U/ml) plus SOD plus ethanol (1 mM). Addition of 700 microM H2O2 also activated AP-1, and catalase at 500 U/ml prevented this activation. This indicates that H2O2 produced as a result of autooxidation of thiols can activate AP-1 but not NF-kappa B. Thus a close association between exposure to reducing agents, activation of NF-kappa B, and elevation of MnSOD gene expression is demonstrated.

scholarly journals Hepatocyte nuclear factor 3/fork head homolog 11 is expressed in proliferating epithelial and mesenchymal cells of embryonic and adult tissues.

1997 ◽  
Vol 17 (3) ◽  
pp. 1626-1641 ◽  
Author(s):  
H Ye ◽  
T F Kelly ◽  
U Samadani ◽  
L Lim ◽  
S Rubio ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Transcription Factor ◽  
Epithelial Cells ◽  
Cell Line ◽  
Mrna Levels ◽  
Nuclear Factor ◽  
Hepatocyte Nuclear Factor ◽  
Winged Helix ◽  
Fork Head ◽  
Ht 29

The hepatocyte nuclear factor 3alpha (HNF-3alpha) and 3beta proteins have homology in the winged helix/fork head DNA binding domain and regulate cell-specific transcription in hepatocytes and in respiratory and intestinal epithelia. In this study, we describe two novel isoforms of the winged helix transcription factor family, HNF-3/fork head homolog 11A (HFH-11A) and HFH-11B, isolated from the human colon carcinoma HT-29 cell line. We show that these isoforms arise via differential splicing and are expressed in a number of epithelial cell lines derived from tumors (HT-29, Caco-2, HepG2, HeLa, A549, and H441). We demonstrate that differentiation of Caco-2 cells toward the enterocyte lineage results in decreased HFH-11 expression and reciprocal increases in HNF-3alpha and HNF-3beta mRNA levels. In situ hybridization of 16 day postcoitus mouse embryos demonstrates that HFH-11 expression is found in the mesenchymal and epithelial cells of the liver, lung, intestine, renal cortex, and urinary tract. Although HFH-11 exhibits a wide cellular expression pattern in the embryo, its adult expression pattern is restricted to epithelial cells of Lieberkühn's crypts of the intestine, the spermatocytes and spermatids of the testis, and the thymus and colon. HFH-11 expression is absent in adult hepatocytes, but its expression is reactivated in proliferating hepatocytes at 4, 24, and 48 h after partial hepatectomy. Consistent with these findings, we demonstrate that HFH-11 mRNA levels are stimulated by intratracheal administration of keratinocyte growth factor in adult lung and its expression in an adult endothelial cell line is reactivated in response to oxidative stress. These experiments show that the HFH-11 transcription factor is expressed in embryonic mesenchymal and epithelial cells and its expression is reactivated in these adult cell types by proliferative signals or oxidative stress.

Identification of Target Genes of an Erythroid Transcription Factor Complex Containing SCL (TAL1).

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4068-4068 ◽  
Author(s):  
Johanna H. Jim ◽  
Pawandeep Dhami ◽  
Amanda King ◽  
Jonathan Cooper ◽  
Dave Vetrie
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
Cell Line ◽  
K562 Cell ◽  
Target Genes ◽  
K562 Cell Line ◽  
Acute Lymphocytic Leukaemia ◽  
Novel Target ◽  
And Function ◽  
Erythroid Development

Abstract Haematopoiesis is the process whereby haematopoietic stem cells give rise to mature blood cell lineages. The SCL (TAL1) gene, originally identified by chromosomal translocations associated with T-cell acute lymphocytic leukaemia, encodes a key transcription factor (TF) which is expressed in various blood lineages and is essential for haematopoietic development. It has been shown that the SCL protein forms a multi-protein complex during erythroid development with other TFs (GATA1, E2A, LDB1, and LMO2) which binds to a sequence-specific motif to regulate the expression of glycophorin A and c-kit. We have used two complementary approaches to identify novel target genes regulated by this TF complex during erythroid development. In the first approach, we have transfected short interfering RNAs (siRNAs) into the K562 cell line to knockdown transiently the TFs of the erythroid complex. For all members of the complex, a knockdown efficiency of at least 70% was confirmed at the mRNA and protein level within 48 hours after transfection. The consequences of the knockdown at the level of gene expression were observed using Affymetrix GeneChips in order to identify downstream events associated with the erythroid complex in transcriptional programmes. In the second approach, chromatin immunoprecipitation (ChIP) was performed for each member of the complex in the K562 cell line and the ChIP material hybridised to a human transcription factor promoter microarray. A number of novel target genes for the SCL erythroid complex have been identified and verified independently using both approaches. Our data shows that members of the erythroid complex are involved in auto-regulation and regulate genes which control chromatin structure and function. These findings demonstrate that the expression of this TF complex is tightly controlled and point to an important role for it in orchestrating fundamental biological processes which have profound effects on gene expression in erythroid development.

scholarly journals Molecular Clues From Testis Involve NHLH2, a DNA Binding Transcription Factor That Controls the Onset of Mini-Puberty via Its Neuronal Activity, in Curative GnRH Treatment of Cryptorchidism Dependent Infertility

2021 ◽  
Vol 5 (Supplement_1) ◽  
pp. A669-A669
Author(s):  
Faruk Hadziselimovic
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
Dna Binding ◽  
Neuronal Activity ◽  
Target Genes ◽  
Hypogonadotropic Hypogonadism ◽  
Hormone Treatment ◽  
Mrna Levels ◽  
Expression Levels ◽  
Rna Profiling

Abstract Introduction: GnRHa treatment following surgery to correct cryptorchidism restores mini-puberty via endocrinological and transcriptional effects and prevents adult infertility in most cases. A large group of genes are important for central hypogonadotropic hypogonadism in mammals, including many that are transcribed both in brain and testis. However, the expression of these genes in prepubertal gonads has not been systematically studied and little is known about the effect of hormone therapy on their testicular and neuronal expression levels. Here, we interpret histological sections, data on hormone levels and RNA profiling data from adult normal testis in comparison to pre-pubertal low infertility risk (LIR) and high infertility risk (HIR) patients randomized for treatment with surgery in combination with GnRHa or only surgery. Major Results: We organize 31 target genes relevant for hypogonadotropic hypogonadism and cryptorchidism into five classes depending on their expression levels in HIR versus LIR samples and their response to GnRHa treatment. Only the mRNA encoding the DNA binding transcription factor NHLH2 is decreased in HIR as compared to LIR samples, increased after GnRHa treatment, and expressed in both brain and testis. GnRHa treatment of cryptorchidism that restores mini-puberty and rescues adult fertility increases NHLH2 mRNA levels in testis from HIR patients. Conclusion: This phenomenon may reflect a broader effect of hormone treatment on gene expression in both testicular and central nervous tissues, which could explain why the hypothalamus-pituitary-testicular axis is permanently restored by the administration of GnRHa.

scholarly journals A CRISPR/Cas9 genome editing pipeline in the EndoC-βH1 cell line to study genes implicated in beta cell function

2020 ◽  
Vol 4 ◽  
pp. 150 ◽  
Author(s):  
Antje K. Grotz ◽  
Fernando Abaitua ◽  
Elena Navarro-Guerrero ◽  
Benoit Hastoy ◽  
Daniel Ebner ◽  
...  
Keyword(s):  
Gene Expression ◽  
Beta Cell ◽  
Er Stress ◽  
Cell Line ◽  
Cell Lines ◽  
Target Genes ◽  
Gene Knockout ◽  
Mrna Levels ◽  
Beta Cell Line ◽  
Human Beta Cell

Type 2 diabetes (T2D) is a global pandemic with a strong genetic component, but most causal genes influencing the disease risk remain unknown. It is clear, however, that the pancreatic beta cell is central to T2D pathogenesis. In vitro gene-knockout (KO) models to study T2D risk genes have so far focused on rodent beta cells. However, there are important structural and functional differences between rodent and human beta cell lines. With that in mind, we have developed a robust pipeline to create a stable CRISPR/Cas9 KO in an authentic human beta cell line (EndoC-βH1). The KO pipeline consists of a dual lentiviral sgRNA strategy and we targeted three genes (INS, IDE, PAM) as a proof of concept. We achieved a significant reduction in mRNA levels and complete protein depletion of all target genes. Using this dual sgRNA strategy, up to 94 kb DNA were cut out of the target genes and the editing efficiency of each sgRNA exceeded >87.5%. Sequencing of off-targets showed no unspecific editing. Most importantly, the pipeline did not affect the glucose-responsive insulin secretion of the cells. Interestingly, comparison of KO cell lines for NEUROD1 and SLC30A8 with siRNA-mediated knockdown (KD) approaches demonstrate phenotypic differences. NEUROD1-KO cells were not viable and displayed elevated markers for ER stress and apoptosis. NEUROD1-KD, however, only had a modest elevation, by 34%, in the pro-apoptotic transcription factor CHOP and a gene expression profile indicative of chronic ER stress without evidence of elevated cell death. On the other hand, SLC30A8-KO cells demonstrated no reduction in KATP channel gene expression in contrast to siRNA silencing. Overall, this strategy to efficiently create stable KO in the human beta cell line EndoC-βH1 will allow for a better understanding of genes involved in beta cell dysfunction, their underlying functional mechanisms and T2D pathogenesis.

Abstract 331: Genome-wide Analysis of Cardiac and Cardiac Fibroblasts Regulatory Elements Reveals Combinatorial Control of Gene Expression

2017 ◽  
Vol 121 (suppl_1) ◽  
Author(s):  
Tal Golan Lagziel ◽  
Lilac Caspi ◽  
Yair Lewis ◽  
Izhak Kehat
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
Target Genes ◽  
Cardiac Fibroblasts ◽  
Cell Types ◽  
Regulatory Elements ◽  
Open Chromatin ◽  
Specific Expression ◽  
Control Of Gene Expression ◽  
Tissue Specific

The mammalian body contains several hundred cell types that share the same genome, but can express distinct gene signatures. This specification of gene expression is achieved through the activity of cis-regulatory genomic elements (CRE), such as enhancers, promoters, and silencers. The Assay for Transposase-Accessible Chromatin followed by sequencing (ATAC-seq) can identify nucleosome evicted open chromatin, an established marker of regulatory regions. Using a differential ATAC-seq approach, coupled with RNA-seq, H3K27ac ChiP-seq, and computational transcription factor (TFs) binding analysis we comprehensively mapped cell-type and condition specific cis regulatory elements for cardiac fibroblasts and cardiomyocytes, and outlined the TFs that control them. We show that in cardiomyocytes six main transcription factor groups, that control their own and each other’s expression, cooperatively bind discrete distal enhancers that are located at a variable distance from the transcription start site of their target genes. None of these factors is entirely tissue specific in expression, yet various combination of binding sites for these factors, densely clustered within a nucleosome length of genomic stretch make these CREs tissue specific. Multiple tissue specific CREs in turn, are clustered around highly tissue specific genes, and multiple factors, acting from the same and from different CREs can converge on these genes to control their tissue specific expression. Together our data puts forward a mechanistic multi-level combinatorial model for cardiac specific genes expression

Codon replacement in the PGK1 gene of Saccharomyces cerevisiae: experimental approach to study the role of biased codon usage in gene expression

1987 ◽  
Vol 7 (8) ◽  
pp. 2914-2924
Author(s):  
A Hoekema ◽  
R A Kastelein ◽  
M Vasser ◽  
H A de Boer
Keyword(s):  
Gene Expression ◽  
Saccharomyces Cerevisiae ◽  
Steady State ◽  
Codon Usage ◽  
Experimental Approach ◽  
Mrna Translation ◽  
Yeast Cells ◽  
Expression Level ◽  
Start Codon ◽  
Mrna Levels

The coding sequences of genes in the yeast Saccharomyces cerevisiae show a preference for 25 of the 61 possible coding triplets. The degree of this biased codon usage in each gene is positively correlated to its expression level. Highly expressed genes use these 25 major codons almost exclusively. As an experimental approach to studying biased codon usage and its possible role in modulating gene expression, systematic codon replacements were carried out in the highly expressed PGK1 gene. The expression of phosphoglycerate kinase (PGK) was studied both on a high-copy-number plasmid and as a single copy gene integrated into the chromosome. Replacing an increasing number (up to 39% of all codons) of major codons with synonymous minor ones at the 5' end of the coding sequence caused a dramatic decline of the expression level. The PGK protein levels dropped 10-fold. The steady-state mRNA levels also declined, but to a lesser extent (threefold). Our data indicate that this reduction in mRNA levels was due to destabilization caused by impaired translation elongation at the minor codons. By preventing translation of the PGK mRNAs by the introduction of a stop codon 3' and adjacent to the start codon, the steady-state mRNA levels decreased dramatically. We conclude that efficient mRNA translation is required for maintaining mRNA stability in S. cerevisiae. These findings have important implications for the study of the expression of heterologous genes in yeast cells.

scholarly journals Genotype-dependent and non-gradient patterns of RSV gene expression

2019 ◽  
Author(s):  
Felipe-Andrés Piedra ◽  
Xueting Qiu ◽  
Michael N. Teng ◽  
Vasanthi Avadhanula ◽  
Annette A. Machado ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcriptional Regulation ◽  
Steady State ◽  
Transcription Initiation ◽  
Mrna Levels ◽  
Viral Pathogen ◽  
Negative Strand ◽  
Gene Start ◽  
Syncytial Virus ◽  
Conserved Gene

AbstractRespiratory syncytial virus (RSV) is a nonsegmented negative-strand (NNS) RNA virus and a leading cause of severe lower respiratory tract illness in infants and the elderly. Transcription of the ten RSV genes proceeds sequentially from the 3’ promoter and requires conserved gene start (GS) and gene end (GE) signals. Previous studies using the prototypical GA1 genotype Long and A2 strains have indicated a gradient of gene transcription. However, recent reports show data that appear inconsistent with a gradient. To better understand RSV transcriptional regulation, mRNA abundances from five RSV genes were measured by quantitative real-time PCR (qPCR) in three cell lines and cotton rats infected with virus isolates belonging to four different genotypes (GA1, ON, GB1, BA). Relative mRNA levels reached steady-state between four and 24 hours post-infection. Steady-state patterns were genotype-specific and non-gradient, where mRNA levels from the G (attachment) gene exceeded those from the more promoter-proximal N (nucleocapsid) gene across isolates. Transcript stabilities could not account for the non-gradient patterns observed, indicating that relative mRNA levels more strongly reflect transcription than decay. While the GS signal sequences were highly conserved, their alignment with N protein in the helical ribonucleocapsid, i.e., N-phase, was variable, suggesting polymerase recognition of GS signal conformation affects transcription initiation. The effect of GS N-phase on transcription efficiency was tested using dicistronic minigenomes. Ratios of minigenome gene expression showed a switch-like dependence on N-phase with a period of seven nucleotides. Our results indicate that RSV gene expression is in part sculpted by polymerases that initiate transcription with a probability dependent on GS signal N-phase.Author SummaryRSV is a major viral pathogen that causes significant morbidity and mortality, especially in young children. Shortly after RSV enters a host cell, transcription from its nonsegmented negative-strand (NNS) RNA genome starts at the 3’ promoter and proceeds sequentially. Transcriptional attenuation is thought to occur at each gene junction, resulting in a gradient of gene expression. However, recent studies showing non-gradient levels of RSV mRNA suggest that transcriptional regulation may have additional mechanisms. We show using RSV isolates belonging to four different genotypes that gene expression is genotype-dependent and one gene (the G or attachment gene) is consistently more highly expressed than an upstream neighbor. We hypothesize that variable alignment of highly conserved gene start (GS) signals with nucleoprotein (i.e., variable GS N-phase) can affect transcription and give rise to non-gradient patterns of gene expression. We show using dicistronic RSV minigenomes wherein the reporter genes differ only in the N-phase of one GS signal that GS N-phase affects gene expression. Our results suggest the existence of a novel mechanism of transcriptional regulation that might play a role in other NNS RNA viruses.

scholarly journals Differential regulation of mRNA fate by the human Ccr4-Not complex is driven by coding sequence composition and mRNA localization

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Sarah L. Gillen ◽  
Chiara Giacomelli ◽  
Kelly Hodge ◽  
Sara Zanivan ◽  
Martin Bushell ◽  
...  
Keyword(s):  
Gene Expression ◽  
Protein Synthesis ◽  
Mrna Stability ◽  
Mrna Localization ◽  
Synthesis Rate ◽  
Protein Synthesis Rate ◽  
Translational Efficiency ◽  
Mrna Levels ◽  
Control Of Gene Expression ◽  
Mrna Fate

Abstract Background Regulation of protein output at the level of translation allows for a rapid adaptation to dynamic changes to the cell’s requirements. This precise control of gene expression is achieved by complex and interlinked biochemical processes that modulate both the protein synthesis rate and stability of each individual mRNA. A major factor coordinating this regulation is the Ccr4-Not complex. Despite playing a role in most stages of the mRNA life cycle, no attempt has been made to take a global integrated view of how the Ccr4-Not complex affects gene expression. Results This study has taken a comprehensive approach to investigate post-transcriptional regulation mediated by the Ccr4-Not complex assessing steady-state mRNA levels, ribosome position, mRNA stability, and protein production transcriptome-wide. Depletion of the scaffold protein CNOT1 results in a global upregulation of mRNA stability and the preferential stabilization of mRNAs enriched for G/C-ending codons. We also uncover that mRNAs targeted to the ER for their translation have reduced translational efficiency when CNOT1 is depleted, specifically downstream of the signal sequence cleavage site. In contrast, translationally upregulated mRNAs are normally localized in p-bodies, contain disorder-promoting amino acids, and encode nuclear localized proteins. Finally, we identify ribosome pause sites that are resolved or induced by the depletion of CNOT1. Conclusions We define the key mRNA features that determine how the human Ccr4-Not complex differentially regulates mRNA fate and protein synthesis through a mechanism linked to codon composition, amino acid usage, and mRNA localization.

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