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      Discovery of a Family of Genomic Sequences Which Interact Specifically with the c-MYC Promoter to Regulate c-MYC Expression

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          Abstract

          G-quadruplex forming sequences are particularly enriched in the promoter regions of eukaryotic genes, especially of oncogenes. One of the most well studied G-quadruplex forming sequences is located in the nuclease hypersensitive element (NHE) III 1 of the c-MYC promoter region. The oncoprotein c-MYC regulates a large array of genes which play important roles in growth regulation and metabolism. It is dysregulated in >70% of human cancers. The silencer NHEIII 1 located upstream of the P1 promoter regulates up-to 80% of c-MYC transcription and includes a G-quadruplex structure (Pu27) that is required for promoter inhibition. We have identified, for the first time, a family of seventeen G-quadruplex-forming motifs with >90% identity with Pu27, located on different chromosomes throughout the human genome, some found near or within genes involved in stem cell maintenance or neural cell development. Notably, all members of the Pu27 family interact specifically with NHEIII 1 sequence, in vitro. Crosslinking studies demonstrate that Pu27 oligonucleotide binds specifically to the C-rich strand of the NHEIII 1 resulting in the G-quadruplex structure stabilization. Pu27 homologous sequences (Pu27-HS) significantly inhibit leukemic cell lines proliferation in culture. Exposure of U937 cells to the Pu27-HS induces cell growth inhibition associated with cell cycle arrest that is most likely due to downregulation of c-MYC expression at the RNA and/or protein levels. Expression of SOX2, another gene containing a Pu27-HS, was affected by Pu27-HS treatment as well. Our data suggest that the oligonucleotides encoding the Pu27 family target complementary DNA sequences in the genome, including those of the c-MYC and SOX2 promoters. This effect is most likely cell type and cell growth condition dependent. The presence of genomic G-quadruplex-forming sequences homologous to Pu27 of c-MYC silencer and the fact that they interact specifically with the parent sequence suggest a common regulatory mechanism for genes whose promoters contain these sequences.

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          Most cited references 85

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          Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method.

           K Livak,  T Schmittgen (2001)
          The two most commonly used methods to analyze data from real-time, quantitative PCR experiments are absolute quantification and relative quantification. Absolute quantification determines the input copy number, usually by relating the PCR signal to a standard curve. Relative quantification relates the PCR signal of the target transcript in a treatment group to that of another sample such as an untreated control. The 2(-Delta Delta C(T)) method is a convenient way to analyze the relative changes in gene expression from real-time quantitative PCR experiments. The purpose of this report is to present the derivation, assumptions, and applications of the 2(-Delta Delta C(T)) method. In addition, we present the derivation and applications of two variations of the 2(-Delta Delta C(T)) method that may be useful in the analysis of real-time, quantitative PCR data. Copyright 2001 Elsevier Science (USA).
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            Identification of c-MYC as a target of the APC pathway.

            The adenomatous polyposis coli gene (APC) is a tumor suppressor gene that is inactivated in most colorectal cancers. Mutations of APC cause aberrant accumulation of beta-catenin, which then binds T cell factor-4 (Tcf-4), causing increased transcriptional activation of unknown genes. Here, the c-MYC oncogene is identified as a target gene in this signaling pathway. Expression of c-MYC was shown to be repressed by wild-type APC and activated by beta-catenin, and these effects were mediated through Tcf-4 binding sites in the c-MYC promoter. These results provide a molecular framework for understanding the previously enigmatic overexpression of c-MYC in colorectal cancers.
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              Direct evidence for a G-quadruplex in a promoter region and its targeting with a small molecule to repress c-MYC transcription.

              The nuclease hypersensitivity element III(1) upstream of the P1 promoter of c-MYC controls 85-90% of the transcriptional activation of this gene. We have demonstrated that the purine-rich strand of the DNA in this region can form two different intramolecular G-quadruplex structures, only one of which seems to be biologically relevant. This biologically relevant structure is the kinetically favored chair-form G-quadruplex, which is destabilized when mutated with a single G --> A transition, resulting in a 3-fold increase in basal transcriptional activity of the c-MYC promoter. The cationic porphyrin TMPyP4, which has been shown to stabilize this G-quadruplex structure, is able to suppress further c-MYC transcriptional activation. These results provide compelling evidence that a specific G-quadruplex structure formed in the c-MYC promoter region functions as a transcriptional repressor element. Furthermore, we establish the principle that c-MYC transcription can be controlled by ligand-mediated G-quadruplex stabilization.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                23 August 2016
                2016
                : 11
                : 8
                Affiliations
                [1 ]James Graham Brown Cancer Center, Department of Medicine, University of Louisville, Louisville Kentucky, United States of America
                [2 ]Department of Computer Engineering and Computer Science, Speed School of Engineering, University of Louisville, Kentucky, United States of America
                Saint Louis University, UNITED STATES
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                • Conceptualization: DMM FR SDT.

                • Formal analysis: FR ECR.

                • Funding acquisition: DMM.

                • Investigation: SDT FR.

                • Methodology: FR SDT.

                • Supervision: DMM.

                • Validation: FR SDT.

                • Writing – original draft: FR.

                • Writing – review & editing: DMM ECR SDT FR.

                PONE-D-16-22090
                10.1371/journal.pone.0161588
                4995011
                27551915
                © 2016 Rezzoug et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                Counts
                Figures: 10, Tables: 2, Pages: 27
                Product
                Funding
                Funded by: U.S. Public Health Service Grants
                Award ID: 5P20RR18733-09
                Award Recipient :
                Funded by: U.S. Public Health Service Grants
                Award ID: 1R21CA191663-01A1
                Award Recipient :
                This work was supported from the National Institute of Health and National Center for Research Resources ( www.nih.gov) Grant # 5P20RR18733-09 to DMM and from National Institute of Health and National Cancer Institute ( www.nih.gov) Grant # 1R21CA191663-01A1 to DMM. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology and Life Sciences
                Biochemistry
                Nucleotides
                Oligonucleotides
                Biology and Life Sciences
                Molecular Biology
                Molecular Biology Techniques
                Sequencing Techniques
                Sequence Analysis
                Sequence Motif Analysis
                Research and Analysis Methods
                Molecular Biology Techniques
                Sequencing Techniques
                Sequence Analysis
                Sequence Motif Analysis
                Biology and Life Sciences
                Genetics
                Gene Expression
                Gene Regulation
                Biology and Life Sciences
                Cell Biology
                Cell Processes
                Cell Cycle and Cell Division
                Research and Analysis Methods
                Biological Cultures
                Cell Cultures
                Cultured Tumor Cells
                Leukemia Cells
                Biology and Life Sciences
                Genetics
                Gene Expression
                Biology and life sciences
                Genetics
                Gene expression
                DNA transcription
                Biology and Life Sciences
                Molecular Biology
                Molecular Biology Techniques
                Artificial Gene Amplification and Extension
                Polymerase Chain Reaction
                Research and Analysis Methods
                Molecular Biology Techniques
                Artificial Gene Amplification and Extension
                Polymerase Chain Reaction
                Custom metadata
                All relevant data are within the paper and its Supporting information files.

                Uncategorized

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