Mechanistic studies for the role of cellular nucleic-acid-binding protein (CNBP) in regulation of c-myc transcription

• Published in: Biochimica et biophysica acta Vol. 1830; no. 10; pp. 4769 - 4777
• Main Authors: Chen, Siqi, Su, Lijuan, Qiu, Jun, Xiao, Nannan, Lin, Jing, Tan, Jia-heng, Ou, Tian-miao, Gu, Lian-quan, Huang, Zhi-shu, Li, Ding
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.10.2013
• Subjects: Amino Acid Sequence; Base Sequence; c-myc; Cellular nucleic-acid-binding protein; Chromatin Immunoprecipitation; Circular Dichroism; DNA Primers; Electrophoretic Mobility Shift Assay; Fluorescence Resonance Energy Transfer; G-quadruplex; Genes, myc; Humans; luciferase; Molecular Sequence Data; NM23 Nucleoside Diphosphate Kinases - metabolism; Nuclease hypersensitive element III1; protein-protein interactions; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; RNA-Binding Proteins - chemistry; RNA-Binding Proteins - metabolism; RNA-Binding Proteins - physiology; Sequence Homology, Amino Acid; Surface Plasmon Resonance; transcription (genetics); transcription factors; Transcription, Genetic; Transcriptional Activation; Transcriptional regulation; Nuclease hypersensitive element III1; CD; Cellular nucleic-acid-binding protein; c-myc; CNBP; G-quadruplex; NHE; Co-IP; ChIP; Transcriptional regulation; TAMRA; FAM; SPR; EMSA; FRET; TMPyP4; RT-qPCR; meso-tetra(N-methyl-4-pyridyl)porphine; circular dichroism; co-immunoprecipitation; chromatin immunoprecipitation; electrophoretic mobility shift assay; surface plasmon resonance; 6-carboxyfluorescein; reverse transcription-quantitative polymerase chain reaction; nuclease hypersensitive element; fluorescence resonance energy transfer; tetramethylrhodamine; Nuclease hypersensitive element III
• ISSN: 0304-4165; 0006-3002; 1872-8006
• DOI: 10.1016/j.bbagen.2013.06.007

Guanine-rich sequence of c-myc nuclease hypersensitive element (NHE) III1 is known to fold in G-quadruplex and subsequently serves as a transcriptional silencer. Cellular nucleic-acid-binding protein (CNBP), a highly conserved zinc-finger protein with multiple biological functions, could bind to c-myc NHE III1 region, specifically to the single strand G-rich sequence. In the present study, a variety of methods, including cloning, expression and purification of protein, EMSA, CD, FRET, Ch-IP, RNA interference, luciferase reporter assay, SPR, co-immunoprecipitation, and co-transfection, were applied to investigate the mechanism for the role of CNBP in regulating c-myc transcription. We found that human CNBP specifically bound to the G-rich sequence of c-myc NHE III1 region both in vitro and in cellulo, and subsequently promoted the formation of G-quadruplex. CNBP could induce a transient decrease followed by an increase in c-myc transcription in vivo. The interaction of CNBP with NM23-H2 was responsible for the increase of c-myc transcription. Based on above experimental results, a new mechanism, involving G-quadruplex related CNBP/NM23-H2 interaction, for the regulation of c-myc transcription was proposed. These findings indicated that the regulation of c-myc transcription through NHE III1 region might be governed by mechanisms involving complex protein–protein interactions, and suggested a new possibility of CNBP as a potential anti-cancer target based on CNBP's biological function in c-myc transcription. •Cellular nucleic-acid-binding protein (CNBP) binds to oncogene c-myc promoter region.•We found that human CNBP bound to c-myc NHE III1 and promoted G-quadruplex formation.•CNBP induced a transient decrease followed by an increase in c-myc transcription.•Interaction of CNBP with NM23-H2 led to the increase of c-myc transcription.•New c-myc regulatory mechanism was proposed suggesting CNBP as an anti-tumor target.