title: Quantitative characterization of the interactions among c-myc transcriptional regulators FUSE, FBP, and FIR.
abstract: Human c-myc is critical for cell homeostasis and growth but is a potent oncogenic factor if improperly regulated. The c-myc far-upstream element (FUSE) melts into single-stranded DNA upon active transcription, and the noncoding strand FUSE recruits an activator [the FUSE-binding protein (FBP)] and a repressor [the FBP-interacting repressor (FIR)] to fine-tune c-myc transcription in a real-time manner. Despite detailed biological experiments describing this unique mode of transcriptional regulation, quantitative measurements of the physical constants regulating the protein-DNA interactions remain lacking. Here, we first demonstrate that the two FUSE strands adopt different conformations upon melting, with the noncoding strand DNA in an extended, linear form. FBP binds to the linear noncoding FUSE with a dissociation constant in the nanomolar range. FIR binds to FUSE more weakly, having its modest dissociation constants in the low micromolar range. FIR is monomeric under near-physiological conditions but upon binding of FUSE dimerizes into a 2:1 FIR(2)-FUSE complex mediated by the RRMs. In the tripartite interaction, our analysis suggests a stepwise addition of FIR onto an activating FBP-FUSE complex to form a quaternary FIR(2)-FBP-FUSE inhibitory complex. Our quantitative characterization enhances understanding of DNA strand preference and the mechanism of the stepwise complex formation in the FUSE-FBP-FIR regulatory system.
authors.affiliation: Department of Pathology, Yale University School of Medicine, New Haven, CT 06520, USA.
authors.forename: Hsin-Hao
authors.initials: HH
authors.lastname: Hsiao
authors.forename: Abhinav
authors.initials: A
authors.lastname: Nath
authors.forename: Chi-Yen
authors.initials: CY
authors.lastname: Lin
authors.forename: Ewa J
authors.initials: EJ
authors.lastname: Folta-Stogniew
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Rhoades
authors.forename: Demetrios T
authors.initials: DT
authors.lastname: Braddock
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism* / Q000502:physiology; D004265:DNA Helicases / Q000737:chemistry / Q000378:metabolism* / Q000502:physiology; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism* / Q000502:physiology; D019281:Dimerization; D020662:Guanine Nucleotide Exchange Factors / Q000737:chemistry / Q000378:metabolism* / Q000502:physiology; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D016271:Proto-Oncogene Proteins c-myc / Q000737:chemistry / Q000378:metabolism* / Q000502:physiology; D000072260:RNA Splicing Factors; D016601:RNA-Binding Proteins; D012097:Repressor Proteins / Q000737:chemistry / Q000378:metabolism* / Q000502:physiology; D064067:Rho Guanine Nucleotide Exchange Factors; D012996:Solutions; D015534:Trans-Activators / Q000737:chemistry / Q000378:metabolism* / Q000502:physiology
chemical_list: D002352:Carrier Proteins; D004268:DNA-Binding Proteins; C577426:FARP2 protein, human; C492258:FUBP1 protein, human; D020662:Guanine Nucleotide Exchange Factors; C489427:MYC protein, human; D016271:Proto-Oncogene Proteins c-myc; D000072260:RNA Splicing Factors; D016601:RNA-Binding Proteins; D012097:Repressor Proteins; D064067:Rho Guanine Nucleotide Exchange Factors; D012996:Solutions; D015534:Trans-Activators; C410595:poly-U binding splicing factor 60KDa; D004265:DNA Helicases