The FUSE/FBP/FIR/TFIIH system is a molecular machine programming a pulse of c-myc expression

Abstract

FarUpStream Element (FUSE) Binding Protein (FBP) binds the human c-myc FUSE in vitro only in single-stranded or supercoiled DNA. Because transcriptionally generated torsion melts FUSE in vitro even in linear DNA, and FBP/ FBP Interacting Repressor (FIR) regulates transcription through TFIIH, these components have been speculated to be the mechanosensor (FUSE) and effectors (FBP/FIR) of a real-time mechanism controlling c-myc transcription. To ascertain whether the FUSE/FBP/FIR system operates according to this hypothesis in vivo, the flux of activators, repressors and chromatin remodeling complexes on the c-myc promoter was monitored throughout the seruminduced pulse of transcription. After transcription was switched on by conventional factors and chromatin regulators, FBP and FIR were recruited and established a dynamically remodeled loop with TFIIH at the P2 promoter. In XPB cells carrying mutant TFIIH, loop formation failed and the serum response was abnormal; RNAi depletion of FIR similarly disabled c-myc regulation. Engineering FUSE into episomal vectors predictably re-programmed metallothionein-promoter-driven reporter expression. The in vitro recruitment of FBP and FIR to dynamically stressed c-myc DNA paralleled the in vivo process.

Far Upstream Element Binding Protein Plays a Crucial Role in Embryonic Development, Hematopoiesis, and Stabilizing Myc Expression Levels

“…As the FUSE first melts in response to the torque generated by ongoing transcription (Figure 10, A and B), 5,12,13 FBP sequence selectively engages DNA via its KH motifs. 2,3,15,22 Then FBP loops and stimulates the p89/ XPB/ERCC3 3 0 to 5 0 helicase/translocase of TFIIH at the promoter increasing transcription.…”

“…6 Similarly, the FBP/FIR/FUSE system acts through this subunit and is proposed to act as a molecular cruise control to dampen fluctuations in MYC expression. 4,5,7,10 If this proposal has merit, then Myc levels in Fubp1 À/À cells should vary more than in WT cells. The relatively uniform expression of Myc in MEFs made it a good substrate to assess the variation elicited on loss of FBP.…”

“…4e6 Positive regulation of MYC is at least in part counteracted by FBP interacting repressor (FIR) that binds FBP and FUSE and opposes TFIIH helicase activity, retarding the transcription cycle. 5,7 Besides MYC, FBP regulates the expression of the cyclin-dependent kinase inhibitor p21, stathmin, and USP29, a powerful stabilizer of p53 induced by oxidative stress. 8e10 Because the FBP/FIR system reads single-stranded DNA sequences exposed by transcriptionally generated dynamic supercoiling, it is proposed that the FBP/ FIR/FUSE system serves as a molecular cruise control that monitors the mechanical output of promoters in real time while providing both positive and negative feedback.…”

Hierarchical mechanisms build the DNA-binding specificity of FUSE binding protein

“…FBP binds to the Far Upstream Element (FUSE), an A/T-rich element located 1.7 kb upstream of the c-myc P2 promoter (9)(10)(11), and modulates its transcription. A molecular machine composed of FUSE, FBP, the FBP Interacting Repressor (FIR), and TFIIH integrates multiple inputs (9) to set the output of the protooncogene.…”

Controlling gene expression by DNA mechanics: emerging insights and challenges

“…Transcription-dependent melting of the far upstream element (FUSE) of the human MYC promoter was seen when this element was included in the excised segment, just as previously hypothesized (He et al 2000). Such transcription-dependent melting licensed the binding to FUSE of FUSE binding protein (FBP) and FBP interacting repressor (FIR) in vitro (Liu et al 2006). FUSE melting is a premier example of the concordance between the prediction of DNA melting using the thermodynamic-based calculations of the supercoil induced duplex destabilization (SIDDs) algorithm and experiment (Benham 1993;Bi and Benham 2004).…”