Truncated G-Quadruplex Isomers Cross-Talk with the Transcription Factors To Maintain Homeostatic Equilibria in c-MYC Transcription.

The nuclease hypersensitive element III1 (NHE III1) upstream c-MYC promoter harbors a transcription-silencing G-quadruplex (Pu27) element. Dynamic turnover of various transcription factors (TFs) across Pu27 to control c-MYC transcription homeostasis is enigmatic. Here, we reveal that native Pu27 evolves truncated G-quadruplex isomers (Pu19, Pu22, Pu24, and Pu25) in cells that are optimal intracellular targets of specific TFs in a sequence- and structure-dependent manner. Nuclear magnetic resonance and isothermal titration calorimetry envisaged that NM23-H2 (nucleoside diphosphate kinase) and nucleolin induce conformational fluctuations in Pu27 to sample specific conformationally restricted conformer(s). Structural investigations revealed that the flanking guanines at 5'-Pu27 control solvent exposure at G-quartets upon NM23-H2 and nucleolin binding driving Pu27 unfolding and folding, respectively. Transient chromatin immunoprecipitations confirmed that NM23-H2 drives the conformation switch to Pu24 that outcompetes nucleolin recruitment. Similarly, nucleolin arrests Pu27 in the Pu22 conformer minimizing NM23-H2 binding at Pu27. hnRNPK (heterogeneous nuclear ribonucleoprotein K) positively regulates NM23-H2 and nucleolin association at Pu27 despite their antagonism. On the basis of these results, we simulated the transcription kinetics in a feed-forward loop in which the transcription output responds to hnRNPK-induced early activation via NM23-H2 association, which favors Pu24 formation at NHE III1 reducing nucleolin occupancy and driving quadruplex unfolding to initiate transcription. NM23-H2 further promotes hnRNPK deposition across NHE III1 altering Pu27 plasticity that finally enriches the nucleolin abundance to drive Pu22 formation and weaken NM23-H2 binding to extinguish transcription. This mechanism involves three positive feedback loops (NM23-H2-hnRNPK, NM23-H2-CNBP, and hnRNPK-nucleolin) and one negative feedback loop (NM23-H2-nucleolin) controlling optimal turnover and residence time of TFs at Pu27 to homeostatically regulate c-MYC transcription.