Gene syntax defines supercoiling-mediated transcriptional feedback [CUT&RUN]

Gene syntax---the order and arrangement of genes and their regulatory elements---shapes the dynamic coordination of both natural and synthetic gene circuits. Transcription at one locus profoundly impacts the transcription of nearby adjacent genes, but the molecular basis of this effect remains poorly understood. Here, using integrated reporter circuits in human cells, we show that the reciprocal effects of transcription and DNA supercoiling, which we term supercoiling-mediated feedback, regulates expression of adjacent genes in a syntax-specific manner. Using a suite of chromatin state assays, we measure syntax- and induction-dependent formation of chromatin structures in human induced pluripotent stem cells. Applying syntax as a design parameter and without altering sequence or copy number, we built compact gene circuits, tuning the expression mean, noise, and stoichiometry across diverse delivery methods and cell types. Integrating supercoiling-mediated feedback into models of gene regulation will expand our understanding of native systems and enhance the design of synthetic gene circuits. Overall design: GapRUN profiling of hiPSC lines containing integrated synthetic gene pairs. Gene pairs with one of the four possible syntaxes (divergent, convergent, upstream tandem, downstream tandem) were integrated homozygously at the CYLBL locus in hiPSCs. The two genes were mTagBFP2-T2A-rtTA expressed constitutively from the CAG promoter, and mScarlet expressed from the inducible TRE promoter. These four lines were split into plus and minus induction conditions. The induced condition was treated with treated with 1000 ng/mL doxycycline for three days. Two days prior to collection, the cells were transduced with a lentivirus expressing EGFP-GapR from an EF1a promoter. Around 500k cells were collected and bound to ConA beads. A fusion protein of MNase and a nanobody targeting EGFP was purified separately and added to the cells. After nanobody binding and wash steps, the MNase was activated to fragment the DNA, as in CUT&RUN. The resulting library was prepared as in CUT&RUN. The GapRUN protocol was derived from dx.doi.org/10.1016/j.molcel.2024.10.007; see the attached work here for protocol details.