(A) In trans MAP-C, the effect of TF knockouts or variants are assessed for their effect on a specific 3D contact by association with barcodes. Barcoded versions of the minimal pairing sequence were ectopically integrated and assessed for pairing with the native HAS1pr-TDA1pr in haploid S. cerevisiae. Red Xs indicate gene knockouts; red boxes indicate pairing sequence (Figure 1—figure supplement 1C); BC indicates barcode; double-headed arrows indicate the presence of chromosomal contacts. (B) Full TF knockout screen identifies Leu3 and Rgt1 as trans requirements for pairing. Histogram of relative abundance of each barcoded gene knockout strain in 3C library compared to the genomic library, excluding strains below a frequency of 0.3% of the pool. Barcode replicates are shown as separate squares in histogram. LEU3, RGT1, and MOT3 are highlighted individually; TF indicates other transcription factors; Nup indicates nuclear pore complex components; Neutral indicates fitness-neutral negative controls (Figure 2—figure supplement 2A). (C) Validation TF knockout screen confirms that Leu3, Sdd4, and Rgt1 are required for pairing. Bar plot of median relative abundance in 3C library compared to the genomic library, with overlaid scatter plot of individual barcoded strains. TF includes MIG1, VHR1, CBF1, and YGR067C. (D) Regions tested by ChIP-qPCR for TF binding. Bars show Leu3, Sdd4, and Rgt1 motif matches in S. cerevisiae HAS1pr-TDA1pr, and lines indicate regions (L and R) used for qPCRs in panel E. Bar heights indicate motif score as fraction of maximum possible score; all motifs with a score of at least 0.45 shown. (E) TFs bind HAS1pr-TDA1pr more strongly in saturated conditions. Chromatin immunoprecipitation qPCR results in exponentially growing (Exp) and saturated (Sat) cultures, normalized by input and to a negative control locus in the PRM1 coding sequence. L and R indicate two primer sets as shown in panel D. Bars indicate mean ± s.e.m. of biological triplicates. Dashed lines indicate a value of 1 (background enrichment). (F). Chromatin immunoprecipitation sequencing data near the HAS1pr-TDA1pr locus (coordinates chrXIII:844,705–862,314 in sacCer3 reference), shown as fold enrichment in IPs over input.