A combination of transcription factors mediates inducible interchromosomal pairing

Remodeling of the three-dimensional organization of a genome has been previously described (e.g. condition-specific pairing or looping), but it remains unknown which factors specify and mediate such shifts in chromosome conformation. Here we describe an assay, MAP-C (Mutation Analysis in Pools by Chromosome conformation capture), that enables the simultaneous characterization of hundreds of cis or trans-acting mutations for their effects on a chromosomal contact or loop. As a proof of concept, we applied MAP-C to systematically dissect the molecular mechanism of inducible interchromosomal pairing between HAS1pr-TDA1pr alleles in Saccharomyces yeast. We identified three transcription factors, Leu3, Sdd4 (Ypr022c), and Rgt1, whose collective binding to nearby DNA sequences is necessary and sufficient for inducible pairing between binding site clusters. Rgt1 contributes to the regulation of pairing, both through changes in expression level and through its interactions with the Tup1/Ssn6 repressor complex. HAS1pr-TDA1pr is the only locus with a cluster of binding site motifs for all three factors in both S. cerevisiae and S. uvarum genomes, but the promoter for HXT3, which contains Leu3 and Rgt1 motifs, also exhibits inducible homolog pairing. Altogether, our results demonstrate that specific combinations of transcription factors can mediate condition-specific interchromosomal contacts, and reveal a molecular mechanism for interchromosomal contacts and mitotic homolog pairing.