GSE118118
A combination of transcription factors mediates inducible interchromosomal contacts
Development of MAP-C (mutation analysis in pools by chromosome conformation capture), which involves performing 3C on a pool of mutants (generated by programmed oligonucleotide pools, error-prone PCR, or existing mutant collections), followed by amplification of 3C DNA using primers specific to a chromosomal contact of interest (with the mutagenized or barcode region included), and as a control, amplifying the mutagenized or barcode region regardless of ligation. The 3C and genomic libraries are amplified with primers that add sequencing and flowcell adapters, and then deep sequenced to quantify the abundance of each mutant/barcode. The ratio of abundance in the 3C compared to the genomic library reflects the extent to which each sequence variant participates in the chromosomal contact of interest. Overall design: Hi-C on a S. cerevisiae x S. uvarum hybrid yeast strain; cis MAP-C to test 178 bp tiling subsequences of the S. cerevisiae HAS1pr-TDA1pr region, error-prone PCR and programmed 3 bp substitution mutants of a minimal pairing region; trans MAP-C to test knockouts of transcription factor and nucleoporins, interaction partners of Rgt1, 10 amino acid deletions of Rgt1, and domain deletion and phosphorylation site mutants of Rgt1; RNA-seq of S. cerevisiae x S. uvarum hybrids with mutations in binding sites for Leu3 or Rgt1; RNA-seq of S. cerevisiae deletion strains for LEU3, SDD4, and RGT1; ChIP-seq for TAP-tagged Leu3, Sdd4, and Rgt1. Each MAP-C experiment was done in 2 or 3 technical replicates beginning with cell lysis, and the Rgt1 deletions were done in two biological replicates beginning with yeast transformation. RNA-seq and ChIP-seq experiments were done in biological triplicate. All FASTA files are available on the series record. Gene KO IDs are included in the processed data files *counts.txt.