­­­The epigenetic reader Phf2 is positioned in the genome by cohesin [ChIP-seq]

Eukaryotic genomes are assembled into chromatin by histones and extruded into loops by cohesin. These mechanisms control important genomic functions, but whether histones and cohesin cooperate in genome regulation is poorly understood. Here we identify Phf2, a member of the Jumonji-C family of histone demethylases, as a cohesin-interacting protein. Phf2 binds to H3K4me3 nucleosomes at active transcription start sites (TSSs) but also co-localizes with cohesin. Cohesin depletion reduces Phf2 binding at sites lacking H3K4me3, and depletion of Wapl and CTCF re-positions Phf2 together with cohesin in the genome, resulting in the accumulation of both proteins in vermicelli and cohesin islands. Conversely, Phf2 depletion reduces cohesin binding at TSSs lacking CTCF, decreases the number of short cohesin loops, but increases the length of heterochromatic B compartments. These results suggest that Phf2 is an ‘epigenetic reader’, which is translocated through the genome by cohesin-mediated DNA loop extrusion, and which recruits cohesin to active TSSs and limits the size of B compartments. These findings reveal an unexpected degree of cooperativity between epigenetic and architectural mechanisms of eukaryotic genome regulation. The objective of this experiment was to investigate the role of cohesin in positioning the epigenetic reader Phf2 by performing ChIP-seq on wild-type (WT) and mutant (Smc3 KO, Phf2 KO, Wapl KO, and CTCF/Wapl dKO) mouse embryonic fibroblast cells (MEFs). For the conditional deletion of floxed Phf2, Wapl, Smc3, or CTCF alleles, we used either the inducible CreERT2 recombinase expressed from the Rosa26 locus (CreER) or Adeno-Cre virus. Each condition was performed in biological duplicates, triplicates, or tetraplicates. ChIP-seq was conducted using Phf2/Smc3 antibodies, and libraries were sequenced on an Illumina platform. The goal is to assess differences in Phf2/cohesin binding patterns between WT and mutant samples, particularly at active TSSs lacking CTCF binding sites.