CTCF-mediated 3D chromatin predetermines the gene expression program in the male germline (RNA-Seq)

Spermatogenesis is a unidirectional differentiation process that generates haploid sperm, but how the gene expression program that directs this process is established is largely unknown. Here we determine the high-resolution 3D chromatin architecture of male germ cells during spermatogenesis and show that CTCF-mediated 3D chromatin predetermines the gene expression program required for spermatogenesis. In undifferentiated spermatogonia, CTCF-mediated chromatin interactions between meiosis-specific super-enhancers (SE) loci and target genes precede activation of meiosis-specific SEs on autosomes. These meiotic SE recruit the master transcription factor A-MYB in meiotic spermatocytes, which strengthens their 3D contacts and instructs a burst of meiotic gene expression. We also find that at the mitosis-to-meiosis transition, the germline-specific Polycomb protein SCML2 resolves chromatin loops that are specific to mitotic spermatogonia. Moreover, SCML2 and A-MYB establish the unique 3D chromatin organization of sex chromosomes during meiotic sex chromosome inactivation. We propose that CTCF-mediated 3D chromatin organization enforces epigenetic priming that directs unidirectional differentiation, thereby determining the cellular identity of the male germline. Overall design: Testis were collected from P17 or 18 A-myb+/- and A-myb-/- mice and pachytene spermatocytes were isolated using fluorescence-activated cell sorting (FACS) with Vybrant DyeCycle Violet Stain (DCV)