H3K9me3 controls epidermis development by repressing RNA Pol II activity on key promoters and enhancers [CUT&Run]

Histone H3 lysine 9 trimethylation (H3K9me3) is a heterochromatin mark important for silencing repetitive DNA elements and for repressing lineage-inappropriate genes during early murine development, but its role in later organogenesis remains understudied. Here, we simultaneously target three histone methylases, Suv39h1, Suv39h2 and Setdb1, to deplete H3K9me3 specifically in developing epidermis. H3K9me3 loss blocks epidermis morphogenesis, resulting in defective cell differentiation, impaired barrier function, disrupted hair follicle formation, and failed Merkel cell specification. Single cell transcriptomic analysis reveals epidermal cells with mixed lineage identity and perturbed cell-type specific gene programs, including cell cycle, extracellular matrix, cytoskeleton, and metabolism. Using our recently developed approach, PReCIS-seq, we present the first cell-type specific, genome-wide landscape of transcriptionally engaged RNA polymerase II (RNA Pol II) in intact tissue following H3K9me3 depletion. We find that H3K9me3-enriched islands restrict inappropriate RNA Pol II activity at select developmentally regulated enhancers and promoters. This work highlights the indispensable role of H3K9me3, mediated by its three major histone methyltransferases, in regulating lineage diversification and developmental progression during mouse organogenesis.