Summary Despite the recent explosion in surveys of cell-type heterogeneity, the mechanisms that specify and stabilize highly related cell subtypes remain poorly understood. Here, focusing initially on exploring quantitative histone mark heterogeneity, we identify two major sub-types of pancreatic β-cells (β HI and β LO ). β HI and β LO cells differ in their size, morphology, cytosolic and nuclear ultrastructure, transcriptional output, epigenomes, cell surface marker, and function. Importantly, β HI and β LO cells can be FACS separated live into CD24 + (β HI ) and CD24 - (β LO ) fractions. From an epigenetic viewpoint, β HI -cells exhibit ∼4-fold higher levels of H3K27me3, more compacted chromatin, and distinct chromatin organization that associates with a specific pattern of transcriptional output. Functionally, β HI cells have increased mitochondrial mass, activity, and insulin secretion both in vivo and ex vivo . Critically, Eed and Jmjd3 loss-of-function studies demonstrate that H3K27me3 dosage is a significant regulator of β HI / β LO cell ratio in vivo, yielding some of the first-ever specific models of β-cell sub-type distortion. β HI and β LO sub-types are conserved in humans with β HI -cells enriched in human Type-2 diabetes. These data identify two novel and fundamentally distinct β-cell subtypes and identify epigenetic dosage as a novel regulator of β-cell subtype specification and heterogeneity. Highlights Quantitative H3K27me3 heterogeneity reveals 2 common β-cell subtypes β HI and β LO cells are stably distinct by 7 independent sets of parameters H3K27me3 dosage controls β HI / β LO ratio in vivo β HI and β LO cells are conserved in humans and enriched in Type-2 diabetes

Identification of two β-cell subtypes by 7 independent criteria