Neurog3-Independent Methylation Is the Earliest Detectable Mark Distinguishing Pancreatic Progenitor Identity

In the developing pancreas, transient Neurog3-expressing progenitors give rise to four major islet cell types: α, β, δ, and γ; when and how the Neurog3⁺ cells choose cell fate is unknown. Using single-cell RNA-seq, trajectory analysis, and combinatorial lineage tracing, we showed here that the Neurog3⁺ cells co-expressing Myt1 (i.e., Myt1⁺Neurog3⁺) were biased toward β cell fate, while those not simultaneously expressing Myt1 (Myt1^-Neurog3⁺) favored α fate. Myt1 manipulation only marginally affected α versus β cell specification, suggesting Myt1 as a marker but not determinant for islet-cell-type specification. The Myt1⁺Neurog3⁺ cells displayed higher Dnmt1 expression and enhancer methylation at Arx, an α-fate-promoting gene. Inhibiting Dnmts in pancreatic progenitors promoted α cell specification, while Dnmt1 overexpression or Arx enhancer hypermethylation favored β cell production. Moreover, the pancreatic progenitors contained distinct Arx enhancer methylation states without transcriptionally definable sub-populations, a phenotype independent of Neurog3 activity. These data suggest that Neurog3-independent methylation on fate-determining gene enhancers specifies distinct endocrine-cell programs.