Highlights d Epigenome dynamics of human-stem-cell-derived islet differentiation and maturation d Pioneer factors coordinate pervasive enhancer priming to steer endocrine cell fates d Core regulatory circuits identify LMX1B as critical for endocrine cell generation d Circadian rhythms trigger islet maturation via clockcontrolled metabolic cycles SUMMARY Stem-cell-derived tissues could transform disease research and therapy, yet most methods generate functionally immature products. We investigate how human pluripotent stem cells (hPSCs) differentiate into pancreatic islets in vitro by profiling DNA methylation, chromatin accessibility, and histone modification changes. We find that enhancer potential is reset upon lineage commitment and show how pervasive epigenetic priming steers endocrine cell fates.Modeling islet differentiation and maturation regulatory circuits reveals genes critical for generating endocrine cells and identifies circadian control as limiting for in vitro islet function. Entrainment to circadian feeding/fasting cycles triggers islet metabolic maturation by inducing cyclic synthesis of energy metabolism and insulin secretion effectors, including antiphasic insulin and glucagon pulses. Following entrainment, hPSC-derived islets gain persistent chromatin changes and rhythmic insulin responses with a raised glucose threshold, a hallmark of functional maturity, and function within days of transplantation. Thus, hPSC-derived tissues are amenable to functional improvement by circadian modulation.

Transcription factors (TFs) direct developmental transitions by binding to target DNA sequences, influencing gene expression and establishing complex gene-regultory networks. To systematically determine the molecular components that enable or constrain TF activity, we investigated the genomic occupancy of FOXA2, GATA4 and OCT4 in several cell types. Despite their classification as pioneer factors, all three TFs exhibit cell-type-specific binding, even when supraphysiologically and ectopically expressed. However, FOXA2 and GATA4 can be distinguished by low enrichment at loci that are highly occupied by these factors in alternative cell types. We find that expression of additional cofactors increases enrichment at a subset of these sites. Finally, FOXA2 occupancy and changes to DNA accessibility can occur in G 1 -arrested cells, but subsequent loss of DNA methylation requires DNA replication.NATuRE GENETiCS | www.nature.com/naturegenetics © 2018 Nature America Inc., part of Springer Nature. All rights reserved. Articles NATure GeNeTiCsBecause primary TF engagement cannot be adequately dissected by using endogenous systems that already express FOXA2 as part of their regulatory circuitry, we engineered a doxycycline (DOX)-inducible system in immortalized foreskin fibroblasts (BJ), which do not normally express FOXA2 or other FOXA family members ( Supplementary Fig. 1d). We derived several clonal cell lines ) cell lines. Cropped western blots of FOXA2 and H3 protein levels in two distinct BJ FOXA2 clones. d, Browser tracks displaying differential binding across ectopic BJ FOXA2 and dEN (chr 18: 19745852-19782939). FOXA2 FPKM values are shown at right. Below the scatter plot, output of DiffBind 42 differential peak-set analysis between dEN and BJ FOXA2 is shown. Red dots, peaks with statistically significant differential enrichment between the two datasets. Axes are reads per kilobase of transcript per million mapped reads (RPKM). e, Read-density heat map (normalized read count) of ectopic FOXA2 enrichment in BJ FOXA2 cells at dEN FOXA2 regions. Vertical side bar, peaks shared between BJ FOXA2 and dEN. Dashed lines mark the start and end of peaks, extended by 2 kb on either side. Most dEN sites still showed low-level enrichment in BJ FOXA2 yet were not called as significantly enriched. f, Read-density heat maps of ectopic OCT4 signal in BJ OCT4 at human ESC occupied regions (n = 22,477) and ectopic GATA4 signal in BJ GATA4 at dEN occupied regions (n = 42,477). Vertical side bar, peaks shared between the ectopic and endogenous context for either factor, as shown in e for FOXA2. In contrast to FOXA2 and GATA4, fewer ESC OCT4 sites showed any notable enrichment in BJ OCT4. g, Density plot displaying FOXA2, OCT4 and GATA4 ectopic enrichment in BJs at union sets of ectopic and endogenous sites (FOXA2, orange; OCT4, navy; GATA4, purple). Dashed lines demarcate regions within the background distribution, sampled sites (shaded) and peaks.NATuRE GENETiCS | www.nature.com/naturegenetics © 2018 Nature America Inc., part of Sprin...