To characterize the epigenetic programs that underlie pancreas differentiation, we have generated genome-scale maps of H3K4me3, H3K4me1 and H3K27me3 patterns by ChIP-seq and determined expression profiles by RNA-seq from undifferentiated human ESCs, three intermediate differentiated stages (definitive endoderm, primitive gut tube, and posterior foregut), pancreatic progenitors and in vitro-differentiated polyhormonal cells. Antibodies against CD142 and CD200 were used to select for targeted pancreatic and endocrine populations at the end of the culture. Pancreatic endoderm was subsequently transplanted for further differentiation into mature insulin-producing beta-cells and compared to sorted polyhormonal cells by RNA-seq and ChIP-seq analysis.

Characterize global gene expression and concomitant changes in chromatin structure during pancreatic lineage progression. Determine how closely the molecular features of hESC-derived beta cells resemble those of their primary human counterparts and which of these features are insufficiently induced in vitro.

Highly purified populations of pancreatic endoderm (PE) and intermediate lineage precursors were generated from hESCs in vitro. Functional endocrine cells (FE) were produced by further differentiation in SCID-Beige mice and compared to polyhormonal cells produced in late stage cultures. Cell populations were characterized by qRT-PCR, immunohistochemistry, and FACS for selected markers. RNA-seq was performed on hESC-derived populations representing embryonic stem cells, definitive endoderm, primitive gut tube, posterior foregut, PE and FE. Bayesian clustering was performed to identify stage-specific signature genes. Bivalency during pancreatic differentiation was studied by ChIP-seq of H3K4me3 and H3K27me3. Functional relevance of H3K27me3 removal during the transition from ES to DE was tested by inhibiting JMJD3 with two shRNAs.

Signature genes were identified for hESC cultured to primitive gut tube, posterior foregut, pancreatic endoderm (PE), and functional endocrine (FE) stages. Also identified were signature genes resolved from bivalent in hESCs to H3K4me3 at the definitive endoderm stage, PE signature genes resolved from bivalent in hESCs to H3K4me3 at the late PE stage, and FE signature genes resolved from bivalent at the PE stage to H3K4me3 after engraftment into mice. FE signature genes with no modification at the PE stage acquiring H3K4me3 after engraftment into mice were identified as well. Comparisons were performed to find genes more highly expressed in FE than in polyhormonal (PH) cells, genes with higher expression in FE retaining H3K27me3 repression during the transition from PE to PH, and genes with higher expression in FE failing to acquire H3K4me3 during the transition from PE to PH. Removal of Polycomb group-mediated repression of stage-specific genes was identified as a key mechanism for the induction of developmental regulators. Silencing of transitory genes during lineage progression associates with reinstatement of Polycomb group-dependent repression.

Bivalency is highly dynamic and tightly associated with activation and silencing of developmental regulators during lineage progression. Chromatin of critical beta cell genes is aberrantly remodeled during endocrine cell differentiation in vitro.

• ES cell differentiation: 2800, 3365, 3425, 3668, 4216, 4314, 4574,