Dynamics of genomic H 3 K 27me3 domains and role of EZH 2 during pancreatic endocrine specification

Abstract

Endoderm cells undergo sequential fate choices to generate insulinsecreting beta cells. Ezh2 of the PRC2 complex, which generates H3K27me3, modulates the transition from endoderm to pancreas progenitors, but the role of Ezh2 and H3K27me3 in the next transition to endocrine progenitors is unknown. We isolated endoderm cells, pancreas progenitors, and endocrine progenitors from different staged mouse embryos and analyzed H3K27me3 genome-wide. Unlike the decline in H3K27me3 domains reported during embryonic stem cell differentiation in vitro, we find that H3K27me3 domains increase in number during endocrine progenitor development in vivo. Genes that lose the H3K27me3 mark typically encode transcriptional regulators, including those for pro-endocrine fates, whereas genes that acquire the mark typically are involved in cell biology and morphogenesis. Deletion of Ezh2 at the pancreas progenitor stage enhanced the production of endocrine progenitors and beta cells. Inhibition of EZH2 in embryonic pancreas explants and in human embryonic stem cell cultures increased endocrine progenitors in vitro. Our studies reveal distinct dynamics in H3K27me3 targets in vivo and a means to modulate beta cell development from stem cells.

Genomic and Proteomic Resolution of Heterochromatin and Its Restriction of Alternate Fate Genes

“…Prior work on the latter focused on H3K9me3 and H3K27me3 marked chromatin (Ezhkova et al, 2009; Hawkins et al, 2010; Mansour et al, 2012; Matoba et al, 2014; Soufi et al, 2012; Xu et al, 2014), yet there was ample evidence, which we affirmed, that H3K9me3 and H3K27me3 domains in mammalian cells could be transcriptionally active or competent for activation (Blahnik et al, 2011; Breiling et al, 2001; Riddle et al, 2012; Vakoc et al, 2005). We find that using sucrose gradients to recover sonication-resistant chromatin, usually excluded from ChIP-seq studies, effectively purifies the subsets of H3K9me3 and H3K27me3 domains that are transcriptionally silent, nuclease resistant, hypermethylated, associated with Lamin B1, and most refractory to cellular reprogramming.…”

Differentially Expressed MicroRNA-483 Confers Distinct Functions in Pancreatic β- and α-Cells

“…In preparation for sorting, isolated islets were hand-picked and dissociated at 37°C by adding 0.05% trypsin-EDTA as described previously (36). Digestion was inactivated by the addition of FCS, and dissociated cells were centrifuged and resuspended in PBS containing 10% FBS for sorting.…”

Epigenetic modifications and long noncoding RNAs influence pancreas development and function

“…Genome-wide ChIP-seq analysis of H3K27me3 in sorted Neurog3+ cells showed increased PRC2-mediated methylation compared to sorted pancreatic progenitors [35]. However, in endocrine cells differentiated from hESCs there appeared to be a global decrease of H3K27me3 [5].…”

“…It has also been shown that Ezh2 suppresses the normal extent of endocrine cell induction; conditional homozygous and heterozygous deletion of Ezh2 in Pdx1+ pancreatic progenitors results in increased numbers of Neurog3+ cells without changes in proliferation [35]. This suggests that the elevated Neurog3+ cell numbers may be due to increased specification at the expense of other lineages, although this has not been directly tested.…”