Derepression of Polycomb targets during pancreatic organogenesis allows insulin-producing beta-cells to adopt a neural gene activity program

Abstract

The epigenome changes that underlie cellular differentiation in developing organisms are poorly understood. To gain insights into how pancreatic beta-cells are programmed, we profiled key histone methylations and transcripts in embryonic stem cells, multipotent progenitors of the nascent embryonic pancreas, purified beta-cells, and 10 differentiated tissues. We report that despite their endodermal origin, beta-cells show a transcriptional and active chromatin signature that is most similar to ectoderm-derived neural tissues. In contrast, the beta-cell signature of trimethylated H3K27, a mark of Polycomb-mediated repression, clusters with pancreatic progenitors, acinar cells and liver, consistent with the epigenetic transmission of this mark from endoderm progenitors to their differentiated cellular progeny. We also identified two H3K27 methylation events that arise in the beta-cell lineage after the pancreatic progenitor stage. One is a wave of cell-selective de novo H3K27 trimethylation in non-CpG island genes. Another is the loss of bivalent and H3K27me3-repressed chromatin in a core program of neural developmental regulators that enables a convergence of the gene activity state of beta-cells with that of neural cells. These findings reveal a dynamic regulation of Polycomb repression programs that shape the identity of differentiated beta-cells.

Figure 1.

Beta-cells and neural tissues share a gene activity program. Euclidean distances of genome-wide gene activity profiles, based on the presence or absence of mRNAs (A) or H3K4me3 (B) in each tissue. In both dendrograms, beta-cells are grouped with cerebral cortex and cerebellum. Similar conclusions were reached with principal component analysis using both purified beta-cells and islets (Supplemental Fig. S1).

Figure 2.

Tissue-specific loss and gain of H3K27 methylation. (A) Although many genes that show H3K27me3 enrichment in differentiated cells are also H3K27me3+ in ES cells, unexpectedly 69% of genes only show H3K27me3 enrichment in differentiated tissues (de novo H3K27me3). The cluster representation shows H3K27me3 (red), H3K4me3 (green), or both (yellow) in the 5248 genes that show H3K27me3 in at least one tissue. Genes with de novo H3K27me3 often lack CpG islands, which are indicated as a black line. (B) Most genes with H3K27me3 are inactive (see also Supplemental Fig. S2d). The mRNA heatmap maintains the same order as A. (C,D) H3K27me3 repression is both gene-specific and cell-specific. (C) Genes that are targeted by H3K27me3 in only few tissues are often also inactive in tissues where they do not show H3K27me3. This effect was more pronounced for non-CpG island genes (Supplemental Fig. S2c). (D) The beta-cell glucose transporter Slc2a2 shows H3K27me3 enrichment in acinar cells, yet is inactive in other tissues that lack H3K27me3. The graph shows posterior probability values ranging from 0–1 for the enrichment of H3K4me3 (green) and H3K27me3 (red).

Figure 3.

H3K27me3 profiles suggest a central role in beta-cell identity. (A) Beta-cell H3K27me3 preferentially targets genes that promote alternate developmental fates. (B) H3K27me3 is also enriched in genes whose expression is known to be deleterious for mature beta-cells.

Figure 4.

The beta-cell H3K27me3 enrichment signature includes pancreatic endoderm and terminal differentiation methylation programs. (A) In 59% of the 1480 genes that are H3K27me3+ in beta-cells, the methylation is acquired de novo, either before the pancreatic progenitor stage or during subsequent differentiation. De novo events occurring during later stages of differentiation preferentially target genes that lack CpG islands. On the other hand, in 67% of genes with H3K27me3 enrichment in beta-cells, this mark is present in progenitors and is then maintained. The cluster diagram depicts H3K27me3 enrichment (in red) in the different stages, and the presence of CpG islands is indicated in black in the adjacent column. (B) The gene profile that is H3K27me3+ in beta-cells is most similar to that of acinar, pancreatic progenitors, and liver. This was determined by Euclidean distances of genome-wide H3K27me3 distributions, as described in Methods. Similar results were observed using principal component analysis (Supplemental Fig. S6b).

Figure 5.

A neural regulatory program shows selective absence of PcG repression in beta-cells. (A) A major fraction of genes with selective absence of PcG repression in beta-cells showed an identical pattern in cortex and cerebellum. The cluster diagram shows the 249 genes that are H3K27me3- H3K4me3+ in beta-cells but are H3K27me3+ in more than five other tissues. A quantitative analysis is shown in Supplemental Figure S6c. (B) Examples of genes with selective absence of PcG repression in beta-cells. (C) Most enriched nonredundant Gene Ontology biological process and molecular function terms (levels 4 and 5) among the 249 genes with beta-cell-selective absence of PcG repression. A more complete list is shown in Supplemental Table S8.

Figure 6.

Pancreatic progenitors suppress regulators of beta-cell differentiation. (A) genes with selective absence of PcG repression in beta-cells often exhibit H3K27me3 enrichment in ES cells or pancreatic progenitors and have CpG islands. The cluster representation shows H3K27me3 (red) in the 249 genes with selective absence of PcG repression in beta-cells in different stages. In the adjacent column, the presence of CpG islands is indicated in black. (B) As expected, genes with selective absence of PcG repression in beta-cells are frequently bivalent in ES cells. In pancreatic progenitors, bivalency in such genes is similar to other differentiated cell types. (C) Genes with selective absence of PcG repression in beta-cells show H3K27me3 repression in both differentiated cell types of distant lineages as well as in pancreatic progenitors. (D) Pancreatic progenitors do not preferentially show a bivalent H3K27me3+ H3K4me3+ state (yellow) for genes with selective absence of PcG repression in their progeny (i.e., acinar and beta-cells), compared with genes with selective absence of PcG repression in distant lineages. (E) Pancreatic multipotent progenitors exhibit H3K27me3 repression of genes involved in pluripotency and alternate endoderm fates, but also in regulators of their progeny.

Figure 7.

PcG repression programs that shape beta-cell identity. Schematic illustrating how PcG-dependent modifications are placed or removed in a highly context-dependent manner during beta-cell differentiation. (A) A subset of H3K27me3+ genes in beta-cells have acquired this mark by the pancreatic endoderm progenitor stage, which is then epigenetically inherited in their cellular progeny. (B) Another subset of genes undergoes de novo H3K27me3 repression during late differentiation. These often lack CpG islands and are highly cell-type-specific H3K27me3 events. In some of these genes, repression is essential for the differentiated function of beta-cells. (C) The selective loss of PcG repression after the pancreatic progenitor stage in a core set of developmental regulators enables a convergence of transcriptional profiles in beta-cells with neuroectoderm-derived tissues.