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

ABSTRACT: 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.

SUBMITTER: Liu J

PROVIDER: S-EPMC6327977 | biostudies-literature | 2019 Jan

REPOSITORIES: biostudies-literature

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

Liu Jing J Banerjee Amrita A Herring Charles A CA Attalla Jonathan J Hu Ruiying R Xu Yanwen Y Shao Qiujia Q Simmons Alan J AJ Dadi Prasanna K PK Wang Sui S Jacobson David A DA Liu Bindong B Hodges Emily E Lau Ken S KS Gu Guoqiang G

Developmental cell 20190101 1

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<s ...[more]

PMID: 30620902

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Project description:ObjectiveMice lacking the bHLH transcription factor (TF) Neurog3 do not form pancreatic islet cells, including insulin-secreting beta cells, the absence of which leads to diabetes. In humans, homozygous mutations of NEUROG3 manifest with neonatal or childhood diabetes. Despite this critical role in islet cell development, the precise function of and downstream genetic programs regulated directly by NEUROG3 remain elusive. Therefore, we mapped genome-wide NEUROG3 occupancy in human induced pluripotent stem cell (hiPSC)-derived endocrine progenitors and determined NEUROG3 dependency of associated genes to uncover direct targets.MethodsWe generated a novel hiPSC line (NEUROG3-HA-P2A-Venus) where NEUROG3 is HA-tagged and fused to a self-cleaving fluorescent VENUS reporter. We used the CUT&RUN technique to map NEUROG3 occupancy and epigenetic marks in pancreatic endocrine progenitors (PEP) that were differentiated from this hiPSC line. We integrated NEUROG3 occupancy data with chromatin status and gene expression in PEPs as well as their NEUROG3-dependence. In addition, we investigated whether NEUROG3 binds type 2 diabetes mellitus (T2DM)-associated variants at the PEP stage.ResultsCUT&RUN revealed a total of 863 NEUROG3 binding sites assigned to 1263 unique genes. NEUROG3 occupancy was found at promoters as well as at distant cis-regulatory elements that frequently overlapped within PEP active enhancers. De novo motif analyses defined a NEUROG3 consensus binding motif and suggested potential co-regulation of NEUROG3 target genes by FOXA or RFX transcription factors. We found that 22% of the genes downregulated in NEUROG3-/- PEPs, and 10% of genes enriched in NEUROG3-Venus positive endocrine cells were bound by NEUROG3 and thus likely to be directly regulated. NEUROG3 binds to 138 transcription factor genes, some with important roles in islet cell development or function, such as NEUROD1, PAX4, NKX2-2, SOX4, MLXIPL, LMX1B, RFX3, and NEUROG3 itself, and many others with unknown islet function. Unexpectedly, we uncovered that NEUROG3 targets genes critical for insulin secretion in beta cells (e.g., GCK, ABCC8/KCNJ11, CACNA1A, CHGA, SCG2, SLC30A8, and PCSK1). Thus, analysis of NEUROG3 occupancy suggests that the transient expression of NEUROG3 not only promotes islet destiny in uncommitted pancreatic progenitors, but could also initiate endocrine programs essential for beta cell function. Lastly, we identified eight T2DM risk SNPs within NEUROG3-bound regions.ConclusionMapping NEUROG3 genome occupancy in PEPs uncovered unexpectedly broad, direct control of the endocrine genes, raising novel hypotheses on how this master regulator controls islet and beta cell differentiation.

Project description:Background & aimsTransient expression of Neurog3 commits intestinal secretory progenitors to become enteroendocrine-biased progenitors and hence drive enteroendocrine differentiation. Loss of Neurog3 in mouse resulted in the depletion of intestinal enteroendocrine cells (EECs) and an increase in goblet cells. Earlier studies in developing mouse pancreas identified a role of Neurog3 gene dosage in endocrine and exocrine cell fate allocation. We aimed to determine whether Neurog3 gene dosage controls fate choice of enteroendocrine progenitors.MethodsWe acquired mutant Neurog3 reporter mice carrying 2, 1, or null Neurog3 alleles to study Neurog3 gene dosage effect by lineage tracing. Cell types arising from Neurog3+ progenitors were determined by immunohistochemistry using antibodies against intestinal lineage-specific markers. RNA sequencing of sorted Neurog3+/+, Neurog3+/-, or bulk intestinal cells were performed and differentially expressed genes were analyzed.ResultsWe identified 2731 genes enriched in sorted Neurog3+/+-derived cells in the Neurog3+/+EYFP mouse intestine when compared with bulk duodenum epithelial cells. In the intestine of Neurog3+/-EGFP heterozygous mouse, we observed a 63% decrease in EEC numbers. Many Neurog3-derived cells stained for goblet marker Mucin 2. RNA sequencing of sorted Neurog3+/- cells uncovered enriched expression of genes characteristic for both goblet and enteroendocrine cells, indicating the mixed lineages arose from Neurog3+ progenitors. Consistent with this hypothesis, deletion of both Neurog3 alleles resulted in the total absence of EECs. All Neurog3+-derived cells stained for Mucin 2.ConclusionsWe identified that the fate of Neurog3+ enteroendocrine progenitors is dependent on Neurog3 gene dosage. High Neurog3 gene dosage enforces the commitment of secretory progenitors to an EE lineage, while constraining their goblet cell lineage potential. Transcriptome profiling data was deposited to Gene Ontology omnibus, accession number: GSE149203.

Dataset Information

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

Publications

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

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