Extensive NEUROG3 occupancy in the human pancreatic endocrine gene regulatory network

Abstract

Objective Mice 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. Methods We 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. Results CUT&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. Conclusion Mapping 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.

Characterization of cell-fate decision landscapes by estimating transcription factor dynamics

“…2J); it has already been pointed out as a critical factor during other differentiation processes such as in thymocyte development 35 . Also, previous studies from our group reported it to be a direct target of Neurog3 36 .…”

“…4D), indicating that in the human in-vitro context, there is a similar switch from MAFB to MAFA, in developing towards fully functional β-cells, to the one reported in rodents 61 . GLIS3, which we previously found as a direct target of NEUROG3 in pancreatic endocrine progenitor cells 36 , was classified as SC-EC (Fig. 4E).…”

“…IRX2 was highly active later on during SC-Fig. 4F); importantly, Gage et al (2015) found it downregulated in hPSC-derived human islet cells lacking ARX 63 , and Schreiber et al (2021) found it as a direct target of NEUROG3 36 . Hence, our predictions -specific novel TF acting downstream NEUROG3.…”

scmTE: multivariate transfer entropy builds interpretable compact gene regulatory networks by reducing false predictions

“…A previous study proposed that Neurog3 can start endocrine development by regulating a set of TFs that are related to the proliferation and maturation of endocrine cells (Napolitano et al 2015). Islet cells cannot be formed in mice lacing Neurog3, leading to diabetes (Schreiber, et al, 2021).…”

“…Pax4 is an important regulator for β cells construction. Experiments show that mice lacking Pax4 die after a few days because they lack mature β cells (Napolitano, et al, 2015;Schreiber, et al, 2021). Insm1 maintains mature β-cell functions (Jia, et al, 2015).…”