Extensive NEUROG3 occupancy in the human pancreatic endocrine gene regulatory network.

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.

[The pre-gastrulation embryonic human development: future models and societal concerns]. / Le développement embryonnaire pré-gastrulatoire humain : modèles d'avenir et enjeux sociétaux.

Infertility, early miscarriages and congenital malformations are major public health issues that are frequent and poorly understood. Until now, what is known about early human development originates from two main sources: studies of human embryos and studies of model animals. Although some molecular mechanisms are conserved, there are specific human features. Thus, it is necessary to study model animals that are close to humans in the phylogenetic classification, which led to the use of pre-established primate cell lineages. Currently, the only human embryos available come from In Vitro Fertilization, which leads to important limitations: these embryos are relatively few and must be destroyed after 14 days. This has led researchers to develop new strategies. Several teams used Embryonic Stem Cells or Induced Pluripotent Stem Cells and their in vitro auto-organization properties to recreate "embryos" and thereby study their development. These new strategies allow a reduced use of human embryos but new questions arise about the legal status of these new research "models". In the future, it would be important to update the different legislations and recommendations of the International Society for Stem Cell Research as science progresses to avoid any failing drift. The respect of recommendations as well as the maintenance of discussions between specialists and the general public will allow a better understanding of early human development and the establishment of innovative strategies to target health challenges.

TITLE: Le développement embryonnaire pré-gastrulatoire humain : modèles d'avenir et enjeux sociétaux. ABSTRACT: L'infertilité, les fausses couches précoces et les malformations congénitales sont des problèmes majeurs de santé publique fréquents et relativement méconnus. Jusqu'à présent ce que l'on sait du développement précoce humain provient de deux sources principales : l'étude d'embryons humains et l'étude d'animaux modèles. Bien que certains mécanismes moléculaires soient conservés, il existe des spécificités liées à l'espèce humaine. Ainsi, il est important d'étudier les animaux modèles les plus proches possibles dans la classification phylogénétique, ce qui a mené à l'utilisation de lignées cellulaires de primates. De nos jours, les seuls embryons humains disponibles sont ceux issus de la Fécondation In Vitro, ils sont donc peu nombreux et doivent être détruits au bout de 14 jours. Cela a poussé les chercheurs à développer de nouvelles stratégies. Différentes équipes ont donc utilisé les cellules souches embryonnaires ou les cellules souches pluripotentes induites et leurs propriétés d'auto-organisation in vitro pour recréer des « embryons ¼ et ainsi étudier leur développement. Ces nouvelles stratégies permettent de limiter l'utilisation d'embryons humains mais de nouvelles questions se posent désormais sur le statut légal de ces nouveaux « modèles ¼. À l'avenir, il sera important de mettre à jour les différentes législations et recommandations de l'International Society for Stem Cell Research (ISSCR) au fur et à mesure des avancées scientifiques pour éviter toute dérive. Un respect des recommandations et le maintien de discussions entre spécialistes et « grand public ¼ permettront une meilleure compréhension du développement précoce humain et la mise en place de stratégies répondant à des enjeux sanitaires.