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Genes differentially expressed between NEUROG3-Venus positive and negative hiPSC line differentiated to pancreatic endocrine progenitors [RNA-Seq]

ABSTRACT: Genes differentially expressed between NEUROG3-Venus positive and negative hiPSC line differentiated to pancreatic endocrine progenitors [RNA-Seq]

PROVIDER: PRJNA743030 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR15014570.fastq.gz	Fastqsanger.gz
	SRR15014571.fastq.gz	Fastqsanger.gz
	SRR15014572.fastq.gz	Fastqsanger.gz
	SRR15014573.fastq.gz	Fastqsanger.gz
	SRR15014574.fastq.gz	Fastqsanger.gz

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Genes differentially expressed between NEUROG3-Venus positive and negative hiPSC line differentiated to pancreatic endocrine progenitors [RNA-Seq]

Project description:Despite this critical role in islet cell development, the precise function and downstream genetic programs regulated directedly by NEUROG3 remain elusive. We therefore mapped genome-wide NEUROG3 occupancy in human induced pluripotent stem cell (iPSC)-derived endocrine progenitors and determined NEUROG3 dependency of associated genes to uncover direct targets. To this aim, 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, an alternative method to CHIP-seq allowing transcription factor profiling from a low cell number, to map NEUROG3 occupancy and epigenetic marks in pancreatic endocrine progenitors (PEP) differentiated from this hiPSC line. To optimize the stringency and relevance of NEUROG3 binding sites, we focused our analysis on regions identified both with HA and NEUROG3 antibodies and integrated NEUROG3 occupancy data with chromatin status and gene expression in PEPs and their NEUROG3-dependence. Mapping of NEUROG3 genome occupancy in PEPs uncovers an unexpectedly broad, direct control of the endocrine gene regulatory network (GRN) and raises novel hypotheses on how this master regulator controls islet and beta cell differentiation.

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Genome wide profiling of NEUROG3 occupancy in human pancreatic endocrine progenitors [CUT&RUN]

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Open chromatin landscape of pancreatic endocrine progenitors

Project description:During embryonic development, islet progenitors are specified from pancreatic duct cells by transient expression of Neurog3, a transcription factor necessary and sufficient for initiation of islet development. To understand the dynamics of Neurog3-dependent endocrine cell fate determination, in this study we used ATAC-Seq to identify accessible genomic regions of purified duct, endocrine progenitor, and endocrine cells isolated from mice with varying Neurog3 dosage

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Neurog3-independant role of Notch signaling in pancreas explants. Microarray of pancreata of Wildtype or Neurog3tTA/tTA (Neurog3-Null) background at E12.5, explanted for 3d + treatment 24h with DAPT/vehicle.

Project description:Since loss of Hes1 and Notch signalling drives progenitors to the endocrine lineage, we set up a pancreas explant system with crosses of heterozygous Neurog3tTA/+, a knock-in allele which makes the homozygote Neurog3tTA/tTA embryos deficient in Neurog3 (hereafter Neurog3-null). Hereby endocrine differentiation is impeded and we can study the Neurog3-independent role of Notch signalling by DAPT inhibition of γ-secretase. E12.5 wildtype and Neurog3-null pancreata were explanted on fibronectin, grown for 3 days, and then treated with vehicle control (0.1% DMSO) or DAPT for 24h. RNA was isolated and subjected to Agilent microarray analysis

2022-02-27 | E-MTAB-6898 | biostudies-arrayexpress

Extensive NEUROG3 occupancy in the human pancreatic endocrine gene regulatory network

Project description:Extensive NEUROG3 occupancy in the human pancreatic endocrine gene regulatory network

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RNA-seq of Human Embryonic Stem Cell derived pancreas progenitor differentiation, Day 13 of wildtype, HES1-/-, NEUROG3-/- and HES1-/-NEUROG3-/- genotypes

Project description:To better understand the mechanism by which HES1 regulates pancreas development we took advantage of recent developments in directed differentiation of human embryonic stem cells (hESCs) to the pancreatic endocrine lineage via a series of progenitor stages (Figure 1 and (Rezania et al., 2014). Using the iCRISPR platform (González et al., 2014), we introduced indels in the HES1 and NEUROG3 genes, either singly or in combination in iCRISPR H1 cells. Using previously described gRNAs (Zhu et al., 2016) to target exon2 of the NEUROG3 gene and exon2 of the HES1 gene we detected by Sanger and RNA-sequencing a 2 bp insertion and a T insertion, respectively, resulting in premature STOP codons in two/multiple clonal cell lines carrying the introduced mutation and the loss of NEUROG3 by immunostaining We then subjected two or more clonal lines of H1-iCRISPR (wildtype), HES1−/−, NEUROG3−/− and HES1−/−NEUROG3−/− (abbreviated H1N3-dKO or H1−/−N3−/−) to differentiation to β-like cells using a modified version of the protocol from Rezania et al. (2014). Marker analysis showed that all cell lines maintained pluripotency (OCT4+) as hESC and were able to differentiate to FOXA2+ and SOX17+ co-positive definitive endoderm (DE, Day 3), PDX1+ cells at the posterior foregut stage (PF, Day 7), to bipotent progenitors marked by PDX1+ and NKX6-1+ at the Pancreatic Endoderm stage (PE, Day 10), and the Endocrine Precursor stage (EP, Day 13)

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RNA-seq in SREBF1-deficient human iPSCs, hiPSC-NSCs and differentiated cells.

Project description:Omics analyses and qRT-PCR time-course during the transition from hiPSC to hiPSC-NSC highlighted the up-regulation of SREBF1, a gene involved in cholesterol biosynthesis and lipid homeostasis, suggesting its potential role in NSC commitment/maintenance. To test this hypothesis, we generated SREBF1-deficient hiPSC lines by co-delivering ribonucleoprotein Cas9 with a pool of sgRNA targeting exon 5 of the SREBF1 gene. Upon isolation of three clones harboring the deletion we performed RNA-seq analysis in hiPSC, hiPSC-NSC and differentiated cultures at 7 and 14 days of differentiation, compared to control cells. This analysis will allow to identify the potential role of SREBF1 in affecting hiPSC-to-hiPSC-NSC transition, hiPSC-NSC maintenance and commitment toward differentiated cell populations.

2024-09-23 | GSE273125 | GEO

Knockdown of Atoh8 in Neurog3-expressing mouse ductal cells mPAC results in altered expression of multiple cell cycle related genes

Project description:Atoh8 is a transcription factor of the basic-helix-loop-helix (bHLH) family that is expressed in multiple tissues during embryonic development but whose specific functions remain unknown. The gene encoding Atoh8 is induced by various lineage- determining bHLH transcription factors in cell culture, suggesting a possible common role of this gene in multiple bHLH-driven differentiation programs. In the pancreas, the pro-endocrine bHLH factors Neurogenin3 (Neurog3) and NeuroD1 activate Atoh8 expression. Moreover, Atoh8 is expressed in the embryonic pancreas confirming its participation in the pancreatic transcriptional cascade. This work aims at gaining insight into the molecular function of Atoh8 during the endocrine differentiation program initiated by Neurog3 in the pancreas. To this aim, we have generated a recombinant adenovirus encoding an Atoh8-specific shRNA (Ad-shAtoh8) and used it to down-regulate expression of the Atoh8 gene in Neurog3-expressing pancreatic ductal cells (mPAC), a cellular model of endocrine cell differentiation. Thus, we have compared global changes in gene expression profiles between cells treated with Ad-Neurog3+shControl and cells treated with Ad-Neurog3+shAtoh8 using Affymetrix microarrays. Our results show that Atoh8 silencing significantly affects the expression of 293 genes in Neurog3-expressing mPAC cells. Gene Ontology analysis has revealed cell cycle as the biological function most significantly represented among the modified genes. These results uncover a potential function of Math6 as a regulator of cell cycle progression and provide novel insights into the link between Neurog3 and the regulation of the cell cycle.

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Single-cell RNA sequencing of human embryonic stem cell-derived pancreatic cells enriched for early endocrine progenitors using a novel dual NEUROG3-reporter

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2022-12-31 | E-MTAB-11208 | biostudies-arrayexpress

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