HNF1A deficiency impairs β-cell fate, granule maturation and function (scRNA-seq of 309 hESC-derived cells: Differentiation day 25)
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ABSTRACT:
PROVIDER: PRJNA532396 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR12813904.fastq.gz | Fastqsanger.gz | |||
| SRR12813905.fastq.gz | Fastqsanger.gz | |||
| SRR12813906.fastq.gz | Fastqsanger.gz | |||
| SRR12813907.fastq.gz | Fastqsanger.gz | |||
| SRR12813908.fastq.gz | Fastqsanger.gz |
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Project description:Mutations in HNF1A cause Maturity Onset Diabetes of the Young type 3, the second most frequent form of diabetes caused by single gene mutation. We generated human pancreatic stem cell-derived endocrine cells with mutations in HNF1A and show that HNF1A deficiency impairs scβ-cell fate, insulin granule maturation and the secretion of insulin in a glucose responsive manner. Single-cell RNA sequencing reveals that HNF1A orchestrates a network of genes involved in glucose metabolism, zinc transport, calcium ion binding and hormone exocytosis. Furthermore, in both patients and stem cell-derived β-cells, HNF1A deficiency altered the stoichiometry of secreted c-peptide to insulin. Sulfonylurea, used in the treatment of these patients, restored both insulin secretion and stoichiometry. Significantly, uncoupling of c-peptide and insulin secretion as described here questions the common practice in using c-peptide as a proxy to evaluate β-cell function. We also demonstrate that correction of the HNF1A locus restores function, providing a path to cell therapy.
2019-04-12 | GSE129653 | GEO
Project description:Mutations in HNF1A cause Maturity Onset Diabetes of the Young type 3, the second most frequent form of diabetes caused by single gene mutation. We generated human pancreatic stem cell-derived endocrine cells with mutations in HNF1A and show that HNF1A deficiency impairs scβ-cell fate, insulin granule maturation and the secretion of insulin in a glucose responsive manner. Single-cell RNA sequencing reveals that HNF1A orchestrates a network of genes involved in glucose metabolism, zinc transport, calcium ion binding and hormone exocytosis. Furthermore, in both patients and stem cell-derived β-cells, HNF1A deficiency altered the stoichiometry of secreted c-peptide to insulin. Sulfonylurea, used in the treatment of these patients, restored both insulin secretion and stoichiometry. Significantly, uncoupling of c-peptide and insulin secretion as described here questions the common practice in using c-peptide as a proxy to evaluate β-cell function. We also demonstrate that correction of the HNF1A locus restores function, providing a path to cell therapy.
2019-03-16 | GSE128331 | GEO
Project description:HNF1A deficiency impairs β-cell fate, granule maturation and function
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Project description:To investigate whether early zygotic miR-309 cluster miRNA expression might contribute to the down-regulation of maternal transcripts during the maternal to zygotic transition in Drosophila, we performed microarray analyses of control and miR-309 cluster mutant embryos at 0-1h and 2-3h of embryonic development. One-hour egg collections of w1118 and miR-309-6delta1 mutant flies at 25?C were either aged (2-3h sample) at 25?C or directly processed (0-1h sample). Six independent collections per genotype and time-point were performed. Using this approach, we found that maternal transcripts were stabilized in the miR-309 cluster mutant, indicating that the miR-309 cluster miRNAs are required for the down-regulation of these transcripts during the maternal to zygotic transition in Drosophila.
2009-01-27 | E-TABM-441 | biostudies-arrayexpress
Project description:The specific N-glycans of glycoproteins play key roles in regulating protein localization, stability and activity. B7H3, an immune checkpoint molecule, is a highly N-glycosylated membrane protein. However, the key glycosylated asparagine (Asn) residues that mediate the function of the B7H3 protein are still unclear. Here we identify that among the four pairs of putative N-glycosylation sites, N-glycans attached to asparagine residues N91/309 and N104/322 are required for proper B7H3 protein folding in the endoplasmic reticulum (ER) and intracellular trafficking to the cell surface membrane. We demonstrate that mutations in these two pairs of N-glycosylation sites induce ER accumulation of B7H3 by blocking its ER-to-Golgi translocation and subsequently promote its degradation via the endoplasmic reticulum-associated protein degradation (ERAD) pathway. Additional evidence suggests that N-glycosylation at N91/309 and N104/322 of B7H3 is essential for its inhibition of T-cell proliferation and activation. More importantly, a monoclonal antibody, Ab-82, targeting B7H3 glycosylated at N91/309 and N104/322 is developed, which exhibits the ability to elicit cytotoxic T lymphocyte-mediated antitumor immunity via B7H3 internalization and degradation. Together, these findings provide novel insights into the functional significance of B7H3 glycosylation and offer a rationale for targeting glycosylated B7H3 as a potential strategy for immunotherapy. To compare the binding partners of WT, 8NQ, and N91/104/309/322Q and N91/104/309/322 B7H3, these B7H3 constructs were stably expressed in MDA-MB-231 cell lines. The B7H3 protein was purified using affinity capture purification, followed by immunoprecipitation and mass spectrometry to identify the B7H3 interactome. Ingenuity Pathway Analysis based on these mass spectrometry data showed that the binding to components of the endoplasmic reticulum-associated degradation (ERAD) pathway was greater for the N91/104/309/322Q and 8NQ mutants than for the N91/104/309/322 mutant and WT B7H3.
2025-03-27 | PXD059437 | Pride
Project description:Purpose: The goal of this study is to conduct and compare NGS-derived transcriptome profiling (RNA-seq) of progenitor lines derived from 3 HNF1A-WT and 3 HNF1A-CRISPR (with p291fsinsC mutation) human induced pluripotent stem cell lines. Methods: mRNA profiles of WT/CRISPR pancreatic progenitor cells obtained after in-vitro differentiation for 14 days were generated by deep sequencing using Illumina HiSeq 2000 sequencer. The raw RNA-sequencing read files were processed using the research computing facility at King’s College London, Rosalind (https://rosalind.kcl.ac.uk). RNA-sequencing reads were trimmed from adapters using Trimmomatic/0.39 (Bolger et al., 2014) and quality control was done using FASTQC/0.11.8 (Andrews, 2010). Reads were mapped to a concatenated genome sequence of human GRCh38/hg38 using STAR/2.4.2 (Dobin and Gingeras, 2015). Transcript abundance was measured using featureCount in Subread package (Liao et al., 2013). Results: The DESeq2 package (Love et al., 2014) was used to identify differentially expressed genes. We found 919 significantly regulated genes (271 upregulated, 648 downregulated), with 16 HNF1B targets and 19 HNF1A specific targets within the significantly dysregulated genes when comparing HNF1A-CRISPR versus HNF1A-WT groups. Conclusions: HNF1A and HNF1B gene targets are disturbed by the p291fsinsC mutation engineered in the CRISPR-Cas9 lines.
2022-01-25 | GSE166822 | GEO
Project description:HNF1A and UTX are putative tumor suppressors in pancreatic cancer. In this study, we have combined mouse genetics, transcriptomics and genome binding studies to link HNF1A and UTX in a molecular mechanism that suppresses pancreatic cancer. In this session, we have profiled UTX, HNF1A, H3K27me3 and H3K27ac in normal and UTX- or HNF1A-deficient mouse pancreas by ChIP-seq experiments. We show that HNF1A recruits UTX to its genomic targets in pancreatic acinar cells, which results in remodeling of the chromatin landscape and activation of a broad transcriptional program of differentiated acinar cells, which in turn indirectly suppresses tumor suppressor pathways.
2019-12-20 | E-MTAB-7944 | biostudies-arrayexpress
Project description:Background: We previously identified an oncogenic role for the transcription factor HNF1A in pancreatic ductal adenocarcinoma (PDAC). However, the role of HNF1A in the metastatic progression of PDAC remains unknown and targeting modalities for HNF1A-dependent phenotypes have yet to be identified. Methods: Transwell chambers were used to assess the effects of HNF1A and FGFR4 modulation on the migration and invasion of ATCC and patient-derived PDAC cells in vitro. An intrasplenic injection xenograft model was used to evaluate the impact of HNF1A knockdown and overexpression on metastatic tumor burden. Single-cell RNA sequencing, tissue microarray (TMA) data, and UMAP spatial profiling were used to identify FGFR4 as an HNF1A target gene upregulated in metastatic cells. RNAi and two FGFR4 inhibiting modalities (H3B-6527 and U3-1784) were utilized to demonstrate the efficacy of FGFR4 inhibiting agents at reducing HNF1A38 driven metastasis. Results: Knockdown of HNF1A significantly decreases and HNF1A overexpression significantly increases PDAC cell migration and invasion. In vivo studies show that HNF1A knockdown significantly abrogates metastasis, while overexpression significantly promotes metastasis. Single-cell RNAseq shows that FGFR4 is upregulated in metastatic PDAC cells and staining for HNF1A and FGFR4 in a PDAC TMA reveals significant correlation between HNF1A and FGFR4 in PDAC patients. Further, knockdown and inhibition of FGFR4 significantly decreases HNF1A45 mediated cell migration and invasion, and blocks HNF1A-driven metastasis in vivo. Conclusions: These findings demonstrate that HNF1A drives PDAC metastasis via upregulation of FGFR4, and FGFR4 inhibition is a potential mechanism to target metastasis in PDAC patients.
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Project description:The HASTER promoter region is a cis-regulatory element that stabilizes the transcription HNF1A. If HASTER is deleted after beta cells have been formed and HNF1A is already stably expressed, HASTER is not required to maintain HNF1A activity, and is only required to provide negative feedback on HNF1A. HNF1A binding to the HASTER promoter mediates the repression of the HNF1A gene itself. To determine whether HNF1A binding to the HASTER promoter remodels 3D chromatin interactions of the HNF1A promoter, we performed UMI-4C experiments in wild type and HASTER-promoter KO EndoC-betaH3 cells with or without doxycycline-induced HNF1A overexpression using the HNF1A promoter as viewpoint.
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