CRISPR Screening Uncovers a Long-Range Enhancer for ONECUT1 in Pancreatic Differentiation and Links a Diabetes Risk Variant [Hi-C]
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ABSTRACT:
PROVIDER: PRJNA1096037 | ENA |
REPOSITORIES: ENA
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Project description:Functional enhancer annotation is a valuable first step for understanding tissue-specific transcriptional regulation and prioritizing disease-associated non-coding variants for investigation. However, unbiased enhancer discovery in physiologically relevant contexts remains a major challenge. To discover regulatory elements pertinent to diabetes, we conducted a CRISPR interference (CRISPRi) screen in the human pluripotent stem cell (hPSC) pancreatic differentiation system. Among the enhancers uncovered, we focused on a long-range enhancer ~664 kb from the ONECUT1 promoter, as coding mutations in ONECUT1 cause pancreatic hypoplasia and neonatal diabetes. Homozygous enhancer deletion in hPSCs was associated with a near-complete loss of ONECUT1 gene expression and compromised pancreatic differentiation. This enhancer contains a confidently fine-mapped type 2 diabetes (T2D) associated variant (rs528350911) which disrupts a GATA motif. Introduction of the risk variant into hPSCs revealed substantially reduced binding of key pancreatic transcription factors (GATA4, GATA6 and FOXA2) on the edited allele, accompanied by a slight reduction of ONECUT1 transcription, supporting a causal role for this risk variant in metabolic disease. This work expands our knowledge about transcriptional regulation in pancreatic development through the characterization of a long-range enhancer and highlights the utility of enhancer discovery in disease-relevant settings for understanding monogenic and complex disease.
2024-04-30 | GSE263174 | GEO
Project description:CRISPR Screening Uncovers a Long-Range Enhancer for ONECUT1 in Pancreatic Differentiation and Links a Diabetes Risk Variant
| PRJNA1111076 | ENA
Project description:Functional enhancer annotation is a valuable first step for understanding tissue-specific transcriptional regulation and prioritizing disease-associated non-coding variants for investigation. However, unbiased enhancer discovery in physiologically relevant contexts remains a major challenge. To discover regulatory elements pertinent to diabetes, we conducted a CRISPR interference (CRISPRi) screen in the human pluripotent stem cell (hPSC) pancreatic differentiation system. Among the enhancers uncovered, we focused on a long-range enhancer ~664 kb from the ONECUT1 promoter, as coding mutations in ONECUT1 cause pancreatic hypoplasia and neonatal diabetes. Homozygous enhancer deletion in hESCs was associated with a near-complete loss of ONECUT1 gene expression and compromised pancreatic differentiation. We then identified a type 2 diabetes (T2D) associated variant (rs528350911) in the enhancer which disrupts a GATA motif. Introduction of the risk variant into hESCs revealed substantially reduced binding of key pancreatic transcription factors (GATA4, GATA6 and FOXA2) on the edited allele, accompanied by a subtle reduction of ONECUT1 transcription, supporting a causal role for this risk variant in metabolic disease. This work expands our knowledge about transcriptional regulation in pancreatic development through the characterization of a long-range enhancer and highlights the utility of enhancer discovery in disease-relevant settings for understanding monogenic and complex disease.
2024-03-29 | GSE261391 | GEO
Project description:Functional enhancer annotation is a valuable first step for understanding tissue-specific transcriptional regulation and prioritizing disease-associated non-coding variants for investigation. However, unbiased enhancer discovery in physiologically relevant contexts remains a major challenge. To discover regulatory elements pertinent to diabetes, we conducted a CRISPR interference (CRISPRi) screen in the human pluripotent stem cell (hPSC) pancreatic differentiation system. Among the enhancers uncovered, we focused on a long-range enhancer ~664 kb from the ONECUT1 promoter, as coding mutations in ONECUT1 cause pancreatic hypoplasia and neonatal diabetes. Homozygous enhancer deletion in hESCs was associated with a near-complete loss of ONECUT1 gene expression and compromised pancreatic differentiation. We then identified a type 2 diabetes (T2D) associated variant (rs528350911) in the enhancer which disrupts a GATA motif. Introduction of the risk variant into hESCs revealed substantially reduced binding of key pancreatic transcription factors (GATA4, GATA6 and FOXA2) on the edited allele, accompanied by a subtle reduction of ONECUT1 transcription, supporting a causal role for this risk variant in metabolic disease. This work expands our knowledge about transcriptional regulation in pancreatic development through the characterization of a long-range enhancer and highlights the utility of enhancer discovery in disease-relevant settings for understanding monogenic and complex disease.
2024-03-29 | GSE261392 | GEO
Project description:Functional enhancer annotation is a valuable first step for understanding tissue-specific transcriptional regulation and prioritizing disease-associated non-coding variants for investigation. However, unbiased enhancer discovery in physiologically relevant contexts remains a major challenge. To discover regulatory elements pertinent to diabetes, we conducted a CRISPR interference (CRISPRi) screen in the human pluripotent stem cell (hPSC) pancreatic differentiation system. Among the enhancers uncovered, we focused on a long-range enhancer ~664 kb from the ONECUT1 promoter, as coding mutations in ONECUT1 cause pancreatic hypoplasia and neonatal diabetes. Homozygous enhancer deletion in hESCs was associated with a near-complete loss of ONECUT1 gene expression and compromised pancreatic differentiation. We then identified a type 2 diabetes (T2D) associated variant (rs528350911) in the enhancer which disrupts a GATA motif. Introduction of the risk variant into hESCs revealed substantially reduced binding of key pancreatic transcription factors (GATA4, GATA6 and FOXA2) on the edited allele, accompanied by a subtle reduction of ONECUT1 transcription, supporting a causal role for this risk variant in metabolic disease. This work expands our knowledge about transcriptional regulation in pancreatic development through the characterization of a long-range enhancer and highlights the utility of enhancer discovery in disease-relevant settings for understanding monogenic and complex disease.
2024-03-29 | GSE261390 | GEO
Project description:CRISPR Screening Uncovers a Long-Range Enhancer for ONECUT1 in Pancreatic Differentiation and Links a Diabetes Risk Variant [long_read_seq]
| PRJNA1086831 | ENA
Project description:CRISPR Screening Uncovers a Long-Range Enhancer for ONECUT1 in Pancreatic Differentiation and Links a Diabetes Risk Variant [H3K27ac_ChIP-seq]
| PRJNA1086836 | ENA
Project description:CRISPR Screening Uncovers a Long-Range Enhancer for ONECUT1 in Pancreatic Differentiation and Links a Diabetes Risk Variant [RNA-seq]
| PRJNA1086827 | ENA
Project description:Genes involved in distinct diabetes types suggest shared disease mechanisms. We show that rare ONECUT1 coding variants cause monogenic recessive diabetes (neonatal or very early-onset, syndromic) in two unrelated patients, and monogenic dominant diabetes (early adult-onset) in heterozygous relatives of these and 13 additional unrelated cases. Patients heterozygous for rare ONECUT1 coding variants define a subgroup of T2D with early-onset diabetes and other features. In addition, common regulatory ONECUT1 variants are associated with multifactorial T2D. Directed differentiation of human pluripotent stem cells to the pancreatic lineage revealed that loss of ONECUT1 impairs pancreatic progenitor formation and a subsequent endocrine program. We uncovered that ONECUT1 activates the pro-endocrine genes NKX6.1 and NKX2.2 through binding to their cis-regulatory elements. Globally, ONECUT1-directed gene transcription occurs in association with major islet transcription factors, at clusters of pancreas- and endocrine-specific enhancers within open chromatin. ONECUT1 regulates a transcriptional and epigenetic machinery critical for proper endocrine pancreatic development, involved in a spectrum of diabetes, monogenic recessive and dominant, and multifactorial.
2021-11-03 | PXD018887 | Pride
Project description:Chromosome conformation capture (3C) and derivative (4C, 5C and Hi-C) methods employ ligation of diluted cross-linked chromatin complexes, intended to favor proximity-dependent, intra-complex ligation. We previously described an alternative Hi-C protocol with ligation in preserved nuclei rather than in solution. Here we directly compare Hi-C methods employing "in-nucleus ligation" and the standard "in-solution ligation". The results show that in-nucleus ligation captures chromatin interactions more consistently over a wider range of distances, and significantly reduces both experimental noise and bias. Thus in-nucleus ligation not only simplifies the experimental procedures, but also produces higher quality data with benefits for the entire range of 3C applications. We created Hi-C libraries by two different methods, in-solution ligation and in-nucleus ligation, from two biological replicates each of mouse foetal liver cells (mouse-1 and mouse-2) and human ES cells (human-1 and human-2) or the mixture of these two species. We also sequenced a random ligation library prepared by reversal of the cross-links and purification of the DNA prior to ligation.
2015-08-12 | E-GEOD-70181 | biostudies-arrayexpress