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(a) Principal component analysis of Patient-1 and his parents compared to reference populations from the 1000 Genomes project10. (b) Principal component analysis of Patient-1 compared to European subpopulations, showing that he clusters within the French subpopulation. (c) Local ancestry analysis of parents from Patient-1 showing chromosome 15. The arrow shows the position of <t>ONECUT1</t> locus on chromosome 15, estimated to be of European ancestry. Admixed American (AMR), East Asian (EAS), European (EUR), South Asian (SAS). (d) ONECUT1 sequencing in Patient-1 and his nuclear family (Family-1) identified a nonsense variant resulting in a truncated protein homozygous in this patient, heterozygous in both parents and one unaffected sibling and absent in the other unaffected sibling. (e) Targeted sequencing of Patient-2 and his nuclear family (Family-2) identified a homozygous missense variant p.E231D in this patient, heterozygous in both parents. (f) Schematic ONECUT1 protein representing rare coding variants identified in neonatal (homozygous) and young-onset (heterozygous) diabetic patients. Green: index patients with neonatal/very-early-onset diabetes (families 1 and 2), red: UDC-T2D population screening, blue: de novo, identified by WES in one patient. (g) T2D and BMI association in ONECUT1 region. P-values for association are based on DIAMANTE GWAS for T2D and T2D adjusted for BMI (T2DadjBMI), and on GIANT-UK Biobank GWAS for BMI, all of which were available on the AMP-T2D site (www.type2diabetesgenetics.org; date 10/2020). Statistics are shown for the 4 credible SNPs for T2D (*) and for three representative SNPs for BMI association. Pairwise linkage disequilibrium between SNPs was estimated using LDLINK in the European population (https://ldlink.nci.nih.gov) 21
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1) Product Images from "ONECUT1 mutations and variants in diabetes"

Article Title: ONECUT1 mutations and variants in diabetes

Journal: Nature medicine

doi: 10.1038/s41591-021-01502-7

(a) Principal component analysis of Patient-1 and his parents compared to reference populations from the 1000 Genomes project10. (b) Principal component analysis of Patient-1 compared to European subpopulations, showing that he clusters within the French subpopulation. (c) Local ancestry analysis of parents from Patient-1 showing chromosome 15. The arrow shows the position of ONECUT1 locus on chromosome 15, estimated to be of European ancestry. Admixed American (AMR), East Asian (EAS), European (EUR), South Asian (SAS). (d) ONECUT1 sequencing in Patient-1 and his nuclear family (Family-1) identified a nonsense variant resulting in a truncated protein homozygous in this patient, heterozygous in both parents and one unaffected sibling and absent in the other unaffected sibling. (e) Targeted sequencing of Patient-2 and his nuclear family (Family-2) identified a homozygous missense variant p.E231D in this patient, heterozygous in both parents. (f) Schematic ONECUT1 protein representing rare coding variants identified in neonatal (homozygous) and young-onset (heterozygous) diabetic patients. Green: index patients with neonatal/very-early-onset diabetes (families 1 and 2), red: UDC-T2D population screening, blue: de novo, identified by WES in one patient. (g) T2D and BMI association in ONECUT1 region. P-values for association are based on DIAMANTE GWAS for T2D and T2D adjusted for BMI (T2DadjBMI), and on GIANT-UK Biobank GWAS for BMI, all of which were available on the AMP-T2D site (www.type2diabetesgenetics.org; date 10/2020). Statistics are shown for the 4 credible SNPs for T2D (*) and for three representative SNPs for BMI association. Pairwise linkage disequilibrium between SNPs was estimated using LDLINK in the European population (https://ldlink.nci.nih.gov) 21
Figure Legend Snippet: (a) Principal component analysis of Patient-1 and his parents compared to reference populations from the 1000 Genomes project10. (b) Principal component analysis of Patient-1 compared to European subpopulations, showing that he clusters within the French subpopulation. (c) Local ancestry analysis of parents from Patient-1 showing chromosome 15. The arrow shows the position of ONECUT1 locus on chromosome 15, estimated to be of European ancestry. Admixed American (AMR), East Asian (EAS), European (EUR), South Asian (SAS). (d) ONECUT1 sequencing in Patient-1 and his nuclear family (Family-1) identified a nonsense variant resulting in a truncated protein homozygous in this patient, heterozygous in both parents and one unaffected sibling and absent in the other unaffected sibling. (e) Targeted sequencing of Patient-2 and his nuclear family (Family-2) identified a homozygous missense variant p.E231D in this patient, heterozygous in both parents. (f) Schematic ONECUT1 protein representing rare coding variants identified in neonatal (homozygous) and young-onset (heterozygous) diabetic patients. Green: index patients with neonatal/very-early-onset diabetes (families 1 and 2), red: UDC-T2D population screening, blue: de novo, identified by WES in one patient. (g) T2D and BMI association in ONECUT1 region. P-values for association are based on DIAMANTE GWAS for T2D and T2D adjusted for BMI (T2DadjBMI), and on GIANT-UK Biobank GWAS for BMI, all of which were available on the AMP-T2D site (www.type2diabetesgenetics.org; date 10/2020). Statistics are shown for the 4 credible SNPs for T2D (*) and for three representative SNPs for BMI association. Pairwise linkage disequilibrium between SNPs was estimated using LDLINK in the European population (https://ldlink.nci.nih.gov) 21

Techniques Used: Sequencing, Variant Assay

(a) Gene identification in Patient-1 using a combined linkage study and selection of candidate genes associated with early gut endoderm development. (b) Schematic protein representation of ONECUT1 mutations: truncated ONECUT1 protein lacking the CUT and homeobox (HOX) DNA binding domains and homozygous missense mutation p.E231D. (c) Extended family tree of Patient-1 (Family-1) showed a high prevalence of T2D or impaired fasting glucose (IFG, light blue) in heterozygous carriers of the ONECUT1-p.E231X variant. Subject 6, sibling of Patient-1 (subject 5), was recruited during the course of the study and did not have neonatal diabetes. Subject 21 died at the age of 1 day from unknown causes. Her diabetic mother (subject 18) also had gestational diabetes and repeated miscarriages. FPG: Fasting plasma glucose. (d) Family tree of Patient-2 (Family-2) showed a high prevalence of T2D and IFG/IGT in parents and grandparents. The father had impaired glucose tolerance (IGT). (e) Family trees of the 13 diabetic patients (UDC-T2D cohort) identified with rare missense ONECUT1 variants suggesting dominant inheritance. Genotypes of these patients at rare ONECUT1 variants and age at diabetes diagnosis are shown. Two of the 13 index cases, indicated by stars (*), were also heterozygous for the low-frequency ONECUT1-p.P75A variant. The mother of patient 11 also suffered from gestational diabetes (GD) at the age of 28 years. Arrows within the family trees indicate the genotyped index patients. (f) Age at diagnosis of UCD-T2D diabetic patients carrying rare missense ONECUT1 variants (+/m) and relatives of patients with rare missense ONECUT1 variants (T2D rel. of +/m) compared to T2D non-carriers (+/+). Boxplots show the median, interquartile range and extreme values. P-values were calculated using non-parametric Wilcoxon rank test. not significant (ns). (g) Kaplan-Meier survival curve analysis of age at onset of diabetes depending on the presence (+/m) or absence (+/+) of rare missense variants in the 2165 UDC-T2D cases.
Figure Legend Snippet: (a) Gene identification in Patient-1 using a combined linkage study and selection of candidate genes associated with early gut endoderm development. (b) Schematic protein representation of ONECUT1 mutations: truncated ONECUT1 protein lacking the CUT and homeobox (HOX) DNA binding domains and homozygous missense mutation p.E231D. (c) Extended family tree of Patient-1 (Family-1) showed a high prevalence of T2D or impaired fasting glucose (IFG, light blue) in heterozygous carriers of the ONECUT1-p.E231X variant. Subject 6, sibling of Patient-1 (subject 5), was recruited during the course of the study and did not have neonatal diabetes. Subject 21 died at the age of 1 day from unknown causes. Her diabetic mother (subject 18) also had gestational diabetes and repeated miscarriages. FPG: Fasting plasma glucose. (d) Family tree of Patient-2 (Family-2) showed a high prevalence of T2D and IFG/IGT in parents and grandparents. The father had impaired glucose tolerance (IGT). (e) Family trees of the 13 diabetic patients (UDC-T2D cohort) identified with rare missense ONECUT1 variants suggesting dominant inheritance. Genotypes of these patients at rare ONECUT1 variants and age at diabetes diagnosis are shown. Two of the 13 index cases, indicated by stars (*), were also heterozygous for the low-frequency ONECUT1-p.P75A variant. The mother of patient 11 also suffered from gestational diabetes (GD) at the age of 28 years. Arrows within the family trees indicate the genotyped index patients. (f) Age at diagnosis of UCD-T2D diabetic patients carrying rare missense ONECUT1 variants (+/m) and relatives of patients with rare missense ONECUT1 variants (T2D rel. of +/m) compared to T2D non-carriers (+/+). Boxplots show the median, interquartile range and extreme values. P-values were calculated using non-parametric Wilcoxon rank test. not significant (ns). (g) Kaplan-Meier survival curve analysis of age at onset of diabetes depending on the presence (+/m) or absence (+/+) of rare missense variants in the 2165 UDC-T2D cases.

Techniques Used: Selection, Binding Assay, Mutagenesis, Variant Assay

Clinical characteristics of diabetic subjects from the UDC-T2D cohort heterozygous for rare missense  ONECUT1  variants
Figure Legend Snippet: Clinical characteristics of diabetic subjects from the UDC-T2D cohort heterozygous for rare missense ONECUT1 variants

Techniques Used: Variant Assay

(a) Overview of ONECUT1 variants derived from gene-edited HUES8 hESCs and fibroblasts reprogrammed toward iPSCs. (b) Schematic outline of the applied pancreatic differentiation strategy of human pluripotent stem cells and subsequent stage-specific large-scale sequencing analysis or correspondingly employed data set52,53. Stages abbreviate as follows: PSC: human pluripotent stem cells; DE: definitive endoderm; GTE: gut tube endoderm; PE: pancreatic endoderm; PP: pancreatic progenitors. (c,d) Differentiation efficiency at the PE and PP stages in ONECUT1 null HUES8 cells. Representative images show immunofluorescence staining of PDX1 and PDX1/NKX6.1 at the PE and PP stage, respectively. Quantification of positive cells was performed by flow cytometry in HUES8 cells and showed at PP stage 69% and 64% reduction of efficiency in HUES8 ONECUT1 trunc and HUES8 KO, respectively (n=4, one-way ANOVA with Tukey’s test). (e) Principal component analysis from RNA-seq of HUES8 ONECUT1 null and WT PE and PP cells. Different subpopulations and developmental trajectories are indicated as borders and arrows, respectively. (f) Pathway enrichment analysis54 of differentially expressed genes with decreased expression in HUES8 ONECUT1 truncated compared to ONECUT1 WT cells at the PP stage. (g) Schematic representation of fluorescence-activated cell sorting (FACS) to purify PP cells. (h) Principal component analysis of RNA-seq comprising HUES8 ONECUT1 null and WT PE and PP cells (bulk) as well as purified PP (PDX1+/NKX6.1+) cells. Dashed circles indicate ONECUT1 null, while continuous circles label WT cells. (i) Gene set enrichment analysis (GSEA)23 of contrasting HUES8 WT vs. KO of purified PP (PDX1+/NKX6.1+) and bulk PP cells on a specific gene set for pancreatic progenitors24 as well as for endocrine development and β-cell function25.
Figure Legend Snippet: (a) Overview of ONECUT1 variants derived from gene-edited HUES8 hESCs and fibroblasts reprogrammed toward iPSCs. (b) Schematic outline of the applied pancreatic differentiation strategy of human pluripotent stem cells and subsequent stage-specific large-scale sequencing analysis or correspondingly employed data set52,53. Stages abbreviate as follows: PSC: human pluripotent stem cells; DE: definitive endoderm; GTE: gut tube endoderm; PE: pancreatic endoderm; PP: pancreatic progenitors. (c,d) Differentiation efficiency at the PE and PP stages in ONECUT1 null HUES8 cells. Representative images show immunofluorescence staining of PDX1 and PDX1/NKX6.1 at the PE and PP stage, respectively. Quantification of positive cells was performed by flow cytometry in HUES8 cells and showed at PP stage 69% and 64% reduction of efficiency in HUES8 ONECUT1 trunc and HUES8 KO, respectively (n=4, one-way ANOVA with Tukey’s test). (e) Principal component analysis from RNA-seq of HUES8 ONECUT1 null and WT PE and PP cells. Different subpopulations and developmental trajectories are indicated as borders and arrows, respectively. (f) Pathway enrichment analysis54 of differentially expressed genes with decreased expression in HUES8 ONECUT1 truncated compared to ONECUT1 WT cells at the PP stage. (g) Schematic representation of fluorescence-activated cell sorting (FACS) to purify PP cells. (h) Principal component analysis of RNA-seq comprising HUES8 ONECUT1 null and WT PE and PP cells (bulk) as well as purified PP (PDX1+/NKX6.1+) cells. Dashed circles indicate ONECUT1 null, while continuous circles label WT cells. (i) Gene set enrichment analysis (GSEA)23 of contrasting HUES8 WT vs. KO of purified PP (PDX1+/NKX6.1+) and bulk PP cells on a specific gene set for pancreatic progenitors24 as well as for endocrine development and β-cell function25.

Techniques Used: Derivative Assay, Sequencing, Immunofluorescence, Staining, Flow Cytometry, RNA Sequencing Assay, Expressing, Fluorescence, FACS, Purification

(a) ONECUT1 sequence analysis of respective ONECUT1 mutated HUES8 and iPSC cells. (b) Representative immunofluorescence stainings of pluripotency markers NANOG and OCT3/4 in ONECUT1 null and WT HUES8 ESCs as well as ONECUT1-p.E231X iPSCs. (c) Western Blot analysis for ONECUT1 and β-Actin in ONECUT1 null and WT HUES8 as well as iPSC differentiated to pancreatic progenitor (PP) cells. Of note, HUES8 heterozygous ONECUT1 KO (het) was included and undifferentiated stem cells serve as control (ESC, PSC). (d,e) Differentiation efficiency of HUES8 and iPSC ONECUT1 null and WT cells to definitive endoderm (DE) was analyzed by markers SOX17 or CXCR4 and c-Kit as shown by representative immunofluorescence images (d) and flow cytometry (e; HUES8: n=4; iPSC: n=3). (f,g) Differentiation efficiency of ONECUT1 null iPSC cells and respective WT cells to pancreatic endoderm (PE) and pancreatic progenitors (PP) was analyzed by markers PDX1 and NKX6.1 as shown by representative immunofluorescence images and flow cytometry with 62% reduction of PP cells in iPSC ONECUT1 E231X (PE: n=2, PP: n=3; with 2 replicates; two-tailed, unpaired t-test).
Figure Legend Snippet: (a) ONECUT1 sequence analysis of respective ONECUT1 mutated HUES8 and iPSC cells. (b) Representative immunofluorescence stainings of pluripotency markers NANOG and OCT3/4 in ONECUT1 null and WT HUES8 ESCs as well as ONECUT1-p.E231X iPSCs. (c) Western Blot analysis for ONECUT1 and β-Actin in ONECUT1 null and WT HUES8 as well as iPSC differentiated to pancreatic progenitor (PP) cells. Of note, HUES8 heterozygous ONECUT1 KO (het) was included and undifferentiated stem cells serve as control (ESC, PSC). (d,e) Differentiation efficiency of HUES8 and iPSC ONECUT1 null and WT cells to definitive endoderm (DE) was analyzed by markers SOX17 or CXCR4 and c-Kit as shown by representative immunofluorescence images (d) and flow cytometry (e; HUES8: n=4; iPSC: n=3). (f,g) Differentiation efficiency of ONECUT1 null iPSC cells and respective WT cells to pancreatic endoderm (PE) and pancreatic progenitors (PP) was analyzed by markers PDX1 and NKX6.1 as shown by representative immunofluorescence images and flow cytometry with 62% reduction of PP cells in iPSC ONECUT1 E231X (PE: n=2, PP: n=3; with 2 replicates; two-tailed, unpaired t-test).

Techniques Used: Sequencing, Immunofluorescence, Western Blot, Flow Cytometry, Two Tailed Test

(a,b) GSEA23 analysis of differentially expressed (DE) genes in HUES8 WT and ONECUT1 KO PP cells (a) as well as PDX1+/NKX6.1+ purified PP cells from HUES8 ONECUT1 KO and WT (b) using a specific gene set for pancreatic progenitors24 as well as genes important for endocrine development and β-cell function25. (c) GSEA enrichment scores contrasting HUES8 WT and KO (or trunc) at PP stage on gene expression signatures of pancreas cells obtained from a single cell RNA-seq study (GSE81547). (d) Correlation of all significant differentially expressed genes (RNA-seq) and proteins (mass spectrometry, MS) in HUES8 ONECUT1 truncated (trunc) cells at the PP stage. (e) Comparison of expression values for depicted genes in HUES8 edited (ONECUT1 truncated and KO) and WT PP cells. Bar graphs represent min and max values with indicated mean normalized to ONECUT1 WT cells (RNA-seq: n=6; qPCR: WT n=4, KO/trunc n=4; MS: n=3; one-way ANOVA with Tukey’s test).
Figure Legend Snippet: (a,b) GSEA23 analysis of differentially expressed (DE) genes in HUES8 WT and ONECUT1 KO PP cells (a) as well as PDX1+/NKX6.1+ purified PP cells from HUES8 ONECUT1 KO and WT (b) using a specific gene set for pancreatic progenitors24 as well as genes important for endocrine development and β-cell function25. (c) GSEA enrichment scores contrasting HUES8 WT and KO (or trunc) at PP stage on gene expression signatures of pancreas cells obtained from a single cell RNA-seq study (GSE81547). (d) Correlation of all significant differentially expressed genes (RNA-seq) and proteins (mass spectrometry, MS) in HUES8 ONECUT1 truncated (trunc) cells at the PP stage. (e) Comparison of expression values for depicted genes in HUES8 edited (ONECUT1 truncated and KO) and WT PP cells. Bar graphs represent min and max values with indicated mean normalized to ONECUT1 WT cells (RNA-seq: n=6; qPCR: WT n=4, KO/trunc n=4; MS: n=3; one-way ANOVA with Tukey’s test).

Techniques Used: Purification, Expressing, RNA Sequencing Assay, Mass Spectrometry

(a) Fine mapping of type II diabetes traits from DIAMANTE GWAS dataset. This region (chr15:53070141–53165681) corresponds to the 99% genetic credible set from Mahajan et al.5 and includes the first exon of ONECUT1 and the non-coding RNA RP11–209K10.2. IGV plot depicts ONECUT1, FOXA1/2, GATA6, PDX1, and NKX6.1 ChIP-seq peaks (PP), ATAC-seq signals and histone modifications. T2D-associated SNPs (“T2D SNPs”) with a p-value < 10−5 are shown in pink, p-value <10−8 in blue. Of those, rs2440374 overlaps with both a ONECUT1 peak and a differential ATAC-seq peak in PE stage. This SNP is localized at the promoter region of the non-coding gene RP11–209K10.2 (ENSEMBL ID ENSG00000259203). (b,c) Tissue-specific expression of ONECUT1 and RP11–209K10.2 obtained from GTEx database showing gene expression in top 10 tissues sorted by median expression. Both genes have high expression specific to pancreas, liver and testis. (d) Motif analysis with RSAT-Var-tools indicates that the SNP disrupts a putative binding sequence of NKX2.2. (e) eQTL analysis with GTEx indicates an association of rs2440374, rs2456530 and rs75332279 with the expression of lncRNA RP11–209K10.2 in pancreas. (f) Expression of lncRNA RP11–209K10.2 in HUES8 WT and ONECUT1 KO PE and PP cells (RNA-seq, n=6; two-tailed, unpaired t-test). (g) Graphical illustration of the proposed mechanism how ONECUT1 loss impairs pancreatic development to cause diabetes.
Figure Legend Snippet: (a) Fine mapping of type II diabetes traits from DIAMANTE GWAS dataset. This region (chr15:53070141–53165681) corresponds to the 99% genetic credible set from Mahajan et al.5 and includes the first exon of ONECUT1 and the non-coding RNA RP11–209K10.2. IGV plot depicts ONECUT1, FOXA1/2, GATA6, PDX1, and NKX6.1 ChIP-seq peaks (PP), ATAC-seq signals and histone modifications. T2D-associated SNPs (“T2D SNPs”) with a p-value < 10−5 are shown in pink, p-value <10−8 in blue. Of those, rs2440374 overlaps with both a ONECUT1 peak and a differential ATAC-seq peak in PE stage. This SNP is localized at the promoter region of the non-coding gene RP11–209K10.2 (ENSEMBL ID ENSG00000259203). (b,c) Tissue-specific expression of ONECUT1 and RP11–209K10.2 obtained from GTEx database showing gene expression in top 10 tissues sorted by median expression. Both genes have high expression specific to pancreas, liver and testis. (d) Motif analysis with RSAT-Var-tools indicates that the SNP disrupts a putative binding sequence of NKX2.2. (e) eQTL analysis with GTEx indicates an association of rs2440374, rs2456530 and rs75332279 with the expression of lncRNA RP11–209K10.2 in pancreas. (f) Expression of lncRNA RP11–209K10.2 in HUES8 WT and ONECUT1 KO PE and PP cells (RNA-seq, n=6; two-tailed, unpaired t-test). (g) Graphical illustration of the proposed mechanism how ONECUT1 loss impairs pancreatic development to cause diabetes.

Techniques Used: ChIP-sequencing, Expressing, Binding Assay, Sequencing, RNA Sequencing Assay, Two Tailed Test

(a) Luciferase reporter assay (HeLa cells, n=8) with WT and ONECUT1 coding variants fused to the transcriptional activator (transactivator) VP16 using a reporter construct consisting of six ONECUT1 binding motifs found in the human FOXA2 promoter region. After binding of ONECUT1 to its binding motif, VP16 is activating transcription independent of the transactivation activity of ONECUT1 variants. Statistical analysis was performed by one-way ANOVA with Dunnett’s test. (b) Proportion of genes with or without restriction to endocrine lineage genes with overlapping binding by ONECUT1 (ChIP-seq, PP) with depicted TFs (ChIP-seq). (c) Pearson correlation between genome-wide binding signals of depicted TFs. (d,e) Co-immunoprecipitation of Flag- or GFP-tagged WT ONECUT1 protein and GFP- or Flag-tagged target proteins. Proteins co-immunoprecipitating with ONECUT1 are highlighted in green, others in orange. WBs on the bottom show successful overexpression of putative interaction partners in HEK293, while WBs on the top were performed after Flag immunoprecipitation. The heavy chain of the Flag-antibody is indicated with an asterisk.
Figure Legend Snippet: (a) Luciferase reporter assay (HeLa cells, n=8) with WT and ONECUT1 coding variants fused to the transcriptional activator (transactivator) VP16 using a reporter construct consisting of six ONECUT1 binding motifs found in the human FOXA2 promoter region. After binding of ONECUT1 to its binding motif, VP16 is activating transcription independent of the transactivation activity of ONECUT1 variants. Statistical analysis was performed by one-way ANOVA with Dunnett’s test. (b) Proportion of genes with or without restriction to endocrine lineage genes with overlapping binding by ONECUT1 (ChIP-seq, PP) with depicted TFs (ChIP-seq). (c) Pearson correlation between genome-wide binding signals of depicted TFs. (d,e) Co-immunoprecipitation of Flag- or GFP-tagged WT ONECUT1 protein and GFP- or Flag-tagged target proteins. Proteins co-immunoprecipitating with ONECUT1 are highlighted in green, others in orange. WBs on the bottom show successful overexpression of putative interaction partners in HEK293, while WBs on the top were performed after Flag immunoprecipitation. The heavy chain of the Flag-antibody is indicated with an asterisk.

Techniques Used: Luciferase, Reporter Assay, Construct, Binding Assay, Activity Assay, ChIP-sequencing, Genome Wide, Immunoprecipitation, Over Expression

(a) Schematic enrichment analysis of ONECUT1-bound genes with either differentially expressed genes or differential open chromatin peaks (HUES8 WT vs. KO). (b) Binding enrichment (z-score) test of ONECUT1 (ChIP-seq, PP stage) in up- and downregulated genes (RNA-seq) at the depicted differentiation stages of ONECUT1 null and WT HUES8 cells. (c) Binding enrichment (z-score) test of ONECUT1 (ChIP-seq, PP stage) in differential open chromatin regions (HUES8 WT vs. KO, ATAC-seq) of the depicted stages. Notably, bars show enrichment in open chromatin (OC) regions lost or gained in ONECUT1-depleted cells. (d) ChIP-seq signals of key TFs at OC peaks lost or gained at the PE and PP stage in HUES8 ONECUT1 KO cells. (e) Differentiation scheme of HUES8 cells toward β-like cells. (f,g) Representative images show immunofluorescence staining of NKX6.1 and C-peptide at stage 6 (f) and quantification of markers was performed by flow cytometry at stage 5 and 6 of ONECUT1 KO and WT HUES8 cells (g, n=3; one-way ANOVA with Tukey’s test). (h) Heatmap depicting relative marker expression in ONECUT1 KO HUES8 cells at stage 5 and 6. Expression values are normalized to HUES8 ONECUT1 WT and scaled by the sum of each row (n=2). (i) Induced insulin secretion of ONECUT1 KO and WT HUES8 cells at stage 6 depicted as fold increase comparing low glucose stimulated insulin secretion with subsequent KCl-stimulated insulin secretion (n=3 with 3 replicates).
Figure Legend Snippet: (a) Schematic enrichment analysis of ONECUT1-bound genes with either differentially expressed genes or differential open chromatin peaks (HUES8 WT vs. KO). (b) Binding enrichment (z-score) test of ONECUT1 (ChIP-seq, PP stage) in up- and downregulated genes (RNA-seq) at the depicted differentiation stages of ONECUT1 null and WT HUES8 cells. (c) Binding enrichment (z-score) test of ONECUT1 (ChIP-seq, PP stage) in differential open chromatin regions (HUES8 WT vs. KO, ATAC-seq) of the depicted stages. Notably, bars show enrichment in open chromatin (OC) regions lost or gained in ONECUT1-depleted cells. (d) ChIP-seq signals of key TFs at OC peaks lost or gained at the PE and PP stage in HUES8 ONECUT1 KO cells. (e) Differentiation scheme of HUES8 cells toward β-like cells. (f,g) Representative images show immunofluorescence staining of NKX6.1 and C-peptide at stage 6 (f) and quantification of markers was performed by flow cytometry at stage 5 and 6 of ONECUT1 KO and WT HUES8 cells (g, n=3; one-way ANOVA with Tukey’s test). (h) Heatmap depicting relative marker expression in ONECUT1 KO HUES8 cells at stage 5 and 6. Expression values are normalized to HUES8 ONECUT1 WT and scaled by the sum of each row (n=2). (i) Induced insulin secretion of ONECUT1 KO and WT HUES8 cells at stage 6 depicted as fold increase comparing low glucose stimulated insulin secretion with subsequent KCl-stimulated insulin secretion (n=3 with 3 replicates).

Techniques Used: Binding Assay, ChIP-sequencing, RNA Sequencing Assay, Immunofluorescence, Staining, Flow Cytometry, Marker, Expressing

(a) Scheme of ONECUT1 variant E231D generated by targeted gene-editing in HUES8 hESCs. (b) Sequence verification of ONECUT1-p.E231D edited HUES8 cells. Of note, sequencing was performed on reverse strand. (c,d) Differentiation efficiency at the PE and PP stages in ONECUT1-p.E231D HUES8 cells. Of note, ILV was omitted after PE stage to better demonstrate small effects in differentiation efficiency of ONECUT1 variants. Representative images show immunofluorescence staining of PDX1 and PDX1/NKX6.1 at the PE and PP stage, respectively. Quantification of positive cells was performed by flow cytometry (PE: n=4; PP: n=3; two-tailed, unpaired t-test). (e) Heatmap depicting relative marker expression in ONECUT1-p.E231D edited HUES8 cells at PP stage. Of note, ILV was omitted after PE stage compared to regular differentiation protocol. Expression values are normalized to HUES8 ONECUT1 WT (n=4, 2 technical replicates) and scaled by the sum of each row. (f) Co-immunoprecipitation of NKX2.2 with ONECUT1 E231D and WT. The asterisk on the blot shows the heavy chain of the Flag antibody. Bottom control western blots show successful overexpression of TFs in HEK293. (g) Quantification relative to NKX2.2 input (n=4; two-tailed, unpaired t-test) shows reduced heterodimerization for ONECUT1 E231D.
Figure Legend Snippet: (a) Scheme of ONECUT1 variant E231D generated by targeted gene-editing in HUES8 hESCs. (b) Sequence verification of ONECUT1-p.E231D edited HUES8 cells. Of note, sequencing was performed on reverse strand. (c,d) Differentiation efficiency at the PE and PP stages in ONECUT1-p.E231D HUES8 cells. Of note, ILV was omitted after PE stage to better demonstrate small effects in differentiation efficiency of ONECUT1 variants. Representative images show immunofluorescence staining of PDX1 and PDX1/NKX6.1 at the PE and PP stage, respectively. Quantification of positive cells was performed by flow cytometry (PE: n=4; PP: n=3; two-tailed, unpaired t-test). (e) Heatmap depicting relative marker expression in ONECUT1-p.E231D edited HUES8 cells at PP stage. Of note, ILV was omitted after PE stage compared to regular differentiation protocol. Expression values are normalized to HUES8 ONECUT1 WT (n=4, 2 technical replicates) and scaled by the sum of each row. (f) Co-immunoprecipitation of NKX2.2 with ONECUT1 E231D and WT. The asterisk on the blot shows the heavy chain of the Flag antibody. Bottom control western blots show successful overexpression of TFs in HEK293. (g) Quantification relative to NKX2.2 input (n=4; two-tailed, unpaired t-test) shows reduced heterodimerization for ONECUT1 E231D.

Techniques Used: Variant Assay, Generated, Sequencing, Immunofluorescence, Staining, Flow Cytometry, Two Tailed Test, Marker, Expressing, Immunoprecipitation, Western Blot, Over Expression

(a) PCA analysis of stage-specific ATAC-seq for differentiation of ONECUT1 null and WT HUES8 lines (left) as well as restricted to PE and PP stages (right). (b) Genomic location of stage-specific ATAC peaks lost or gained in ONECUT1 null (KO) HUES8 line. TTS: transcriptional termination site. (c) Heatmap depicting chromatin accessibility signals (+/− 2kb of peak center) of OC peaks lost upon ONECUT1 KO in HUES8 and ordered by ONECUT1 ChIP-seq peak strength at the PE and PP stage. (d) Enrichment analysis (GREAT26) of OC peaks lost upon ONECUT1 KO at the PE stage. (e) Significance of overlap (log10 p-value) of open chromatin (OC) for different tissues as well as OC lost in HUES8 KO at PE or PP stage and ONECUT1 ChIP-seq peaks. (f) Scatter plot depicting the footprint-based activity score (strength of binding) of TFs in PE state (y-axis) versus the difference of the activity score upon ONECUT1 KO at the PE state (ATAC-seq, HUES8). (g) HNF family factors have the highest loss in activity followed by PAX family, SOX9 and PDX1 factors upon ONECUT1 KO. Representative examples of footprints.
Figure Legend Snippet: (a) PCA analysis of stage-specific ATAC-seq for differentiation of ONECUT1 null and WT HUES8 lines (left) as well as restricted to PE and PP stages (right). (b) Genomic location of stage-specific ATAC peaks lost or gained in ONECUT1 null (KO) HUES8 line. TTS: transcriptional termination site. (c) Heatmap depicting chromatin accessibility signals (+/− 2kb of peak center) of OC peaks lost upon ONECUT1 KO in HUES8 and ordered by ONECUT1 ChIP-seq peak strength at the PE and PP stage. (d) Enrichment analysis (GREAT26) of OC peaks lost upon ONECUT1 KO at the PE stage. (e) Significance of overlap (log10 p-value) of open chromatin (OC) for different tissues as well as OC lost in HUES8 KO at PE or PP stage and ONECUT1 ChIP-seq peaks. (f) Scatter plot depicting the footprint-based activity score (strength of binding) of TFs in PE state (y-axis) versus the difference of the activity score upon ONECUT1 KO at the PE state (ATAC-seq, HUES8). (g) HNF family factors have the highest loss in activity followed by PAX family, SOX9 and PDX1 factors upon ONECUT1 KO. Representative examples of footprints.

Techniques Used: ChIP-sequencing, Activity Assay, Binding Assay

(a) Overview of ONECUT1 WT and mutated protein variants used in overexpression experiments. (b) Representative images of mutated ONECUT1 fused to GFP, overexpressed in HeLa cells. (c-e) Electromobility shift assay (EMSA) and super shift assay of selected WT and mutated ONECUT1 proteins fused to a Flag-tag using a probe consisting of a ONECUT1 binding motif (label A). Additional Flag antibody binding the complex leads to a further shift (label B). Unspecific binding complexes are indicated with an asterisk. In addition, in vitro translated ONECUT1 proteins (TnT Transcription/Translation System) are detected by ONECUT1 or Flag antibody (WB: α-ONECUT1 control).
Figure Legend Snippet: (a) Overview of ONECUT1 WT and mutated protein variants used in overexpression experiments. (b) Representative images of mutated ONECUT1 fused to GFP, overexpressed in HeLa cells. (c-e) Electromobility shift assay (EMSA) and super shift assay of selected WT and mutated ONECUT1 proteins fused to a Flag-tag using a probe consisting of a ONECUT1 binding motif (label A). Additional Flag antibody binding the complex leads to a further shift (label B). Unspecific binding complexes are indicated with an asterisk. In addition, in vitro translated ONECUT1 proteins (TnT Transcription/Translation System) are detected by ONECUT1 or Flag antibody (WB: α-ONECUT1 control).

Techniques Used: Binding Assay, Over Expression, Electro Mobility Shift Assay, Super-Shift Assay, FLAG-tag, In Vitro

(a) Subcellular localization of WT and mutated ONECUT1 proteins fused to GFP (HeLa cells). (b) Electromobility shift assay (EMSA) of WT and ONECUT1 variants using a ONECUT1 binding motif (TRANSFAC T03257) as probe. (c) TNT-ONECUT1 proteins as WB control for (b). (d) Luciferase reporter assay with WT and indicated diabetes-associated (G30S, E231D, E231X, H33Q, G81D, P215R, V242A) and control (D26E, K412R) variants of ONECUT1 (n=6 for G30S, E231D, E231X, H33Q, G81D; n=10 for D26E, K412R, P215R, V242A; one-way ANOVA with Dunnett’s test). (e,f) Co-immunoprecipitation (top) of FLAG-tagged ONECUT1 and interacting factors NKX6.1 (e) and NKX2.2 (f) confirming physical interaction after FLAG immunoprecipitation. Control western blots (bottom) show successful overexpression of respective factors in HEK293 cells. (g,h) Relative expression of NKX6.1 and NKX2.2 in HUES8 WT and ONECUT1 KO at the PP stage and in purified PP (PDX1+/NKX6.1+) cells from RNA-seq (n=6; two-tailed, unpaired t-test). (i) Activity of the E1-NKX6.1 enhancer during pancreatic differentiation using a GFP reporter construct. Images show a GFP reporter signal as well as staining for PP stage marker NKX6.1 in CyT49 cells. (j) Activity of the E1-NKX6.1 enhancer in a GFP reporter construct using a β-cell line (MIN6) and α-cell line (αTC). (k) Luciferase reporter assay with selected NKX6.1 and NKX2.2 enhancer regions overexpressing WT or ONECUT1 variants alone or together with NKX2.2 in HeLa cells (n=6; one-way ANOVA with Tukey’s test). (l) Significance of overlap of variants associated with T2D acquired from DIAMANTE GWAS dataset (P < 10−20) and ATAC-seq (regions with loss or gain of OC upon ONECUT1 KO) as well as ChIP-seq peaks.
Figure Legend Snippet: (a) Subcellular localization of WT and mutated ONECUT1 proteins fused to GFP (HeLa cells). (b) Electromobility shift assay (EMSA) of WT and ONECUT1 variants using a ONECUT1 binding motif (TRANSFAC T03257) as probe. (c) TNT-ONECUT1 proteins as WB control for (b). (d) Luciferase reporter assay with WT and indicated diabetes-associated (G30S, E231D, E231X, H33Q, G81D, P215R, V242A) and control (D26E, K412R) variants of ONECUT1 (n=6 for G30S, E231D, E231X, H33Q, G81D; n=10 for D26E, K412R, P215R, V242A; one-way ANOVA with Dunnett’s test). (e,f) Co-immunoprecipitation (top) of FLAG-tagged ONECUT1 and interacting factors NKX6.1 (e) and NKX2.2 (f) confirming physical interaction after FLAG immunoprecipitation. Control western blots (bottom) show successful overexpression of respective factors in HEK293 cells. (g,h) Relative expression of NKX6.1 and NKX2.2 in HUES8 WT and ONECUT1 KO at the PP stage and in purified PP (PDX1+/NKX6.1+) cells from RNA-seq (n=6; two-tailed, unpaired t-test). (i) Activity of the E1-NKX6.1 enhancer during pancreatic differentiation using a GFP reporter construct. Images show a GFP reporter signal as well as staining for PP stage marker NKX6.1 in CyT49 cells. (j) Activity of the E1-NKX6.1 enhancer in a GFP reporter construct using a β-cell line (MIN6) and α-cell line (αTC). (k) Luciferase reporter assay with selected NKX6.1 and NKX2.2 enhancer regions overexpressing WT or ONECUT1 variants alone or together with NKX2.2 in HeLa cells (n=6; one-way ANOVA with Tukey’s test). (l) Significance of overlap of variants associated with T2D acquired from DIAMANTE GWAS dataset (P < 10−20) and ATAC-seq (regions with loss or gain of OC upon ONECUT1 KO) as well as ChIP-seq peaks.

Techniques Used: Electro Mobility Shift Assay, Binding Assay, Luciferase, Reporter Assay, Immunoprecipitation, Western Blot, Over Expression, Expressing, Purification, RNA Sequencing Assay, Two Tailed Test, Activity Assay, Construct, Staining, Marker, ChIP-sequencing

(a) Homo- and heterodimerization of ONECUT1 proteins was analyzed by co-immunoprecipitation of GFP-tagged ONECUT1 and Flag-tagged ONECUT1 WT or variant in HEK293. The heavy chain of the Flag-antibody is indicated with an asterisk. Note that the ONECUT1 PTV (p.E231X) did not bind to WT ONECUT1 protein. (b,c) Co-immunoprecipitation (top) of NKX6.1 (b) and NKX2.2 (c) with ONECUT1 WT and E231X. Heterodimerization only in ONECUT1 WT and NKX6.1/NKX2.2. The asterisk on the blot shows the heavy chain of the Flag antibody. Bottom control western blots show successful overexpression of TFs in HEK293.
Figure Legend Snippet: (a) Homo- and heterodimerization of ONECUT1 proteins was analyzed by co-immunoprecipitation of GFP-tagged ONECUT1 and Flag-tagged ONECUT1 WT or variant in HEK293. The heavy chain of the Flag-antibody is indicated with an asterisk. Note that the ONECUT1 PTV (p.E231X) did not bind to WT ONECUT1 protein. (b,c) Co-immunoprecipitation (top) of NKX6.1 (b) and NKX2.2 (c) with ONECUT1 WT and E231X. Heterodimerization only in ONECUT1 WT and NKX6.1/NKX2.2. The asterisk on the blot shows the heavy chain of the Flag antibody. Bottom control western blots show successful overexpression of TFs in HEK293.

Techniques Used: Immunoprecipitation, Variant Assay, Western Blot, Over Expression

(a) NKX6.2 expression in HUES8 WT and ONECUT1 KO PP bulk and PDX1+/NKX6.1+ purified cells (RNA-seq, n=6; two-tailed, unpaired t-test). (b,c) ATAC-seq, histone modifications and ONECUT1 ChIP-seq signals around NKX6.1, NKX6.2, and NKX2.2 locus. Red traced squares indicate enhancer regions expanded in (c). Below, the region selected for luciferase assay and reporter assay are shown. (d) Luciferase reporter assay with selected NKX6.2 enhancer region overexpressing WT or ONECUT1 variants alone or together with NKX2.2 in HeLa cells (n=6; one-way ANOVA with Tukey’s test).
Figure Legend Snippet: (a) NKX6.2 expression in HUES8 WT and ONECUT1 KO PP bulk and PDX1+/NKX6.1+ purified cells (RNA-seq, n=6; two-tailed, unpaired t-test). (b,c) ATAC-seq, histone modifications and ONECUT1 ChIP-seq signals around NKX6.1, NKX6.2, and NKX2.2 locus. Red traced squares indicate enhancer regions expanded in (c). Below, the region selected for luciferase assay and reporter assay are shown. (d) Luciferase reporter assay with selected NKX6.2 enhancer region overexpressing WT or ONECUT1 variants alone or together with NKX2.2 in HeLa cells (n=6; one-way ANOVA with Tukey’s test).

Techniques Used: Expressing, Purification, RNA Sequencing Assay, Two Tailed Test, ChIP-sequencing, Luciferase, Reporter Assay


Structured Review

Promega onecut1 proteins
(a) Principal component analysis of Patient-1 and his parents compared to reference populations from the 1000 Genomes project10. (b) Principal component analysis of Patient-1 compared to European subpopulations, showing that he clusters within the French subpopulation. (c) Local ancestry analysis of parents from Patient-1 showing chromosome 15. The arrow shows the position of <t>ONECUT1</t> locus on chromosome 15, estimated to be of European ancestry. Admixed American (AMR), East Asian (EAS), European (EUR), South Asian (SAS). (d) ONECUT1 sequencing in Patient-1 and his nuclear family (Family-1) identified a nonsense variant resulting in a truncated protein homozygous in this patient, heterozygous in both parents and one unaffected sibling and absent in the other unaffected sibling. (e) Targeted sequencing of Patient-2 and his nuclear family (Family-2) identified a homozygous missense variant p.E231D in this patient, heterozygous in both parents. (f) Schematic ONECUT1 protein representing rare coding variants identified in neonatal (homozygous) and young-onset (heterozygous) diabetic patients. Green: index patients with neonatal/very-early-onset diabetes (families 1 and 2), red: UDC-T2D population screening, blue: de novo, identified by WES in one patient. (g) T2D and BMI association in ONECUT1 region. P-values for association are based on DIAMANTE GWAS for T2D and T2D adjusted for BMI (T2DadjBMI), and on GIANT-UK Biobank GWAS for BMI, all of which were available on the AMP-T2D site (www.type2diabetesgenetics.org; date 10/2020). Statistics are shown for the 4 credible SNPs for T2D (*) and for three representative SNPs for BMI association. Pairwise linkage disequilibrium between SNPs was estimated using LDLINK in the European population (https://ldlink.nci.nih.gov) 21
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1) Product Images from "ONECUT1 mutations and variants in diabetes"

Article Title: ONECUT1 mutations and variants in diabetes

Journal: Nature medicine

doi: 10.1038/s41591-021-01502-7

(a) Principal component analysis of Patient-1 and his parents compared to reference populations from the 1000 Genomes project10. (b) Principal component analysis of Patient-1 compared to European subpopulations, showing that he clusters within the French subpopulation. (c) Local ancestry analysis of parents from Patient-1 showing chromosome 15. The arrow shows the position of ONECUT1 locus on chromosome 15, estimated to be of European ancestry. Admixed American (AMR), East Asian (EAS), European (EUR), South Asian (SAS). (d) ONECUT1 sequencing in Patient-1 and his nuclear family (Family-1) identified a nonsense variant resulting in a truncated protein homozygous in this patient, heterozygous in both parents and one unaffected sibling and absent in the other unaffected sibling. (e) Targeted sequencing of Patient-2 and his nuclear family (Family-2) identified a homozygous missense variant p.E231D in this patient, heterozygous in both parents. (f) Schematic ONECUT1 protein representing rare coding variants identified in neonatal (homozygous) and young-onset (heterozygous) diabetic patients. Green: index patients with neonatal/very-early-onset diabetes (families 1 and 2), red: UDC-T2D population screening, blue: de novo, identified by WES in one patient. (g) T2D and BMI association in ONECUT1 region. P-values for association are based on DIAMANTE GWAS for T2D and T2D adjusted for BMI (T2DadjBMI), and on GIANT-UK Biobank GWAS for BMI, all of which were available on the AMP-T2D site (www.type2diabetesgenetics.org; date 10/2020). Statistics are shown for the 4 credible SNPs for T2D (*) and for three representative SNPs for BMI association. Pairwise linkage disequilibrium between SNPs was estimated using LDLINK in the European population (https://ldlink.nci.nih.gov) 21
Figure Legend Snippet: (a) Principal component analysis of Patient-1 and his parents compared to reference populations from the 1000 Genomes project10. (b) Principal component analysis of Patient-1 compared to European subpopulations, showing that he clusters within the French subpopulation. (c) Local ancestry analysis of parents from Patient-1 showing chromosome 15. The arrow shows the position of ONECUT1 locus on chromosome 15, estimated to be of European ancestry. Admixed American (AMR), East Asian (EAS), European (EUR), South Asian (SAS). (d) ONECUT1 sequencing in Patient-1 and his nuclear family (Family-1) identified a nonsense variant resulting in a truncated protein homozygous in this patient, heterozygous in both parents and one unaffected sibling and absent in the other unaffected sibling. (e) Targeted sequencing of Patient-2 and his nuclear family (Family-2) identified a homozygous missense variant p.E231D in this patient, heterozygous in both parents. (f) Schematic ONECUT1 protein representing rare coding variants identified in neonatal (homozygous) and young-onset (heterozygous) diabetic patients. Green: index patients with neonatal/very-early-onset diabetes (families 1 and 2), red: UDC-T2D population screening, blue: de novo, identified by WES in one patient. (g) T2D and BMI association in ONECUT1 region. P-values for association are based on DIAMANTE GWAS for T2D and T2D adjusted for BMI (T2DadjBMI), and on GIANT-UK Biobank GWAS for BMI, all of which were available on the AMP-T2D site (www.type2diabetesgenetics.org; date 10/2020). Statistics are shown for the 4 credible SNPs for T2D (*) and for three representative SNPs for BMI association. Pairwise linkage disequilibrium between SNPs was estimated using LDLINK in the European population (https://ldlink.nci.nih.gov) 21

Techniques Used: Sequencing, Variant Assay

(a) Gene identification in Patient-1 using a combined linkage study and selection of candidate genes associated with early gut endoderm development. (b) Schematic protein representation of ONECUT1 mutations: truncated ONECUT1 protein lacking the CUT and homeobox (HOX) DNA binding domains and homozygous missense mutation p.E231D. (c) Extended family tree of Patient-1 (Family-1) showed a high prevalence of T2D or impaired fasting glucose (IFG, light blue) in heterozygous carriers of the ONECUT1-p.E231X variant. Subject 6, sibling of Patient-1 (subject 5), was recruited during the course of the study and did not have neonatal diabetes. Subject 21 died at the age of 1 day from unknown causes. Her diabetic mother (subject 18) also had gestational diabetes and repeated miscarriages. FPG: Fasting plasma glucose. (d) Family tree of Patient-2 (Family-2) showed a high prevalence of T2D and IFG/IGT in parents and grandparents. The father had impaired glucose tolerance (IGT). (e) Family trees of the 13 diabetic patients (UDC-T2D cohort) identified with rare missense ONECUT1 variants suggesting dominant inheritance. Genotypes of these patients at rare ONECUT1 variants and age at diabetes diagnosis are shown. Two of the 13 index cases, indicated by stars (*), were also heterozygous for the low-frequency ONECUT1-p.P75A variant. The mother of patient 11 also suffered from gestational diabetes (GD) at the age of 28 years. Arrows within the family trees indicate the genotyped index patients. (f) Age at diagnosis of UCD-T2D diabetic patients carrying rare missense ONECUT1 variants (+/m) and relatives of patients with rare missense ONECUT1 variants (T2D rel. of +/m) compared to T2D non-carriers (+/+). Boxplots show the median, interquartile range and extreme values. P-values were calculated using non-parametric Wilcoxon rank test. not significant (ns). (g) Kaplan-Meier survival curve analysis of age at onset of diabetes depending on the presence (+/m) or absence (+/+) of rare missense variants in the 2165 UDC-T2D cases.
Figure Legend Snippet: (a) Gene identification in Patient-1 using a combined linkage study and selection of candidate genes associated with early gut endoderm development. (b) Schematic protein representation of ONECUT1 mutations: truncated ONECUT1 protein lacking the CUT and homeobox (HOX) DNA binding domains and homozygous missense mutation p.E231D. (c) Extended family tree of Patient-1 (Family-1) showed a high prevalence of T2D or impaired fasting glucose (IFG, light blue) in heterozygous carriers of the ONECUT1-p.E231X variant. Subject 6, sibling of Patient-1 (subject 5), was recruited during the course of the study and did not have neonatal diabetes. Subject 21 died at the age of 1 day from unknown causes. Her diabetic mother (subject 18) also had gestational diabetes and repeated miscarriages. FPG: Fasting plasma glucose. (d) Family tree of Patient-2 (Family-2) showed a high prevalence of T2D and IFG/IGT in parents and grandparents. The father had impaired glucose tolerance (IGT). (e) Family trees of the 13 diabetic patients (UDC-T2D cohort) identified with rare missense ONECUT1 variants suggesting dominant inheritance. Genotypes of these patients at rare ONECUT1 variants and age at diabetes diagnosis are shown. Two of the 13 index cases, indicated by stars (*), were also heterozygous for the low-frequency ONECUT1-p.P75A variant. The mother of patient 11 also suffered from gestational diabetes (GD) at the age of 28 years. Arrows within the family trees indicate the genotyped index patients. (f) Age at diagnosis of UCD-T2D diabetic patients carrying rare missense ONECUT1 variants (+/m) and relatives of patients with rare missense ONECUT1 variants (T2D rel. of +/m) compared to T2D non-carriers (+/+). Boxplots show the median, interquartile range and extreme values. P-values were calculated using non-parametric Wilcoxon rank test. not significant (ns). (g) Kaplan-Meier survival curve analysis of age at onset of diabetes depending on the presence (+/m) or absence (+/+) of rare missense variants in the 2165 UDC-T2D cases.

Techniques Used: Selection, Binding Assay, Mutagenesis, Variant Assay

Clinical characteristics of diabetic subjects from the UDC-T2D cohort heterozygous for rare missense  ONECUT1  variants
Figure Legend Snippet: Clinical characteristics of diabetic subjects from the UDC-T2D cohort heterozygous for rare missense ONECUT1 variants

Techniques Used: Variant Assay

(a) Overview of ONECUT1 variants derived from gene-edited HUES8 hESCs and fibroblasts reprogrammed toward iPSCs. (b) Schematic outline of the applied pancreatic differentiation strategy of human pluripotent stem cells and subsequent stage-specific large-scale sequencing analysis or correspondingly employed data set52,53. Stages abbreviate as follows: PSC: human pluripotent stem cells; DE: definitive endoderm; GTE: gut tube endoderm; PE: pancreatic endoderm; PP: pancreatic progenitors. (c,d) Differentiation efficiency at the PE and PP stages in ONECUT1 null HUES8 cells. Representative images show immunofluorescence staining of PDX1 and PDX1/NKX6.1 at the PE and PP stage, respectively. Quantification of positive cells was performed by flow cytometry in HUES8 cells and showed at PP stage 69% and 64% reduction of efficiency in HUES8 ONECUT1 trunc and HUES8 KO, respectively (n=4, one-way ANOVA with Tukey’s test). (e) Principal component analysis from RNA-seq of HUES8 ONECUT1 null and WT PE and PP cells. Different subpopulations and developmental trajectories are indicated as borders and arrows, respectively. (f) Pathway enrichment analysis54 of differentially expressed genes with decreased expression in HUES8 ONECUT1 truncated compared to ONECUT1 WT cells at the PP stage. (g) Schematic representation of fluorescence-activated cell sorting (FACS) to purify PP cells. (h) Principal component analysis of RNA-seq comprising HUES8 ONECUT1 null and WT PE and PP cells (bulk) as well as purified PP (PDX1+/NKX6.1+) cells. Dashed circles indicate ONECUT1 null, while continuous circles label WT cells. (i) Gene set enrichment analysis (GSEA)23 of contrasting HUES8 WT vs. KO of purified PP (PDX1+/NKX6.1+) and bulk PP cells on a specific gene set for pancreatic progenitors24 as well as for endocrine development and β-cell function25.
Figure Legend Snippet: (a) Overview of ONECUT1 variants derived from gene-edited HUES8 hESCs and fibroblasts reprogrammed toward iPSCs. (b) Schematic outline of the applied pancreatic differentiation strategy of human pluripotent stem cells and subsequent stage-specific large-scale sequencing analysis or correspondingly employed data set52,53. Stages abbreviate as follows: PSC: human pluripotent stem cells; DE: definitive endoderm; GTE: gut tube endoderm; PE: pancreatic endoderm; PP: pancreatic progenitors. (c,d) Differentiation efficiency at the PE and PP stages in ONECUT1 null HUES8 cells. Representative images show immunofluorescence staining of PDX1 and PDX1/NKX6.1 at the PE and PP stage, respectively. Quantification of positive cells was performed by flow cytometry in HUES8 cells and showed at PP stage 69% and 64% reduction of efficiency in HUES8 ONECUT1 trunc and HUES8 KO, respectively (n=4, one-way ANOVA with Tukey’s test). (e) Principal component analysis from RNA-seq of HUES8 ONECUT1 null and WT PE and PP cells. Different subpopulations and developmental trajectories are indicated as borders and arrows, respectively. (f) Pathway enrichment analysis54 of differentially expressed genes with decreased expression in HUES8 ONECUT1 truncated compared to ONECUT1 WT cells at the PP stage. (g) Schematic representation of fluorescence-activated cell sorting (FACS) to purify PP cells. (h) Principal component analysis of RNA-seq comprising HUES8 ONECUT1 null and WT PE and PP cells (bulk) as well as purified PP (PDX1+/NKX6.1+) cells. Dashed circles indicate ONECUT1 null, while continuous circles label WT cells. (i) Gene set enrichment analysis (GSEA)23 of contrasting HUES8 WT vs. KO of purified PP (PDX1+/NKX6.1+) and bulk PP cells on a specific gene set for pancreatic progenitors24 as well as for endocrine development and β-cell function25.

Techniques Used: Derivative Assay, Sequencing, Immunofluorescence, Staining, Flow Cytometry, RNA Sequencing Assay, Expressing, Fluorescence, FACS, Purification

(a) ONECUT1 sequence analysis of respective ONECUT1 mutated HUES8 and iPSC cells. (b) Representative immunofluorescence stainings of pluripotency markers NANOG and OCT3/4 in ONECUT1 null and WT HUES8 ESCs as well as ONECUT1-p.E231X iPSCs. (c) Western Blot analysis for ONECUT1 and β-Actin in ONECUT1 null and WT HUES8 as well as iPSC differentiated to pancreatic progenitor (PP) cells. Of note, HUES8 heterozygous ONECUT1 KO (het) was included and undifferentiated stem cells serve as control (ESC, PSC). (d,e) Differentiation efficiency of HUES8 and iPSC ONECUT1 null and WT cells to definitive endoderm (DE) was analyzed by markers SOX17 or CXCR4 and c-Kit as shown by representative immunofluorescence images (d) and flow cytometry (e; HUES8: n=4; iPSC: n=3). (f,g) Differentiation efficiency of ONECUT1 null iPSC cells and respective WT cells to pancreatic endoderm (PE) and pancreatic progenitors (PP) was analyzed by markers PDX1 and NKX6.1 as shown by representative immunofluorescence images and flow cytometry with 62% reduction of PP cells in iPSC ONECUT1 E231X (PE: n=2, PP: n=3; with 2 replicates; two-tailed, unpaired t-test).
Figure Legend Snippet: (a) ONECUT1 sequence analysis of respective ONECUT1 mutated HUES8 and iPSC cells. (b) Representative immunofluorescence stainings of pluripotency markers NANOG and OCT3/4 in ONECUT1 null and WT HUES8 ESCs as well as ONECUT1-p.E231X iPSCs. (c) Western Blot analysis for ONECUT1 and β-Actin in ONECUT1 null and WT HUES8 as well as iPSC differentiated to pancreatic progenitor (PP) cells. Of note, HUES8 heterozygous ONECUT1 KO (het) was included and undifferentiated stem cells serve as control (ESC, PSC). (d,e) Differentiation efficiency of HUES8 and iPSC ONECUT1 null and WT cells to definitive endoderm (DE) was analyzed by markers SOX17 or CXCR4 and c-Kit as shown by representative immunofluorescence images (d) and flow cytometry (e; HUES8: n=4; iPSC: n=3). (f,g) Differentiation efficiency of ONECUT1 null iPSC cells and respective WT cells to pancreatic endoderm (PE) and pancreatic progenitors (PP) was analyzed by markers PDX1 and NKX6.1 as shown by representative immunofluorescence images and flow cytometry with 62% reduction of PP cells in iPSC ONECUT1 E231X (PE: n=2, PP: n=3; with 2 replicates; two-tailed, unpaired t-test).

Techniques Used: Sequencing, Immunofluorescence, Western Blot, Flow Cytometry, Two Tailed Test

(a,b) GSEA23 analysis of differentially expressed (DE) genes in HUES8 WT and ONECUT1 KO PP cells (a) as well as PDX1+/NKX6.1+ purified PP cells from HUES8 ONECUT1 KO and WT (b) using a specific gene set for pancreatic progenitors24 as well as genes important for endocrine development and β-cell function25. (c) GSEA enrichment scores contrasting HUES8 WT and KO (or trunc) at PP stage on gene expression signatures of pancreas cells obtained from a single cell RNA-seq study (GSE81547). (d) Correlation of all significant differentially expressed genes (RNA-seq) and proteins (mass spectrometry, MS) in HUES8 ONECUT1 truncated (trunc) cells at the PP stage. (e) Comparison of expression values for depicted genes in HUES8 edited (ONECUT1 truncated and KO) and WT PP cells. Bar graphs represent min and max values with indicated mean normalized to ONECUT1 WT cells (RNA-seq: n=6; qPCR: WT n=4, KO/trunc n=4; MS: n=3; one-way ANOVA with Tukey’s test).
Figure Legend Snippet: (a,b) GSEA23 analysis of differentially expressed (DE) genes in HUES8 WT and ONECUT1 KO PP cells (a) as well as PDX1+/NKX6.1+ purified PP cells from HUES8 ONECUT1 KO and WT (b) using a specific gene set for pancreatic progenitors24 as well as genes important for endocrine development and β-cell function25. (c) GSEA enrichment scores contrasting HUES8 WT and KO (or trunc) at PP stage on gene expression signatures of pancreas cells obtained from a single cell RNA-seq study (GSE81547). (d) Correlation of all significant differentially expressed genes (RNA-seq) and proteins (mass spectrometry, MS) in HUES8 ONECUT1 truncated (trunc) cells at the PP stage. (e) Comparison of expression values for depicted genes in HUES8 edited (ONECUT1 truncated and KO) and WT PP cells. Bar graphs represent min and max values with indicated mean normalized to ONECUT1 WT cells (RNA-seq: n=6; qPCR: WT n=4, KO/trunc n=4; MS: n=3; one-way ANOVA with Tukey’s test).

Techniques Used: Purification, Expressing, RNA Sequencing Assay, Mass Spectrometry

(a) Fine mapping of type II diabetes traits from DIAMANTE GWAS dataset. This region (chr15:53070141–53165681) corresponds to the 99% genetic credible set from Mahajan et al.5 and includes the first exon of ONECUT1 and the non-coding RNA RP11–209K10.2. IGV plot depicts ONECUT1, FOXA1/2, GATA6, PDX1, and NKX6.1 ChIP-seq peaks (PP), ATAC-seq signals and histone modifications. T2D-associated SNPs (“T2D SNPs”) with a p-value < 10−5 are shown in pink, p-value <10−8 in blue. Of those, rs2440374 overlaps with both a ONECUT1 peak and a differential ATAC-seq peak in PE stage. This SNP is localized at the promoter region of the non-coding gene RP11–209K10.2 (ENSEMBL ID ENSG00000259203). (b,c) Tissue-specific expression of ONECUT1 and RP11–209K10.2 obtained from GTEx database showing gene expression in top 10 tissues sorted by median expression. Both genes have high expression specific to pancreas, liver and testis. (d) Motif analysis with RSAT-Var-tools indicates that the SNP disrupts a putative binding sequence of NKX2.2. (e) eQTL analysis with GTEx indicates an association of rs2440374, rs2456530 and rs75332279 with the expression of lncRNA RP11–209K10.2 in pancreas. (f) Expression of lncRNA RP11–209K10.2 in HUES8 WT and ONECUT1 KO PE and PP cells (RNA-seq, n=6; two-tailed, unpaired t-test). (g) Graphical illustration of the proposed mechanism how ONECUT1 loss impairs pancreatic development to cause diabetes.
Figure Legend Snippet: (a) Fine mapping of type II diabetes traits from DIAMANTE GWAS dataset. This region (chr15:53070141–53165681) corresponds to the 99% genetic credible set from Mahajan et al.5 and includes the first exon of ONECUT1 and the non-coding RNA RP11–209K10.2. IGV plot depicts ONECUT1, FOXA1/2, GATA6, PDX1, and NKX6.1 ChIP-seq peaks (PP), ATAC-seq signals and histone modifications. T2D-associated SNPs (“T2D SNPs”) with a p-value < 10−5 are shown in pink, p-value <10−8 in blue. Of those, rs2440374 overlaps with both a ONECUT1 peak and a differential ATAC-seq peak in PE stage. This SNP is localized at the promoter region of the non-coding gene RP11–209K10.2 (ENSEMBL ID ENSG00000259203). (b,c) Tissue-specific expression of ONECUT1 and RP11–209K10.2 obtained from GTEx database showing gene expression in top 10 tissues sorted by median expression. Both genes have high expression specific to pancreas, liver and testis. (d) Motif analysis with RSAT-Var-tools indicates that the SNP disrupts a putative binding sequence of NKX2.2. (e) eQTL analysis with GTEx indicates an association of rs2440374, rs2456530 and rs75332279 with the expression of lncRNA RP11–209K10.2 in pancreas. (f) Expression of lncRNA RP11–209K10.2 in HUES8 WT and ONECUT1 KO PE and PP cells (RNA-seq, n=6; two-tailed, unpaired t-test). (g) Graphical illustration of the proposed mechanism how ONECUT1 loss impairs pancreatic development to cause diabetes.

Techniques Used: ChIP-sequencing, Expressing, Binding Assay, Sequencing, RNA Sequencing Assay, Two Tailed Test

(a) Luciferase reporter assay (HeLa cells, n=8) with WT and ONECUT1 coding variants fused to the transcriptional activator (transactivator) VP16 using a reporter construct consisting of six ONECUT1 binding motifs found in the human FOXA2 promoter region. After binding of ONECUT1 to its binding motif, VP16 is activating transcription independent of the transactivation activity of ONECUT1 variants. Statistical analysis was performed by one-way ANOVA with Dunnett’s test. (b) Proportion of genes with or without restriction to endocrine lineage genes with overlapping binding by ONECUT1 (ChIP-seq, PP) with depicted TFs (ChIP-seq). (c) Pearson correlation between genome-wide binding signals of depicted TFs. (d,e) Co-immunoprecipitation of Flag- or GFP-tagged WT ONECUT1 protein and GFP- or Flag-tagged target proteins. Proteins co-immunoprecipitating with ONECUT1 are highlighted in green, others in orange. WBs on the bottom show successful overexpression of putative interaction partners in HEK293, while WBs on the top were performed after Flag immunoprecipitation. The heavy chain of the Flag-antibody is indicated with an asterisk.
Figure Legend Snippet: (a) Luciferase reporter assay (HeLa cells, n=8) with WT and ONECUT1 coding variants fused to the transcriptional activator (transactivator) VP16 using a reporter construct consisting of six ONECUT1 binding motifs found in the human FOXA2 promoter region. After binding of ONECUT1 to its binding motif, VP16 is activating transcription independent of the transactivation activity of ONECUT1 variants. Statistical analysis was performed by one-way ANOVA with Dunnett’s test. (b) Proportion of genes with or without restriction to endocrine lineage genes with overlapping binding by ONECUT1 (ChIP-seq, PP) with depicted TFs (ChIP-seq). (c) Pearson correlation between genome-wide binding signals of depicted TFs. (d,e) Co-immunoprecipitation of Flag- or GFP-tagged WT ONECUT1 protein and GFP- or Flag-tagged target proteins. Proteins co-immunoprecipitating with ONECUT1 are highlighted in green, others in orange. WBs on the bottom show successful overexpression of putative interaction partners in HEK293, while WBs on the top were performed after Flag immunoprecipitation. The heavy chain of the Flag-antibody is indicated with an asterisk.

Techniques Used: Luciferase, Reporter Assay, Construct, Binding Assay, Activity Assay, ChIP-sequencing, Genome Wide, Immunoprecipitation, Over Expression

(a) Schematic enrichment analysis of ONECUT1-bound genes with either differentially expressed genes or differential open chromatin peaks (HUES8 WT vs. KO). (b) Binding enrichment (z-score) test of ONECUT1 (ChIP-seq, PP stage) in up- and downregulated genes (RNA-seq) at the depicted differentiation stages of ONECUT1 null and WT HUES8 cells. (c) Binding enrichment (z-score) test of ONECUT1 (ChIP-seq, PP stage) in differential open chromatin regions (HUES8 WT vs. KO, ATAC-seq) of the depicted stages. Notably, bars show enrichment in open chromatin (OC) regions lost or gained in ONECUT1-depleted cells. (d) ChIP-seq signals of key TFs at OC peaks lost or gained at the PE and PP stage in HUES8 ONECUT1 KO cells. (e) Differentiation scheme of HUES8 cells toward β-like cells. (f,g) Representative images show immunofluorescence staining of NKX6.1 and C-peptide at stage 6 (f) and quantification of markers was performed by flow cytometry at stage 5 and 6 of ONECUT1 KO and WT HUES8 cells (g, n=3; one-way ANOVA with Tukey’s test). (h) Heatmap depicting relative marker expression in ONECUT1 KO HUES8 cells at stage 5 and 6. Expression values are normalized to HUES8 ONECUT1 WT and scaled by the sum of each row (n=2). (i) Induced insulin secretion of ONECUT1 KO and WT HUES8 cells at stage 6 depicted as fold increase comparing low glucose stimulated insulin secretion with subsequent KCl-stimulated insulin secretion (n=3 with 3 replicates).
Figure Legend Snippet: (a) Schematic enrichment analysis of ONECUT1-bound genes with either differentially expressed genes or differential open chromatin peaks (HUES8 WT vs. KO). (b) Binding enrichment (z-score) test of ONECUT1 (ChIP-seq, PP stage) in up- and downregulated genes (RNA-seq) at the depicted differentiation stages of ONECUT1 null and WT HUES8 cells. (c) Binding enrichment (z-score) test of ONECUT1 (ChIP-seq, PP stage) in differential open chromatin regions (HUES8 WT vs. KO, ATAC-seq) of the depicted stages. Notably, bars show enrichment in open chromatin (OC) regions lost or gained in ONECUT1-depleted cells. (d) ChIP-seq signals of key TFs at OC peaks lost or gained at the PE and PP stage in HUES8 ONECUT1 KO cells. (e) Differentiation scheme of HUES8 cells toward β-like cells. (f,g) Representative images show immunofluorescence staining of NKX6.1 and C-peptide at stage 6 (f) and quantification of markers was performed by flow cytometry at stage 5 and 6 of ONECUT1 KO and WT HUES8 cells (g, n=3; one-way ANOVA with Tukey’s test). (h) Heatmap depicting relative marker expression in ONECUT1 KO HUES8 cells at stage 5 and 6. Expression values are normalized to HUES8 ONECUT1 WT and scaled by the sum of each row (n=2). (i) Induced insulin secretion of ONECUT1 KO and WT HUES8 cells at stage 6 depicted as fold increase comparing low glucose stimulated insulin secretion with subsequent KCl-stimulated insulin secretion (n=3 with 3 replicates).

Techniques Used: Binding Assay, ChIP-sequencing, RNA Sequencing Assay, Immunofluorescence, Staining, Flow Cytometry, Marker, Expressing

(a) Scheme of ONECUT1 variant E231D generated by targeted gene-editing in HUES8 hESCs. (b) Sequence verification of ONECUT1-p.E231D edited HUES8 cells. Of note, sequencing was performed on reverse strand. (c,d) Differentiation efficiency at the PE and PP stages in ONECUT1-p.E231D HUES8 cells. Of note, ILV was omitted after PE stage to better demonstrate small effects in differentiation efficiency of ONECUT1 variants. Representative images show immunofluorescence staining of PDX1 and PDX1/NKX6.1 at the PE and PP stage, respectively. Quantification of positive cells was performed by flow cytometry (PE: n=4; PP: n=3; two-tailed, unpaired t-test). (e) Heatmap depicting relative marker expression in ONECUT1-p.E231D edited HUES8 cells at PP stage. Of note, ILV was omitted after PE stage compared to regular differentiation protocol. Expression values are normalized to HUES8 ONECUT1 WT (n=4, 2 technical replicates) and scaled by the sum of each row. (f) Co-immunoprecipitation of NKX2.2 with ONECUT1 E231D and WT. The asterisk on the blot shows the heavy chain of the Flag antibody. Bottom control western blots show successful overexpression of TFs in HEK293. (g) Quantification relative to NKX2.2 input (n=4; two-tailed, unpaired t-test) shows reduced heterodimerization for ONECUT1 E231D.
Figure Legend Snippet: (a) Scheme of ONECUT1 variant E231D generated by targeted gene-editing in HUES8 hESCs. (b) Sequence verification of ONECUT1-p.E231D edited HUES8 cells. Of note, sequencing was performed on reverse strand. (c,d) Differentiation efficiency at the PE and PP stages in ONECUT1-p.E231D HUES8 cells. Of note, ILV was omitted after PE stage to better demonstrate small effects in differentiation efficiency of ONECUT1 variants. Representative images show immunofluorescence staining of PDX1 and PDX1/NKX6.1 at the PE and PP stage, respectively. Quantification of positive cells was performed by flow cytometry (PE: n=4; PP: n=3; two-tailed, unpaired t-test). (e) Heatmap depicting relative marker expression in ONECUT1-p.E231D edited HUES8 cells at PP stage. Of note, ILV was omitted after PE stage compared to regular differentiation protocol. Expression values are normalized to HUES8 ONECUT1 WT (n=4, 2 technical replicates) and scaled by the sum of each row. (f) Co-immunoprecipitation of NKX2.2 with ONECUT1 E231D and WT. The asterisk on the blot shows the heavy chain of the Flag antibody. Bottom control western blots show successful overexpression of TFs in HEK293. (g) Quantification relative to NKX2.2 input (n=4; two-tailed, unpaired t-test) shows reduced heterodimerization for ONECUT1 E231D.

Techniques Used: Variant Assay, Generated, Sequencing, Immunofluorescence, Staining, Flow Cytometry, Two Tailed Test, Marker, Expressing, Immunoprecipitation, Western Blot, Over Expression

(a) PCA analysis of stage-specific ATAC-seq for differentiation of ONECUT1 null and WT HUES8 lines (left) as well as restricted to PE and PP stages (right). (b) Genomic location of stage-specific ATAC peaks lost or gained in ONECUT1 null (KO) HUES8 line. TTS: transcriptional termination site. (c) Heatmap depicting chromatin accessibility signals (+/− 2kb of peak center) of OC peaks lost upon ONECUT1 KO in HUES8 and ordered by ONECUT1 ChIP-seq peak strength at the PE and PP stage. (d) Enrichment analysis (GREAT26) of OC peaks lost upon ONECUT1 KO at the PE stage. (e) Significance of overlap (log10 p-value) of open chromatin (OC) for different tissues as well as OC lost in HUES8 KO at PE or PP stage and ONECUT1 ChIP-seq peaks. (f) Scatter plot depicting the footprint-based activity score (strength of binding) of TFs in PE state (y-axis) versus the difference of the activity score upon ONECUT1 KO at the PE state (ATAC-seq, HUES8). (g) HNF family factors have the highest loss in activity followed by PAX family, SOX9 and PDX1 factors upon ONECUT1 KO. Representative examples of footprints.
Figure Legend Snippet: (a) PCA analysis of stage-specific ATAC-seq for differentiation of ONECUT1 null and WT HUES8 lines (left) as well as restricted to PE and PP stages (right). (b) Genomic location of stage-specific ATAC peaks lost or gained in ONECUT1 null (KO) HUES8 line. TTS: transcriptional termination site. (c) Heatmap depicting chromatin accessibility signals (+/− 2kb of peak center) of OC peaks lost upon ONECUT1 KO in HUES8 and ordered by ONECUT1 ChIP-seq peak strength at the PE and PP stage. (d) Enrichment analysis (GREAT26) of OC peaks lost upon ONECUT1 KO at the PE stage. (e) Significance of overlap (log10 p-value) of open chromatin (OC) for different tissues as well as OC lost in HUES8 KO at PE or PP stage and ONECUT1 ChIP-seq peaks. (f) Scatter plot depicting the footprint-based activity score (strength of binding) of TFs in PE state (y-axis) versus the difference of the activity score upon ONECUT1 KO at the PE state (ATAC-seq, HUES8). (g) HNF family factors have the highest loss in activity followed by PAX family, SOX9 and PDX1 factors upon ONECUT1 KO. Representative examples of footprints.

Techniques Used: ChIP-sequencing, Activity Assay, Binding Assay

(a) Overview of ONECUT1 WT and mutated protein variants used in overexpression experiments. (b) Representative images of mutated ONECUT1 fused to GFP, overexpressed in HeLa cells. (c-e) Electromobility shift assay (EMSA) and super shift assay of selected WT and mutated ONECUT1 proteins fused to a Flag-tag using a probe consisting of a ONECUT1 binding motif (label A). Additional Flag antibody binding the complex leads to a further shift (label B). Unspecific binding complexes are indicated with an asterisk. In addition, in vitro translated ONECUT1 proteins (TnT Transcription/Translation System) are detected by ONECUT1 or Flag antibody (WB: α-ONECUT1 control).
Figure Legend Snippet: (a) Overview of ONECUT1 WT and mutated protein variants used in overexpression experiments. (b) Representative images of mutated ONECUT1 fused to GFP, overexpressed in HeLa cells. (c-e) Electromobility shift assay (EMSA) and super shift assay of selected WT and mutated ONECUT1 proteins fused to a Flag-tag using a probe consisting of a ONECUT1 binding motif (label A). Additional Flag antibody binding the complex leads to a further shift (label B). Unspecific binding complexes are indicated with an asterisk. In addition, in vitro translated ONECUT1 proteins (TnT Transcription/Translation System) are detected by ONECUT1 or Flag antibody (WB: α-ONECUT1 control).

Techniques Used: Binding Assay, Over Expression, Electro Mobility Shift Assay, Super-Shift Assay, FLAG-tag, In Vitro

(a) Subcellular localization of WT and mutated ONECUT1 proteins fused to GFP (HeLa cells). (b) Electromobility shift assay (EMSA) of WT and ONECUT1 variants using a ONECUT1 binding motif (TRANSFAC T03257) as probe. (c) TNT-ONECUT1 proteins as WB control for (b). (d) Luciferase reporter assay with WT and indicated diabetes-associated (G30S, E231D, E231X, H33Q, G81D, P215R, V242A) and control (D26E, K412R) variants of ONECUT1 (n=6 for G30S, E231D, E231X, H33Q, G81D; n=10 for D26E, K412R, P215R, V242A; one-way ANOVA with Dunnett’s test). (e,f) Co-immunoprecipitation (top) of FLAG-tagged ONECUT1 and interacting factors NKX6.1 (e) and NKX2.2 (f) confirming physical interaction after FLAG immunoprecipitation. Control western blots (bottom) show successful overexpression of respective factors in HEK293 cells. (g,h) Relative expression of NKX6.1 and NKX2.2 in HUES8 WT and ONECUT1 KO at the PP stage and in purified PP (PDX1+/NKX6.1+) cells from RNA-seq (n=6; two-tailed, unpaired t-test). (i) Activity of the E1-NKX6.1 enhancer during pancreatic differentiation using a GFP reporter construct. Images show a GFP reporter signal as well as staining for PP stage marker NKX6.1 in CyT49 cells. (j) Activity of the E1-NKX6.1 enhancer in a GFP reporter construct using a β-cell line (MIN6) and α-cell line (αTC). (k) Luciferase reporter assay with selected NKX6.1 and NKX2.2 enhancer regions overexpressing WT or ONECUT1 variants alone or together with NKX2.2 in HeLa cells (n=6; one-way ANOVA with Tukey’s test). (l) Significance of overlap of variants associated with T2D acquired from DIAMANTE GWAS dataset (P < 10−20) and ATAC-seq (regions with loss or gain of OC upon ONECUT1 KO) as well as ChIP-seq peaks.
Figure Legend Snippet: (a) Subcellular localization of WT and mutated ONECUT1 proteins fused to GFP (HeLa cells). (b) Electromobility shift assay (EMSA) of WT and ONECUT1 variants using a ONECUT1 binding motif (TRANSFAC T03257) as probe. (c) TNT-ONECUT1 proteins as WB control for (b). (d) Luciferase reporter assay with WT and indicated diabetes-associated (G30S, E231D, E231X, H33Q, G81D, P215R, V242A) and control (D26E, K412R) variants of ONECUT1 (n=6 for G30S, E231D, E231X, H33Q, G81D; n=10 for D26E, K412R, P215R, V242A; one-way ANOVA with Dunnett’s test). (e,f) Co-immunoprecipitation (top) of FLAG-tagged ONECUT1 and interacting factors NKX6.1 (e) and NKX2.2 (f) confirming physical interaction after FLAG immunoprecipitation. Control western blots (bottom) show successful overexpression of respective factors in HEK293 cells. (g,h) Relative expression of NKX6.1 and NKX2.2 in HUES8 WT and ONECUT1 KO at the PP stage and in purified PP (PDX1+/NKX6.1+) cells from RNA-seq (n=6; two-tailed, unpaired t-test). (i) Activity of the E1-NKX6.1 enhancer during pancreatic differentiation using a GFP reporter construct. Images show a GFP reporter signal as well as staining for PP stage marker NKX6.1 in CyT49 cells. (j) Activity of the E1-NKX6.1 enhancer in a GFP reporter construct using a β-cell line (MIN6) and α-cell line (αTC). (k) Luciferase reporter assay with selected NKX6.1 and NKX2.2 enhancer regions overexpressing WT or ONECUT1 variants alone or together with NKX2.2 in HeLa cells (n=6; one-way ANOVA with Tukey’s test). (l) Significance of overlap of variants associated with T2D acquired from DIAMANTE GWAS dataset (P < 10−20) and ATAC-seq (regions with loss or gain of OC upon ONECUT1 KO) as well as ChIP-seq peaks.

Techniques Used: Electro Mobility Shift Assay, Binding Assay, Luciferase, Reporter Assay, Immunoprecipitation, Western Blot, Over Expression, Expressing, Purification, RNA Sequencing Assay, Two Tailed Test, Activity Assay, Construct, Staining, Marker, ChIP-sequencing

(a) Homo- and heterodimerization of ONECUT1 proteins was analyzed by co-immunoprecipitation of GFP-tagged ONECUT1 and Flag-tagged ONECUT1 WT or variant in HEK293. The heavy chain of the Flag-antibody is indicated with an asterisk. Note that the ONECUT1 PTV (p.E231X) did not bind to WT ONECUT1 protein. (b,c) Co-immunoprecipitation (top) of NKX6.1 (b) and NKX2.2 (c) with ONECUT1 WT and E231X. Heterodimerization only in ONECUT1 WT and NKX6.1/NKX2.2. The asterisk on the blot shows the heavy chain of the Flag antibody. Bottom control western blots show successful overexpression of TFs in HEK293.
Figure Legend Snippet: (a) Homo- and heterodimerization of ONECUT1 proteins was analyzed by co-immunoprecipitation of GFP-tagged ONECUT1 and Flag-tagged ONECUT1 WT or variant in HEK293. The heavy chain of the Flag-antibody is indicated with an asterisk. Note that the ONECUT1 PTV (p.E231X) did not bind to WT ONECUT1 protein. (b,c) Co-immunoprecipitation (top) of NKX6.1 (b) and NKX2.2 (c) with ONECUT1 WT and E231X. Heterodimerization only in ONECUT1 WT and NKX6.1/NKX2.2. The asterisk on the blot shows the heavy chain of the Flag antibody. Bottom control western blots show successful overexpression of TFs in HEK293.

Techniques Used: Immunoprecipitation, Variant Assay, Western Blot, Over Expression

(a) NKX6.2 expression in HUES8 WT and ONECUT1 KO PP bulk and PDX1+/NKX6.1+ purified cells (RNA-seq, n=6; two-tailed, unpaired t-test). (b,c) ATAC-seq, histone modifications and ONECUT1 ChIP-seq signals around NKX6.1, NKX6.2, and NKX2.2 locus. Red traced squares indicate enhancer regions expanded in (c). Below, the region selected for luciferase assay and reporter assay are shown. (d) Luciferase reporter assay with selected NKX6.2 enhancer region overexpressing WT or ONECUT1 variants alone or together with NKX2.2 in HeLa cells (n=6; one-way ANOVA with Tukey’s test).
Figure Legend Snippet: (a) NKX6.2 expression in HUES8 WT and ONECUT1 KO PP bulk and PDX1+/NKX6.1+ purified cells (RNA-seq, n=6; two-tailed, unpaired t-test). (b,c) ATAC-seq, histone modifications and ONECUT1 ChIP-seq signals around NKX6.1, NKX6.2, and NKX2.2 locus. Red traced squares indicate enhancer regions expanded in (c). Below, the region selected for luciferase assay and reporter assay are shown. (d) Luciferase reporter assay with selected NKX6.2 enhancer region overexpressing WT or ONECUT1 variants alone or together with NKX2.2 in HeLa cells (n=6; one-way ANOVA with Tukey’s test).

Techniques Used: Expressing, Purification, RNA Sequencing Assay, Two Tailed Test, ChIP-sequencing, Luciferase, Reporter Assay


Structured Review

Promega onecut1 proteins
(a) Principal component analysis of Patient-1 and his parents compared to reference populations from the 1000 Genomes project10. (b) Principal component analysis of Patient-1 compared to European subpopulations, showing that he clusters within the French subpopulation. (c) Local ancestry analysis of parents from Patient-1 showing chromosome 15. The arrow shows the position of <t>ONECUT1</t> locus on chromosome 15, estimated to be of European ancestry. Admixed American (AMR), East Asian (EAS), European (EUR), South Asian (SAS). (d) ONECUT1 sequencing in Patient-1 and his nuclear family (Family-1) identified a nonsense variant resulting in a truncated protein homozygous in this patient, heterozygous in both parents and one unaffected sibling and absent in the other unaffected sibling. (e) Targeted sequencing of Patient-2 and his nuclear family (Family-2) identified a homozygous missense variant p.E231D in this patient, heterozygous in both parents. (f) Schematic ONECUT1 protein representing rare coding variants identified in neonatal (homozygous) and young-onset (heterozygous) diabetic patients. Green: index patients with neonatal/very-early-onset diabetes (families 1 and 2), red: UDC-T2D population screening, blue: de novo, identified by WES in one patient. (g) T2D and BMI association in ONECUT1 region. P-values for association are based on DIAMANTE GWAS for T2D and T2D adjusted for BMI (T2DadjBMI), and on GIANT-UK Biobank GWAS for BMI, all of which were available on the AMP-T2D site (www.type2diabetesgenetics.org; date 10/2020). Statistics are shown for the 4 credible SNPs for T2D (*) and for three representative SNPs for BMI association. Pairwise linkage disequilibrium between SNPs was estimated using LDLINK in the European population (https://ldlink.nci.nih.gov) 21
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1) Product Images from "ONECUT1 mutations and variants in diabetes"

Article Title: ONECUT1 mutations and variants in diabetes

Journal: Nature medicine

doi: 10.1038/s41591-021-01502-7

(a) Principal component analysis of Patient-1 and his parents compared to reference populations from the 1000 Genomes project10. (b) Principal component analysis of Patient-1 compared to European subpopulations, showing that he clusters within the French subpopulation. (c) Local ancestry analysis of parents from Patient-1 showing chromosome 15. The arrow shows the position of ONECUT1 locus on chromosome 15, estimated to be of European ancestry. Admixed American (AMR), East Asian (EAS), European (EUR), South Asian (SAS). (d) ONECUT1 sequencing in Patient-1 and his nuclear family (Family-1) identified a nonsense variant resulting in a truncated protein homozygous in this patient, heterozygous in both parents and one unaffected sibling and absent in the other unaffected sibling. (e) Targeted sequencing of Patient-2 and his nuclear family (Family-2) identified a homozygous missense variant p.E231D in this patient, heterozygous in both parents. (f) Schematic ONECUT1 protein representing rare coding variants identified in neonatal (homozygous) and young-onset (heterozygous) diabetic patients. Green: index patients with neonatal/very-early-onset diabetes (families 1 and 2), red: UDC-T2D population screening, blue: de novo, identified by WES in one patient. (g) T2D and BMI association in ONECUT1 region. P-values for association are based on DIAMANTE GWAS for T2D and T2D adjusted for BMI (T2DadjBMI), and on GIANT-UK Biobank GWAS for BMI, all of which were available on the AMP-T2D site (www.type2diabetesgenetics.org; date 10/2020). Statistics are shown for the 4 credible SNPs for T2D (*) and for three representative SNPs for BMI association. Pairwise linkage disequilibrium between SNPs was estimated using LDLINK in the European population (https://ldlink.nci.nih.gov) 21
Figure Legend Snippet: (a) Principal component analysis of Patient-1 and his parents compared to reference populations from the 1000 Genomes project10. (b) Principal component analysis of Patient-1 compared to European subpopulations, showing that he clusters within the French subpopulation. (c) Local ancestry analysis of parents from Patient-1 showing chromosome 15. The arrow shows the position of ONECUT1 locus on chromosome 15, estimated to be of European ancestry. Admixed American (AMR), East Asian (EAS), European (EUR), South Asian (SAS). (d) ONECUT1 sequencing in Patient-1 and his nuclear family (Family-1) identified a nonsense variant resulting in a truncated protein homozygous in this patient, heterozygous in both parents and one unaffected sibling and absent in the other unaffected sibling. (e) Targeted sequencing of Patient-2 and his nuclear family (Family-2) identified a homozygous missense variant p.E231D in this patient, heterozygous in both parents. (f) Schematic ONECUT1 protein representing rare coding variants identified in neonatal (homozygous) and young-onset (heterozygous) diabetic patients. Green: index patients with neonatal/very-early-onset diabetes (families 1 and 2), red: UDC-T2D population screening, blue: de novo, identified by WES in one patient. (g) T2D and BMI association in ONECUT1 region. P-values for association are based on DIAMANTE GWAS for T2D and T2D adjusted for BMI (T2DadjBMI), and on GIANT-UK Biobank GWAS for BMI, all of which were available on the AMP-T2D site (www.type2diabetesgenetics.org; date 10/2020). Statistics are shown for the 4 credible SNPs for T2D (*) and for three representative SNPs for BMI association. Pairwise linkage disequilibrium between SNPs was estimated using LDLINK in the European population (https://ldlink.nci.nih.gov) 21

Techniques Used: Sequencing, Variant Assay

(a) Gene identification in Patient-1 using a combined linkage study and selection of candidate genes associated with early gut endoderm development. (b) Schematic protein representation of ONECUT1 mutations: truncated ONECUT1 protein lacking the CUT and homeobox (HOX) DNA binding domains and homozygous missense mutation p.E231D. (c) Extended family tree of Patient-1 (Family-1) showed a high prevalence of T2D or impaired fasting glucose (IFG, light blue) in heterozygous carriers of the ONECUT1-p.E231X variant. Subject 6, sibling of Patient-1 (subject 5), was recruited during the course of the study and did not have neonatal diabetes. Subject 21 died at the age of 1 day from unknown causes. Her diabetic mother (subject 18) also had gestational diabetes and repeated miscarriages. FPG: Fasting plasma glucose. (d) Family tree of Patient-2 (Family-2) showed a high prevalence of T2D and IFG/IGT in parents and grandparents. The father had impaired glucose tolerance (IGT). (e) Family trees of the 13 diabetic patients (UDC-T2D cohort) identified with rare missense ONECUT1 variants suggesting dominant inheritance. Genotypes of these patients at rare ONECUT1 variants and age at diabetes diagnosis are shown. Two of the 13 index cases, indicated by stars (*), were also heterozygous for the low-frequency ONECUT1-p.P75A variant. The mother of patient 11 also suffered from gestational diabetes (GD) at the age of 28 years. Arrows within the family trees indicate the genotyped index patients. (f) Age at diagnosis of UCD-T2D diabetic patients carrying rare missense ONECUT1 variants (+/m) and relatives of patients with rare missense ONECUT1 variants (T2D rel. of +/m) compared to T2D non-carriers (+/+). Boxplots show the median, interquartile range and extreme values. P-values were calculated using non-parametric Wilcoxon rank test. not significant (ns). (g) Kaplan-Meier survival curve analysis of age at onset of diabetes depending on the presence (+/m) or absence (+/+) of rare missense variants in the 2165 UDC-T2D cases.
Figure Legend Snippet: (a) Gene identification in Patient-1 using a combined linkage study and selection of candidate genes associated with early gut endoderm development. (b) Schematic protein representation of ONECUT1 mutations: truncated ONECUT1 protein lacking the CUT and homeobox (HOX) DNA binding domains and homozygous missense mutation p.E231D. (c) Extended family tree of Patient-1 (Family-1) showed a high prevalence of T2D or impaired fasting glucose (IFG, light blue) in heterozygous carriers of the ONECUT1-p.E231X variant. Subject 6, sibling of Patient-1 (subject 5), was recruited during the course of the study and did not have neonatal diabetes. Subject 21 died at the age of 1 day from unknown causes. Her diabetic mother (subject 18) also had gestational diabetes and repeated miscarriages. FPG: Fasting plasma glucose. (d) Family tree of Patient-2 (Family-2) showed a high prevalence of T2D and IFG/IGT in parents and grandparents. The father had impaired glucose tolerance (IGT). (e) Family trees of the 13 diabetic patients (UDC-T2D cohort) identified with rare missense ONECUT1 variants suggesting dominant inheritance. Genotypes of these patients at rare ONECUT1 variants and age at diabetes diagnosis are shown. Two of the 13 index cases, indicated by stars (*), were also heterozygous for the low-frequency ONECUT1-p.P75A variant. The mother of patient 11 also suffered from gestational diabetes (GD) at the age of 28 years. Arrows within the family trees indicate the genotyped index patients. (f) Age at diagnosis of UCD-T2D diabetic patients carrying rare missense ONECUT1 variants (+/m) and relatives of patients with rare missense ONECUT1 variants (T2D rel. of +/m) compared to T2D non-carriers (+/+). Boxplots show the median, interquartile range and extreme values. P-values were calculated using non-parametric Wilcoxon rank test. not significant (ns). (g) Kaplan-Meier survival curve analysis of age at onset of diabetes depending on the presence (+/m) or absence (+/+) of rare missense variants in the 2165 UDC-T2D cases.

Techniques Used: Selection, Binding Assay, Mutagenesis, Variant Assay

Clinical characteristics of diabetic subjects from the UDC-T2D cohort heterozygous for rare missense  ONECUT1  variants
Figure Legend Snippet: Clinical characteristics of diabetic subjects from the UDC-T2D cohort heterozygous for rare missense ONECUT1 variants

Techniques Used: Variant Assay

(a) Overview of ONECUT1 variants derived from gene-edited HUES8 hESCs and fibroblasts reprogrammed toward iPSCs. (b) Schematic outline of the applied pancreatic differentiation strategy of human pluripotent stem cells and subsequent stage-specific large-scale sequencing analysis or correspondingly employed data set52,53. Stages abbreviate as follows: PSC: human pluripotent stem cells; DE: definitive endoderm; GTE: gut tube endoderm; PE: pancreatic endoderm; PP: pancreatic progenitors. (c,d) Differentiation efficiency at the PE and PP stages in ONECUT1 null HUES8 cells. Representative images show immunofluorescence staining of PDX1 and PDX1/NKX6.1 at the PE and PP stage, respectively. Quantification of positive cells was performed by flow cytometry in HUES8 cells and showed at PP stage 69% and 64% reduction of efficiency in HUES8 ONECUT1 trunc and HUES8 KO, respectively (n=4, one-way ANOVA with Tukey’s test). (e) Principal component analysis from RNA-seq of HUES8 ONECUT1 null and WT PE and PP cells. Different subpopulations and developmental trajectories are indicated as borders and arrows, respectively. (f) Pathway enrichment analysis54 of differentially expressed genes with decreased expression in HUES8 ONECUT1 truncated compared to ONECUT1 WT cells at the PP stage. (g) Schematic representation of fluorescence-activated cell sorting (FACS) to purify PP cells. (h) Principal component analysis of RNA-seq comprising HUES8 ONECUT1 null and WT PE and PP cells (bulk) as well as purified PP (PDX1+/NKX6.1+) cells. Dashed circles indicate ONECUT1 null, while continuous circles label WT cells. (i) Gene set enrichment analysis (GSEA)23 of contrasting HUES8 WT vs. KO of purified PP (PDX1+/NKX6.1+) and bulk PP cells on a specific gene set for pancreatic progenitors24 as well as for endocrine development and β-cell function25.
Figure Legend Snippet: (a) Overview of ONECUT1 variants derived from gene-edited HUES8 hESCs and fibroblasts reprogrammed toward iPSCs. (b) Schematic outline of the applied pancreatic differentiation strategy of human pluripotent stem cells and subsequent stage-specific large-scale sequencing analysis or correspondingly employed data set52,53. Stages abbreviate as follows: PSC: human pluripotent stem cells; DE: definitive endoderm; GTE: gut tube endoderm; PE: pancreatic endoderm; PP: pancreatic progenitors. (c,d) Differentiation efficiency at the PE and PP stages in ONECUT1 null HUES8 cells. Representative images show immunofluorescence staining of PDX1 and PDX1/NKX6.1 at the PE and PP stage, respectively. Quantification of positive cells was performed by flow cytometry in HUES8 cells and showed at PP stage 69% and 64% reduction of efficiency in HUES8 ONECUT1 trunc and HUES8 KO, respectively (n=4, one-way ANOVA with Tukey’s test). (e) Principal component analysis from RNA-seq of HUES8 ONECUT1 null and WT PE and PP cells. Different subpopulations and developmental trajectories are indicated as borders and arrows, respectively. (f) Pathway enrichment analysis54 of differentially expressed genes with decreased expression in HUES8 ONECUT1 truncated compared to ONECUT1 WT cells at the PP stage. (g) Schematic representation of fluorescence-activated cell sorting (FACS) to purify PP cells. (h) Principal component analysis of RNA-seq comprising HUES8 ONECUT1 null and WT PE and PP cells (bulk) as well as purified PP (PDX1+/NKX6.1+) cells. Dashed circles indicate ONECUT1 null, while continuous circles label WT cells. (i) Gene set enrichment analysis (GSEA)23 of contrasting HUES8 WT vs. KO of purified PP (PDX1+/NKX6.1+) and bulk PP cells on a specific gene set for pancreatic progenitors24 as well as for endocrine development and β-cell function25.

Techniques Used: Derivative Assay, Sequencing, Immunofluorescence, Staining, Flow Cytometry, RNA Sequencing Assay, Expressing, Fluorescence, FACS, Purification

(a) ONECUT1 sequence analysis of respective ONECUT1 mutated HUES8 and iPSC cells. (b) Representative immunofluorescence stainings of pluripotency markers NANOG and OCT3/4 in ONECUT1 null and WT HUES8 ESCs as well as ONECUT1-p.E231X iPSCs. (c) Western Blot analysis for ONECUT1 and β-Actin in ONECUT1 null and WT HUES8 as well as iPSC differentiated to pancreatic progenitor (PP) cells. Of note, HUES8 heterozygous ONECUT1 KO (het) was included and undifferentiated stem cells serve as control (ESC, PSC). (d,e) Differentiation efficiency of HUES8 and iPSC ONECUT1 null and WT cells to definitive endoderm (DE) was analyzed by markers SOX17 or CXCR4 and c-Kit as shown by representative immunofluorescence images (d) and flow cytometry (e; HUES8: n=4; iPSC: n=3). (f,g) Differentiation efficiency of ONECUT1 null iPSC cells and respective WT cells to pancreatic endoderm (PE) and pancreatic progenitors (PP) was analyzed by markers PDX1 and NKX6.1 as shown by representative immunofluorescence images and flow cytometry with 62% reduction of PP cells in iPSC ONECUT1 E231X (PE: n=2, PP: n=3; with 2 replicates; two-tailed, unpaired t-test).
Figure Legend Snippet: (a) ONECUT1 sequence analysis of respective ONECUT1 mutated HUES8 and iPSC cells. (b) Representative immunofluorescence stainings of pluripotency markers NANOG and OCT3/4 in ONECUT1 null and WT HUES8 ESCs as well as ONECUT1-p.E231X iPSCs. (c) Western Blot analysis for ONECUT1 and β-Actin in ONECUT1 null and WT HUES8 as well as iPSC differentiated to pancreatic progenitor (PP) cells. Of note, HUES8 heterozygous ONECUT1 KO (het) was included and undifferentiated stem cells serve as control (ESC, PSC). (d,e) Differentiation efficiency of HUES8 and iPSC ONECUT1 null and WT cells to definitive endoderm (DE) was analyzed by markers SOX17 or CXCR4 and c-Kit as shown by representative immunofluorescence images (d) and flow cytometry (e; HUES8: n=4; iPSC: n=3). (f,g) Differentiation efficiency of ONECUT1 null iPSC cells and respective WT cells to pancreatic endoderm (PE) and pancreatic progenitors (PP) was analyzed by markers PDX1 and NKX6.1 as shown by representative immunofluorescence images and flow cytometry with 62% reduction of PP cells in iPSC ONECUT1 E231X (PE: n=2, PP: n=3; with 2 replicates; two-tailed, unpaired t-test).

Techniques Used: Sequencing, Immunofluorescence, Western Blot, Flow Cytometry, Two Tailed Test

(a,b) GSEA23 analysis of differentially expressed (DE) genes in HUES8 WT and ONECUT1 KO PP cells (a) as well as PDX1+/NKX6.1+ purified PP cells from HUES8 ONECUT1 KO and WT (b) using a specific gene set for pancreatic progenitors24 as well as genes important for endocrine development and β-cell function25. (c) GSEA enrichment scores contrasting HUES8 WT and KO (or trunc) at PP stage on gene expression signatures of pancreas cells obtained from a single cell RNA-seq study (GSE81547). (d) Correlation of all significant differentially expressed genes (RNA-seq) and proteins (mass spectrometry, MS) in HUES8 ONECUT1 truncated (trunc) cells at the PP stage. (e) Comparison of expression values for depicted genes in HUES8 edited (ONECUT1 truncated and KO) and WT PP cells. Bar graphs represent min and max values with indicated mean normalized to ONECUT1 WT cells (RNA-seq: n=6; qPCR: WT n=4, KO/trunc n=4; MS: n=3; one-way ANOVA with Tukey’s test).
Figure Legend Snippet: (a,b) GSEA23 analysis of differentially expressed (DE) genes in HUES8 WT and ONECUT1 KO PP cells (a) as well as PDX1+/NKX6.1+ purified PP cells from HUES8 ONECUT1 KO and WT (b) using a specific gene set for pancreatic progenitors24 as well as genes important for endocrine development and β-cell function25. (c) GSEA enrichment scores contrasting HUES8 WT and KO (or trunc) at PP stage on gene expression signatures of pancreas cells obtained from a single cell RNA-seq study (GSE81547). (d) Correlation of all significant differentially expressed genes (RNA-seq) and proteins (mass spectrometry, MS) in HUES8 ONECUT1 truncated (trunc) cells at the PP stage. (e) Comparison of expression values for depicted genes in HUES8 edited (ONECUT1 truncated and KO) and WT PP cells. Bar graphs represent min and max values with indicated mean normalized to ONECUT1 WT cells (RNA-seq: n=6; qPCR: WT n=4, KO/trunc n=4; MS: n=3; one-way ANOVA with Tukey’s test).

Techniques Used: Purification, Expressing, RNA Sequencing Assay, Mass Spectrometry

(a) Fine mapping of type II diabetes traits from DIAMANTE GWAS dataset. This region (chr15:53070141–53165681) corresponds to the 99% genetic credible set from Mahajan et al.5 and includes the first exon of ONECUT1 and the non-coding RNA RP11–209K10.2. IGV plot depicts ONECUT1, FOXA1/2, GATA6, PDX1, and NKX6.1 ChIP-seq peaks (PP), ATAC-seq signals and histone modifications. T2D-associated SNPs (“T2D SNPs”) with a p-value < 10−5 are shown in pink, p-value <10−8 in blue. Of those, rs2440374 overlaps with both a ONECUT1 peak and a differential ATAC-seq peak in PE stage. This SNP is localized at the promoter region of the non-coding gene RP11–209K10.2 (ENSEMBL ID ENSG00000259203). (b,c) Tissue-specific expression of ONECUT1 and RP11–209K10.2 obtained from GTEx database showing gene expression in top 10 tissues sorted by median expression. Both genes have high expression specific to pancreas, liver and testis. (d) Motif analysis with RSAT-Var-tools indicates that the SNP disrupts a putative binding sequence of NKX2.2. (e) eQTL analysis with GTEx indicates an association of rs2440374, rs2456530 and rs75332279 with the expression of lncRNA RP11–209K10.2 in pancreas. (f) Expression of lncRNA RP11–209K10.2 in HUES8 WT and ONECUT1 KO PE and PP cells (RNA-seq, n=6; two-tailed, unpaired t-test). (g) Graphical illustration of the proposed mechanism how ONECUT1 loss impairs pancreatic development to cause diabetes.
Figure Legend Snippet: (a) Fine mapping of type II diabetes traits from DIAMANTE GWAS dataset. This region (chr15:53070141–53165681) corresponds to the 99% genetic credible set from Mahajan et al.5 and includes the first exon of ONECUT1 and the non-coding RNA RP11–209K10.2. IGV plot depicts ONECUT1, FOXA1/2, GATA6, PDX1, and NKX6.1 ChIP-seq peaks (PP), ATAC-seq signals and histone modifications. T2D-associated SNPs (“T2D SNPs”) with a p-value < 10−5 are shown in pink, p-value <10−8 in blue. Of those, rs2440374 overlaps with both a ONECUT1 peak and a differential ATAC-seq peak in PE stage. This SNP is localized at the promoter region of the non-coding gene RP11–209K10.2 (ENSEMBL ID ENSG00000259203). (b,c) Tissue-specific expression of ONECUT1 and RP11–209K10.2 obtained from GTEx database showing gene expression in top 10 tissues sorted by median expression. Both genes have high expression specific to pancreas, liver and testis. (d) Motif analysis with RSAT-Var-tools indicates that the SNP disrupts a putative binding sequence of NKX2.2. (e) eQTL analysis with GTEx indicates an association of rs2440374, rs2456530 and rs75332279 with the expression of lncRNA RP11–209K10.2 in pancreas. (f) Expression of lncRNA RP11–209K10.2 in HUES8 WT and ONECUT1 KO PE and PP cells (RNA-seq, n=6; two-tailed, unpaired t-test). (g) Graphical illustration of the proposed mechanism how ONECUT1 loss impairs pancreatic development to cause diabetes.

Techniques Used: ChIP-sequencing, Expressing, Binding Assay, Sequencing, RNA Sequencing Assay, Two Tailed Test

(a) Luciferase reporter assay (HeLa cells, n=8) with WT and ONECUT1 coding variants fused to the transcriptional activator (transactivator) VP16 using a reporter construct consisting of six ONECUT1 binding motifs found in the human FOXA2 promoter region. After binding of ONECUT1 to its binding motif, VP16 is activating transcription independent of the transactivation activity of ONECUT1 variants. Statistical analysis was performed by one-way ANOVA with Dunnett’s test. (b) Proportion of genes with or without restriction to endocrine lineage genes with overlapping binding by ONECUT1 (ChIP-seq, PP) with depicted TFs (ChIP-seq). (c) Pearson correlation between genome-wide binding signals of depicted TFs. (d,e) Co-immunoprecipitation of Flag- or GFP-tagged WT ONECUT1 protein and GFP- or Flag-tagged target proteins. Proteins co-immunoprecipitating with ONECUT1 are highlighted in green, others in orange. WBs on the bottom show successful overexpression of putative interaction partners in HEK293, while WBs on the top were performed after Flag immunoprecipitation. The heavy chain of the Flag-antibody is indicated with an asterisk.
Figure Legend Snippet: (a) Luciferase reporter assay (HeLa cells, n=8) with WT and ONECUT1 coding variants fused to the transcriptional activator (transactivator) VP16 using a reporter construct consisting of six ONECUT1 binding motifs found in the human FOXA2 promoter region. After binding of ONECUT1 to its binding motif, VP16 is activating transcription independent of the transactivation activity of ONECUT1 variants. Statistical analysis was performed by one-way ANOVA with Dunnett’s test. (b) Proportion of genes with or without restriction to endocrine lineage genes with overlapping binding by ONECUT1 (ChIP-seq, PP) with depicted TFs (ChIP-seq). (c) Pearson correlation between genome-wide binding signals of depicted TFs. (d,e) Co-immunoprecipitation of Flag- or GFP-tagged WT ONECUT1 protein and GFP- or Flag-tagged target proteins. Proteins co-immunoprecipitating with ONECUT1 are highlighted in green, others in orange. WBs on the bottom show successful overexpression of putative interaction partners in HEK293, while WBs on the top were performed after Flag immunoprecipitation. The heavy chain of the Flag-antibody is indicated with an asterisk.

Techniques Used: Luciferase, Reporter Assay, Construct, Binding Assay, Activity Assay, ChIP-sequencing, Genome Wide, Immunoprecipitation, Over Expression

(a) Schematic enrichment analysis of ONECUT1-bound genes with either differentially expressed genes or differential open chromatin peaks (HUES8 WT vs. KO). (b) Binding enrichment (z-score) test of ONECUT1 (ChIP-seq, PP stage) in up- and downregulated genes (RNA-seq) at the depicted differentiation stages of ONECUT1 null and WT HUES8 cells. (c) Binding enrichment (z-score) test of ONECUT1 (ChIP-seq, PP stage) in differential open chromatin regions (HUES8 WT vs. KO, ATAC-seq) of the depicted stages. Notably, bars show enrichment in open chromatin (OC) regions lost or gained in ONECUT1-depleted cells. (d) ChIP-seq signals of key TFs at OC peaks lost or gained at the PE and PP stage in HUES8 ONECUT1 KO cells. (e) Differentiation scheme of HUES8 cells toward β-like cells. (f,g) Representative images show immunofluorescence staining of NKX6.1 and C-peptide at stage 6 (f) and quantification of markers was performed by flow cytometry at stage 5 and 6 of ONECUT1 KO and WT HUES8 cells (g, n=3; one-way ANOVA with Tukey’s test). (h) Heatmap depicting relative marker expression in ONECUT1 KO HUES8 cells at stage 5 and 6. Expression values are normalized to HUES8 ONECUT1 WT and scaled by the sum of each row (n=2). (i) Induced insulin secretion of ONECUT1 KO and WT HUES8 cells at stage 6 depicted as fold increase comparing low glucose stimulated insulin secretion with subsequent KCl-stimulated insulin secretion (n=3 with 3 replicates).
Figure Legend Snippet: (a) Schematic enrichment analysis of ONECUT1-bound genes with either differentially expressed genes or differential open chromatin peaks (HUES8 WT vs. KO). (b) Binding enrichment (z-score) test of ONECUT1 (ChIP-seq, PP stage) in up- and downregulated genes (RNA-seq) at the depicted differentiation stages of ONECUT1 null and WT HUES8 cells. (c) Binding enrichment (z-score) test of ONECUT1 (ChIP-seq, PP stage) in differential open chromatin regions (HUES8 WT vs. KO, ATAC-seq) of the depicted stages. Notably, bars show enrichment in open chromatin (OC) regions lost or gained in ONECUT1-depleted cells. (d) ChIP-seq signals of key TFs at OC peaks lost or gained at the PE and PP stage in HUES8 ONECUT1 KO cells. (e) Differentiation scheme of HUES8 cells toward β-like cells. (f,g) Representative images show immunofluorescence staining of NKX6.1 and C-peptide at stage 6 (f) and quantification of markers was performed by flow cytometry at stage 5 and 6 of ONECUT1 KO and WT HUES8 cells (g, n=3; one-way ANOVA with Tukey’s test). (h) Heatmap depicting relative marker expression in ONECUT1 KO HUES8 cells at stage 5 and 6. Expression values are normalized to HUES8 ONECUT1 WT and scaled by the sum of each row (n=2). (i) Induced insulin secretion of ONECUT1 KO and WT HUES8 cells at stage 6 depicted as fold increase comparing low glucose stimulated insulin secretion with subsequent KCl-stimulated insulin secretion (n=3 with 3 replicates).

Techniques Used: Binding Assay, ChIP-sequencing, RNA Sequencing Assay, Immunofluorescence, Staining, Flow Cytometry, Marker, Expressing

(a) Scheme of ONECUT1 variant E231D generated by targeted gene-editing in HUES8 hESCs. (b) Sequence verification of ONECUT1-p.E231D edited HUES8 cells. Of note, sequencing was performed on reverse strand. (c,d) Differentiation efficiency at the PE and PP stages in ONECUT1-p.E231D HUES8 cells. Of note, ILV was omitted after PE stage to better demonstrate small effects in differentiation efficiency of ONECUT1 variants. Representative images show immunofluorescence staining of PDX1 and PDX1/NKX6.1 at the PE and PP stage, respectively. Quantification of positive cells was performed by flow cytometry (PE: n=4; PP: n=3; two-tailed, unpaired t-test). (e) Heatmap depicting relative marker expression in ONECUT1-p.E231D edited HUES8 cells at PP stage. Of note, ILV was omitted after PE stage compared to regular differentiation protocol. Expression values are normalized to HUES8 ONECUT1 WT (n=4, 2 technical replicates) and scaled by the sum of each row. (f) Co-immunoprecipitation of NKX2.2 with ONECUT1 E231D and WT. The asterisk on the blot shows the heavy chain of the Flag antibody. Bottom control western blots show successful overexpression of TFs in HEK293. (g) Quantification relative to NKX2.2 input (n=4; two-tailed, unpaired t-test) shows reduced heterodimerization for ONECUT1 E231D.
Figure Legend Snippet: (a) Scheme of ONECUT1 variant E231D generated by targeted gene-editing in HUES8 hESCs. (b) Sequence verification of ONECUT1-p.E231D edited HUES8 cells. Of note, sequencing was performed on reverse strand. (c,d) Differentiation efficiency at the PE and PP stages in ONECUT1-p.E231D HUES8 cells. Of note, ILV was omitted after PE stage to better demonstrate small effects in differentiation efficiency of ONECUT1 variants. Representative images show immunofluorescence staining of PDX1 and PDX1/NKX6.1 at the PE and PP stage, respectively. Quantification of positive cells was performed by flow cytometry (PE: n=4; PP: n=3; two-tailed, unpaired t-test). (e) Heatmap depicting relative marker expression in ONECUT1-p.E231D edited HUES8 cells at PP stage. Of note, ILV was omitted after PE stage compared to regular differentiation protocol. Expression values are normalized to HUES8 ONECUT1 WT (n=4, 2 technical replicates) and scaled by the sum of each row. (f) Co-immunoprecipitation of NKX2.2 with ONECUT1 E231D and WT. The asterisk on the blot shows the heavy chain of the Flag antibody. Bottom control western blots show successful overexpression of TFs in HEK293. (g) Quantification relative to NKX2.2 input (n=4; two-tailed, unpaired t-test) shows reduced heterodimerization for ONECUT1 E231D.

Techniques Used: Variant Assay, Generated, Sequencing, Immunofluorescence, Staining, Flow Cytometry, Two Tailed Test, Marker, Expressing, Immunoprecipitation, Western Blot, Over Expression

(a) PCA analysis of stage-specific ATAC-seq for differentiation of ONECUT1 null and WT HUES8 lines (left) as well as restricted to PE and PP stages (right). (b) Genomic location of stage-specific ATAC peaks lost or gained in ONECUT1 null (KO) HUES8 line. TTS: transcriptional termination site. (c) Heatmap depicting chromatin accessibility signals (+/− 2kb of peak center) of OC peaks lost upon ONECUT1 KO in HUES8 and ordered by ONECUT1 ChIP-seq peak strength at the PE and PP stage. (d) Enrichment analysis (GREAT26) of OC peaks lost upon ONECUT1 KO at the PE stage. (e) Significance of overlap (log10 p-value) of open chromatin (OC) for different tissues as well as OC lost in HUES8 KO at PE or PP stage and ONECUT1 ChIP-seq peaks. (f) Scatter plot depicting the footprint-based activity score (strength of binding) of TFs in PE state (y-axis) versus the difference of the activity score upon ONECUT1 KO at the PE state (ATAC-seq, HUES8). (g) HNF family factors have the highest loss in activity followed by PAX family, SOX9 and PDX1 factors upon ONECUT1 KO. Representative examples of footprints.
Figure Legend Snippet: (a) PCA analysis of stage-specific ATAC-seq for differentiation of ONECUT1 null and WT HUES8 lines (left) as well as restricted to PE and PP stages (right). (b) Genomic location of stage-specific ATAC peaks lost or gained in ONECUT1 null (KO) HUES8 line. TTS: transcriptional termination site. (c) Heatmap depicting chromatin accessibility signals (+/− 2kb of peak center) of OC peaks lost upon ONECUT1 KO in HUES8 and ordered by ONECUT1 ChIP-seq peak strength at the PE and PP stage. (d) Enrichment analysis (GREAT26) of OC peaks lost upon ONECUT1 KO at the PE stage. (e) Significance of overlap (log10 p-value) of open chromatin (OC) for different tissues as well as OC lost in HUES8 KO at PE or PP stage and ONECUT1 ChIP-seq peaks. (f) Scatter plot depicting the footprint-based activity score (strength of binding) of TFs in PE state (y-axis) versus the difference of the activity score upon ONECUT1 KO at the PE state (ATAC-seq, HUES8). (g) HNF family factors have the highest loss in activity followed by PAX family, SOX9 and PDX1 factors upon ONECUT1 KO. Representative examples of footprints.

Techniques Used: ChIP-sequencing, Activity Assay, Binding Assay

(a) Overview of ONECUT1 WT and mutated protein variants used in overexpression experiments. (b) Representative images of mutated ONECUT1 fused to GFP, overexpressed in HeLa cells. (c-e) Electromobility shift assay (EMSA) and super shift assay of selected WT and mutated ONECUT1 proteins fused to a Flag-tag using a probe consisting of a ONECUT1 binding motif (label A). Additional Flag antibody binding the complex leads to a further shift (label B). Unspecific binding complexes are indicated with an asterisk. In addition, in vitro translated ONECUT1 proteins (TnT Transcription/Translation System) are detected by ONECUT1 or Flag antibody (WB: α-ONECUT1 control).
Figure Legend Snippet: (a) Overview of ONECUT1 WT and mutated protein variants used in overexpression experiments. (b) Representative images of mutated ONECUT1 fused to GFP, overexpressed in HeLa cells. (c-e) Electromobility shift assay (EMSA) and super shift assay of selected WT and mutated ONECUT1 proteins fused to a Flag-tag using a probe consisting of a ONECUT1 binding motif (label A). Additional Flag antibody binding the complex leads to a further shift (label B). Unspecific binding complexes are indicated with an asterisk. In addition, in vitro translated ONECUT1 proteins (TnT Transcription/Translation System) are detected by ONECUT1 or Flag antibody (WB: α-ONECUT1 control).

Techniques Used: Binding Assay, Over Expression, Electro Mobility Shift Assay, Super-Shift Assay, FLAG-tag, In Vitro

(a) Subcellular localization of WT and mutated ONECUT1 proteins fused to GFP (HeLa cells). (b) Electromobility shift assay (EMSA) of WT and ONECUT1 variants using a ONECUT1 binding motif (TRANSFAC T03257) as probe. (c) TNT-ONECUT1 proteins as WB control for (b). (d) Luciferase reporter assay with WT and indicated diabetes-associated (G30S, E231D, E231X, H33Q, G81D, P215R, V242A) and control (D26E, K412R) variants of ONECUT1 (n=6 for G30S, E231D, E231X, H33Q, G81D; n=10 for D26E, K412R, P215R, V242A; one-way ANOVA with Dunnett’s test). (e,f) Co-immunoprecipitation (top) of FLAG-tagged ONECUT1 and interacting factors NKX6.1 (e) and NKX2.2 (f) confirming physical interaction after FLAG immunoprecipitation. Control western blots (bottom) show successful overexpression of respective factors in HEK293 cells. (g,h) Relative expression of NKX6.1 and NKX2.2 in HUES8 WT and ONECUT1 KO at the PP stage and in purified PP (PDX1+/NKX6.1+) cells from RNA-seq (n=6; two-tailed, unpaired t-test). (i) Activity of the E1-NKX6.1 enhancer during pancreatic differentiation using a GFP reporter construct. Images show a GFP reporter signal as well as staining for PP stage marker NKX6.1 in CyT49 cells. (j) Activity of the E1-NKX6.1 enhancer in a GFP reporter construct using a β-cell line (MIN6) and α-cell line (αTC). (k) Luciferase reporter assay with selected NKX6.1 and NKX2.2 enhancer regions overexpressing WT or ONECUT1 variants alone or together with NKX2.2 in HeLa cells (n=6; one-way ANOVA with Tukey’s test). (l) Significance of overlap of variants associated with T2D acquired from DIAMANTE GWAS dataset (P < 10−20) and ATAC-seq (regions with loss or gain of OC upon ONECUT1 KO) as well as ChIP-seq peaks.
Figure Legend Snippet: (a) Subcellular localization of WT and mutated ONECUT1 proteins fused to GFP (HeLa cells). (b) Electromobility shift assay (EMSA) of WT and ONECUT1 variants using a ONECUT1 binding motif (TRANSFAC T03257) as probe. (c) TNT-ONECUT1 proteins as WB control for (b). (d) Luciferase reporter assay with WT and indicated diabetes-associated (G30S, E231D, E231X, H33Q, G81D, P215R, V242A) and control (D26E, K412R) variants of ONECUT1 (n=6 for G30S, E231D, E231X, H33Q, G81D; n=10 for D26E, K412R, P215R, V242A; one-way ANOVA with Dunnett’s test). (e,f) Co-immunoprecipitation (top) of FLAG-tagged ONECUT1 and interacting factors NKX6.1 (e) and NKX2.2 (f) confirming physical interaction after FLAG immunoprecipitation. Control western blots (bottom) show successful overexpression of respective factors in HEK293 cells. (g,h) Relative expression of NKX6.1 and NKX2.2 in HUES8 WT and ONECUT1 KO at the PP stage and in purified PP (PDX1+/NKX6.1+) cells from RNA-seq (n=6; two-tailed, unpaired t-test). (i) Activity of the E1-NKX6.1 enhancer during pancreatic differentiation using a GFP reporter construct. Images show a GFP reporter signal as well as staining for PP stage marker NKX6.1 in CyT49 cells. (j) Activity of the E1-NKX6.1 enhancer in a GFP reporter construct using a β-cell line (MIN6) and α-cell line (αTC). (k) Luciferase reporter assay with selected NKX6.1 and NKX2.2 enhancer regions overexpressing WT or ONECUT1 variants alone or together with NKX2.2 in HeLa cells (n=6; one-way ANOVA with Tukey’s test). (l) Significance of overlap of variants associated with T2D acquired from DIAMANTE GWAS dataset (P < 10−20) and ATAC-seq (regions with loss or gain of OC upon ONECUT1 KO) as well as ChIP-seq peaks.

Techniques Used: Electro Mobility Shift Assay, Binding Assay, Luciferase, Reporter Assay, Immunoprecipitation, Western Blot, Over Expression, Expressing, Purification, RNA Sequencing Assay, Two Tailed Test, Activity Assay, Construct, Staining, Marker, ChIP-sequencing

(a) Homo- and heterodimerization of ONECUT1 proteins was analyzed by co-immunoprecipitation of GFP-tagged ONECUT1 and Flag-tagged ONECUT1 WT or variant in HEK293. The heavy chain of the Flag-antibody is indicated with an asterisk. Note that the ONECUT1 PTV (p.E231X) did not bind to WT ONECUT1 protein. (b,c) Co-immunoprecipitation (top) of NKX6.1 (b) and NKX2.2 (c) with ONECUT1 WT and E231X. Heterodimerization only in ONECUT1 WT and NKX6.1/NKX2.2. The asterisk on the blot shows the heavy chain of the Flag antibody. Bottom control western blots show successful overexpression of TFs in HEK293.
Figure Legend Snippet: (a) Homo- and heterodimerization of ONECUT1 proteins was analyzed by co-immunoprecipitation of GFP-tagged ONECUT1 and Flag-tagged ONECUT1 WT or variant in HEK293. The heavy chain of the Flag-antibody is indicated with an asterisk. Note that the ONECUT1 PTV (p.E231X) did not bind to WT ONECUT1 protein. (b,c) Co-immunoprecipitation (top) of NKX6.1 (b) and NKX2.2 (c) with ONECUT1 WT and E231X. Heterodimerization only in ONECUT1 WT and NKX6.1/NKX2.2. The asterisk on the blot shows the heavy chain of the Flag antibody. Bottom control western blots show successful overexpression of TFs in HEK293.

Techniques Used: Immunoprecipitation, Variant Assay, Western Blot, Over Expression

(a) NKX6.2 expression in HUES8 WT and ONECUT1 KO PP bulk and PDX1+/NKX6.1+ purified cells (RNA-seq, n=6; two-tailed, unpaired t-test). (b,c) ATAC-seq, histone modifications and ONECUT1 ChIP-seq signals around NKX6.1, NKX6.2, and NKX2.2 locus. Red traced squares indicate enhancer regions expanded in (c). Below, the region selected for luciferase assay and reporter assay are shown. (d) Luciferase reporter assay with selected NKX6.2 enhancer region overexpressing WT or ONECUT1 variants alone or together with NKX2.2 in HeLa cells (n=6; one-way ANOVA with Tukey’s test).
Figure Legend Snippet: (a) NKX6.2 expression in HUES8 WT and ONECUT1 KO PP bulk and PDX1+/NKX6.1+ purified cells (RNA-seq, n=6; two-tailed, unpaired t-test). (b,c) ATAC-seq, histone modifications and ONECUT1 ChIP-seq signals around NKX6.1, NKX6.2, and NKX2.2 locus. Red traced squares indicate enhancer regions expanded in (c). Below, the region selected for luciferase assay and reporter assay are shown. (d) Luciferase reporter assay with selected NKX6.2 enhancer region overexpressing WT or ONECUT1 variants alone or together with NKX2.2 in HeLa cells (n=6; one-way ANOVA with Tukey’s test).

Techniques Used: Expressing, Purification, RNA Sequencing Assay, Two Tailed Test, ChIP-sequencing, Luciferase, Reporter Assay