Similar Datasets
Project description:OBJECTIVE:Hundreds of missense mutations in the coding region of PDX1 exist; however, if these mutations predispose to diabetes mellitus is unknown. METHODS:In this study, we screened a large cohort of subjects with increased risk for diabetes and identified two subjects with impaired glucose tolerance carrying common, heterozygous, missense mutations in the PDX1 coding region leading to single amino acid exchanges (P33T, C18R) in its transactivation domain. We generated iPSCs from patients with heterozygous PDX1P33T/+, PDX1C18R/+ mutations and engineered isogenic cell lines carrying homozygous PDX1P33T/P33T, PDX1C18R/C18R mutations and a heterozygous PDX1 loss-of-function mutation (PDX1+/-). RESULTS:Using an in vitro β-cell differentiation protocol, we demonstrated that both, heterozygous PDX1P33T/+, PDX1C18R/+ and homozygous PDX1P33T/P33T, PDX1C18R/C18R mutations impair β-cell differentiation and function. Furthermore, PDX1+/- and PDX1P33T/P33T mutations reduced differentiation efficiency of pancreatic progenitors (PPs), due to downregulation of PDX1-bound genes, including transcription factors MNX1 and PDX1 as well as insulin resistance gene CES1. Additionally, both PDX1P33T/+ and PDX1P33T/P33T mutations in PPs reduced the expression of PDX1-bound genes including the long-noncoding RNA, MEG3 and the imprinted gene NNAT, both involved in insulin synthesis and secretion. CONCLUSIONS:Our results reveal mechanistic details of how common coding mutations in PDX1 impair human pancreatic endocrine lineage formation and β-cell function and contribute to the predisposition for diabetes.
| S-EPMC6531841 | biostudies-literature
Project description:Missense mutations in coding region of PDX1 predispose to type-2 diabetes mellitus as well as cause MODY through largely unexplored mechanisms. Here, we screened a large cohort of subjects with increased risk for diabetes and identified two subjects with impaired glucose tolerance carrying heterozygous missense mutations in the PDX1 coding region leading to single amino acid exchanges (P33T, C18R) in its transactivation domain. We generated iPSCs from patients with heterozygous PDX1P33T/+, PDX1C18R/+ mutations and engineered isogenic cell lines carrying homozygous PDX1P33T/P33T, PDX1C18R/C18R mutations and a heterozygous PDX1 loss-of-function mutation (PDX1+/-). Using an in vitro β-cell differentiation protocol, we demonstrated that both PDX1P33T/+, PDX1C18R/+ and PDX1P33T/P33T, PDX1C18R/C18R mutations impair β-cell differentiation and function. Furthermore, PDX1+/- and PDX1P33T/P33T mutations reduced differentiation efficiency of pancreatic progenitors (PPs), due to downregulation of PDX1-bound genes, including transcription factors MNX1 and PDX1 as well as insulin resistance gene CES1. Additionally, both PDX1P33T/+ and PDX1P33T/P33T mutations in PPs reduced the expression of PDX1-bound genes including the long-noncoding RNA, MEG3 and the imprinted gene NEURONATIN, both involved in insulin synthesis and secretion. Our results reveal mechanistic details of how diabetes-associated PDX1 point mutations impair human pancreatic endocrine lineage formation and β-cell function and contribute to pre-disposition for diabetes.
2019-06-19 | GSE125769 | GEO
Project description:Missense mutations in coding region of PDX1 predispose to type-2 diabetes mellitus as well as cause MODY through largely unexplored mechanisms. Here, we screened a large cohort of subjects with increased risk for diabetes and identified two subjects with impaired glucose tolerance carrying heterozygous missense mutations in the PDX1 coding region leading to single amino acid exchanges (P33T, C18R) in its transactivation domain. We generated iPSCs from patients with heterozygous PDX1P33T/+, PDX1C18R/+ mutations and engineered isogenic cell lines carrying homozygous PDX1P33T/P33T, PDX1C18R/C18R mutations and a heterozygous PDX1 loss-of-function mutation (PDX1+/-). Using an in vitro β-cell differentiation protocol, we demonstrated that both PDX1P33T/+, PDX1C18R/+ and PDX1P33T/P33T, PDX1C18R/C18R mutations impair β-cell differentiation and function. Furthermore, PDX1+/- and PDX1P33T/P33T mutations reduced differentiation efficiency of pancreatic progenitors (PPs), due to downregulation of PDX1-bound genes, including transcription factors MNX1 and PDX1 as well as insulin resistance gene CES1. Additionally, both PDX1P33T/+ and PDX1P33T/P33T mutations in PPs reduced the expression of PDX1-bound genes including the long-noncoding RNA, MEG3 and the imprinted gene NEURONATIN, both involved in insulin synthesis and secretion. Our results reveal mechanistic details of how diabetes-associated PDX1 point mutations impair human pancreatic endocrine lineage formation and β-cell function and contribute to pre-disposition for diabetes.
2019-06-19 | GSE125768 | GEO
Project description:Missense mutations in coding region of PDX1 predispose to type-2 diabetes mellitus as well as cause MODY through largely unexplored mechanisms. Here, we screened a large cohort of subjects with increased risk for diabetes and identified two subjects with impaired glucose tolerance carrying heterozygous missense mutations in the PDX1 coding region leading to single amino acid exchanges (P33T, C18R) in its transactivation domain. We generated iPSCs from patients with heterozygous PDX1P33T/+, PDX1C18R/+ mutations and engineered isogenic cell lines carrying homozygous PDX1P33T/P33T, PDX1C18R/C18R mutations and a heterozygous PDX1 loss-of-function mutation (PDX1+/-). Using an in vitro β-cell differentiation protocol, we demonstrated that both PDX1P33T/+, PDX1C18R/+ and PDX1P33T/P33T, PDX1C18R/C18R mutations impair β-cell differentiation and function. Furthermore, PDX1+/- and PDX1P33T/P33T mutations reduced differentiation efficiency of pancreatic progenitors (PPs), due to downregulation of PDX1-bound genes, including transcription factors MNX1 and PDX1 as well as insulin resistance gene CES1. Additionally, both PDX1P33T/+ and PDX1P33T/P33T mutations in PPs reduced the expression of PDX1-bound genes including the long-noncoding RNA, MEG3 and the imprinted gene NEURONATIN, both involved in insulin synthesis and secretion. Our results reveal mechanistic details of how diabetes-associated PDX1 point mutations impair human pancreatic endocrine lineage formation and β-cell function and contribute to pre-disposition for diabetes. Methods: In this study, we have generated a novel induced pluripotent stem cell (iPSC) line that efficiently differentiates into human pancreatic progenitors (PPs). Furthermore, PDX1 and H3K27ac chromatin immunoprecipitation sequencing (ChIP-seq) was used to identify PDX1 transcriptional targets and active enhancer and promoter regions. To address potential differences in the function of PDX1 during development and adulthood, we compared PDX1 binding profiles from PPs and adult islets. Moreover, combining ChIP-seq and GWAS meta-analysis data we identified T2DM-associated SNPs in PDX1 binding sites and active chromatin regions.
2019-06-19 | GSE125590 | GEO
Project description:Point mutations in the PDX1 transactivation domain impair human β-cell development and function
| PRJNA517470 | ENA
Project description:Point mutations in the PDX1 transactivation domain impair human β-cell development and function (ChIP-Seq)
| PRJNA517474 | ENA
Project description:Point mutations in the PDX1 transactivation domain impair human β-cell development and function (RNA-Seq)
| PRJNA517478 | ENA
Project description:Point mutations in the PDX1 transactivation domain impair human β-cell development and function (mRNA microarray)
| PRJNA516880 | ENA
Project description:RNA polymerase III recognizes and pauses at its terminator, an oligo(dT) tract in non-template DNA, terminates 3' oligo(rU) synthesis within this sequence, and releases the RNA. The pol III subunit Rpc11p (C11) mediates RNA 3'-5' cleavage in the catalytic center of pol III during pausing. The amino and carboxyl regions of C11 are homologous to domains of the pol II subunit Rpb9p, and the pol II elongation and RNA cleavage factor, TFIIS, respectively. We isolated C11 mutants from Schizosaccharomyces pombe that cause pol III to readthrough terminators in vivo. Mutant RNA confirmed the presence of terminator readthrough transcripts. A predominant mutation site, F32, resides in the C11 Rpb9-like domain. Another mutagenic approach confirmed the F32 mutation and also isolated I34 and Y30 mutants. Modeling Y30, F32 and I34 of C11 in available cryoEM pol III structures predicts a hydrophobic patch that may interface with C53/37. Another termination mutant, Rpc2-T455I, appears to reside internally, near the RNA-DNA hybrid. We show that the Rpb9 and TFIIS homologous mutants of C11 reflect distinct activities, that differentially affect terminator recognition and RNA 3' cleavage. We propose that these C11 domains integrate action at the upper jaw and center of pol III during termination.
| S-EPMC3152337 | biostudies-literature
Project description:Liver X receptor alpha (LXRα) is crucial for the maintenance of lipid and cholesterol homeostasis. Ligand binding and dimerization with retinoid X receptor (RXR) or peroxisome proliferator-activated receptor (PPAR) is required for forming active DNA binding complexes leading to gene regulation. Structure based prediction and solvent accessibility of LXRα LBD shows that residues H383, E387, H390, L414, and R415 which are located in helices 9 and 10 may be critical for mediating protein-protein interactions. In this study, LXRα interface residues were individually mutated to determine their effects on ligand binding, protein-protein association, subcellular localization, and transactivation activity. LXRα L414R and R415A lacked binding to T-0901317, but retained binding to 25-Hydroxycholesterol. In vitro assay and a cell based assay demonstrated that LXRα L414R was specifically impaired for interactions with RXRα but not PPARα suggesting that charge reversal at the interface provides selectivity to LXRα dimerization. Furthermore, binding of LXRα L414R or R415A with PPARα exhibited minimal conformational changes in the dimer secondary structure. Interestingly, all LXRα mutants exhibited lower levels of ligand dependent luciferase activity driven by the SREBP-1c or ApoA1 promoter. Taken together, our data demonstrates that intact hydrophobic interactions and salt bridges at the interface mediate efficient ligand-dependent transactivation activities.
| S-EPMC5630223 | biostudies-literature