3-LB: The Type 2 Diabetes-Associated Transcription Factor RREB1 Affects Beta-Cell Function and Development

Abstract

Genome-wide association studies have identified multiple independent signals for type 2 diabetes risk at the RREB1 locus. Coding variant fine-mapping (D1171N, rs9379084) indicated RREB1 as the effector transcript. To investigate the role of this transcription factor (TF) in beta cell function and development, we performed transcriptome profiling and cellular phenotyping of genome-edited EndoC-βH1 beta cells and human induced pluripotent stem cells (hiPSC). CRISPR/Cas9-mediated knockout (KO) of RREB1 in EndoC-βH1 cells reduced INS expression (log2FC=-0.4, q=0.01) and insulin content (-44.2%, p=2x10-4). Motif activity response analysis (ISMARA) of RREB1 KO and wild type (WT) transcriptome expression data highlighted RFX2 and RFX3 as key TFs mediating gene expression variation across samples. RFX2 expression was upregulated in RREB1 KO cells at the transcript (log2FC=0.4,q=6.5x10-4) and protein level (+125.7%, p=8x10-4). Consistent with this, RFX2/3 target genes CAMK2A (log2FC=3.9, q=8.7x10-109) and GPR56 (log2FC=3.1, q=5.2x10-283) were among the strongest upregulated differentially expressed genes (DEG). Transcriptome profiling of CRISPR/Cas9-generated RREB1 KO hiPSC, differentiated along the pancreatic endocrine lineage, revealed that RREB1 was expressed throughout development (CV=10.7%). In total 5476 DEG (q<0.01) were identified across all 7 differentiation stages between 4 RREB1 KO and 3 WT lines. ISMARA again predicted RFX2/3 as the most important regulatory TFs driving variation between KO and WT lines. Motif activity gradually increased from definitive endoderm to a peak in the endocrine precursor stage. Endocrine progenitor markers (NEUROG3, NEUROD1, NKX2.2; p=4x10-83) were enriched among DEG upregulated at the pancreatic endoderm stage in the KO lines, suggesting RREB1 as a potential regulator of beta cell development. In conclusion, our combined data support a novel role for RREB1 in both beta cell function and development likely via RFX2/3. Disclosure K.K. Mattis: None. F. Abaitua: None. A. Grotz: None. A. Wesolowska-Andersen: None. M. Perez-Alcantara: None. M. McCarthy: Advisory Panel; Self; European Association for the Study of Diabetes, Pfizer Inc. Consultant; Self; Eli Lilly and Company, Merck & Co., Inc. Consultant; Spouse/Partner; Merck & Co., Inc. Research Support; Self; AbbVie Inc., Boehringer Ingelheim International GmbH. Research Support; Spouse/Partner; Diabetes UK. Research Support; Self; Janssen Pharmaceuticals, Inc., Merck & Co., Inc., National Institutes of Health. Research Support; Spouse/Partner; National Institutes of Health. Research Support; Self; Novo Nordisk A/S. Research Support; Spouse/Partner; Novo Nordisk A/S. Research Support; Self; Novo Nordisk Foundation, Roche Pharma, Sanofi-Aventis, Servier, Takeda Pharmaceutical Company Limited. A. Mahajan: None. B. Davies: None. A.L. Gloyn: Consultant; Spouse/Partner; Eli Lilly and Company, Merck & Co., Inc. Consultant; Self; Merck & Co., Inc. Consultant; Spouse/Partner; Novo Nordisk A/S, Pfizer Inc. Research Support; Self; Novo Nordisk A/S. Speaker’s Bureau; Self; Novo Nordisk A/S. Other Relationship; Self; Diabetes UK, European Foundation for the Study of Diabetes. Funding UK Wellcome Trust