The role of RREB1 in human pancreatic beta cell development and function

Human genetics can be used as a tool to gain insights into fundamental aspects of human development, physiology and pathophysiology. Genome-wide association studies have uncovered multiple independent signals which alter risk for type 2 diabetes (T2D) and influence related glycaemic traits at the RREB1 locus. Fine-mapping revealed that one of these signals is driven by a common coding variant rs9379084 (p.D1171N), highlighting RREB1 as the causal transcript. Little is known about the function of the zinc finger transcription factor (TF) RREB1 in glucose homeostasis and how changes in its expression and/or activity might alter diabetes risk. The aim of my thesis was to determine the role of RREB1 in human beta cell development and function through transcriptomic and cellular phenotyping of genome-edited human induced pluripotent stem cells (hiPSCs) and EndoC-βH1 beta cells.
To investigate the effect of loss of RREB1 on islet cell development I generated RREB1 knockout (KO) hiPSC lines using CRISPR/Cas9 and differentiated them along the pancreatic endocrine lineage. Gene expression profiling revealed that loss of RREB1 had a positive impact on the generation of endocrine precursors (EPs). Endocrine progenitor markers (NEUROG3, NKX2.2, NEUROD1) were significantly up-regulated, while pancreas progenitor marker genes (CPA2, NOTCH1) were markedly down-regulated in hiPSC-derived RREB1 KO EPs.
RREB1-depleted EndoC-βH1 cells, generated using either RNA interference-mediated knock-down or CRISPR/Cas9-mediated KO of RREB1, were characterised by reduced INS expression and insulin content, whilst, gene expression profiling pointed to a positive effect of loss of RREB1 on the differentiation state of mature beta cells. Significant up-regulation of genes implicated in beta cell function, connectivity and maturity (GCK, SNAP25, GJD2, UCN3 ) was accompanied by simultaneous down-regulation of beta cell disallowed genes (PDGFRA, IGFBP4). RREB1 ChIP-Seq analysis in EndoC-βH1 cells identified a subset of these as direct RREB1 target genes and revealed an enrichment of RREB1 binding sites in islet active promoters, highlighting RREB1 as novel transcriptional activator and repressor in endocrine cells. RREB1 cis-regulated genes were enriched for target genes of the beta cell forbidden TF REST, pointing to a potential cooperative action of these two TFs in endocrine gene regulation.
Transcriptional activities of RFX2 and RFX3 were significantly up-regulated in developing and mature RREB1 KO beta cells. While RFX2 was identified as direct RREB1 target gene showing markedly increased transcript levels in RREB1-deficient beta cells, the mechanism underlying up-regulation of RFX3 motif activity remained unclear.
To explore the impact of the RREB1 T2D-associated alleles on beta cell development, I generated allele-specific hiPSC lines using CRISPR/Cas9. Characterisation of differentiated hiPSCs homozygous for the RREB1 T2D-associated coding variant p.N1171 suggested that the T2D protective allele acted as a gain-of-function allele, negatively affecting beta cell differentiation. How this is compatible with a protective effect on T2D risk requires further investigation.
Overall, characterisation of two complimentary RREB1 KO models showed a novel role for RREB1 in beta cell development and function, contributing to the growing list of studies aiming to facilitate biological insights from T2D-associated genes.

Keywords: Diabetes
Gene editing
Stem cells
Advisor: Gloyn, A
Davies, B