Identification of Novel Regulatory Mechanisms Driving Human beta-Cell Maturation and Function.

Abstract Type 1 Diabetes mellitus (T1D) is an autoimmune disease targeting pancreatic β-cells that affects over 8 million people worldwide. Standard treatment for T1D is limited to administration of exogenous insulin, which can result in hypoglycemic episodes, and fails to fully prevent micro- and macro- vascular complications. Thus, deriving glucose-responsive insulin-producing β-cells from renewable sources is an index goal of regenerative medicine. Yet, current human stem cell-derived β-cells produce insulin at lower levels than native β-cells and fail to recapitulate regulated insulin secretion typical of mature adult islet β-cells. This limits their therapeutic potential and reflects a significant gap in our knowledge regarding mechanisms regulating human islet maturation. Exciting recent studies identified human-specific age-dependent gene expression changes, including novel transcription factors, such as RXRG, not expressed in mouse or immature human β-like cells, that might control functional maturation of human islet cells. My Preliminary Data shows that RXRG loss in human adult β-cells results in impaired glucose-stimulated insulin secretion. Recent studies also showed that gradual turnover of enhancers drives lineage progression and maturation. I have found a presumptive enhancer regulatory element of RXRG that becomes differentially accessible with age in adult β-cells compared to immature β-cells and a-cells, as measured by H3K27Ac occupancy. My objective here is to discover mechanisms that induce maturation of human β-cells, using two approaches. In Aim 1, I will investigate the regulatory roles of the novel transcriptional regulator of β-cell maturation RXRG. This will involve knock down of RXRG, and then β-cell specific RNA-seq and CUT&RUN to identify RXRG downstream targets. In addition, I will activate RXRG endogenous promoter using the CRISPRa system and evaluate chromatin accessibility changes induced by activation of this maturation-required transcription factor. In Aim 2, I will study the non-coding regulatory mechanisms activating RXRG expression to induce β-cell maturation. Since these mechanisms do not occur in mouse or stem cell-derived human β-cells, and are specific to adult β-cells, the studies I proposed will be performed in adult human islets using the pseudoislet-genetics platform I developed during my postdoctoral studies. Here, I will use novel technological advancements, including CRISPR-Cas9 ribonucleoprotein (RNP) editing of human islet cells, to delete the presumptive enhancer element of RXRG and evaluate its role in regulating RXRG expression. Finally, I will activate RXRG expression in juvenile islets using CRISPRa to activate RXRG promoter, enhancer or both, and evaluate if RXRG expression enhances coordinated glucose-dependent insulin secretion in adulthood. Altogether, findings from this proposal will uncover novel pathways leading to β- cell maturation, that could be exploited towards efforts with β-cells from renewable sources. Equally importantly, these studies provide a novel toolkit for genetically manipulating previously “unstudiable” or inaccessible non- replicating mature and immature β-cells.