The Bevacqua Lab

The vast preponderance of genetic risk for diabetes mellitus (at >400 risk loci) maps to non-coding DNA, mostly in presumptive regulatory regions. In other cell systems such as neurons, licensing and decommissioning of thousands of cis-regulatory enhancer elements mediates postnatal maturation. Whether in neurons or islets, we still lack understanding of the target genes of most of these enhancers, and of their dynamic age-dependent regulation. Recent developments by Dr. Bevacqua, including CRISPR-based systems to study non-coding regulatory sequences in primary non-dividing cells (Bevacqua et al. 2021a) and CUT&RUN -Cleavage Under Targets and Release Using Nucleases- from low cell inputs, such as those available from human islets (Bevacqua et al. 2021b) introduce unprecedented possibilities to identify and study the function of these regulatory elements in a bona fide islet context. Identification of enhancer regions that are differentially accessible throughout islet maturation and disease will contribute to our understanding of the genetic and epigenetic mechanisms that contribute to islet β-cell dysfunction in diabetes.

Postnatal maturation of human islet cells occurs over a prolonged period from birth to early adulthood, developmental stages that coincide with changes in diet, nutrient metabolism, and hormone levels. However, the signaling basis of this fascinating example of cellular development remains poorly understood. Based on recent publications identifying differential regulation of feeding-induced enhancers with age in human islet cells, we hypothesize that nutrient- and hormone-linked islet enhancers might be enriched for motifs recognized by TFs that show differential expression with age, including those “priming” functional islet maturation. We plan to study these mechanisms using reverse genetics in primary human islets and mouse models with mutations in signal-linked-TF binding motifs, combined with exposure to environmental stimuli. These studies could provide valuable information about how to generate functionally mature β-cells and to restore function to diseased β-cells.

Technological advances foster discovery. We are currently developing novel CRISPR approaches, including using electroporation of ribonucleoproteins (RNPs) to delete non-coding regulatory DNA elements, without integration of lenti-vectors. This approach has multiple advantages, including simplicity, scalability, avoidance of lentiviral integration or toxicity, and adaptation for multiplexing, relevant to diabetes modeling. In addition, we are working to adapt additional dCas9- based approaches, that will allow the study of epigenetic mechanisms relevant to maturation and disease.