Biology subjects

Bevacqua, R. J.

Publications and source records attributed to Bevacqua, R. J..

4 recordsLinked to original sources

bioRxiv2023

Multiplexed CRISPR gene editing in primary human islet cells with Cas9 ribonucleoprotein

Successful genome editing in primary human islets could reveal features of the genetic regulatory landscape underlying {beta} cell function and diabetes risk. Here, we describe a CRISPR-based strategy to interrogate functions of predicted regulatory DNA elements using electroporation of a complex of Cas9 ribonucleoprotein (Cas9 RNP) and guide RNAs into primary human islet cells. We successfully targeted coding regions including the PDX1 exon 1, and non-coding DNA linked to diabetes susceptibility. CRISPR/Cas9 RNP approaches revealed genetic targets of regulation by DNA elements containing candidate diabetes risk SNPs, including an in vivo enhancer of the MPHOSPH9 gene. CRISPR/Cas9 RNP multiplexed targeting of two cis-regulatory elements linked to diabetes risk in PCSK1, which encodes an endoprotease crucial for insulin processing, also demonstrated efficient simultaneous editing of PCSK1 regulatory elements, resulting in impaired {beta} cell PCSK1 regulation and insulin secretion. Multiplex CRISPR/Cas9 RNP provides powerful approaches to investigate and elucidate human islet cell gene regulation in health and diabetes.

cell biology↗

bioRxiv2022

HNF1α transcriptional activation and repression maintain human islet α and β cell function

HNF1A haploinsufficiency underlies the most common form of human monogenic diabetes (HNF1A-MODY) and hypomorphic HNF1A variants confer type 2 diabetes risk, but a lack of experimental systems has limited our understanding of how the transcription factor HNF1 regulates adult human islet function. Here, we combined human islet genetics, RNA sequencing, Cleavage Under Targets & Release Using Nuclease (CUT&RUN) chromatin mapping, patch-clamp electrophysiology and transplantation-based assays to elucidate HNF1-regulated mechanisms in mature pancreatic and {beta} cells. shRNA-mediated suppression of HNF1A in primary human pseudoislets led to blunted insulin output and dysregulated glucagon secretion both in vitro and after transplantation into immunocompromised mice, recapitulating phenotypes observed in HNF1A-MODY patients. These deficits corresponded with altered expression of genes encoding factors critical for hormone secretion, including calcium channel subunits, ATP-transporters and extracellular matrix constituents. Additionally, HNF1A loss led to upregulation of transcriptional repressors, providing evidence for a mechanism of transcriptional de-repression through HNF1. CUT&RUN mapping of HNF1 DNA-binding sites in primary human islets verified that a subset of HNF1-regulated genes were direct targets. These data provide unprecedented mechanistic links between HNF1A loss and diabetic phenotypes in mature human and {beta} cells.

genetics↗

bioRxiv2021

A genome-wide CRISPR screen identifies regulators of beta cell function involved in type 2 diabetes risk

Identification of the genes and processes mediating genetic association signals for complex disease represents a major challenge. Since many of the genetic signals for type 2 diabetes exert their effects through pancreatic islet-cell dysfunction, we performed a genome-wide pooled CRISPR loss-of- function screen in human pancreatic beta cells. We focused on the regulation of insulin content as a disease-relevant readout of beta cell function. We identified 580 genes influencing this phenotype: integration with genetic and genomic data provided experimental support for 20 candidate type 2 diabetes effector transcripts including the autophagy receptor CALCOCO2. Our study highlights how cellular screens can augment existing multi-omic efforts to accelerate biological and translational inference at GWAS loci.

genomics↗

bioRxiv2020

SIX2 and SIX3 coordinately regulate functional maturity and fate of human pancreatic β-cells

The physiological functions of many vital tissues and organs continue to mature after birth, but the genetic mechanisms governing this postnatal maturation remain an unsolved mystery. Human pancreatic {beta}-cells produce and secrete insulin in response to physiological cues like glucose, and these hallmark functions improve in the years after birth. This coincides with expression of the transcription factors SIX2 and SIX3, whose functions in native human {beta}-cells remain unknown. Here, we show that shRNA-mediated SIX2 or SIX3 suppression in human pancreatic adult islets impairs insulin secretion. However, transcriptome studies revealed that SIX2 and SIX3 regulate distinct targets. Loss of SIX2 markedly impaired expression of genes governing {beta}-cell insulin processing and output, glucose sensing, and electrophysiology, while SIX3 loss led to inappropriate expression of genes normally expressed in fetal {beta}-cells, adult a-cells and other non-{beta}-cells. Chromatin accessibility studies identified genes directly regulated by SIX2. Moreover, {beta}-cells from diabetic humans with impaired insulin secretion also had reduced SIX2 transcript levels. Revealing how SIX2 and SIX3 govern functional maturation and maintain developmental fate in native human {beta}-cells should advance {beta}-cell replacement and other therapeutic strategies for diabetes.

developmental biology↗