HNF1α maintains pancreatic α and β cell functions in primary human islets

Abstract

HNF1A haploinsufficiency underlies the most common form of human monogenic diabetes (HNF1A-maturity onset diabetes of the young [HNF1A-MODY]), and hypomorphic HNF1A variants confer type 2 diabetes risk. But a lack of experimental systems for interrogating mature human islets has limited our understanding of how the transcription factor HNF1α regulates adult islet function. Here, we combined conditional genetic targeting in human islet cells, RNA-Seq, chromatin mapping with cleavage under targets and release using nuclease (CUT&RUN), and transplantation-based assays to determine HNF1α-regulated mechanisms in adult human pancreatic α and β cells. Short hairpin RNA-mediated (shRNA-mediated) suppression of HNF1A in primary human pseudoislets led to blunted insulin output and dysregulated glucagon secretion after transplantation in mice, recapitulating phenotypes observed in patients with diabetes. These deficits corresponded with altered expression of genes encoding factors critical for hormone secretion, including calcium channel subunits, ATPase transporters, and extracellular matrix constituents. Additionally, HNF1A loss led to upregulation of transcriptional repressors, providing evidence for a mechanism of transcriptional derepression through HNF1α. CUT&RUN mapping of HNF1α DNA binding sites in primary human islets imputed a subset of HNF1α-regulated genes as direct targets. These data elucidate mechanistic links between HNF1A loss and diabetic phenotypes in mature human α and β cells.

Keywords: Beta cells; Diabetes; Endocrinology; Genetics; Monogenic diseases.

Figure 1. shRNA-mediated KD of HNF1A in primary human islets.

(A) Formation of pseudoislets for downstream assays; transduction with lentivirus, followed by reaggregation over 5 days in culture. (B) Schematic of lentiviral constructs coding for shRNA and a GFP reporter (tGFP). Control-shRNA, nontargeting (“control”); HNF1A-shRNA, HNF1A-targeting (“HNF1AKD”). (C and D) Blue light (488 nm) images of human control (C) and HNF1AKD (D) pseudoislets. Scale bars: 1,000 μm. (E and F) mRNA expression of HNF1A (E) and putative HNF1α (F) targets in HNF1AKD relative to control pseudoislets; statistics performed on ΔCT values (n = 4–8 donors per gene). (G) Western blot analysis of HNF1α protein expression in control (CTL) and HNF1AKD (KD) pseudoislets (n = 3). (H) Quantification of blot intensities normalized to the housekeeping gene β-actin (ACTB). Data are presented as mean values ± SEM. Two-tailed t tests were used to generate P values; *P < 0.05, **P < 0.01, ***P < 0.00001.

Figure 2. HNF1A suppression leads to dysregulated insulin and glucagon secretion after 1 month in vivo.

(A) Experimental approach for control and HNF1AKD pseudoislet transplantation under kidney capsules of NSG mice and characterization of graft phenotypes; 1,000 pseudoislets were transplanted per mouse. (B–D) Blood glucose, plasma human insulin levels, and AUC of insulin excursion upon i.p. glucose challenge after transplantation of pseudoislets to NSG mice (n = 4 mice, 3 human donors). (E) Schematic of pseudoislet transplantation to glucagon-KO mice on an NSG background (GKO-NSG) for characterization of glucagon phenotypes; 1,000 pseudoislets were transplanted per mouse. (F–K) Blood glucose, plasma glucagon levels, and AUC of glucagon excursion upon i.p. glucose (F–H) or insulin (I–K) challenge after transplantation to GKO-NSG mice (n = 4 mice, 4 human donors). Data are mean values ± SEM. Two-tailed t tests were used to generate P values; *P < 0.05, **P < 0.01.

Figure 3. RNA-Seq of HNF1AKD β cells shows that HNF1α regulates insulin secretion, metabolism, developmental pathways, and cell-to-cell signaling in β cells.

(A) Schematic of FACS scheme for isolation of transduced live β cells (HPi2⁺GFP⁺NTPDase3⁺) from control and HNF1AKD pseudoislets for downstream RNA-Seq (n = 4 donors). (B) Fraction of endocrine (HPi2⁺) cells expressing GFP in sorted samples. (C) HNF1A transcripts per million (TPM) in sequenced samples. (D) Differential expression analysis revealed significantly up- and downregulated genes after HNF1AKD in β cells. Fold change (FC) = 1.5, adjusted P = 0.05. (E) Heatmap of DEGs in β cells after HNF1AKD. (F–H) Significantly downregulated (F) and upregulated (G) Gene Ontology (GO) pathways and downregulated KEGG pathways (H) in HNF1AKD relative to control β cells. *P < 0.05.

Figure 4. RNA-Seq of HNF1AKD α cells identifies dysregulation of calcium channel complexes and ATPase-coupled transmembrane transport as well as hormone secretion pathways shared with β cells.

(A) Schematic of methods for isolation of transduced α cells (HPi2⁺GFP⁺CD26⁺) from control and HNF1AKD pseudoislets for downstream RNA-Seq (n = 4 donors); left image is bright-field, and right image is blue light (488 nm) of human HNF1AKD pseudoislets. Scale bars: 1,000 μm. (B) HNF1A transcripts per million (TPM) in sequenced α cell samples. (C) DEG analysis revealed significantly up- and downregulated genes after HNF1AKD in α cells. FC = 1.5, adjusted P = 0.05. (D) Venn diagram comparing α versus β cell DEGs revealed shared and cell-specific consequences of HNF1AKD. (E and F) Gene Ontology (GO) pathways of shared (E) and α cell (F) enriched DEG sets. (G) Box plots displaying TPM of select DEGs. *P < 0.05.

Figure 5. CUT&RUN identifies direct binding targets of HNF1α in primary human islet cells.

(A) Schematic of methods. Pseudoislets expressing HNF1α-FLAG were used for CUT&RUN with anti-FLAG or anti-IgG (control) antibody (n = 3 donors). (B) Enriched motifs in the HNF1α-FLAG CUT&RUN peaks (versus IgG controls). (C) Venn diagram of genes associated with HNF1α-FLAG peaks identified by CUT&RUN (HNF1α-FLAG CUT&RUN) versus HNF1AKD DEGs in primary islet cells identified by RNA-Seq (HNF1AKD RNA-Seq). (D) Gene Ontology (GO) pathway analysis of overlapping genes from C, subset into genes that were downregulated or upregulated in RNA-Seq analysis. (E and F) UCSC Genome Browser tracks showing genomic regions associated with HNF1α-FLAG CUT&RUN peaks near the genes TM4SF4 (E) and CACNA1D (F); HNF1α-FLAG CUT&RUN enriched peaks identified by Genomic Regions Enrichment of Annotations Tool (GREAT) are highlighted in dashed boxes, and regulated genes are depicted below IgG control tracks. Accessible chromatin regions in human islets are shown by ATAC-Seq and ChIP-Seq (H3K427ac, H3K4me1, and H3Kme3) tracks (27). (G) Schematic depicting HNF1α’s dual role as a transcriptional activator and repressor in pancreatic islet cells.

Figure 6. Comparison of HNF1α targets in primary human islets with HNF1A-MODY adult donor data sets demonstrates conserved HNF1α regulatory pathways that are critical for mature islet cell function.

(A and B) Heatmaps showing relative expression of genes in α (A) and β (B) cells isolated from an HNF1A^+/T260M donor (MODY) versus healthy control donors (C1-C5) (13); genes depicted were top DEGs in primary islet HNF1AKD RNA-Seq data also identified in HNF1α-FLAG CUT&RUN data (putative adult HNF1α targets).