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

Abstract

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 β 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 β 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 β-cell insulin processing and output, glucose sensing, and electrophysiology, while SIX3 loss led to inappropriate expression of genes normally expressed in fetal β cells, adult α cells, and other non-β cells. Chromatin accessibility studies identified genes directly regulated by SIX2. Moreover, β 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 β cells should advance β-cell replacement and other therapeutic strategies for diabetes.

Keywords: diabetes mellitus; islet; pancreas; transcription factor; β cells.

Figure 1.

shRNA-mediated suppression of SIX2 and SIX3 in primary human islets results in impaired glucose-stimulated insulin secretion. (A) Schematics of the lentiviral constructs coding for a short hairpin RNA (shRNA) and GFP. (B) Schematic detailing the pseudoislet technique. (C) SIX2 mRNA expression in primary human islets. (Gray bar) Control, (green bar) SIX2^kd. n = 9 independent donor repetitions. (D) SIX3 mRNA expression in primary human islets. (Gray bar) Control, (blue bar) SIX3^kd. n = 11 independent donor repetitions. SIX3 mRNA were not altered by SIX2^kd (n = 8 independent donor repetitions) (E), and SIX2 mRNA levels were not affected by SIX3^kd (n = 11 independent donor repetitions) (F). (G,I) In vitro glucose-stimulated insulin secretion from human pseudoislets control, SIX2^kd (n = 9 independent donor repetitions) (G), or SIX3^kd (n = 12 independent donor repetitions) (I). Secreted insulin normalized to insulin content. (Black lines) Significant differences within the control, (red lines) significant differences within the KD groups, (green lines) significant differences between control and KD conditions. (H,J) Total insulin from human pseudoislets after transduction with SIX2^kd (n = 9 independent donor repetitions) (H) or SIX3^kd (n = 12 independent donor repetitions) (J). Data presented as mean; error bars represent the standard error. Two-tailed t-tests used to generate P-values: (*) P < 0.05, (**) P < 0.01, (***) P < 0.0001.

Figure 2.

RNA-seq of SIX2^kd β cells reveals genes regulated by SIX2 in primary human islets. (A,B) SIX2^kd human pseudoislets. (A) Bright field. (B) Blue light (488 nm). Scale bars, 500 µm. (C) FACS scheme used to sort GFP+ β cells. (D,E) Normalized transcript levels of SIX2 (D) and SIX3 (E) in GFP+ β−cells. (Gray bar) Control, (green bar) SIX2^kd. n = 4. (F) GO term enrichment in genes deregulated in β cells post-SIX2^kd. (G,H) KEGG pathway enrichment in genes up-regulated (G) or down-regulated (H) in β cells post-SIX2^kd (n = 4). (I) Heat map showing all differentially expressed (DE) genes in β cells post-SIX2^kd. The data are presented as mean; error bars represent the standard error. (*) P < 0.05

Figure 3.

SIX2^kd in primary β cells results in down-regulation of genes enriched in adult β-cell. (A) Venn diagram showing adult β-cell genes down-regulated post-SIX2^kd. (B) Heat map of adult down-regulated genes and juvenile up-regulated genes in adult β cells post-SIX2^kd.

Figure 4.

Identification of presumptive SIX2 genetic targets in primary human β cells using CUT&RUN. (A) Schematic of the CUT&RUN approach: Pseudoislets overexpressing SIX2-FLAG under the RIP promoter were used for CUT&RUN with anti-FLAG antibody (n = 3 independent donors). (B) Heat map showing enrichment of peak read densities at the center of the peak for the CUT&RUN libraries generated with FLAG antibody, but not for IgG. Peaks were called using HOMER. (C) Enriched motifs in the differential peaks were identified by HOMER. (D) Overlap of the SIX2-associated genomic regions and the SIX2^kd DE genes. (E,F) Tracks showing SIX2-FLAG genomic regions associated with GCGR (E) or CHGA (F). Accessible chromatin regions in human islets are shown by ATAC-seq, H3K4me3, and H3K27ac ChiP-seq tracks. SIX2-FLAG CUT&RUN peaks are shown in pink boxes (note: for GCGR, two peaks are shown), and regulated genes are highlighted in green boxes.

Figure 5.

RNA-seq of SIX3^kd β cells reveals a distinct gene set regulated by SIX3. (A) Normalized transcript levels of SIX3 and SIX2 in GFP-expressing β cells. (Gray bar) Control, (blue bar) SIX3^kd . n = 3. (B) Heat map of the sample-to-sample distances for all the samples used in this experiment. (C) GO term enrichment in genes deregulated in β cells post-SIX3^kd. (D) KEGG pathways enriched in genes up-regulated in β cells post-SIX3^kd (n = 3). (E) Heat map showing all up-regulated genes upon SIX3^kd in β cells. (F) Overlapped DE genes in adult β cells post SIX2^kd and SIX3^kd. (G) Fold transcript levels of non-β-cell genes significantly altered in β cells post-SIX3^kd (n = 3). The data are presented as mean; error bars represent the standard error. (*) P < 0.05.

Figure 6.

SIX3 represses non-β-cell programs in the adult β-cell. (A) Venn diagram showing juvenile β-cell genes up-regulated post-SIX3^kd in adult β cells. (B) Heat map of adult down-regulated and juvenile up-regulated genes in adult β cells post-SIX3^kd. (C) Schematic detailing the juvenile pseudoislet technique used to overexpress SIX3-FLAG (SIX3-ox) in juvenile pseudoislets (n = 2) and of the constructs used to overexpress SIX3 in juvenile pseudoislets: FACS was used to sort FLAG+ β cells. (D) SIX3, INS, and GCG mRNA expression in FLAG+-expressing β cells post FACS. (White bar) Control , (red bar) SIX3-ox. n = 2. (E) Schematics showing proposed coordinated regulation of maturation by SIX2 and SIX3 in the β-cell. The data are presented as mean; error bars represent the standard error. (*) P < 0.05.

Figure 7.

SIX2 expression is reduced in β cells from T2D donors. (A) Gene expression levels in whole islets from nondiabetic (ND; gray bars) (n = 5) or type 2 diabetic (T2D; red bar) (n = 3). (B) Heat map showing DE genes in nondiabetic versus T2 diabetic β cells. (C,D) Box plots displaying TPM counts of SIX2 (C) and SIX3 (D) in nondiabetic (ND) (n = 5) β cells (dark-gray bars) and α cells (light-gray bars) or type 2 diabetic (T2D) β cells (red bars) and α cells (dark-red bars) (n = 3). (E–G) Box plots displaying TPM GCGR (E), MAFB (F), and CDKN1C (G) in β-cells of ND (n = 5) and T2D (n = 3), the expression of which is DE in adult β cells post-SIX2^kd. (Gray bars) Çontrol, (green bars) SIX2^kd. (H) Insulin secretion of ND (n = 5) versus T2D (n = 3; see the Materials and Methods). (I) Plot of the total area under the curve of the released insulin. The data in A are presented as mean; error bars represent the standard error. Box plots show the mean. (Red *) P ≤ 0.01, (black *) P < 0.05.