Swi-independent 3a and 3b (Sin3a and Sin3b) are paralogous transcriptional coregulators that direct cellular differentiation, survival, and function. Here, we report that mouse Sin3a and Sin3b are coproduced in most pancreatic cells during embryogenesis but become much more enriched in endocrine cells in adults, implying continued essential roles in mature endocrine cell function. Mice with loss of Sin3a in endocrine progenitors were normal during early postnatal stages but gradually developed diabetes before weaning. These physiological defects were preceded by the compromised survival, insulin-vesicle packaging, insulin secretion, and nutrient-induced Ca2+ influx of Sin3a-deficient β-cells. RNA sequencing coupled with candidate chromatin immunoprecipitation assays revealed several genes that could be directly regulated by Sin3a in β-cells, which modulate Ca2+/ion transport, cell survival, vesicle/membrane trafficking, glucose metabolism, and stress responses. Finally, mice with loss of both Sin3a and Sin3b in multipotent embryonic pancreatic progenitors had significantly reduced islet cell mass at birth, caused by decreased endocrine progenitor production and increased β-cell death. These findings highlight the stage-specific requirements for the presumed “general” coregulators Sin3a and Sin3b in islet β-cells, with Sin3a being dispensable for differentiation but required for postnatal function and survival.

Islet β-cell differentiation starts during early embryogenesis when a subset of pancreatic multipotent progenitor cells (MPCs) activates the expression of transcription factor (TF) neurogenin 3 (Ngn3 or Neurog3) and subsequently dozens of other islet cell TFs (1). During perinatal growth, the differentiated β-cells proliferate to increase their overall mass while also fine-tuning gene expression profiles to become mature, functional cells (2). Besides TFs, several transcriptional coregulators, including the mixed-lineage leukemia 3 and 4 methyltransferases (3) and Swi/Snf complexes (4), are essential for the production and/or maturation of β-cells. The standard model is that specific combinations of coregulators and TFs organize the chromatin landscape to shape the transcriptomic profiles of progenitors for cell development and function (4).

Swi-independent 3 (Sin3) is a well-established coregulator conserved from yeast to human cells (5). It does not bind DNA but uses several highly conserved amphipathic α-helices to interact with a variety of TFs and other coregulators, including P53, Mad1, Myc, REST, and ESET (5,6). In addition, Sin3 contains binding sites for histone deacetylases, histone lysine methylases, and demethylases (79). Thus, Sin3 primarily acts as a scaffold protein to assemble chromatin-modifying complexes that regulate gene transcription, and its target gene selectivity is determined by the DNA-binding TFs that recruit Sin3 (79). Notably, although Sin3 was commonly known as a “corepressor,” several studies suggest that it can coactivate gene expression in some cellular contexts (10).

Sin3 has two paralogs in mammalian cells, Sin3a and Sin3b, with evidence showing overlapping yet distinct functions (5,10). In mice, Sin3a, but not Sin3b, is essential in cell survival and/or differentiation during embryogenesis. Nullizygous Sin3a−/− mouse embryos died shortly after implantation, whereas Sin3b-null mice were born with seemingly normal organs but died immediately after birth (5). Consequently, Sin3a was reported to be required for the development and/or survival of embryonic stem (ES) cells (11,12), muscle cells (13), male germ cells (14), lung progenitors (15), and some skin cells (16). In addition, gene expression and protein-DNA interaction studies showed that Sin3a could directly regulate molecules involved in cell proliferation, survival, metabolism, and stress responses (7,17,18), yet how Sin3a functions in postnatal organs has not been examined.

In pancreatic cells, Sin3a was detected in transcriptional complexes containing Myt, Mafa, and/or Foxo TFs, which are all required for β-cell function (3,19,20). In this study, we assessed the roles and mechanisms of Sin3, focusing on Sin3a, in embryonic development and postnatal function of mouse islet β-cells. We show that Sin3a is dispensable for islet cell differentiation but required for β-cell function and survival, that is, their postnatal “fitness.” In addition, although Sin3 activity, sufficiently provided by either Sin3a or Sin3b, is essential for endocrine specification from MPCs, it is not needed for the differentiation of endocrine progenitors into islet hormone-positive cells. Thus, our data reveal stage- and cell-type–specific roles of the Sin3 complex, with Sin3a being particularly important for postnatal β-cell fitness.

Mice

Mouse usage was supervised by the Vanderbilt University institutional animal care and use committee in compliance with Association for Assessment and Accreditation of Laboratory Animal Care regulations. Sin3aF, Sin3a, Sin3bF, and Sin3b mice were previously described (17,21). Pdx1Cre [Tg(Pdx1Cre)89.1Dam], Ai9 [Cg-Gt(ROSA)26Sortm9(CAG-tdTomato)Hze/J], and Neurog3-Cre [Tg(Neurog3-Cre)C1Able/J] mice were from The Jackson Laboratory. ICR (CD1) mice were from Charles River Laboratories. All analyzed mice had a mixed genetic background (∼25% CD1, ∼37.5% C57BL/6, and ∼37.5% 129/Sv, as estimated from crossing history). The day of vaginal plug appearance was counted as embryonic day 0.5 (E0.5), and the day of birth was counted as postnatal day 1 (P1). PCR-based genotyping used oligos listed in Supplementary Table 1.

Tissue Preparation, Immunofluorescence Detection, and Imaging

Tissue preparation and immunofluorescence (IF) analysis followed routine protocols using 10-μm-thick frozen sections (19). Primary antibodies (1:500 to 1:2,000 dilution) were as follows: rabbit anti-Sin3a (LS-C331555; Lifespan Biosciences), rabbit anti-Sin3b (ab101841; Abcam), rabbit anti-Mafa (NBP1-00121; Novus Biologicals), rabbit anti-Mafb (IHC-00351; Bethyl Laboratories), rabbit anti-Nkx6.1 (gift from Palle Serup, University of Copenhagen, Copenhagen, Denmark), goat anti-Pdx1 (ab47303 [Abcam] or 06-13850 [Millipore]) (22), goat anti-Neurog3 (this laboratory, available upon request) (23), rabbit anti-Glut2 (GT21-A; Alpha Diagnostic), rabbit anti-Ucn3 (ab79121; Abcam), guinea pig anti-insulin (A0564; Dako), rabbit anti-glucagon (ab92517; Abcam), goat anti-somatostatin (sc-7819; Santa Cruz Biotechnology), rabbit anti–cleaved caspase-3 (#9661; Cell Signaling Technology), and rabbit anti-Ki67 (ab15580; Abcam). Secondary antibodies (1:500) used included the following: Alexa Fluor 488 donkey anti-guinea pig (706-545-148), Cy3 donkey anti-rabbit (711-165-152), Cy3 donkey anti-goat (705-165-147), Alexa Fluor 647 donkey anti-guinea pig (706-605-148), Alexa Fluor 647 donkey anti-rabbit (711-605-152), and Alexa Fluor 647 donkey anti-goat (705-605-147), all from Jackson ImmunoResearch.

Images were taken using a Nikon spinning disk confocal microscope and quantified with ImageJ 1.51j 14 software (National Institutes of Health [NIH]) under double-blind settings using 16-bit images. The presented results are the average intensity per pixel within the region of interest. The region of interest was manually selected to encircle the plasma membrane (for Glut2), cytoplasm (Ucn3), or nuclei (Mafa) of β-cells. We then normalized the relative levels of control cells to 1.0 for comparison. For each sample, 12–15 islet sections were examined. Transmission electron microscopy (TEM) and quantification followed published protocols (19).

Islet Cell Mass Measurement

One of every 12 sections (∼120 μm apart) of entire P1 and P7 pancreata and 1 of every 18 sections (∼180 μm apart) of P14 pancreata were scored. The sections were costained with DAPI and hormone antibodies to identify all cells and islet cells, respectively. Tissue sections were scanned/analyzed using Aperio ImageScope software (Leica Biosystems). The islet cell mass was calculated as follows: islet cell mass (mg) = (hormone + area / DAPI-positive area) × pancreas weight (mg).

Blood Glucose/Insulin Secretion Assays, Insulin Tolerance Test, and Intraperitoneal Glucose Tolerance Test

Blood glucose was measured through tail snipping. Plasma insulin/glucagon assays used blood from the retro-orbital collection. Islet isolation and secretion assays followed published protocol (19). Handpicked islets, after overnight recovery in RPMI medium (10% v/v FBS and 11.0 mmol/L glucose), were incubated in Krebs-Ringer buffer (KRB), containing 2.8 mmol/L glucose, for 1 h, with 2.8 mmol/L glucose for 45 min, then in KRB with 20 mmol/L glucose for 45 min, and lastly in KRB with 25 mmol/L KCl and 2.8 mmol/L glucose for 45 min. Insulin was measured using an ELISA kit from ALPCO and glucagon with a kit from Mercodia (Uppsala, Sweden) in the Vanderbilt Hormone Assay and Analytical Services Core. Insulin tolerance test and intraperitoneal glucose tolerance test followed that in Huang et al. (19).

Ca2+ Imaging

Ca2+ imaging followed published protocols (24). P5 islets were attached to poly-lysine–coated dishes overnight in RPMI medium (10% FBS and 11.0 mmol/L glucose). Islets were incubated in RPMI medium/10% FBS/2.8 mmol/L glucose basal media with 2.0 mmol/L Fura-2 AM (Invitrogen) for 20 min, followed by 20 min of washing in basal media. Ca2+ imaging was performed in recording solution (119 mmol/L NaCl, 2.5 mmol/L CaCl2, 4.7 mmol/L KCl, 10 mmol/L HEPES, 1.2 mmol/L MgSO4, and 1.2 mmol/L KH2PO4). The islets were perfused at 2.0 mL/min at 37°C with 2.8 mmol/L glucose (0–195 s), 20 mmol/L glucose (196–915 s), 2.8 mmol/L glucose (916–1,800 s), and 25 mmol/L KCl with 2.8 mmol/L glucose (1,801–2,090 s). Fluorescence images, excited at 340 and 380 nm (F340/F380), were taken/measured every 5 s with a Nikon Eclipse Ti2 microscope and Photometrics Prime 95B 25mm sCMOS camera.

Quantitative RT-PCR and Single-Cell RNA Sequencing

Quantitative RT-PCR (qRT-PCR) followed routine procedures using Universal SYBR Green Supermix (Bio-Rad) on a Bio-Rad CFX96 thermal cycler. Transcript abundance was normalized to Gapdh. Oligos are listed in Supplementary Table 1.

For single-cell RNA sequencing (scRNA-seq), two batches of P4 Sin3aF/+ and Sin3aΔendo islets (two to four mice of each genotype used for each batch) were isolated and dissociated into single cells using trypsin-EDTA. After live cell selection, the inDrop platform (1CellBio) was used to encapsulate and barcode single cells used for CEL-Seq–based library preparation and sequencing (NextSeq 500; Illumina). Reads were assigned to individual cells. After adaptor sequences were trimmed and cell doublets corrected, batch alignment assigned the reads to specific gene loci or unique molecular identifiers. We next identified β-cells based on insulin 1 and insulin 2 gene expression, which were then compared with the Mouse Cell Atlas to verify their cell identity (25). Afterward, the number of reads of each unique molecular identifier within each sample were combined and analyzed as one bulk sample using DESeq2. Log(fold change [FC]) was calculated as log(2){[(level in mutant) − (level in control)] / (level in control)}. Statistical analyses used the Wilcoxon rank sum test, and the adjusted P values were derived using the Bonferroni posttest correction on the basis of the total number of genes in the data set. Gene set enrichment assays (GSEAs) followed that in Subramanian et al. (26). Gene ontology–based Database for Annotation, Visualization, and Integrated Discovery analysis of potential Sin3a-binding genes followed that in Huang et al. (27).

Chromatin Immunoprecipitation Assays

A Magna ChIP-HiSens Chromatin Immunoprecipitation Kit (MilliporeSigma) was used. MIN6 cells (passage #38–43) were fixed for 20 min using 2.0 mmol/L disuccinimidyl glutarate followed by an additional 12 min with 1% formaldehyde. Sheared chromatin (200–500 bp) was prepared using a Bioruptor Pico (Diagenode) (∼30 cycles of 30 s sonication + 30 s rest). For each immunoprecipitation, 2.0 μg of chromatin and 2.0 μg of antibodies were used. The normal rabbit IgG was from Cell Signaling Technology (Cat# 2729). The rabbit anti-Sin3a (LS-C331555) was from Lifespan Biosciences.

Statistics for Non-scRNA-Seq Data

SPSS version 25 statistical software (IBM Corporation) was used. Data are shown as mean ± SD. Student t test or one-way repeated-measures ANOVA was used to compare two groups or more than two groups of data, respectively. P < 0.05 was considered significant.

Data and Resource Availability

The data sets generated and/or analyzed during the current study are deposited in Gene Expression Omnibus (GEO) under GSE146474 and are freely available for all. The data are also available from the corresponding author upon reasonable request.

Pan-Pancreatic Sin3a/Sin3b Expression During Embryogenesis Becomes Enriched in Adult Endocrine Cells

At E10.5, E15.5, and P1, both Sin3a and Sin3b proteins were detected in most pancreatic cells (Fig. 1A–C), including Neurog3+ endocrine progenitors (Fig. 1B) and hormone-positive islet cells (Fig. 1C). After P1, their expression further increased, and they became highly enriched in islet cells (Fig. 1D and E). However, both Sin3a and Sin3b remained detectable in some nonislet cells in the 3-month-old pancreas (Fig. 1E).

Figure 1

Sin3a and Sin3b are produced in pancreatic progenitors and islet cells. DAPI was used to mark nuclei in some panels. A: IF of Sin3a or Sin3b, costained with Pdx1 at E10.5. For Sin3a IF, both dorsal pancreatic bud (DB) and ventral pancreatic bud (VB) are shown. For Sin3b, only DB is shown. Arrowheads indicate Pdx1+ cells that also coexpress Sin3a or Sin3b. B: Costaining of Sin3a/Sin3b, Neurog3, and Pdx1 at E15.5. Arrowheads indicate Neurog3+ cells that also express Sin3a or Sin3b. CE: IF of Sin3a, Sin3b, insulin, and glucagon in P1, P21, and P90 pancreata. Arrows in E point to several nonislet cells that express Sin3a or Sin3b. Scale bars = 20 μm.

Figure 1

Sin3a and Sin3b are produced in pancreatic progenitors and islet cells. DAPI was used to mark nuclei in some panels. A: IF of Sin3a or Sin3b, costained with Pdx1 at E10.5. For Sin3a IF, both dorsal pancreatic bud (DB) and ventral pancreatic bud (VB) are shown. For Sin3b, only DB is shown. Arrowheads indicate Pdx1+ cells that also coexpress Sin3a or Sin3b. B: Costaining of Sin3a/Sin3b, Neurog3, and Pdx1 at E15.5. Arrowheads indicate Neurog3+ cells that also express Sin3a or Sin3b. CE: IF of Sin3a, Sin3b, insulin, and glucagon in P1, P21, and P90 pancreata. Arrows in E point to several nonislet cells that express Sin3a or Sin3b. Scale bars = 20 μm.

Loss of Sin3a Causes Late-Onset Diabetes

Sin3aF/−; Neurog3-Cre mice (termed Sin3aΔendo) were derived to inactivate Sin3a in the endocrine progenitors and some neuronal cells (28). IF and qRT-PCR assays supported the efficient deletion of Sin3a in Sin3aΔendo islets (Fig. 2A and B). We examined male and female Sin3aF/−, Sin3aF/+, and Sin3aF/+; Neurog3-Cre mice. No significant differences were observed in body weight, ad lib–fed blood glucose, islet structure, or expression of several diagnostic endocrine markers (e.g., Mafa, Pdx1, Nkx6.1, hormones), except that Sin3aF/− female mice had slightly lower body weights compared with Sin3aF/+ females at 2 weeks of age (Supplementary Fig. 1A–F). Thus, we used Sin3aF/− mice as controls for most studies unless noted.

Figure 2

Loss of Sin3a causes late-onset diabetes. A: IF detection of Sin3a in P1 and P7 control and Sin3aΔendo pancreata, highlighting insulin-positive β-cells. DAPI counterstaining marked nuclei. Scale bars = 20 μm. B: RT-PCR detection of Sin3a transcripts in P7 control and Sin3aΔendo islets, amplifying the floxed exon that should be deleted. Note that the islet samples include mesenchymal and endothelial cells as well, which also express Sin3a. CI: Phenotyping of control and Sin3aΔendo mice, including body weight (n = 7–10) (C), random-fed (n = 7–10) (D), or fasting (n = 5–14) (E) blood glucose, glucose tolerance (n = 7–8) (F), insulin tolerance (n = 5–6) (G), and plasma insulin (n = 4–5) (H), or glucagon (n = 4) (I). *P < 0.05, **P < 0.01, ***P < 0.001. IPGTT, intraperitoneal glucose tolerance test; ITT, insulin tolerance test; wk, week.

Figure 2

Loss of Sin3a causes late-onset diabetes. A: IF detection of Sin3a in P1 and P7 control and Sin3aΔendo pancreata, highlighting insulin-positive β-cells. DAPI counterstaining marked nuclei. Scale bars = 20 μm. B: RT-PCR detection of Sin3a transcripts in P7 control and Sin3aΔendo islets, amplifying the floxed exon that should be deleted. Note that the islet samples include mesenchymal and endothelial cells as well, which also express Sin3a. CI: Phenotyping of control and Sin3aΔendo mice, including body weight (n = 7–10) (C), random-fed (n = 7–10) (D), or fasting (n = 5–14) (E) blood glucose, glucose tolerance (n = 7–8) (F), insulin tolerance (n = 5–6) (G), and plasma insulin (n = 4–5) (H), or glucagon (n = 4) (I). *P < 0.05, **P < 0.01, ***P < 0.001. IPGTT, intraperitoneal glucose tolerance test; ITT, insulin tolerance test; wk, week.

Up to P14, both male and female Sin3aΔendo mice had body weights indistinguishable from control littermates but showed significant growth retardation afterward (Fig. 2C). The Sin3aΔendo mice showed normal glycemia at P7 but significantly higher blood glucose levels afterward (Fig. 2D). The temporal phenotypic development is similar in both male and female mice (Fig. 2C and D). We therefore used both sexes interchangeably and presented our findings together for the rest of the studies unless noted.

Sin3aΔendo mice had significantly higher fasting glucose levels on P28 but not on P14 (Fig. 2E). Compromised glucose tolerance was obvious in P14 Sin3aΔendo mice (Fig. 2F), which had normal insulin sensitivity on P14 but increased sensitivity on P21 (Fig. 2G). P7 and P28 Sin3aΔendo mice showed reduced plasma insulin (Fig. 2H), and plasma glucagon levels in P7 Sin3aΔendo mice were reduced (Fig. 2I). These combined findings suggest that reduced circulating insulin, but not increased glucagon or compromised insulin response, causes the hyperglycemic phenotype of Sin3aΔendo mice. Thus, our following studies focused on β-cells.

Sin3a Is Required for Postnatal β-Cell Function and Survival

We assayed insulin secretion of P7 Sin3aΔendo islets, the oldest stage when intact mutant islets could be readily isolated. These islets secreted more insulin under 2.8 mmol/L glucose (Fig. 3A) but less under 20 mmol/L glucose (Fig. 3B) or 25 mmol/L KCl (Fig. 3C). Sin3aΔendo β-cells had compromised Ca2+ influx induced by glucose or KCl, with higher levels of basal but a lower stimulated Ca2+ influx (Fig. 3D–F). In addition, Sin3aΔendo β-cells had more vesicles per unit of cytoplasmic area and a decreased size of the insulin dense core within each vesicle and more vesicle-like membrane structures that lacked detectable insulin crystals (Fig. 3G–I).

Figure 3

Sin3a promotes insulin secretion and granule biosynthesis. AC: Insulin secretion in P7 Sin3aF/− control and Sin3aΔendo islets (n = 6), presented as % of insulin secretion within a 45-min window. DF: Quantification of Ca2+ influx from P5 control and Sin3aΔendo islets, with representative Ca2+ recording (D), the overall Ca2+ influx at 2.8 mmol/L glucose (area under the curve 0–195 s) (E) and the highest Ca2+ influx amplitude at 20 mmol/L glucose and 25 mmol/L KCl stimulation (n = 3 mice) (F). G: TEM images of P7 β-cells. Arrows indicate normal insulin vesicles; arrowheads indicate empty vesicles. H and I: Vesicular quantification of P7 control and mutant β-cells (n = 40 images from two batches of mice), including vesicle density (H) and size (I). Scale bars = 500 nm. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 3

Sin3a promotes insulin secretion and granule biosynthesis. AC: Insulin secretion in P7 Sin3aF/− control and Sin3aΔendo islets (n = 6), presented as % of insulin secretion within a 45-min window. DF: Quantification of Ca2+ influx from P5 control and Sin3aΔendo islets, with representative Ca2+ recording (D), the overall Ca2+ influx at 2.8 mmol/L glucose (area under the curve 0–195 s) (E) and the highest Ca2+ influx amplitude at 20 mmol/L glucose and 25 mmol/L KCl stimulation (n = 3 mice) (F). G: TEM images of P7 β-cells. Arrows indicate normal insulin vesicles; arrowheads indicate empty vesicles. H and I: Vesicular quantification of P7 control and mutant β-cells (n = 40 images from two batches of mice), including vesicle density (H) and size (I). Scale bars = 500 nm. *P < 0.05, **P < 0.01, ***P < 0.001.

P7 and P14, but not P1, Sin3aΔendo mice had significantly reduced β-cell mass (Fig. 4A and B) accompanied by increased apoptosis starting from P1 (Fig. 4C and D) but no change of proliferation (Fig. 4E and F). In addition, Sin3aΔendo mice had reduced α-cell mass but no change in δ-cell mass in P14 (Supplementary Fig. 2). These combined findings suggest that Sin3a is not required for β-cell differentiation (i.e., producing insulin-positive cells) during embryogenesis but is required for their fitness after birth. This conclusion led us to examine the detailed molecular mechanisms on how Sin3a regulates this fitness.

Figure 4

Sin3a is required for postnatal β-cell survival. A and B: Quantification of β-cell mass using IF assays (n = 3–5). Scale bar = 200 μm. CF: Co-IF staining of insulin with cleaved caspase-3 (cCasp3) or Ki67 and the quantification (n = 3). Arrows in E indicate cCasp3+ (C) or Ki67+ β-cells. Scale bars = 20 μm. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 4

Sin3a is required for postnatal β-cell survival. A and B: Quantification of β-cell mass using IF assays (n = 3–5). Scale bar = 200 μm. CF: Co-IF staining of insulin with cleaved caspase-3 (cCasp3) or Ki67 and the quantification (n = 3). Arrows in E indicate cCasp3+ (C) or Ki67+ β-cells. Scale bars = 20 μm. *P < 0.05, **P < 0.01, ***P < 0.001.

Sin3a Regulates the Expression of Maturation and Function Genes in Postnatal β-Cells

We used a candidate approach and examined the expression of several TFs (Mafa, Mafb, Pdx1, and Nkx6.1) that are important for β-cell differentiation and maturation markers (Glut2 and Ucn3) that are important for function. Pdx1, Nkx6.1, or Mafb levels showed no difference in Sin3aΔendo β-cells compared with controls (Supplementary Fig. 3), but Glut2, Ucn3, and Mafa expression was progressively reduced. Specifically, Ucn3 expression was significantly downregulated by P4, while Glut2 and Mafa expression was downregulated by P7 at both protein and mRNA levels (Fig. 5 and Supplementary Fig. 4). However, because the glycemic defects of Ucn3- or Mafa-mutant mice developed much later than the Sin3aΔendo animals (29,30) and the increased β-cell death was observed before the downregulation of Ucn3, Mafa, and Slc2a2, these three genes are unlikely the major mediators of Sin3a function. Thus, we comprehensively defined the Sin3a-regulated genes in β-cells by RNA-seq approaches.

Figure 5

Postnatal Sin3a-deficient β-cells lack the expression of several functional genes. AE: Glut2 staining and quantification in islets of different ages with or without Sin3a inactivation. FJ and KO: The same as above expect Ucn3 and Mafa protein levels were measured, respectively (n = 3 mice for all assays). Scale bars = 20 μm. Broken lines encircle the islet areas, highlighting the signal intensity. Refer to the insulin IF in Supplementary Fig. 4 to see how β-cells were located. Inset in N shows an example of corresponding insulin signals. P and Q: Relative expression of Slc2a2 (encoding Glut2), Ucn3, and Mafa in P4 and P7 islets, assayed using qRT-PCR. The gene expression levels were normalized to Gapdh (n = 3 mice). *P < 0.05, **P < 0.01, ***P < 0.001. A.U., arbitrary unit.

Figure 5

Postnatal Sin3a-deficient β-cells lack the expression of several functional genes. AE: Glut2 staining and quantification in islets of different ages with or without Sin3a inactivation. FJ and KO: The same as above expect Ucn3 and Mafa protein levels were measured, respectively (n = 3 mice for all assays). Scale bars = 20 μm. Broken lines encircle the islet areas, highlighting the signal intensity. Refer to the insulin IF in Supplementary Fig. 4 to see how β-cells were located. Inset in N shows an example of corresponding insulin signals. P and Q: Relative expression of Slc2a2 (encoding Glut2), Ucn3, and Mafa in P4 and P7 islets, assayed using qRT-PCR. The gene expression levels were normalized to Gapdh (n = 3 mice). *P < 0.05, **P < 0.01, ***P < 0.001. A.U., arbitrary unit.

Sin3a Regulates Genes Involved in Ion Transport, Cell Death, Vesicular Production/Secretion, Glucose Metabolism, and Stress Response

scRNA-seq was used to examine gene expression of P4 control and Sin3aΔendo islets, allowing us to distinguish β-cells from other islet cell types. P4 Sin3aΔendo mice had no recognizable physiological defects, avoiding complications imposed by hyperglycemia, but had increased β-cell death, underscoring the existing molecular defects in the P4 Sin3aΔendo β-cells.

Two highly reiterative scRNA-seq data sets (Fig. 6A) were obtained, which showed clear islet cell type separations (Supplementary Fig. 5). Differences in expression were seen between control and Sin3aΔendo β-cells, with both downregulated and upregulated genes (e.g., Ucn3 that recapitulates qRT-PCR results, Hspe1 that represents newly identified Sin3a-regulated genes, respectively) (Fig. 6B).

Figure 6

scRNA-seq identifies Sin3a-dependent genes and pathways in islet β-cells. A: t-Distributed stochastic neighbor embedding (tSNE)–aided visualization of α-cell (green circles) and β-cell (red circles) clusters in two duplicate experiments, with both control and Sin3aΔendo-mutant cells. Note that cells from each experiment are represented by dots of different colors (black and blue for control samples, red and yellow for mutant cells). B: The expression of Ins1, Ucn3, and Hspe1 in β-cells on the tSNE map. Note that in this panel, the color of dots indicates the relative gene expression level, with white indicating the highest expression level and black the lowest expression level. C: GSEA results of all differentially expressed genes in control and Sin3aΔendo β-cells. Pathways with false discovery rate ≤0.05 are shown. D: Gene ontology clustering of differentially expressed genes that were also reported to be direct Sin3a targets in several cell types. Only the top 15 upregulated terms (enrichment score >4.8) and top 5 downregulated terms (enrichment score >2.4) are listed.

Figure 6

scRNA-seq identifies Sin3a-dependent genes and pathways in islet β-cells. A: t-Distributed stochastic neighbor embedding (tSNE)–aided visualization of α-cell (green circles) and β-cell (red circles) clusters in two duplicate experiments, with both control and Sin3aΔendo-mutant cells. Note that cells from each experiment are represented by dots of different colors (black and blue for control samples, red and yellow for mutant cells). B: The expression of Ins1, Ucn3, and Hspe1 in β-cells on the tSNE map. Note that in this panel, the color of dots indicates the relative gene expression level, with white indicating the highest expression level and black the lowest expression level. C: GSEA results of all differentially expressed genes in control and Sin3aΔendo β-cells. Pathways with false discovery rate ≤0.05 are shown. D: Gene ontology clustering of differentially expressed genes that were also reported to be direct Sin3a targets in several cell types. Only the top 15 upregulated terms (enrichment score >4.8) and top 5 downregulated terms (enrichment score >2.4) are listed.

To minimize the saturation and scarcity issues inherent to scRNA-seq (31), we analyzed the scRNA-seq results by combining identical cell types into bulk expression data within each sample (32). This revealed 772 downregulated and 3,668 upregulated genes in Sin3aΔendo β-cells, with adjusted P < 0.05 and at least a onefold increase or difference in expression (i.e., the expression level in one sample is at least twice as much as the other; see the table “Sin3 candidate gene list.xlsm” in GEO [GSE146474]). We observed the changed transcript level of Ucn3 but not Mafa, Nkx6.1, or Pdx1, consistent with IF (Supplementary Fig. 3) and qRT-PCR results (Fig. 5P). GSEA of the scRNA-seq data set revealed several altered molecular pathways (Fig. 6C). The terms include protein/membrane transport/endoplasmic reticulum function, tricarboxylic acid cycle/mitochondrial activities, and oxidative stress responses.

Sin3a Is Enriched in 5′ Regulatory Regions of Several β-Cell Function/Survival Genes

Sin3a was reported to associate with several widely expressed DNA-binding proteins, including P53, Myc, Mad1, and Foxo1 (20,33). Sin3a was also detected in a Mafa-containing transcriptional complex, highly expressed in islet β-cells (3). We postulated that Sin3a shares common target genes among different cell types. Bioinformatics analyses were used to identify putative Sin3 target genes in β-cells by comparing the list of Sin3a-dependent genes with published Sin3a-based chromatin immunoprecipitation sequencing (ChIP-seq) results in mouse ES cells (34), epiblast stem cells (35), and muscle cells (13). We also included the Mafa-bound enhancers in mouse islets, postulating that the Sin3a associates with these sites through Mafa (35,36). This analysis revealed 2,847 gene loci having at least one reported Sin3a/Mafa-binding site, with 335 being downregulated and 2,512 being upregulated in Sin3aΔendo β-cells (see the table “Sin3 candidate gene list.xlsm” in GEO [GSE146474]). Gene ontology–based analyses (Database for Annotation, Visualization, and Integrated Discovery) revealed that these Sin3a-associated genes likely regulate processes in mitochondrion, endoplasmic reticulum/protein transport, ribosome, cell redox homeostasis, and synapses (Fig. 6C and D), similar to pathways revealed by GSEA analysis of all differentially expressed genes.

ChIP-PCR was used to verify whether some of these predicted genes are direct targets of Sin3a in MIN6 β-cells. We prioritized several genes that have established molecular functions that correlated with the cellular defects of Sin3aΔendo β-cells (Table 1). These include downregulated Ins1 and Kcnh2 required for insulin production and cell membrane polarization (37). They also include upregulated genes Esyt1 and Calr (related to defective Ca2+ homeostasis) (38,39); Bnip3, Casp3, and Ing1 (cell death) (40,41); Arl6, Cltb, Ergic3, and Rab11a (lipid transport and vesicular biosynthesis) (4245); Aldoa and Idh3a (glucose metabolism); and Hsp90b1 and Hspe1 (stress responses) (46). Mafa was also included because Sin3a enrichment was detected on its putative regulatory elements (Table1). Ucn3, Slc2a2, Gcg, Gapdh, and albumin were included as controls because Sin3a was not reported to directly regulate these genes (Fig. 6E and Supplementary Fig. 6).

Table 1

Several candidate genes studied by ChIP-PCR and qRT-PCR assays

Sin3a bindingMafa site
Function categoryGeneLogFCPercentage of mutantPercentage of controlAdjusted P valueEpiblast (35)ES Ab1* (34)ES Ab2* (34)Muscle (13)Islets (36)
Hormone Ins1 −0.838 0.998 1.00 4.20E-120     Yes 
Function/maturation Slc2a2 −0.002 0.911 0.843 1.00      
 Mafa −0.007 0.002 0.005 1.00 Yes Yes  Yes  
 Ucn3 −1.041 0.373 0.715 1.65E-106      
 Pdx1 −0.143 0.531 0.544 1.00      
Channel Kcnh2 −0.728 0.394 0.541 8.89E-40 Yes Yes Yes   
Ca2+ homeostasis Esyt1 0.588 0.764 0.355 4.10E-133  Yes Yes   
Calr 0.344 0.985 0.923 3.09E-70  Yes Yes   
Cell death Bnip3 0.572 0.777 0.363 1.32E-113  Yes    
 Casp3 0.617 0.723 0.277 1.89E-131  Yes    
 Ing1 0.683 0.770 0.332 3.65E-140  Yes  Yes  
Lipid transport Arl1 0.324 0.397 0.125 2.32E-57 Yes Yes Yes   
 Cltb 0.346 0.854 0.608 3.02E-55  Yes Yes   
 Ergic3 0.335 0.760 0.491 8.56E-48  Yes Yes Yes  
 Rab11a 0.324 0.790 0.522 7.11E-49  Yes Yes   
Metabolism Aldoa 0.609 0.757 0.368 5.93E-123 Yes Yes Yes   
 Idh3a 0.542 0.794 0.318 1.59E-105  Yes Yes Yes  
Stress response Hsp90b1 0.596 0.982 0.883 9.66E-171   Yes Yes  
 Hspe1 0.709 0.885 0.552 3.48E-151  Yes Yes   
Paralog Sin3b 0.198 0.437 0.225 2.85E-33      
Sin3a bindingMafa site
Function categoryGeneLogFCPercentage of mutantPercentage of controlAdjusted P valueEpiblast (35)ES Ab1* (34)ES Ab2* (34)Muscle (13)Islets (36)
Hormone Ins1 −0.838 0.998 1.00 4.20E-120     Yes 
Function/maturation Slc2a2 −0.002 0.911 0.843 1.00      
 Mafa −0.007 0.002 0.005 1.00 Yes Yes  Yes  
 Ucn3 −1.041 0.373 0.715 1.65E-106      
 Pdx1 −0.143 0.531 0.544 1.00      
Channel Kcnh2 −0.728 0.394 0.541 8.89E-40 Yes Yes Yes   
Ca2+ homeostasis Esyt1 0.588 0.764 0.355 4.10E-133  Yes Yes   
Calr 0.344 0.985 0.923 3.09E-70  Yes Yes   
Cell death Bnip3 0.572 0.777 0.363 1.32E-113  Yes    
 Casp3 0.617 0.723 0.277 1.89E-131  Yes    
 Ing1 0.683 0.770 0.332 3.65E-140  Yes  Yes  
Lipid transport Arl1 0.324 0.397 0.125 2.32E-57 Yes Yes Yes   
 Cltb 0.346 0.854 0.608 3.02E-55  Yes Yes   
 Ergic3 0.335 0.760 0.491 8.56E-48  Yes Yes Yes  
 Rab11a 0.324 0.790 0.522 7.11E-49  Yes Yes   
Metabolism Aldoa 0.609 0.757 0.368 5.93E-123 Yes Yes Yes   
 Idh3a 0.542 0.794 0.318 1.59E-105  Yes Yes Yes  
Stress response Hsp90b1 0.596 0.982 0.883 9.66E-171   Yes Yes  
 Hspe1 0.709 0.885 0.552 3.48E-151  Yes Yes   
Paralog Sin3b 0.198 0.437 0.225 2.85E-33      

Shown are the reported function, logFC of mutant over control β-cells, percentage of mutant and control β-cells that expressed each gene, and adjusted P values. Also indicated are whether Sin3a or Mafa was enriched in putative cis-RRs of each gene, from published data. Ab, antibody.

*The data were from a single publication, with two Abs used for ChIP-seq.

For ChIP-PCR, we selected several putative regulatory regions (RRs) in the 5′ region of each gene with putative Myc/P53-binding sites (47) (Supplementary Table 1). We found significantly enriched Sin3a occupancy in the 5′ RRs of Kcnh2, Esyt1, Bnip3, Casp3, Ing1, Cltb, Rab11a, Aldoa, Hsp90b1, and Hspe1 (Fig. 7A). Corresponding to these results, qRT-PCR assays detected significantly decreased transcription of Kcnh2 but increased expression of Bnip3, Casp3, Ing1, Rab11a, Aldoa, and Hspe1 in P4 Sin3aΔendo islets, matching the scRNA-seq results (Fig. 7B and Table 1). We did not detect Sin3a enrichment in the tested regions of Ins1, Gcg, Calr, Arl6, Ergic3, and Ucn3 genes (Supplementary Fig. 6). Intriguingly, we also found enriched Sin3a binding to the 5′ regions of Slc2a2 and Mafa, although their expression levels remain unchanged in P4 Sin3aΔendo β-cells (Table 1). It is not clear whether Sin3a directly regulates these two genes in older β-cells.

Figure 7

ChIP-PCR reveals several potential Sin3a target genes in MIN6 β-cells. A: ChIP-PCR assays of Sin3a-associated DNA RRs of several genes. Percentage of chromatin recovery is shown (n = 4 batches of chromatin preparations). Results of two RRs for each gene are presented. Normal IgG was used as control. The selected genes were grouped according to their reported functions (see text). B: qRT-PCR assays of gene transcription in P4 islets (n = 5 batches of islets, including 1–3 individual mice for each batch). The results were normalized against that of Gapdh. We then artificially set the relative expression level in control islets to 1.0 for comparison.

Figure 7

ChIP-PCR reveals several potential Sin3a target genes in MIN6 β-cells. A: ChIP-PCR assays of Sin3a-associated DNA RRs of several genes. Percentage of chromatin recovery is shown (n = 4 batches of chromatin preparations). Results of two RRs for each gene are presented. Normal IgG was used as control. The selected genes were grouped according to their reported functions (see text). B: qRT-PCR assays of gene transcription in P4 islets (n = 5 batches of islets, including 1–3 individual mice for each batch). The results were normalized against that of Gapdh. We then artificially set the relative expression level in control islets to 1.0 for comparison.

Sin3a and Sin3b Are Required for Endocrine Cell Specification

An intriguing finding is the upregulation of Sin3b in the absence of Sin3a (Table 1). We therefore tested whether inactivating Sin3b could further exacerbate the defects in Sin3aΔendo β-cells. Inactivating Sin3b alone in Sin3bF/−; Neurog3-Cre mice produced no detectable phenotypes (data not shown), yet Sin3aF/−; Sin3bF/−; Neurog3-Cre (Sin3a/3bΔendo) mice became diabetic before P5, at least 1 week earlier than Sin3aΔendo mice (Supplementary Fig. 7A), accompanied by largely reduced β-cell production (Supplementary Fig. 7B). These findings support the redundant function of Sin3a and Sin3b for the production/survival of islet β-cells.

Finally, we tested the roles of Sin3a and Sin3b in the pancreatic MPCs. Sin3aF/F; Sin3bF/F; Pdx1Cre (yielding Sin3a/3bΔPanc mice), and Sin3aF/F; Sin3bF/F; Pdx1Cre; Ai9 mice were derived. The Ai9 Cre-reporter allele in the latter marked Sin3a/Sin3b-deficient pancreatic cells with tdTomato (23). At E15.5 and P1, there was substantially reduced production of β- and α-cells in Sin3a/3bΔPanc pancreata (Supplementary Fig. 7C–E), accompanied by reduced Neurog3+ cell production (E15.5) (Supplementary Fig. 7F) and increased β-cell death (P1) (Supplementary Fig. 7G). Notably, the majority of pancreatic cells in Sin3aF/F; Sin3bF/F; Pdx1Cre; Ai9 mice expressed tdTomato, suggesting that Sin3a/3b-deficient pancreatic cells do not die immediately after Sin3 inactivation (Supplementary Fig. 7H).

The mammalian Sin3a and Sin3b paralogs are scaffold proteins for the overall Sin3 coregulator complex that can associate with common TFs, such as Foxo, Mad1, Myc, and P53, and β-cell TFs Mafa and Myt (3). These associations recruit histone deacetylases, histone lysine methylases, and demethylase to regulate gene transcription in a highly cell context–dependent manner (5). We report here that inactivating Sin3a singly in the early mouse pancreatic endocrine lineage has little effect on islet cell differentiation but substantially reduces postnatal β-cell fitness without affecting the survival of δ-cells. The Sin3a-deficient β-cells showed defective insulin secretion, cell survival, Ca2+ influx, and insulin vesicle biogenesis. We further showed that Sin3a associates with putative enhancers of several genes involved in ion transport/Ca2+ homeostasis, cell death, membrane trafficking, glucose metabolism, and stress response in β-cells. Intriguingly, inactivating both Sin3a and Sin3b in the same endocrine lineage resulted in further reduction of endocrine cell numbers by birth, while coinactivating both Sin3a and Sin3b in the early pancreatic MPCs substantially reduced production of endocrine progenitors without preventing their differentiation into hormone-positive islet cells.

Our discoveries highlight a few features of Sin3 function in pancreatic cells. First, although Sin3a but not Sin3b is required for producing postnatal β-cells with similar “fitness qualities,” both paralogs can prevent cell death. Thus, inactivating Sin3b expedited islet cell loss in Sin3a-null islets. It is possible that Sin3a and Sin3b regulate a similar set of molecular targets, yet Sin3a contributes a higher proportion of such activity because of a higher affinity for transcriptional effectors. Alternatively, Sin3a and Sin3b may regulate different sets of target genes that have similar functions. Future comparisons of transcriptomic alterations between Sin3a-null and Sin3b-null β-cells together with comprehensive ChIP-seq analyses should address these possibilities. In either case, it appears that the total Sin3 activity has to achieve a certain threshold to maintain normal β-cell fitness.

The second feature is that different islet cell types have different levels of Sin3a dependency, with α- and β-cells, but not δ-cells, requiring Sin3a for postnatal survival. It is possible that Sin3a regulates similar gene sets in all islet cell types but that the cellular context dictates resistance to cell death pathways. Alternatively, different islet cell types may use different Sin3a-TF complexes to regulate transcription. Follow-up identification of these complexes using proteomic studies could address this question.

Third, although Sin3 (either Sin3a or Sin3b) is required in MPCs to promote endocrine specification, their overall activities are dispensable for the differentiation of committed endocrine progenitors into hormone-expressing islet cells. Therefore, our collective findings, combined with Sin3a’s reported roles in the differentiation of several nonpancreatic cells (1315,17,48), highlight the idea that pancreatic cells use the Sin3-TF complexes differentially in a stage- and/or cell type–specific manner for differentiation, function, and survival.

The compromised β-cell fitness in Sin3a-null mice is consistent with Sin3a being a coregulator recruited by Mafa and Foxo1, both of which interact with Sin3a and are essential for β-cell fitness (20,49). However, Sin3a-null mice developed overt diabetes before weaning, while Mafa- and Foxo1-deficient mice only did so a significant period afterward. An implication is that Sin3a inactivation may be equivalent to coinactivation of Mafa and Foxo1. Alternatively, Sin3a may also mediate the function of additional TFs required for β-cell function and/or survival (e.g., Myc and P53), which are ubiquitous in most cell types. Our ChIP-PCR assays detected Sin3a enrichment in putative Myc/P53-binding enhancers of several genes in β-cells whose products regulate cell death, ion transport, lipid trafficking/vesicular biosynthesis, metabolism, and stress responses (Table 1). Thus, our collective findings are consistent with the idea that Myc/P53 could recruit Sin3a in β-cells to promote β-cell fitness by ensuring insulin vesicle biosynthesis, stimulus secretion coupling, and survival. Note that the complete list of key Sin3a/Sin3b targets in β-cells remains unknown. Future genome-wide ChIP-seq studies of β-cells using specific Sin3a and Sin3b antibodies, combined with transcriptomic analysis of the few Sin3a/3bΔEndo and/or Sin3a/3bΔPanc β-cells that are formed, may shed more light on how Sin3a and Sin3b regulate β-cells.

Sin3 has been functionally diagnosed as both a coactivator and a corepressor. Consistent with this notion, we found both downregulated and upregulated genes in Sin3a-deficient β-cells, with many being reported as directly regulated by Sin3a in other cell types. Our candidate ChIP-PCR assays corroborated enrichment of Sin3a in putative regulatory regions of Sin3-repressed (e.g., Hspe1, Casp3) and -activated genes (Kcnh2) in β-cells, underscoring the bidirectional regulatory roles of Sin3a.

In summary, our findings show that the Sin3 coregulator plays essential roles in islet-cell production and postnatal β-cell fitness, with Sin3a being the major contributor. Thus, modulating Sin3a levels or activities could be explored to protect β-cell fitness and to control diabetes initiation and progression.

This article contains supplementary material online at https://doi.org/10.2337/db20-4567/suppl.12042828.

Acknowledgments. The authors thank Dr. Roland Stein (Vanderbilt University) for useful discussions about the Sin3 activities, Drs. Chin Chiang and Lei Chen (Vanderbilt University) for help with ChIP-PCR, and William Gu (Montgomery Bell Academy, Nashville, TN) for text editing.

Funding. This study is supported by grants from the National Institute of Diabetes and Digestive and Kidney Diseases (DK-106228 to I.K. and G.G., DK-103831 and CA-095103 to K.S.L., and DK-065949 to G.G.) and JDRF (1-2009-371 to G.G.). Confocal and TEM imaging was performed with Vanderbilt University Cell Imaging Shared Resource (supported by National Institutes of Health [NIH] grants CA-68485, DK-20593, 500 DK-58404, DK-59637, and EY-08126). Vanderbilt University Medical Center Hormone Assay and Analytical Services Core was supported by NIH grants DK-059637 (Mouse Metabolic Phenotyping Center) and DK-020593 (Diabetes Research and Training Center) and the Islet Procurement & Analysis Core by DK-020593. DNA sequencing was done by Vanderbilt Technologies for Advanced Genomics, supported by the Vanderbilt Ingram Cancer Center (P30-CA-68485), the Vanderbilt Vision Center (P30-EY-08126), and NIH/National Center for Research Resources (G20-RR-030956).

Duality of Interest. No potential conflicts of interest relevant to this article were reported.

Author Contributions. X.Y., S.M.G., K.-H.H., D.A.J., K.S.L., and G.G. analyzed data. X.Y., S.M.G., A.J.S., and G.G. performed the experiments. X.Y., D.A.J., I.K., C.V.E.W., K.S.L., and G.G. designed the experiments. X.Y., K.S.L., and G.G. prepared figures. C.N.H., B.C., A.N.S-S., and K.S.L. performed the scRNA-seq analysis. G.D. provided the mice harboring Sin3a/Sin3b floxed alleles. All authors helped with data interpretation, participated in manuscript writing/proofreading, and the approved final version of the manuscript. G.G. is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Prior Presentation. Parts of this study were presented at the 11th Annual Midwest Islet Club, St. Louis, MO, 14-15 May 2018, and 12th Annual Midwest Islet Club, Ann Arbor, MI, 19–20 May 2019.

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