GLIS3 binds pancreatic beta cell regulatory regions alongside other islet transcription factors

in Journal of Endocrinology
Authors:
David W Scoville Cell Biology Group, Immunity, Inflammation and Disease Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina, USA

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Kristin Lichti-Kaiser Cell Biology Group, Immunity, Inflammation and Disease Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina, USA

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Sara A Grimm Integrative Bioinformatics Support Group, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina, USA

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Anton M Jetten Cell Biology Group, Immunity, Inflammation and Disease Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina, USA

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Correspondence should be addressed to A M Jetten: jetten@niehs.nih.gov
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The Krüppel-like zinc finger transcription factor Gli-similar 3 (GLIS3) plays a critical role in the regulation of pancreatic beta cells, with global Glis3-knockout mice suffering from severe hyperglycemia and dying by post-natal day 11. In addition, GLIS3 has been shown to directly regulate the early endocrine marker Ngn3, as well as Ins2 gene expression in mature beta cells. We hypothesize that GLIS3 regulates several other genes critical to beta cell function, in addition to Ins2, by directly binding to regulatory regions. We therefore generated a pancreas-specific Glis3 deletion mouse model (Glis3Δ panc ) using a Pdx1-driven Cre mouse line. Roughly 20% of these mice develop hyperglycemia by 8 weeks and lose most of their insulin expression. However, this did not appear to be due to loss of the beta cells themselves, as no change in cell death was observed. Indeed, presumptive beta cells appeared to persist as PDX1+/INS/MAFA/GLUT2 cells. Islet RNA-seq analysis combined with GLIS3 ChIP-seq analysis revealed apparent direct regulation of a variety of diabetes-related genes, including Slc2a2 and Mafa. GLIS3 binding near these genes coincided with binding for other islet-enriched transcription factors, indicating these are distinct regulatory hubs. Our data indicate that GLIS3 regulates not only insulin expression, but also several additional genes critical for beta cell function.

Abstract

The Krüppel-like zinc finger transcription factor Gli-similar 3 (GLIS3) plays a critical role in the regulation of pancreatic beta cells, with global Glis3-knockout mice suffering from severe hyperglycemia and dying by post-natal day 11. In addition, GLIS3 has been shown to directly regulate the early endocrine marker Ngn3, as well as Ins2 gene expression in mature beta cells. We hypothesize that GLIS3 regulates several other genes critical to beta cell function, in addition to Ins2, by directly binding to regulatory regions. We therefore generated a pancreas-specific Glis3 deletion mouse model (Glis3Δ panc ) using a Pdx1-driven Cre mouse line. Roughly 20% of these mice develop hyperglycemia by 8 weeks and lose most of their insulin expression. However, this did not appear to be due to loss of the beta cells themselves, as no change in cell death was observed. Indeed, presumptive beta cells appeared to persist as PDX1+/INS/MAFA/GLUT2 cells. Islet RNA-seq analysis combined with GLIS3 ChIP-seq analysis revealed apparent direct regulation of a variety of diabetes-related genes, including Slc2a2 and Mafa. GLIS3 binding near these genes coincided with binding for other islet-enriched transcription factors, indicating these are distinct regulatory hubs. Our data indicate that GLIS3 regulates not only insulin expression, but also several additional genes critical for beta cell function.

Introduction

GLIS3 belongs to the GLIS subfamily of Krüppel-like proteins that are most closely related to members of the GLI and ZIC families (Jetten 2018). It plays a critical role in the regulation of various physiological processes, including pancreas development, osteogenesis, kidney function and thyroid hormone regulation and is implicated in several major human pathologies (e.g., diabetes, hypothyroidism, cancer, cystic kidney disease) (Dimitri et al. 2011, Scoville et al. 2017, Wen & Yang 2017, Jetten 2018). GLIS3 contains five Krüppel-like zinc finger motifs that recognize specific DNA sequences in regulatory regions of target genes allowing it to function as a transcriptional activator and/or repressor (Kang et al. 2010, Lichti-Kaiser et al. 2012). Genetic aberrations in human GLIS3 are associated with a rare syndrome characterized by neonatal diabetes and congenital hypothyroidism (NDH). Depending on the nature of the mutation, additional features have included hepatic fibrosis, congenital glaucoma, polycystic kidney disease, facial dysmorphism and osteopenia (Senee et al. 2006, Dimitri et al. 2011, Habeb et al. 2012). Additional evidence for a role of GLIS3 in diabetes comes from genome-wide association studies (GWAS) that link GLIS3 variants to aberrant glucose regulation and reduced beta cell function and identified GLIS3 as a risk locus for both type-1 and type-2 diabetes (Barrett et al. 2009, Dupuis et al. 2010, Cho et al. 2011, Liu et al. 2011, Rees et al. 2011).

GLIS3 protein expression in the mouse pancreas is first observed at embryonic day 13.5 in bipotent pancreatic progenitor cells (i.e. cells capable of differentiating into ductal or endocrine cells; Zhou et al. 2007), and its expression is maintained in both lineages into adulthood (Kang et al. 2016). Within the mature endocrine population, GLIS3 is restricted to beta cells and pancreatic polypeptide cells (Kang et al. 2016). We and others have previously demonstrated that global Glis3-null mice die by post-natal day 11 (PND11) due to neonatal diabetes (Kang et al. 2009, Watanabe et al. 2009, Yang et al. 2009). Islet size and the expression of several beta cell markers, including Ins2, the glucose transporter Slc2a2, Mafa and Nkx6.1 were dramatically decreased in Glis3-null mice. Indeed, GLIS3 is critical for Ins2 expression in mature beta cells through its interaction with other known regulators of Ins2, such as PDX1, NEUROD1 and MAFA (Kang et al. 2009, Yang et al. 2009, Zeruth et al. 2013). However, the precise role of GLIS3 in the maintenance of functional beta cells, aside from regulating insulin expression, remains poorly defined.

To gain greater insights into the regulatory functions of GLIS3 in the pancreas, we generated a pancreas-specific Glis3-knockout mouse model. Glis3fl/fl mice were crossed with mice expressing Cre recombinase under the control of the Pdx1 gene promoter to generate a pancreas-specific knockout model (Glis3Δ panc ). We were thus able to separate the pancreatic phenotype from other organs affected by global Glis3 loss, such as the kidney and thyroid. The major objective of this study was to obtain a better understanding of GLIS3-mediated regulation of gene transcription in pancreatic islets; we therefore performed gene expression analysis on islets from these mice, as well as GLIS3 ChIP-seq analysis. In addition, we compared these results to publicly available data on the genomic binding of several other islet-enriched transcription factors, in order to determine whether GLIS3 regulates pancreatic beta cell gene transcription by binding to distinct regulatory hubs with other islet transcription factors (Hu & Tee 2017). Our study shows that GLIS3 plays a critical role in directly regulating a number of genes associated with beta cell identity, maturation and function and suggests that GLIS3 performs these regulatory functions in association with other islet-enriched transcription factors.

Materials and methods

Generation of pancreas-specific Glis3-knockout mice

A conditional knockout allele for the Glis3 gene was generated in C57BL/6J mice using homologous recombination in mouse embryonic stem cells and subsequent blastocyst injection of the appropriate targeted ES cells (C57BL/6-Glis3<tm2Amj>). The vector contained loxP sites flanking exon 5 of Glis3, which included part of the DNA-binding domain (Fig. 1A). Previous work has shown that deletion of any part of the DNA-binding domain produces a non-functional protein (Beak et al. 2008). Mice were bred with a strain expressing Cre recombinase under the control of the Pdx1 promoter, which produces pancreas-specific deletion by e10.5 (Jackson Laboratories Stock No. 014647) (Hingorani et al. 2003). Animal studies followed guidelines outlined by the NIH Guide for the Care and Use of Laboratory Animals, and protocols were approved by the Institutional Animal Care and Use Committee at the NIEHS.

Figure 1
Figure 1

Glis3Δ panc mice exhibit a loss of insulin-producing beta cells. (A) Schematic of WT and recombinant Glis3 allele. A conditional knockout allele for the Glis3 gene was generated in C57BL/6 mice containing loxP sites (triangles) surrounding exon 5 of Glis3. Exons 4–6 encode the DNA-binding domain (DBD). Mice were bred with a strain expressing Cre recombinase under the control of the Pdx1 promoter. (B) Random blood glucose levels of mice at 8 weeks of age. Glis3Δ panc mice were grouped as either euglycemic (<250 mg/dL) or hyperglycemic (>250 mg/dL) and compared to control littermates (WT) (n = 11 WT, n = 5 Glis3Δ panc euglycemic, n = 4 Glis3Δ panc hyperglycemic). Immunofluorescence staining revealed a reduction in the number of INS+PDX1+ beta cells (C) and pancreatic polypeptide+ cells (D), with no apparent effect on (E) glucagon or (F) somatostatin expression. Nuclei were visualized by DAPI staining. Representative images are shown. * indicates P < 0.05.

Citation: Journal of Endocrinology 243, 1; 10.1530/JOE-19-0182

Blood glucose measurements

Tail blood was collected from WT (Glis3fl/fl ) and Glis3Δ panc mice at indicated time points. Glucose levels were measured with Nova Max glucose monitoring system (Nova Biomedical; Waltham, MA, USA).

Immunohistochemical analysis

Mouse pancreases were fixed for 4 h in 4% paraformaldehyde in PBS and embedded in OCT (Tissue Tek; Hatfield, PA, USA) prior to cryogenic sectioning. Primary antibodies are listed in Supplementary Table 1 (see section on supplementary data given at the end of this article). DBA-Fluorescein was purchased from Vector Labs (Burlingame, CA, USA). Alexa Fluor-conjugated secondary antibodies were purchased from Molecular Probes. Nuclei were stained with DAPI Prolong Diamond from Invitrogen. Fluorescent images were captured using a Zeiss LSM 880 confocal microscope and ZEN software.

Proliferation and TUNEL staining

Two-week-old pancreases were stained with antibodies against PDX1 and either Ki67 or the DeadEnd™ Fluorometric TUNEL System (Promega #G3250) following manufacturer’s protocol. At least six sections >200 µ apart per mouse from three mice per genotype were examined using confocal microscopy (Zeiss LSM 880 with Airyscan), and co-localization quantified manually using ImageJ (NIH).

Islet isolation and qRT-PCR analysis

Pancreatic islets were isolated via collagenase injection through the common bile duct into the pancreas, followed by digestion and hand-picking. RNA was isolated using TRIzol reagent (Life Technologies) followed by DNAse I digestion and additional purification via Ambion RNAqueous micro kit (Ambion). cDNA was synthesized using a High-Capacity DNA Reverse Transcription Kit (Applied Biosystems). qRT-PCR was carried out in triplicate in a StepOnePlus Real Time PCR System (Applied Biosystems). All results were normalized to Gapdh expression and are shown relative to control using the 2ΔΔct method. Primer sequences are listed in Supplementary Table 1.

RNA sequencing

Islets were isolated from 4-week-old Glis3Δ panc and control littermates (Glis3fl/fl ). ~10 ng of RNA was converted to cDNA using the high-capacity cDNA reverse transcription kit (Applied Biosystems), and islet RNA from mice with a 78–91% removal of Glis3 (as measured by qRT-PCR of the deleted exon) were submitted for RNA-seq analysis at the NIEHS Epigenomics Core (with control littermates). Briefly, RNA was converted to cDNA using the NextFlex Rapid RNA-seq kit (Illumina) and sequenced as paired-end 76mers on a NextSeq500 (Ilumina) for each sample (n = 4 Glis3fl/fl , 3 Glis3Δ panc ). Read pairs were filtered to retain only those with a mean base quality score of at least 20 for each end. Adapter was clipped from the end of each read via Cutadapt v1.2.1 (Martin 2011) (parameters: -a AGATCGGAAGAG -O 5 -q 0), then read pairs were filtered to retain only those at least 30 nt in length after adapter removal. Trimmed and filtered read pairs were aligned against the mm10 reference assembly via STAR v2.5 (Dobin et al. 2013) (parameters: --alignSJoverhangMin 8 --limitBAMsortRAM 55000000000 --outSAMstrandField intronMotif --outFilterIntronMotifs RemoveNoncanonical). Counts per gene were determined by Subread featureCounts v1.5.0-p1 (Liao et al. 2014) (parameters: -s0 -Sfr -p) for a set of gene models defined by RefSeq transcripts as downloaded from the UCSC Table Browser (http://genome.ucsc.edu/cgi-bin/hgTables) as of November 7, 2017. Differentially expressed genes were identified via DESeq2 v1.10.1 (Love et al. 2014) at an FDR threshold of 0.05, and a complete list is provided in Supplementary Table 2. For RNA-seq genome browser views, replicates were merged and normalized to 50 million mappable reads.

ChIP sequencing

Islets from 8-week-old GLIS3-GFP mice (Kang et al. 2016) were isolated and flash frozen at −80°C. ChIP-seq analysis was performed by Active Motif (Carlsbad, CA, USA). ChIP was performed using a GFP antibody and ~1800 islets. Input and ChIP samples were run as single-end 75mers on a NextSeq500 (Illumina); 42–43 million reads each were obtained for each. Raw data are available from gene expression omnibus (GSE122120). For comparison to other published ChIP-seq data, raw sequencing data were obtained from GEO for NKX2.2 (GSE79785), MAFA and NEUROD1 (GSE30298), PDX1 and FOXA2 (SRA008281), NKX6.1 (GSE40975), ISL1 and LDB1 (GSE84759) and from ArrayExpress for PDX1 (E-MTAB-1143). For consistency, all ChIP-seq datasets were processed by the same workflow, as follows. Reads were filtered to retain only those with a mean base quality score of at least 20. Adapter was clipped from the end of each read via Cutadapt v1.2.1 (Martin 2011) (parameters: -a AGATCGGAAGAG -O 5 -q 0), and then reads were filtered to retain only those at least 30nt in length after adapter removal. Trimmed and filtered reads were aligned against the mm10 reference assembly via Bowtie v1.2 (Langmead et al. 2009), retaining only uniquely mapped reads (parameter: -m 1). For samples with multiple sequencing runs, replicates were combined with MergeSamFiles.jar from the Picard tool suite v1.110. Duplicate reads were removed with MarkDuplicates.jar from the Picard tool suite v1.110. For visualization of mapped read depth on the UCSC genome browser, uniquely mapped non-duplicate reads were extended to 200 nt, converted to bedGraph format using BEDTools v2.24.0 genomeCoverageBed (Quinlan & Hall 2010), and then normalized to 10 million reads per sample. Peak calling was performed with HOMER v4.9.1 (Heinz et al. 2010), with parameters: -style factor -fdr 0.00001. The matched input sample was used as background if available. For public samples with no available matched input, a surrogate input of 20 million reads was generated by aggregating randomly selected (uniquely mapped non-duplicate) reads from the mouse islet input samples from GSE40975, GSE84759, E-MTAB-1143 and our current dataset. Called peaks from each dataset were re-defined as 200mers centered on the midpoint of the original peak call. A complete list of nearest genes bound by GLIS3 is included in Supplementary Table 3.

Pathway analysis

Pathway analysis was performed via DAVID tools (v6.8) for KEGG pathway analysis (Huang da et al. 2009a ,b ). Full list of pathways and genes are included in Supplementary Tables 4, 5 and 6. Pathview analysis was performed using the online tool, as described (Luo et al. 2017).

Luciferase reporter assay

HEK293T cells were grown in DMEM containing 10% fetal bovine serum (FBS) and penicillin/streptomycin. Cells were transfected using Lipofectamine 2000 (Invitrogen) with 100 ng of luciferase reporter plasmid (pGL4.27) under the control of the Mafa region 3 regulatory sequence (Raum et al. 2006), 50 ng of CMV-driven beta-galactosidase plasmid and 100 ng of either CMV-driven mouse GLIS3 or a control plasmid. Twenty-four hours after transfection, cells were lysed with Passive Lysis Buffer (Promega), and luciferase activity was measured with a Luciferase Assay System (Promega) and beta-galactosidase measured with a Luminescent Beta-Galactosidase Detection kit II (TaKaRa) according to the manufacturer’s protocols. Transfections were performed in triplicate, with each experiment performed at least three times.

Statistical analysis

Where indicated, statistics were calculated using a two-tailed Student t-test. Significance indicates a P value <0.05, unless otherwise specified.

Results

Glis3 Δpanc mice develop hyperglycemia due to loss of insulin expression

In order to investigate the role of GLIS3 in the pancreas, we generated a pancreas-specific Glis3 deletion mouse model. Glis3fl/fl mice containing LoxP sites flanking exon 5 (Fig. 1A), which encodes a portion of the DNA-binding domain, were generated and crossed with a mouse strain expressing Cre recombinase under regulation of the Pdx1 promoter, expressed in the early stages of pancreatic development in the common pancreatic progenitor cells. Glis3Δ panc mice did not develop neonatal diabetes like the global Glis3 mutant mice, as their blood glucose levels were not significantly different at 2 weeks (data not shown). However, by 8 weeks of age, ~20% of Glis3Δ panc mice developed severe hyperglycemia (>500 mg/dL blood glucose), while the remaining Glis3Δ panc mice exhibited glucose levels comparable to those of control littermates (Fig. 1B). The development of hyperglycemia appears to relate to the efficiency of Glis3 exon 5 removal, as mice with >75% deletion displayed mild hyperglycemia at 4 weeks (Supplementary Fig. 1), consistent with Pdx1-Cre mosaic expression in pancreatic progenitor cells (Hingorani et al. 2003). Another investigator utilizing a similar Glis3-knockout strategy with a Pdx1-Cre model reported hyperglycemia in only a subset of Glis3-knockout mice (Yang et al. 2017). In addition, partial Glis3 deletion in Glis3Δ panc euglycemic mice appears to phenocopy Glis3 heterozygous mice, which are euglycemic, but are more susceptible to impaired glucose tolerance in high-fat diet-induced obesity (Yang et al. 2013).

Immunofluorescent staining of pancreas sections from euglycemic and hyperglycemic Glis3Δ panc mice and control littermates at 8 weeks of age (Fig. 1C, D, E and F) revealed a loss of insulin expression in PDX1+ presumptive beta cells, with a more severe loss of insulin observed in hyperglycemic animals (Fig. 1C). We presume these cells are former beta cells due to their PDX1 expression and central location within the islet. Pancreatic polypeptide levels also appeared decreased (Fig. 1D), consistent with our recent discovery of GLIS3 expression in pancreatic polypeptide cells (Kang et al. 2016). Glucagon and somatostatin expression appeared unaffected (Fig. 1E and F), likely reflecting the lack of GLIS3 expression in the mature alpha and delta cells (Kang et al. 2016).

Glis3 Δpanc islet beta cells do not undergo apoptosis or dedifferentiation

In order to further study these PDX1+ presumptive beta cells, we stained for MAFA, a marker of mature beta cells in both mice and humans, and GLUT2, a beta cell-specific glucose transporter. Expression of these markers was lost in most but not all PDX1+ cells by 8-weeks of age (Fig. 2A and B). Talchai et al. suggested that, under stress conditions, beta cells may dedifferentiate into an earlier point in endocrine lineage, as marked by NGN3 expression (Talchai et al. 2012). However, we did not detect re-expression of NGN3 at 8 weeks in the PDX1+ cells (Supplementary Fig. 2). Additionally, it is possible that the loss of insulin expression was due to decreased proliferation or increased apoptosis at an earlier timepoint. We therefore examined proliferation and apoptosis at 2 weeks of age, prior to the secondary effects of hyperglycemia seen at 8 weeks. Ki67+PDX1+ cells were slightly increased, indicating a slight increase in proliferation, whereas no increase in TUNEL staining was observed (Fig. 2C, D, E and F). Correspondingly, we saw a slight, non-significant increase in the total number of PDX1+ cells in Glis3Δ panc mice (data not shown). This indicates that while islet PDX1+ cells in Glis3Δ panc mice no longer express insulin and are likely non-functional, these cells remain present within the islet in a dysfunctional state.

Figure 2
Figure 2

Beta cells lose key markers, but do not apoptose in Glis3Δ panc mice. Pancreas sections from 8-week-old Glis3Δ panc mice were stained for PDX1 and MAFA (A) or GLUT2 (B). Proliferation was measured via Ki67 and PDX1 double staining of 2-week-old Glis3Δ panc mice (C) and quantified (D). Apoptosis was measured via TUNEL and PDX1 double staining of 2-week-old Glis3Δ panc mice (E) and quantified (F). Results from n = 3 mice for each genotype, error bars = s.e.m., * indicates P < 0.05. Arrows indicate MAFA+PDX1+ (A), GLUT2+PDX1+ (B), Ki67+PDX1+ (C) or TUNEL+PDX1+ (D) cells. Representative images are shown.

Citation: Journal of Endocrinology 243, 1; 10.1530/JOE-19-0182

GLIS3 regulates many genes critical for beta cell identity and function

In order to establish a global network of GLIS3-regulated genes, we performed RNA-seq analysis on isolated islets from Glis3Δ panc mice at 4 weeks of age, prior to the onset of severe hyperglycemia (>500 mg/dL) so as to limit its secondary effects. In total, 1,162 genes were found to be upregulated >1.5-fold, and 612 genes were found to be downregulated >1.5-fold upon loss of GLIS3, consistent with GLIS3’s role as both an activator and repressor of gene expression (Supplementary Table 2). KEGG pathway analysis indicated that the downregulated genes were involved in a variety of pathways, including Maturity Onset Diabetes of the Young (MODY) and insulin secretion, as well as a variety of metabolic pathways (Fig. 3A). Examination of the insulin secretion pathway via Pathview analysis indicated that the expression of several insulin pathway-related genes was regulated by GLIS3, including downregulation of the GLP1 receptor (Glp1r) and IP3 receptor (Itpr1), along with Mafa and Slc2a2 (Fig. 3B). The UCSC genome browser tracks in Fig. 3C and D illustrate the significant downregulation of both Mafa and Slc2a2 mRNA expression, consistent with the immunofluorescent staining (Fig. 2A and B). Downregulation of Ins2 was also observed, consistent with the observations of other models of Glis3 deletion (Supplementary Fig. 3) (Kang et al. 2009, Watanabe et al. 2009, Yang et al. 2009, 2011, 2013, 2017, Nogueira et al. 2013, Amin et al. 2018).

Figure 3
Figure 3

Gene expression analysis of Glis3Δ panc mice. RNA-seq analysis of Glis3Δ panc islets at 4 weeks was performed. (A) KEGG pathway analysis of genes downregulated >1.5-fold and with an FDR of <0.05. Numbers behind bars indicate number of genes in the respective pathway. (B) Modified Pathview analysis of the insulin secretion pathway genes, with green representing downregulated genes and red representing upregulated genes. RNA-seq analysis of (C) MafA and (D) Slc2a2, the gene that encodes the GLUT2 protein. Data displayed via the UCSC genome browser. n = 3 Glis3Δ panc , n = 4 WT.

Citation: Journal of Endocrinology 243, 1; 10.1530/JOE-19-0182

GLIS3 binds to beta cell genes in mouse islets

In order to establish which of these differentially expressed genes were directly regulated by GLIS3, we performed ChIP-seq analysis of GLIS3 in adult mouse islets. As no reliable GLIS3 antibody currently exists, we utilized a mouse model where the C-terminus of GLIS3 is fused to an enhanced GFP tag (GLIS3-GFP), thus allowing for precipitation with a GFP antibody. One of the top consensus-binding sequences detected in our ChIP-seq analysis matched that of Zic/Gli/Glis family members, similar to the consensus of the GLIS3 binding site previously identified in the thyroid gland (Fig. 4A) (Kang et al. 2017), indicating the ChIP-seq was successful in detecting specific GLIS3 binding. A complete list of bound genes (i.e. nearest gene to GLIS3 binding peak) is provided in Supplementary Table 3. GLIS3 binding was most highly enriched at introns within the gene body and promoter regions, consistent with what was observed in GLIS3 ChIP-seq experiments in the thyroid (Fig. 4B) (Kang et al. 2017). Similar to previous reports by our group (Zeruth et al. 2013), GLIS3 binding was observed at the Ins2 promoter (Fig. 4C), as well as an enhancer for Slc2a2 (Fig. 4D). Interestingly, all of the genes examined with a known role in beta cell function display GLIS3 binding within 2kb of their transcriptional start site (Table 1). However, only some of these genes were downregulated in Glis3Δ panc mice (Table 1), indicating that GLIS3 may not be essential for regulation of a fraction of these genes or that their regulation by GLIS3 may be dependent upon upstream signaling events absent under the tested conditions. We also combined our ChIP-seq and gene expression data to examine more closely those genes directly regulated by GLIS3 (i.e. GLIS3-bound genes that are differentially expressed in Glis3Δ panc mice). KEGG pathway analysis of these genes identified many of the same pathways as found by analyzing gene expression data alone (Fig. 3A and Supplementary Fig. 4). This highlights the important role GLIS3 binding plays in regulating proper beta cell function, as indicated by the hyperglycemic phenotype of the Glis3Δ panc mice.

Figure 4
Figure 4

Global GLIS3 binding analysis in mouse islets reveals Slc2a2 direct regulation. (A) De novo motif analysis was performed on ChIP-seq data using HOMER. (B) Binding analysis of Glis3 ChIP-seq data indicated a preference for introns and promoters. Upstream = −10 kb to −1 kb relative to TSS, TSS proximal = −1 kb to TSS, gene body = TSS to TES, intergenic = remaining sequences. (C and D) ChIP-seq binding on the Ins2 promoter, and the Slc2a2 promoter and enhancer. Peaks indicate the peaks called using HOMER.

Citation: Journal of Endocrinology 243, 1; 10.1530/JOE-19-0182

Table 1

Islet-specific genes are bound by GLIS3.

Gene Fold change FDR Glis3 ChIP-seq signal
Hormones
Ins1 −2.116517315 0.00506878 Yes
Ins2 −2.839350013 3.35E-13 Yes
Ppy −8.154862239 1.87E-19 Yes
Gcg −1.234894439 NS No
Sst 1.083293292 NS No
Ghrl 1.095231496 NS No
Beta cell function
Slc2a2 −5.112554821 9.58E-33 Yes
Kcnj11 −1.299797953 0.01104388 Yes
Abcc8 1.444296553 0.00016462 Yes
Glp1r −1.848329213 0.02182045 Yes
Chga −2.13350168 3.53E-22 Yes
Ucn3 −1.588987707 NS Yes
Gjd2 −1.120393715 NS Yes
Transcription factors
MafA −2.693922579 0.00026979 Yes
Nkx6.1 −2.239512577 1.03E-09 Yes
Rfx6 1.349799675 0.00446909 Yes
Isl1 1.640589346 4.17E-10 Yes
Ldb1 −1.279981837 0.00500245 Yes
MafB −1.496356376 0.00324023 Yes
FoxA2 −1.281876971 0.045365 Yes
Pdx1 −1.061699976 NS Yes
Tshz1 1.084110443 NS Yes
Mnx1 1.114782737 NS Yes
InsM1 1.102778676 NS Yes
Ngn3 −1.731654424 NS Yes
Nkx2.2 −1.211137492 NS Yes

GLIS3 binding overlaps with that of other islet-enriched transcription factors

Previous reports have suggested that islet-enriched transcription factors often bind in close proximity to one another (Ediger et al. 2017). Moreover, immunoprecipitation assays indicated that GLIS3 is associated with PDX1, NEUROD1 and MAFA protein complexes (Yang et al. 2009). Indeed, motif analysis of GLIS3 ChIP-seq identified a number of additional motifs, including motifs for NEUROD1, FOX and RFX family members (FOXM1 and RFX5), and LHX2, which contains a homeobox DNA-binding domain similar to PDX1, NKX6.1, ISL1 and NKX2.2 (Fig. 4A). In order to determine whether GLIS3 co-localizes with other islet-enriched transcription factors on the same regulatory regions, we compared GLIS3 binding to that of PDX1, ISL1, NKX6.1, NKX2.2, NEUROD1, MAFA and FOXA2 (Fig. 5A). We also included the ISL1 coactivator LDB1, a critical co-regulator of ISL1 function (Hunter et al. 2013, Ediger et al. 2017). It is important to note that the quality of the ChIP-seq data compared varied greatly as is typical among various transcription factors. Nevertheless, our analysis revealed that GLIS3 is associated with many of the same promoter/enhancer regions as the transcription factors mentioned above (Fig. 5B). In addition to a global analysis, we examined the binding of these factors near the Ins2, Slc2a2 and Mafa genes. Fig. 5C, D and E demonstrate the co-localization of GLIS3 binding with that of several other critical islet transcription factors to regulatory regions near the Ins2, Slc2a2 and Mafa genes, indicating potential coordination between these transcription factors. GLIS3’s transcriptional regulation of Mafa was further supported by luciferase assays of the Mafa region 3 upstream regulatory element. GLIS3 stimulated Mafa region 3 transcriptional activity, similar to what has been found for other factors (Supplementary Fig. 5) (Raum et al. 2006). ChIP-seq-binding data showed overlapping binding for almost all of the transcription factors examined, indicating there is likely a high level of coordination between GLIS3 and other islet-enriched transcription factors in regulating genes that are critical for normal beta cell function (Fig. 5F).

Figure 5
Figure 5

GLIS3 binding shows partial overlap with other islet-enriched transcription factors. (A) Heatmap of the 2 kb region centered on each of the 21,253 GLIS3-binding peaks with ChIP-seq signal normalized to 10 million reads for GLIS3, LDB1, ISL1, PDX1 (from two independent labs), NKX6.1, FOXA2, NKX2.2, NEUROD1 and MAFA. Color intensity indicates read depth, as shown by the scale below each TF panel. (B) Number of peaks called for each ChIP, as well as percentage of TF ChIP peaks colocalizing with GLIS3 peaks with signal above threshold cutoff. ChIP peak co-localization near the Ins2 (C), Slc2a2 (D) and Mafa (E) genes. Data displayed via the UCSC genome browser. (F) Model of GLIS3 binding. Glis3 interacts with islet-enriched transcription factors on regulatory hubs to control genes related to diabetes and interacts with other unknown factors to recruit either coactivator or corepressor complexes to control genes related to other processes.

Citation: Journal of Endocrinology 243, 1; 10.1530/JOE-19-0182

Discussion

Global Glis3-mutant mice have a short lifespan, develop polycystic kidney disease, hypothyroidism and exhibit a defect in pancreatic beta cell development that leads to neonatal diabetes (Kang et al. 2009, 2017, Watanabe et al. 2009, Yang et al. 2009). These defects are similar to what is observed in humans with mutations in GLIS3 (Jetten 2018). In order to study the function of GLIS3 in the pancreas while limiting secondary effects from other tissues, we developed a conditional mouse model in which GLIS3 function was impaired specifically in the pancreas. Since ubiquitous Glis3-knockout mice develop hypothyroidism that might influence the pancreatic phenotype (Chen et al. 2018), our model of Glis3 deletion allows us to examine the role of GLIS3 in beta cell function, while minimizing secondary effects caused by hyperglycemia and hypothyroidism.

Unlike global Glis3 mutants, which die by PND11 likely due to severe hyperglycemia, Glis3Δ panc mice begin to develop hyperglycemia around 4 weeks of age dependent upon the efficiency of Glis3 deletion (Supplementary Fig. 1), with almost all Glis3Δ panc mice surviving until at least 8 weeks of age. As GLIS3 is essential for Ins2 expression, this indicates that only a subset of functional beta cells (those lacking Cre expression and thus still expressing GLIS3) appear necessary for maintaining euglycemia. Unfortunately, we were unable to test for the persistence of GLIS3 expression due to the lack of a working antibody. However, our interpretation is consistent with a study by Yang et al. showing that in adult mice a ~66% deletion of Glis3 exon 4, using an tamoxifen-inducible mouse insulin promoter-driven Cre (MIP-CreERT), resulted in euglycemic mice, whereas a tamoxifen-inducible β-cell-specific Pdx1-CreERT deletion model with a >90% deletion of Glis3, produced hyperglycemic mice (Yang et al. 2017). This conclusion is further supported by a Diptheria toxin-based beta cell ablation model, where a ~50% loss of beta cells does not cause hyperglycemia, whereas a ~99% ablation does (Thorel et al. 2010). Furthermore, the persistence of some INSULIN+ (Fig. 1C) or MAFA+ cells (Fig. 2A) is consistent with the mosaicism of the Pdx1-Cre that has been observed in other deletion models, such as the Pdx1-Cre deletion of Mafa (Hang et al. 2014), where a subset of SLC30A8+ and GLUT2+ cells persist in the islets, likely due to the continued expression of Mafa in those cells.

Our analysis of Glis3Δ panc mice revealed no increase in apoptosis at 2 weeks (i.e. prior to hyperglycemia), while proliferation was very slightly increased, possibly in response to an increased demand for insulin+ cells (Fig. 2D and F). This stands in contrast to a previous study of GLIS3 function in the thyroid, which demonstrated that GLIS3 does play a role in stimulating proliferation in that tissue (Kang et al. 2017). Moreover, pancreatic ducts in ubiquitous Glis3 knockout mice, as well as Glis3Δ panc mice (Supplementary Fig. 6), did display a cystic phenotype that involves increased proliferation, suggesting that the effect of GLIS3 on proliferation is cell type dependent. Previous reports suggested that GLIS3 suppresses apoptosis in a pancreatic beta cell line by regulating Srsf6 (referred to as Srp55 in Nogueira et al. 2013) and in human stem cells differentiated into beta like cells through the TGFβ signaling pathway (Amin et al. 2018). However, in our in vivo model, we did not observe an increase in apoptosis, Srsf6 expression was not changed, nor were apoptotic or TGFβ pathways upregulated in our gene expression analysis (Fig. 2F, Supplementary Fig. 3A and Supplementary Table 2). It is possible that apoptosis is increased at some later time in our model; however, it would be difficult to distinguish this from the secondary effects of hyperglycemia. Instead, we observed changes in genes involved in a variety of metabolic pathways and a downregulation of genes involved in beta cell function (Fig. 3A). These results highlight the important, but underappreciated differences that exist between cell line models and in vivo mouse systems, which we believe may more accurately reflect the in vivo role of GLIS3 in humans.

In addition to a lack of apoptosis, we did not detect expression of the progenitor marker NGN3 in Glis3Δ panc pancreas sections (Supplementary Fig. 2), which has been reported to be re-expressed in dedifferentiated β cells (Talchai et al. 2012). This may be expected as GLIS3 is known to function as a positive regulator of Ngn3 transcription (Kang et al. 2009, Yang et al. 2011, Kim et al. 2012). We also did not observe increased expression of several other dedifferentiation-associated genes, such as L-myc, Oct4, or Nanog, in Glis3Δ panc islets (Supplementary Table 2) (Talchai et al. 2012). These observations suggest that impaired GLIS3 function does not cause dedifferentiation of beta cells; however, we cannot rule out that GLIS3 itself may be required for dedifferentiation. Nevertheless, we did detect a ~12-fold upregulation of Aldh1a3, a gene that has been reported to be associated with not only dedifferentiation, but also with beta cell dysfunction (Supplementary Table 2) (Kim-Muller et al. 2016). The expression of Igfbp4 and Cxcl12, ‘disallowed’ genes in pancreatic islets (Pullen et al. 2010, Thorrez et al. 2011), were also upregulated in Glis3Δ panc islets. However, the vast majority of disallowed genes were not induced, including Ldha, Oat, Cat, Arhgdib, Mct1, Maf, Zfp36l1, Itih5, Pdgfra, Lmo4, Zyx, Smad3, Acot7, Cox5a, Fam59a, Gas6, Mgst1, Nfib, Plec1, Rpl36, Tgm2, Tst and Zdhh. These findings are consistent with the concept that Glis3Δ panc beta cells do not exhibit a dedifferentiated phenotype, but remain as non-insulin-producing, dysfunctional β-like cells.

The coordinated binding of transcription factors at ‘super’ enhancer regions, regions bound by several tissue-specific transcription factors for control of tissue-specific genes, has been well documented (Loven et al. 2013, Whyte et al. 2013). Within the pancreatic islet, such enhancers have linked not only the binding of islet-enriched transcription factors with histone marks of enhancers, but also with mutations related to the development of diabetes and dysregulated fasting glycemia (Stitzel et al. 2010, Pasquali et al. 2014). Analysis of the consensus GLIS3 binding motif suggested co-localization with motifs of other transcription factors important for the regulation of pancreatic β cell functions, such as NEUROD1, FOX and RFX family members and the homeobox containing Lhx2 (similar to PDX1, NKX6.1, ISL1 and NKX2.2) (Fig. 4A). We therefore sought to characterize GLIS3 binding in the context of other islet-enriched transcription factors. Ediger et al. recently described the binding of ISL1 in pancreatic islets, where ISL1 largely co-localized with other islet-enriched transcription factors (Ediger et al. 2017). Indeed, we observed that 50% of ISL1 sites overlapped with GLIS3 binding (Fig. 5B). In contrast, we observed that GLIS3 only co-localizes with other islet-enriched transcription factors at a fraction of their target sites. A likely explanation for this is the significantly larger number of ChIP-seq peaks called in our GLIS3 analysis, as compared to the ISL1, MAFA, NEUROD1 and FOXA2 ChIP-seq data analysis (Fig. 5B). However, even when comparing GLIS3 ChIP-seq data to transcription factors with more peaks (PDX1 and NKX6.1), overlap was limited to <25%. This suggests that islet-enriched transcription factors, including GLIS3, display context-specific co-localization, possibly reflecting tissue-specific function. Indeed, genes with nearby overlapping peaks for GLIS3, PDX1 and NKX6.1 were associated particularly with Maturity Onset Diabetes of Young (MODY), insulin resistance, insulin signaling, type 2 diabetes and insulin secretion pathways (Supplementary Fig. 7). This supports a model in which a selective set of genes directly regulated by GLIS3 are co-regulated with other islet-enriched transcription factors through their interaction on the same distinct regulatory regions in beta cell-specific genes (Fig. 5F). GLIS3 and other islet-specific transcription factors may then coordinate their transcriptional regulation through coactivator protein complexes recruited to these regulatory hubs.

In summary, our study provides the first in-depth characterization of the role of GLIS3 in gene regulation within the pancreatic beta cell. GLIS3 has many functions within the beta cell, from regulating aspects of several metabolic pathways and intracellular signaling pathways, to regulating other transcription factors. Indeed, some of the effects on beta cell function could be attributed to GLIS3’s direct regulation of Mafa through its region 3 regulatory domain (Fig. 5E). MAFA has been shown to control the expression of Ins2, Slc2a2, Slc30a8, Sytl4 and Stxbp1 all of which are downregulated in Glis3Δ panc mice (Hang et al. 2014). Unlike Glis3Δ panc mice, the main defect in MafAΔ panc mice appears to be in glucose-stimulated insulin secretion, as they do not display overt hyperglycemia. Additionally, GLIS3 regulates a variety of genes not apparently regulated by MAFA, such as Ngn3 and Nkx6.1. GLIS3 is also expressed in variety of tissues which do not express MAFA, indicative of GLIS3’s role in controlling pathways not directly related to beta cell function.

Recent reports have highlighted the importance of phosphorylation in mediating NGN3’s reprogramming effects (Azzarelli et al. 2018), and MAFA’s interaction with other transcription factors (Han et al. 2016). As GLIS3 is highly phosphorylated, it is likely that GLIS3 transcriptional activity is controlled by upstream and tissue-specific signaling pathways. What these signals are, and how GLIS3 integrates them into co-regulatory interactions or activation/repression functions, remains to be discovered. Understanding how GLIS3 coordinates with other factors, and specifically which upstream signals control GLIS3 activity and guide protein–protein interactions in vivo, might provide new therapies for diabetes treatment. Future studies will hopefully build upon the findings outlined here to distinguish how GLIS3 differentially regulates disease-relevant genes in different tissues, which upstream signaling pathways control GLIS3 activity and provide insight into how these pathways may be targeted in disease treatment.

Supplementary data

This is linked to the online version of the paper at https://doi.org/10.1530/JOE-19-0182.

Declaration of interest

The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

Funding

This research was supported by the Intramural Research Program of the NIEHS, NIH Z01-ES-101585.

Acknowledgments

The authors would like to thank Molly Cook, Nicole Reeves and the Epigenomics Core Laboratory at NIEHS for their assistance in high-throughput sequencing. They would also like to thank Dr Chad Hunter at the University of Alabama-Birmingham for assistance in review of this manuscript, and the Cell Biology group at NIEHS for thoughtful comments throughout.

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  • Glis3Δ panc mice exhibit a loss of insulin-producing beta cells. (A) Schematic of WT and recombinant Glis3 allele. A conditional knockout allele for the Glis3 gene was generated in C57BL/6 mice containing loxP sites (triangles) surrounding exon 5 of Glis3. Exons 4–6 encode the DNA-binding domain (DBD). Mice were bred with a strain expressing Cre recombinase under the control of the Pdx1 promoter. (B) Random blood glucose levels of mice at 8 weeks of age. Glis3Δ panc mice were grouped as either euglycemic (<250 mg/dL) or hyperglycemic (>250 mg/dL) and compared to control littermates (WT) (n = 11 WT, n = 5 Glis3Δ panc euglycemic, n = 4 Glis3Δ panc hyperglycemic). Immunofluorescence staining revealed a reduction in the number of INS+PDX1+ beta cells (C) and pancreatic polypeptide+ cells (D), with no apparent effect on (E) glucagon or (F) somatostatin expression. Nuclei were visualized by DAPI staining. Representative images are shown. * indicates P < 0.05.

  • Beta cells lose key markers, but do not apoptose in Glis3Δ panc mice. Pancreas sections from 8-week-old Glis3Δ panc mice were stained for PDX1 and MAFA (A) or GLUT2 (B). Proliferation was measured via Ki67 and PDX1 double staining of 2-week-old Glis3Δ panc mice (C) and quantified (D). Apoptosis was measured via TUNEL and PDX1 double staining of 2-week-old Glis3Δ panc mice (E) and quantified (F). Results from n = 3 mice for each genotype, error bars = s.e.m., * indicates P < 0.05. Arrows indicate MAFA+PDX1+ (A), GLUT2+PDX1+ (B), Ki67+PDX1+ (C) or TUNEL+PDX1+ (D) cells. Representative images are shown.

  • Gene expression analysis of Glis3Δ panc mice. RNA-seq analysis of Glis3Δ panc islets at 4 weeks was performed. (A) KEGG pathway analysis of genes downregulated >1.5-fold and with an FDR of <0.05. Numbers behind bars indicate number of genes in the respective pathway. (B) Modified Pathview analysis of the insulin secretion pathway genes, with green representing downregulated genes and red representing upregulated genes. RNA-seq analysis of (C) MafA and (D) Slc2a2, the gene that encodes the GLUT2 protein. Data displayed via the UCSC genome browser. n = 3 Glis3Δ panc , n = 4 WT.

  • Global GLIS3 binding analysis in mouse islets reveals Slc2a2 direct regulation. (A) De novo motif analysis was performed on ChIP-seq data using HOMER. (B) Binding analysis of Glis3 ChIP-seq data indicated a preference for introns and promoters. Upstream = −10 kb to −1 kb relative to TSS, TSS proximal = −1 kb to TSS, gene body = TSS to TES, intergenic = remaining sequences. (C and D) ChIP-seq binding on the Ins2 promoter, and the Slc2a2 promoter and enhancer. Peaks indicate the peaks called using HOMER.

  • GLIS3 binding shows partial overlap with other islet-enriched transcription factors. (A) Heatmap of the 2 kb region centered on each of the 21,253 GLIS3-binding peaks with ChIP-seq signal normalized to 10 million reads for GLIS3, LDB1, ISL1, PDX1 (from two independent labs), NKX6.1, FOXA2, NKX2.2, NEUROD1 and MAFA. Color intensity indicates read depth, as shown by the scale below each TF panel. (B) Number of peaks called for each ChIP, as well as percentage of TF ChIP peaks colocalizing with GLIS3 peaks with signal above threshold cutoff. ChIP peak co-localization near the Ins2 (C), Slc2a2 (D) and Mafa (E) genes. Data displayed via the UCSC genome browser. (F) Model of GLIS3 binding. Glis3 interacts with islet-enriched transcription factors on regulatory hubs to control genes related to diabetes and interacts with other unknown factors to recruit either coactivator or corepressor complexes to control genes related to other processes.