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
DOI:
https://doi.org/10.1530/JOE-19-0182
Volume/Issue:
Volume 243: Issue 1
Article Type:
Research Article
Page Range:
1–14
Online Publication Date:
Oct 2019
Copyright:
© 2019 Society for Endocrinology 2019
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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.

Supplementary Materials

    • Supplementary Figure 1. Glis3Δpanc hyperglycemia is dependent on deletion efficiency. (A) Random blood glucose levels of mice at 4-weeks of age (N=9 WT, N=8 Glis3Δpanc). (B) Random blood glucose levels of mice at 4-weeks of age compared to the efficiency of removal of Glis3 in isolated islets from the same mice. Red dots are individual Glis3Δpanc mice, and blue dots are WT mice. (C) Random blood glucose levels of mice at 4 weeks of age with less than 75% removal (<75%) or greater than 75% removal (>75%). Efficiency of removal is based on qRT-PCR for the deleted Glis3 exon. Error bars indicate SEM. * p < 0.05.
    • Supplementary Figure 2. NGN3 is not expressed in Glis3Δpanc mice. Immunostaining for NGN3 at 8-weeks of age is shown, as well as in WT mice at e15.5 as a positive control. Images shown are representative.
    • Supplementary Figure 3. Ins2 expression is decreased in Glis3Δpanc mice. Islet RNAseq data for Ins2 is shown in Glis3Δpanc mice and control littermates at 4-weeks of age.
    • Supplementary Figure 4. Pathway analysis of direct GLIS3 target genes. KEGG pathway analysis was performed on genes that were downregulated >1.5 fold, with an FDR of <0.05, and that had at least 1 nearby peak for GLIS3 binding.
    • Supplementary Figure 5. GLIS3 stimulates MafA Region 3 activity in 293T cells. MafA Region 3 luciferase activity was calculated relative to CMV-driven beta-galactosidase activity, and normalized to empty pGL4.27 vector. Transcriptional activity was stimulated by the addition of Glis3. Representative experiment shown, error bars indicate standard deviation for experiment run in triplicate. * p<0.01 comparing pGL4.27-MafA Reg3 with or without Glis3.
    • Supplementary Figure 6. Glis3Δpanc mice contain cystic ducts. Pancreas sections from 2-week and 10-week old Glis3Δpanc mice were stained with DBA-Fluorescein and DAPI. Representative images are shown.
    • Supplementary Figure 7. Pathway analysis of genes bound by GLIS3, PDX1, and NKX6.1. KEGG pathway analysis was performed on genes that had at least 1 nearby peak for GLIS3, PDX1, and NKX6.1 binding.
    • Supplementary Table 1. Primers and Antibodies.
    • Supplementary Table 2. Glis3Δpanc RNAseq data in 4-week islets.
    • Supplementary Table 3. GLIS3 ChIP-seq in mouse islets at 8-weeks.
    • Supplementary Table 4. KEGG pathway analysis of Glis3Δpanc downregulated genes.
    • Supplementary Table 5. KEGG pathway analysis of GLIS3 direct target genes.
    • Supplementary Table 6. KEGG pathway analysis of genes co-bound by GLIS3, PDX1, and NKX6.1.

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Print ISSN:
0022-0795
Online ISSN:
1479-6805
Copyright:
© 2019 Society for Endocrinology 2019
Page(s):
14
Article Type:
Research Article
Received Date:
19 Jul 2019
Accepted Date:
24 Jul 2019
Print Publication Date:
01 Oct 2019
Online Publication Date:
Oct 2019
Keywords:
GLIS3; beta cells; RNA-seq; ChIP-seq; transcription factor; gene regulation