PAX6 maintains β cell identity by repressing genes of alternative islet cell types
Avital Swisa, Dana Avrahami, Noa Eden, Jia Zhang, Eseye Feleke, Tehila Dahan, Yamit Cohen-Tayar, Miri Stolovich-Rain, Klaus H. Kaestner, Benjamin Glaser, Ruth Ashery-Padan, Yuval Dor
Avital Swisa, Dana Avrahami, Noa Eden, Jia Zhang, Eseye Feleke, Tehila Dahan, Yamit Cohen-Tayar, Miri Stolovich-Rain, Klaus H. Kaestner, Benjamin Glaser, Ruth Ashery-Padan, Yuval Dor
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Research Article Endocrinology Genetics

PAX6 maintains β cell identity by repressing genes of alternative islet cell types

Abstract

Type 2 diabetes is thought to involve a compromised β cell differentiation state, but the mechanisms underlying this dysfunction remain unclear. Here, we report a key role for the TF PAX6 in the maintenance of adult β cell identity and function. PAX6 was downregulated in β cells of diabetic db/db mice and in WT mice treated with an insulin receptor antagonist, revealing metabolic control of expression. Deletion of Pax6 in β cells of adult mice led to lethal hyperglycemia and ketosis that were attributed to loss of β cell function and expansion of α cells. Lineage-tracing, transcriptome, and chromatin analyses showed that PAX6 is a direct activator of β cell genes, thus maintaining mature β cell function and identity. In parallel, we found that PAX6 binds promoters and enhancers to repress alternative islet cell genes including ghrelin, glucagon, and somatostatin. Chromatin analysis and shRNA-mediated gene suppression experiments indicated a similar function of PAX6 in human β cells. We conclude that reduced expression of PAX6 in metabolically stressed β cells may contribute to β cell failure and α cell dysfunction in diabetes.

Authors

Avital Swisa, Dana Avrahami, Noa Eden, Jia Zhang, Eseye Feleke, Tehila Dahan, Yamit Cohen-Tayar, Miri Stolovich-Rain, Klaus H. Kaestner, Benjamin Glaser, Ruth Ashery-Padan, Yuval Dor

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Figure 6

PAX6 ChIP-seq analysis in β cells.

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PAX6 ChIP-seq analysis in β cells. (A) Venn diagram showing the overlap ...
(A) Venn diagram showing the overlap of genes regulated by PAX6 (from the RNA-seq analysis in βPAX6 cells) with PAX6-bound genes (from ChIP-seq analysis in Min6 cells). (B) Top enriched motifs in all PAX6 peaks (left) and motifs differentially enriched in activated versus repressed genes in βPAX6 mice (right). (C) Maps of representative key β cell loci demonstrating the integration of PAX6 ChIP-seq data with other epigenomic data sets. Shown are PAX6-binding sites in the insulin and glucagon promoters and in Foxa2 and Slc2a2 (encoding GLUT2) enhancers (Enh, highlighted in rectangles; the distance from the transcription start site [TSS] is indicated). Note the active histone marks in insulin, Foxa2, and Slc2a2 versus the absence of active marks in the glucagon (Gcg) promoter. (D) Luciferase activity upon cloning of different PAX6-bound elements to a pGL4.23 luciferase reporter vector and transfection into Min6 cells. n = 3 per element. *P < 0.05, **P < 0.01, and ***P < 0.001, by 2-tailed Student’s t test. (E) Integrated map of histone marks and β cell TFs binding at PAX6-bound regions found by luciferase assay (see Figure 6D) to have enhancer (Pdx1) and silencer (Nkx2.2) activity. Note the higher enrichment of H3K27ac in the Pdx1 enhancer region.