The CCR4-NOT Complex Maintains ß-Cell Identity through the Repression of ß-Cell Disallowed Genes

Loss of β-cell identity, dedifferentiation and reprogramming are recognized as mechanisms of β-cell dysfunction in diabetes. Molecular β-cell identity is not only defined by the expression of signature functional β-cell specific genes but also the repression of several genes recently identified in β-cell research as β-cell disallowed genes. The molecular mechanisms involved in the repression of β-cell disallowed genes are largely unknown. Here, we show that the CCR4-NOT complex, a major deadenylase conserved in eukaryotes, is involved in post-transcriptional regulation of β-cell disallowed genes. β-cell specific depletion of CNOT3 (CNOT3βKO), a CCR4-NOT complex subunit, in mice promotes deceased insulin secretion, early impaired glucose tolerance and the development of overt diabetes at 12 weeks of age. CNOT3βKO islets display decreased insulin content and decreased glucose responsiveness. Furthermore, CNOT3 depleted ß cells exhibit ultrastructural abnormalities including: degranulated β cells, increased immature insulin granules and abnormal mitochondria. RNAseq and qRT-PCR analyses revealed that CNOT3βKO islets have decreased expression of β-cell functional genes: (Ins1, Ins2, MafA, Pcsk2 and Cpe) and increased expression of 9 of the 11 core β-cell disallowed genes: (Slc16A1, Ldha, Pdgfra, Cxcl12, Igfbp4, Oat, Maf, Smad3 and Cd302) and progenitor-like cells genes: (Nanog and Ngn3) supporting the loss of β-cell identity. Using the total RNAseq data, we assessed mRNA stability by calculating the relative amounts of introns and exons for a given mRNA. RNA immunoprecipitation revealed that the CCR4-NOT complex directly binds several upregulated stabilized β-cell disallowed genes: Slc16A1, Tgm2, Cat and Parp3 genes. The present data highlight deadenylation as a novel molecular mechanism regulating β-cell disallowed genes. Thus, we further propose that the CCR4-NOT complex is essential for maintaining β-cell identity by repressing β-cell disallowed genes.