Identification of a novel lncRNA (G3R1) regulated by GLIS3 in pancreatic β-cells

Abstract

Recent advances in high throughput RNA sequencing have revealed that, in addition to messenger RNAs (mRNAs), long non-coding RNAs (lncRNAs) play an important role in the regulation of many cell functions and of organ development. While a number of lncRNAs have been identified in pancreatic islets, their function remains largely undetermined. Here, we identify a novel long ncRNA regulated by the transcription factor GLIS3, which we refer to as GLIS3 regulated 1 (G3R1). This lncRNA was identified for its significant loss of expression in GLIS3 knockout mouse pancreatic islets. G3R1 appears to be specifically expressed in mouse pancreatic β-cells and in a β-cell line (βTC-6). ChIP-seq analysis indicated that GLIS3 and other islet-enriched transcription factors bind near the G3R1 gene, suggesting they directly regulate G3R1 transcription. Similarly, an apparent human homolog of G3R1 displays a similar expression pattern, with additional expression seen in human brain. In order to determine the function of G3R1 in mouse pancreatic β-cells, we utilized CRISPR to develop a knockout mouse where ~80% of G3R1 sequence is deleted. Phenotypic analysis of these mice did not reveal any impairment in β-cell function or glucose regulation, indicating the complexity underlying the study of lncRNA function.

Keywords: GLIS3; beta-cells; islets; lncRNA; pancreas.

Fig 1.. G3R1 is an islet-enriched long non-coding RNA controlled by GLIS3.

(A) RNA-seq analysis of islets from a pancreas-specific deletion of Glis3 (Glis3^Δpanc) revealed downregulation of G3R1. (B) Expression analysis of G3R1 across multiple mouse tissues, normalized to pancreas expression. Error bars indicate standard error of the mean, N=3.

Fig 2.. G3R1 is β-cell specific, cytoplasmic lncRNA within the islet.

In situ hybridization of 2-week old (A), e15.5 (B), and e17.5 (C) mouse pancreas for G3R1 (white) and Ins2 (green), with DAPI (blue) nuclear stain. Images shown are representative. (D) RT-qPCR analysis of nuclear and cytoplasmic RNA for G3R1, Glis3 (an mRNA), and Malat1 (a nuclear lncRNA). Cytoplasmic/Nuclear ratio was determined by calculating the relative amount of RNA in cytoplasmic and nuclear fractions of equal volume. Error bars indicate standard error of the mean, N=5.

Fig 3.. GLIS3 and other Islet transcription factors regulate G3R1 expression.

(A) ChIP-seq analysis of islet enriched transcription factors GLIS3, PDX1 (performed independently by two different labs), MAFA, NEUROD1, NKX6.1, NKX2.2, ISL1, LDB1, and FOXA2 revealed binding upstream of G3R1. Data is displayed via UCSC genome browser. (B) Luciferase assay of G3R1-URR1 in βTC-6 cells in the presence of overexpressed GLIS3, PDX1, NEUROD1, ISL1, NKX6.1, NKX2.2, PAX6, and MAFA. Error bars indicate standard deviation of a representative experiment.

Fig 4.. Two apparent human homologs of G3R1 display a similar tissue expression pattern.

(A) RT-qPCR expression analysis using primers that recognized both LOC105372871 and LOC105377190. Error bars indicate standard deviation of technical triplicates. ChIP-seq analysis of binding for FOXA2, MAFB, NKX2.2, NKX6.1, and PDX1 near (B) LOC105372871 and (C) LOC105377190.

Fig 5.. G3R1 knockout mice lack a detectable islet phenotype.

(A) CRISPR strategy for deletion of G3R1 exon 2, and genotyping indicating successful creation of a mouse lines with >80% deletion of G3R1. WT = Wild type genotyping, KO = Knockout genotyping. (B) Intraperitoneal glucose tolerance test of G3R1-KO mice, and both wild type and heterozygous littermates at 8-weeks of age. +/+ = 8, +/− = 4, −/− = 4. (C) Random blood glucose levels measured at 8-weeks of age. +/+ = 5, +/− = 5, −/− = 7. Error bars indicate standard error of the mean. Immunofluorescence staining of 8-week old pancreas from WT and G3R1-KO mice for (D) INSULIN and GLUCAGON, (E) INSULIN and MAFA, and (F) SYNAPTOPHYSIN and PDX1, as well as DAPI. Images shown are representative.