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Loss of TSC complex enhances gluconeogenesis via upregulation of Dlk1-Dio3 locus miRNAs

ABSTRACT: Loss of the tumor suppressor tuberous sclerosis complex 1 (Tsc1) in the liver promotes gluconeogenesis and glucose intolerance. We asked whether this could be attributed to aberrant expression of small RNAs. We preformed small-RNA sequencing on liver of Tsc1 knock-out mice, and found that miRNAs of the delta like homolog 1 (Dlk1) – deiodinase iodothyronine type III (Dio3) locus are upregulated in an mTORC1-dependent manner. Sustained mTORC1 signaling during development prevented CpG methylation and silencing of the Dlk1-Dio3 locus, thereby increasing miRNA transcription. Deletion of miRNAs encoded by the Dlk1-Dio3 locus reduced gluconeogenesis, glucose intolerance and fasting blood glucose levels. Thus, miRNAs contribute to the metabolic effects observed upon loss of TSC1 and hyperactivation of mTORC1 in the liver. Furthermore, we show that miRNA is a downstream effector of hyperactive mTORC1 signaling.

ORGANISM(S): Mus musculus

PROVIDER: GSE141361 | GEO | 2019/12/04

REPOSITORIES: GEO

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Project description:Our previous study demonstrated a significant upregulation of a large set of miRNAs at the genomic imprinted Dlk1-Dio3 locus in lymphocytes of diverse murine lupus-prone strains. The upregulation of Dlk1-Dio3 miRNAs in lupus-prone mice is correlated with the global DNA hypomethylation. In this study, by performing genome-wide DNA methylation analysis, we reported that Dlk1-Dio3 genomic region in CD4+ T cells of MRL/lpr mice was hypomethylated, further linking hypomethylation to the increased expression of Dlk1-Dio3 miRNAs in lupus. Then, we assessed the gene expression levels of enzymes that either write (DNA methyltransferases, DNMTs) or erase DNA methylation (Ten-eleven translation proteins, TETs) to understand the molecular contributor to the DNA hypomethylation in MRL/lpr CD4+ T cells. The expression levels of Dnmt1, Dnmt3b, Tet1, and Tet2 were significantly increased in CD4+ T cells of MRL/lpr mice, as well as in B6/lpr and B6.sle123 mice, compared to their respective control mice. These data indicate the significant involvement of the TETs-mediated active demethylation pathway rather than reduced DNMTs-mediated passive demethylation pathway in the hypomethylation of murine lupus CD4+ T cells. The transcription factor, early growth response 2 (EGR2) is critically involved in regulating T cell functions and autoimmunity. In this research, we found that Egr2 deletion in B6/lpr mice notably reduced methylation-sensitive Dlk1-Dio3 cluster miRNAs expression in CD4+ T cells. Surprisingly, even though EGR2 has been shown to induce DNA demethylation by recruiting TET2, we found that deleting Egr2 in B6/lpr mice induced a higher number of hypomethylated DMRs than hypermethylated DMRs at either whole genome or the Dlk1-Dio3 locus in CD4+ T cells of B6/lpr mice. These data are the first finding on the positive role of EGR2 on the expression of Dlk1-Dio3 cluster miRNAs in lupus mice. Given that Dlk1-Dio3 miRNAs target the major signaling pathways in autoimmunity, these data provide a new perspective in understanding the potential pathogenic role of upregulated EGR2 in lupus.

Project description:Epithelial-to-mesenchymal transition (EMT) and its reversed process mesenchymal-to-epithelial transition (MET) play a critical role in epithelial plasticity during development and cancer progression. Among important regulators of these cellular processes are non-coding RNAs (ncRNAs). The imprinted DLK1-DIO3 locus, containing numerous maternally expressed ncRNAs including the lncRNA maternally expressed gene 3 (MEG3) and a cluster of over 50 miRNAs, has been shown to be a modulator of stemness in embryonic stem cells and in cancer progression, potentially through the tumor suppressor role of MEG3. In this study we analyzed the expression pattern and functional role of ncRNAs from the DLK1-DIO3 locus in epithelial plasticity of the breast. We studied their expression in various cell types of breast tissue and revisit the role of the locus in EMT/MET using a breast epithelial progenitor cell line (D492) and its isogenic mesenchymal derivative (D492M). Marked upregulation of ncRNAs from the DLK1-DIO3 locus was seen after EMT induction in two cell line models of EMT. In addition, the expression of MEG3 and the maternally expressed ncRNAs was higher in stromal cells compared to epithelial cell types in primary breast tissue. We also show that expression of MEG3 is concomitant with the expression of the ncRNAs from the DLK1-DIO3 locus and its expression is therefore likely indicative of activation of all ncRNAs at the locus. MEG3 expression is correlated with stromal markers in normal tissue and breast cancer tissue and negatively correlated with the survival of breast cancer patients in two different cohorts. Overexpression of MEG3 using CRISPR activation in a breast epithelial cell line induced partial EMT and enriched for a basal-like phenotype. Conversely, knock down of MEG3 using CRISPR inhibition in a mesenchymal cell line reduced the mesenchymal and basal-like phenotype of the cell line. In summary our study shows that maternally expressed ncRNAs are markers of EMT and suggests that MEG3 is a novel regulator of EMT/MET in breast tissue. Nevertheless, further studies are needed to fully dissect the molecular pathways influenced by non-coding RNAs at the DLK1-DIO3 locus in breast tissue.

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Loss of TSC complex enhances gluconeogenesis via upregulation of Dlk1-Dio3 locus miRNAs

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