The Dysregulation of the DLK1-MEG3 Locus in Islets From Patients With Type 2 Diabetes Is Mimicked by Targeted Epimutation of Its Promoter With TALE-DNMT Constructs

Abstract

Type 2 diabetes mellitus (T2DM) is characterized by the inability of the insulin-producing β-cells to overcome insulin resistance. We previously identified an imprinted region on chromosome 14, the DLK1-MEG3 locus, as being downregulated in islets from humans with T2DM. In this study, using targeted epigenetic modifiers, we prove that increased methylation at the promoter of Meg3 in mouse βTC6 β-cells results in decreased transcription of the maternal transcripts associated with this locus. As a result, the sensitivity of β-cells to cytokine-mediated oxidative stress was increased. Additionally, we demonstrate that an evolutionarily conserved intronic region at the MEG3 locus can function as an enhancer in βTC6 β-cells. Using circular chromosome conformation capture followed by high-throughput sequencing, we demonstrate that the promoter of MEG3 physically interacts with this novel enhancer and other putative regulatory elements in this imprinted region in human islets. Remarkably, this enhancer is bound in an allele-specific manner by the transcription factors FOXA2, PDX1, and NKX2.2. Overall, these data suggest that the intronic MEG3 enhancer plays an important role in the regulation of allele-specific expression at the imprinted DLK1-MEG3 locus in human β-cells, which in turn impacts the sensitivity of β-cells to cytokine-mediated oxidative stress.

DNA damage dependent hypomethylation regulates the pro-angiogenic LncRNA MEG9

“…Kamesmaran et al confirmed how susceptible the DLK1-DIO3 cluster is to methylation by targeting the specific promoter of lncRNA MEG3 in islets from type 2 Diabetes patients [24]. We have observed in our profiles that MEG9 and MEG3 respond in opposite directions to DNA damage and growth factor responses.…”

MicroRNA Networks in Pancreatic Islet Cells: Normal Function and Type 2 Diabetes

“…Also, as mentioned above, expression of miRNAs from this region is downregulated in T2D islets (16). Interestingly, experiments performed in mouse insulinoma bTC6 cells in which DNA methyltransferases in this region were activated resulted in increased methylation, reduced miRNA expression, and increased sensitivity to cytokine-induced b-cell death (43). Altogether, the DLK1-MEG3 region and the miRNAs involved have the capacity to promote b-cell expansion and protect against b-cell death prior to diabetes development; failure leads to full-blown diabetes.…”

Epigenetic regulation of insulin action and secretion – role in the pathogenesis of type 2 diabetes

“…Several guide systems exist including transcription activator‐like effector (TALE), zink‐finger proteins and Clustered regularly interspaced palindromic repeats (CRISPR) – catalytically inactive Cas9 (dCas9) (CRISPR‐dCas9) RNA‐guided DNA targeting. Only a few studies have used this approach in cell types of importance for diabetes [67,72,73]. Ou et al [67] used the TALE‐TET1 system to demethylate the imprinted control region 2 (ICR2) near CDKN1C.…”

“…Liu et al [72] used targeted demethylation of the distal MyoD enhancer by dCas9‐Tet1 and managed to reprogram fibroblasts into myoblasts. Moreover, using this approach, Kameswaran et al identified an intronic enhancer that seems to regulate allele‐specific expression at the imprinted DLK1‐MEG3 locus [73]. Combining this approach with the identification of epigenetic changes in humans will help answering whether identified changes of specific genomic regions cause T2D‐associated phenotypes such as insulin resistance and decreased insulin secretion.…”