Regulation of nuclear transcription by mitochondrial RNA in endothelial cells.

Chromatin-associated RNAs (caRNAs) form a relatively poorly recognized layer of the epigenome. The caRNAs reported to date are transcribed from the nuclear genome. Here, leveraging a recently developed assay for detection of caRNAs and their genomic association, we report that mitochondrial RNAs (mtRNAs) are attached to the nuclear genome and constitute a subset of caRNA, thus termed mt-caRNA. In four human cell types analyzed, mt-caRNAs preferentially attach to promoter regions. In human endothelial cells (ECs), the level of mt-caRNA-promoter attachment changes in response to environmental stress that mimics diabetes. Suppression of a non-coding mt-caRNA in ECs attenuates stress-induced nascent RNA transcription from the nuclear genome, including that of critical genes regulating cell adhesion, and abolishes stress-induced monocyte adhesion, a hallmark of dysfunctional ECs. Finally, we report increased nuclear localization of multiple mtRNAs in the ECs of human diabetic donors, suggesting many mtRNA translocate to the nucleus in a cell stress and disease-dependent manner. These data nominate mt-caRNAs as messenger molecules responsible for mitochondrial-nuclear communication and connect the immediate product of mitochondrial transcription with the transcriptional regulation of the nuclear genome.

Inhibition of DNMT1 methyltransferase activity via glucose-regulated O-GlcNAcylation alters the epigenome.

The DNA methyltransferase activity of DNMT1 is vital for genomic maintenance of DNA methylation. We report here that DNMT1 function is regulated by O-GlcNAcylation, a protein modification that is sensitive to glucose levels, and that elevated O-GlcNAcylation of DNMT1 from high glucose environment leads to alterations to the epigenome. Using mass spectrometry and complementary alanine mutation experiments, we identified S878 as the major residue that is O-GlcNAcylated on human DNMT1. Functional studies in human and mouse cells further revealed that O-GlcNAcylation of DNMT1-S878 results in an inhibition of methyltransferase activity, resulting in a general loss of DNA methylation that preferentially occurs at partially methylated domains (PMDs). This loss of methylation corresponds with an increase in DNA damage and apoptosis. These results establish O-GlcNAcylation of DNMT1 as a mechanism through which the epigenome is regulated by glucose metabolism and implicates a role for glycosylation of DNMT1 in metabolic diseases characterized by hyperglycemia.

How their environment influences endothelial cells.

Changes in gene expression in cultured endothelial cells can be partially reversed by simulating in vivo conditions.

Vascular Regulation by Super Enhancer-Derived LINC00607.

Vascular endothelial cells (ECs) play a pivotal role in whole body homeostasis. Recent advances have revealed enhancer-associated long non-coding RNAs (lncRNAs) as essential regulators in EC function. We investigated LINC00607, a super enhancer-derived lncRNA (SE-lncRNA) in human arteries with an emphasis on ECs. Based on public databases and our single cell RNA-sequencing (scRNA-seq) data from human arteries collected from healthy and diabetic donors, we found that LINC00607 is abundantly expressed in the arteries and its level is increased in diabetic humans. Using RNA-sequencing, we characterized the transcriptomes regulated by LINC00607 in ECs and vascular smooth muscle cells (VSMCs) and in basal and diabetic conditions in ECs. Furthermore, through transcriptomic and promoter analysis, we identified c-Myc as an upstream transcription factor of LINC00607. Finally, using scRNA-seq, we demonstrated that modified antisense oligonucleotide inhibitor of LINC00607 can reverse dysfunctional changes induced by high glucose and TNFα in ECs. Collectively, our study demonstrates a multi-pronged approach to characterize LINC00607 in vascular cells and its gene regulatory networks in ECs and VSMCs. Our findings provide new insights into the regulation and function of SE-derived lncRNAs in both vascular homeostasis and dysfunction in a cell-type and context-dependent manner, which could have a significant impact on our understanding of epigenetic regulation implicated in cardiovascular health and diseases like diabetes.

Endothelium-gut communication: IGF-1Rs crosstalk with microbiota.

The gut, with its extensive microbiota, plays a fundamental role in metabolism. While alterations of the gut microbiota can induce dysfunction of the endothelium, it remains unclear whether the endothelium can directly impact the gut microbiota. To answer this question, in this issue of EMBO Reports Haywood and colleagues deployed a murine model with endothelial-specific overexpression of human insulin-like growth factor-1 receptor (IGF-1R), termed hIGFREO mice (Haywood et al, 2021). When fed a high-fat diet, hIGFREO mice gained less weight and adiposity, with improved glucose tolerance, as compared to their wild-type littermates. Such protection was attributed to the difference in gut microbiota, exemplified by an increase in the beneficial genus Akkermansia. Furthermore, depletion of microbiota through broad-spectrum antibiotics nullified the advantageous metabolic phenotype observed. Collectively, these findings demonstrate a novel communication axis between the endothelium and the gut wall, specifically through endothelial IGF-1R modulation of gut microbiota, that promotes whole body metabolic homeostasis.

Mapping RNA-chromatin interactions by sequencing with iMARGI.

RNA-chromatin interactions represent an important aspect of the transcriptional regulation of genes and transposable elements. However, analyses of chromatin-associated RNAs (caRNAs) are often limited to one caRNA at a time. Here, we describe the iMARGI (in situ mapping of RNA-genome interactome) technique, which is used to discover caRNAs and reveal their respective genomic interaction loci. iMARGI starts with in situ crosslinking and genome fragmentation, followed by converting each proximal RNA-DNA pair into an RNA-linker-DNA chimeric sequence. These chimeric sequences are subsequently converted into a sequencing library suitable for paired-end sequencing. A standardized bioinformatic software package, iMARGI-Docker, is provided to decode the paired-end sequencing data into caRNA-DNA interactions. Compared to its predecessor MARGI (mapping RNA-genome interactions), the number of input cells for iMARGI is 3-5 million (a 100-fold reduction), experimental time is reduced, and clear checkpoints have been established. It takes a few hours a day and a total of 8 d to complete the construction of an iMARGI sequencing library and 1 d to carry out data processing with iMARGI-Docker.