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Report

Teaser, summary, work performed and final results

Periodic Reporting for period 2 - TransGen RNA (Transgenerational regulation of glucose metabolism by noncoding RNAs )

Teaser

Summary

Obesity and Type 2 Diabetes are multifactorial diseases caused by epistatic interaction of genetic risk alleles and an obesogenic lifestyle. The discovery of >10,000s genes that do not encode for proteins (so-called \'long noncoding RNAs, lncRNAs\') extends the numbers of genes potentially involved in metabolic disease. Although scientific findings showed first functions for long noncoding RNAs in metabolic regulation, no comprehensive study to date addressed how obesity affects lncRNAs expression in metabolic tissues like livers or adipose tissue in mice as well as their functional role in metabolism.

In the first part of this project, we are interested in studying obesity-associated lncRNAs in liver and fat tissue. For this, we carried out next generation RNA sequencing (RNA-Seq) in livers from two different mouse models of obesity and identified first lncRNAs that exhibit positive or negative correlation with obesity. We devised a software pipeline for better detecting lncRNAs in RNA-Seq datasets. We now try to better understand the function of obesity lncRNAs with regards to their role in insulin sensitivity and glucose metabolism. We applied so-called \'gene knockdown\' approaches, where obesity lncRNAs are removed by intravenous injection of small-molecule lncRNA inhibitors. We also investigate those lncRNAs that are shared between laboratory mice and humans by measuring the amount of these evolutionarily conserved lncRNAs in liver biopsies from obese patients. To understand how and if obesity lncRNAs are relevant for glucose metabolism in mice, we generated mice in which we removed them using the so-called CRISPR-Cas9 technique and study their metabolic fitness.

In the second part of this project, we aim to understand why obesity strongly affects the expression of lncRNAs in sperm cells of obese mice. We previously found that diet-induced obesity in mice upregulates many lncRNAs in sperm. As lncRNAs are increasingly appreciated as potent regulators of gene activity, but also other regulatory processes in cells, we hypothesize that altered lncRNA levels might contribute on the peculiar observation that sperm from obese mice fosters offsprings with impaired metabolism.

If succesful, our studies could pave the way for mechanistically understanding why metabolically sick mice and humans give rise to metabolically impaired progeny. In case we reveal a sperm gene signature of obesity, our findings might even be utilized to identify sperm biopsies from potential fathers that carry risk of begetting metabolically compromised kids.

Work performed

The project is fully progressing as anticipated in the original proposal with early exiting scientific discoveries:

A first postdoctoral fellow with computational expertise was hired after beginning of the project and setup analysis pipelines for detecting lncRNA detection in RNA-seq data. He quantified lncRNA in adipose tissue and liver from two mouse models of obesity and type 2 diabetes and from human obesity liver samples. Using this data, we identified two candidate lncRNAs to investigate further. We also devised a software pipeline that increases the numbers of detected lncRNAs in RNA-seq data which will be of great benefit to the whole project (unpublished). Lastly, we created computational approaches that allow unexperienced users to compare their RNA-Seq data in an automated fashion alongside thousands of publicly available NGS datasets (obtained from ENCODE project, https://www.encodeproject.org/vv) using novel visualisation techniques. This software suite (called \'sonar; sâ€¢nR) was published recently (Klemm et al. BMC Genomics, 2019).

A second postdoc obtained crucial and existing biological insights into the physiology controlling lncRNA regulation in metabolic disease. By exposing mice to fasting/refeeding paradigms and performing RNA-seq, we identified a hitherto poorly understood signaling pathway in liver cells that represses many lncRNAs in humans and mice. Interestingly, we could not observe the same downregulation of protein-coding genes in obesity. A novel role for this novel signaling axis in metabolism could be shown in primary hepatocytes but also in mice. Intriguingly, we identified an lncRNA that we call \'lincIRS2\' which is repressed by this signaling axis in primary liver cells but also in liver and, when knocked out, triggered metabolic impairments already in lean knockout (KO) mice.

We also established techniques for generation of lncRNA KO mouse lines using CRISPR/Cas9. A manuscript describing the methodology was published in the journal \'Non-Coding RNA\' (Hansmeier et al.). These mouse ressources, in conjunction with ongoing lncRNA inhibition experiments in mice will dissect the functional role and the molecular nature of these lncRNAs. Further, these genetic mouse models constitute a great tool for procuring primary knockout cells for downstream functional studies. Collectively, the scientific milestones described in Aim 1.1-1.3 were successfully concluded, first lncRNA candidates identified and functional studies performed both in vitro and in vivo as described in Aim 2.1. Findings from these studies are currently under peer review at an interdisciplinary scientific journal.

In the last part of our ERC projects, where we are interested in sperm noncoding RNA expression, we hired an expert in mouse physiology, DNA methylation and gremline epigenetics. We tested and established protocols for isolation and RNA-Seq of motile, mature sperm from lean and obese mice. First rounds of sperm RNA-Seq from lean and obese mice is underway to test which lncRNAs in sperm are affected by metabolic disease.