Periodic Reporting for period 4 - TransGen RNA (Transgenerational regulation of glucose metabolism by noncoding RNAs )
Periodo di rendicontazione: 2021-01-01 al 2021-12-31
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 successful, 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.
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 successful, 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.
The project fully progressed as anticipated in the original proposal with early exiting scientific discoveries:
A 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 first candidate lncRNAs to investigate . We 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. 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 Visual Analytic visualisation principles. This software suite (called 'sonar; s•nR) was published recently (Klemm et al. BMC Genomics, 2019).
Another wet-lab postdoc obtained crucial and existing biological insights into the physiology controlling lncRNA regulation in metabolic disease. By exposing mice to fasting/re-feeding paradigms and performing RNA-seq, we identified a novel signaling pathway in liver cells that represses many lncRNAs in humans and mice. Interestingly, we could not observe the same down-regulation of protein-coding genes in obesity. A role for this specific transcription factor TF) termed MAFG, and MAFG-dependent roles in metabolism were shown in primary hepatocytes and mice. We also identified a MAFG-dependent 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 knockout mice. The findings from this study were published recently (Pradas-Juni et al Nat Commun 2020). We also established techniques for generation of lncRNA KO mouse lines using CRISPR/Cas9. A manuscript describing the methodology was published recently (Hansmeier et al. Noncoding RNA 2019).
We also identified another LncRNA termed H19, which exerted instrumental roles in controlling thermogenic activation in (brown) adipocytes, a cell type with anti-obesity properties. We found that H19 drives brown adipose tissue (BAT) catabolism by epigenetic regulation of mono-allelically expressed ('imprinted') genes in BAT. We found that human H19 in human adipose tissue also inversely correlated with parameters of metabolic health (e.g. BMI). A manuscript describing these findings were recently published (Schmidt et al. Nat Commun 2018).
In the last part of our ERC projects, where we are interested in sperm noncoding RNA expression and the epigenetic effects of paternal (from the father) obesity on progeny metabolic health, we hired an expert in mouse physiology, DNA methylation and gametic (sperm) epigenetics. We established protocols for isolation and RNA-Seq of mature sperm from lean and obese mice and found that paternal obesity (F0 generation) is instrumental for controlling metabolism in offsprings (F1 generation) . Based on multi -omic studies (transcriptomics, metabolomics, proteomics, small noncoding transcriptomics) in liver and adipose tissue from F0 and F1 generation, we could delineate a novel signaling axis where obesity-associated induction of miRNA miR-Let7 in obese adipocytes and sperm is coupled to metabolic deterioration in F0 and F1. The findings are currently prepared for publication in a high-impact journal in the field.
A 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 first candidate lncRNAs to investigate . We 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. 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 Visual Analytic visualisation principles. This software suite (called 'sonar; s•nR) was published recently (Klemm et al. BMC Genomics, 2019).
Another wet-lab postdoc obtained crucial and existing biological insights into the physiology controlling lncRNA regulation in metabolic disease. By exposing mice to fasting/re-feeding paradigms and performing RNA-seq, we identified a novel signaling pathway in liver cells that represses many lncRNAs in humans and mice. Interestingly, we could not observe the same down-regulation of protein-coding genes in obesity. A role for this specific transcription factor TF) termed MAFG, and MAFG-dependent roles in metabolism were shown in primary hepatocytes and mice. We also identified a MAFG-dependent 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 knockout mice. The findings from this study were published recently (Pradas-Juni et al Nat Commun 2020). We also established techniques for generation of lncRNA KO mouse lines using CRISPR/Cas9. A manuscript describing the methodology was published recently (Hansmeier et al. Noncoding RNA 2019).
We also identified another LncRNA termed H19, which exerted instrumental roles in controlling thermogenic activation in (brown) adipocytes, a cell type with anti-obesity properties. We found that H19 drives brown adipose tissue (BAT) catabolism by epigenetic regulation of mono-allelically expressed ('imprinted') genes in BAT. We found that human H19 in human adipose tissue also inversely correlated with parameters of metabolic health (e.g. BMI). A manuscript describing these findings were recently published (Schmidt et al. Nat Commun 2018).
In the last part of our ERC projects, where we are interested in sperm noncoding RNA expression and the epigenetic effects of paternal (from the father) obesity on progeny metabolic health, we hired an expert in mouse physiology, DNA methylation and gametic (sperm) epigenetics. We established protocols for isolation and RNA-Seq of mature sperm from lean and obese mice and found that paternal obesity (F0 generation) is instrumental for controlling metabolism in offsprings (F1 generation) . Based on multi -omic studies (transcriptomics, metabolomics, proteomics, small noncoding transcriptomics) in liver and adipose tissue from F0 and F1 generation, we could delineate a novel signaling axis where obesity-associated induction of miRNA miR-Let7 in obese adipocytes and sperm is coupled to metabolic deterioration in F0 and F1. The findings are currently prepared for publication in a high-impact journal in the field.
The main progress beyond state-of-the-art was our finding of hitherto unknown and pivotal roles for adipocyte and gametic miRNAs in shaping epigenetic inheritance of metabolic health. We hypothesize that physical miRNA transfer, potentially via circulating microvesicles termed 'exosomes', between adipocytes and sperm might occur, which would explain the unusually concordant regulation of miRNAs in obese adipocytes and (transcriptionally silent) mature sperm cells. The existent of such a signaling axis would be ground-breaking and would emphasize the notion of adipocytes being main 'sentinal and interlocator' cell types involve in shaping epigenetic hereditary responses and function of the reproductive apparatus. This concept is the foundation for an ERC Consolidator Grant application in 2021 termed EXO-SOMA and whose main hypotheses are illustrated in the attached figure.
This project has been concluded as of 12/2021.
This project has been concluded as of 12/2021.