Open chromatin mapping and nucleosome positioning in human adipose tissue depots from lean vs. obese individuals
Prosjekt
- Prosjektnummer
- 2017079
- Ansvarlig person
- Yvonne Böttcher
- Institusjon
- Akershus universitetssykehus HF
- Prosjektkategori
- Postdoktorstipend
- Helsekategori
- Metabolic and Endocrine
- Forskningsaktivitet
- 1. Underpinning
Rapporter
Obesity is a major health problem and increases the risk for morbidity and mortality worldwide. Individual fat distribution can be a more accurate risk predictor for metabolic comorbidities than overall fat mass. Storing fat predominantly in the visceral adipose tissue strongly increases risk of developing metabolic diseases. Our research shall contribute to identifying depot-specific differences of adipose tissue that contribute to development of co-morbidities in obesity.
The importance of genetic and epigenetic regulation in obesity, which we reviewed (Metabolism 2019, 92:37-50), have prompted us to focus on the adipose regulome – the 1-2% of accessible (open) chromatin that controls gene regulation. For regulome investigations, we perform ‘Assay for Transposase-Accessible Chromatin’ combined with deep sequencing (ATAC-seq) with primary tissue and cells derived from human subcutaneous and visceral adipose depots. Our ATAC-seq data from pre-adipocytes are in good agreement with publicly available data for chromatin accessibility in adipocytes (ENCODE) generated by a preceding method detecting DNase I hypersensitivity by deep sequencing (DHS-seq). In order to comparatively analyze ATAC seq reads in visceral vs subcutaneous adipose tissue, we applied, amongst several protocols a method reported earlier (Nat Methods 2017,14(10):959-962) to obtain nuclei of high purity. Our optimized protocol has allowed us to generate genome-wide seq results for both subcutaneous and visceral adipose tissue. Further, we have successfully set up a complete analysis pipeline for a comprehensive bioinformatic analysis within a secure environment at UiO (service for sensitive data TSD) providing us with a closed platform. Our analyses resulted for the first time in an unrivaled dataset providing us with genome wide ATAC seq data from visceral and subcutaneous depots. The results of our bioinformatic analysis show that in principle component analyses differentially accessible regions reside in two different groups belonging to the different fat depots. This is in agreement with our previous results with respect to DNA methylation and gene expression, which significantly differ between the two depots. Further, when categorizing differentially accessible, open chromatin regions into various genomic features we find most of the differentially accessible regions in regulatory regions (such as promoter regions /UTRs) but also in intronic and intergenic distal regions. By using the R package limma for paired differential analysis on a genome wide basis we find that most of the differentially accessible regions are more accessible in one of the two tissue types whilst a smaller number of regions seems to be differentially regulated and thereby less open when compared to the other depot. These regions include also top hits we have identified earlier, but also new candidate genes including several demethylases which are promising candidate for future studies. Moreover, when comparing visceral and subcutaneous adipose tissue in insulin resistant vs insulin sensitive individuals we find that the strongest differences in chromatin accessibility results from depot specificity rather from metabolic stability.
Collaborations with leading obesity experts from Oslo (Ahus/Aker Hospital/UiO), Tønsberg (Vestfold Hospital), and Bergen (UiB/Haukeland Hospital) have been established in this project and strengthened at several annual national meetings in Norway (e. g. at ‘Fedmeforskningsdagene’ 2017, 2018, and 2019) and at international conferences (e. g. at EASD 2018,2019, and at the eGMD/3rd LIMIOR 2019 obesity meeting co-organized by the PI). Due to the corona virus pandemic, no international conferences have been attended in 2020.
Our study provides novel knowledge about the open chromatin landscape in different depots of human adipose tissue and can help to better understand how fat distribution and depot specificity is related to metabolic co-morbidities.
Our study shall help to better understand why individuals storing adipose tissue more predominantly in visceral compartments have a higher risk for metabolic comorbidities. Here, we have for the first time, analyzed the open chromatin landscape in two of the main adipose tissue depots and have compared these to each other.
Treating obesity and its sequelae causes a high cost-burden in Norway annually, challenging the national health care system. Therefore, a better understanding of function and differences of adipose tissue depots can help to develop personal therapies in the future (“Good Health”, goal 3 of 17 UN goals). We work on identifying further mechanisms that may contribute to developing obesity-related complications. Our project can therefore significantly contribute to a better understanding for the origin of these diseases in the short term and will help in the long-term to identify reasons that can be used as targets to hamper susceptibility to developing obesity-related co-morbidities. The here described research project is therefore of significant impact to patients with obesity.
NO
Obesity is a leading risk for morbidity and mortality worldwide and storing fat in adipose tissue of the visceral compartment strongly increases risk of developing metabolic disease. We aim at identifying depot-specific differences of adipose tissues that contribute to development of co-morbidities in obesity.The importance of genetic and epigenetic regulation in obesity, which we reviewed recently (Metabolism 2019, 92:37-50), have prompted us to focus more on the adipocyte regulome – the 1-2% of accessible (open) chromatin that cell type-specifically control gene regulation. For regulome investigations, we are performing ‘Assay for Transposase-Accessible Chromatin’ combined with deep sequencing (ATAC-seq) with primary tissue and cells derived from the subcutaneous and visceral adipose depots. The technique is clinically compatible with minute sample amounts and has the advantage of robustly identifying accessible chromatin regions (Nat Methods 2017,14(10):959-962; Nat Methods 2013,10(12):1213-8). Our initial work focused on optimizing culture conditions for primary preadipocyte growth and differentiation and harvest and isolation of cell fractions (e.g. protein, RNA) to be used for analyses parallel to ATAC-seq. Importantly, the ATAC-seq procedure provided us with the first genome-wide view of the regulatory landscape that poises preadipocytes for adipogenic differentiation. Our ATAC-seq data are in good agreement with publicly available data for chromatin accessibility in adipocytes (ENCODE) generated by a preceding method detecting DNase I hypersensitivity by deep sequencing (DHS-seq). Among other genomic locations of interest, we observe multiple accessible sites in the Hox gene clusters that play an emerging role in adipose development and depot-specificity. Our efforts are now focused on ATAC-seq in visceral and subcutaneous adipose tissues from morbidly obese patients undergoing bariatric surgery and control patients with normal weight subjected to cholecystectomy. In preparation for this, we have among several protocols tested out a novel protocol by Corces et al. (Nat Methods 2017,14(10):959-962) on frozen and fresh material to obtain nuclei of high purity. Our optimized protocol has allowed us to generate genome-wide sequencing results for both subcutaneous and visceral fat from bariatric and cholecystectomy patients. These data are to be analyzed by our own pipeline established in the service for sensitive data (TSD) environment, a secure and closed platform for sensitive data provided by the University of Oslo.
Regarding specimens from human subjects, several steps have been taken to obtain adipose tissue biobank material and establish our own biobank from bariatric surgery patient (with established protocols for DNA investigations and RNA and protein expression analyses that are not trivial procedures in adipose tissues). Collaborations with leading obesity experts from Oslo (Ahus/Aker Hospital/UiO), Tønsberg (Vestfold Hospital), and Bergen (UiB/Haukeland Hospital) have been established and strengthened at annual national meetings (e.g. at ‘Fedmeforskningsdagene’ 2019) and at international conferences (e.g. at EASD 2019, and at the recent eGMD/3rd LIMIOR 2019 obesity meeting co-organized by the PI). In addition to this, we have established isolation procedures for adipocytes and stromal-vascular cells (SVCs) that are subjected to ATAC-seq and RNA-seq.
Our study will provide unprecedented information about defining features that dictate depot-specific differences between fat depots in lean versus obese individuals. It will contribute to identify factors in visceral fat that can be targeted to stall progression of obesity and/or its comorbidities including diabetes and cardiovascular diseases.
No
Obesity is a leading risk for morbidity and mortality worldwide and storing fat in adipose tissue of the visceral compartment strongly increases risk to develop metabolic dysfunction (compared to subcutaneous fat storage). We aim at identifying depot-specific differences of adipose tissue that contribute to co-morbidities.For this, we are applying an array of genome-wide methods allowing scrutiny of the genetic and epigenetic features that make up the chromatin landscape of adipocytes. Previous and ongoing work in our lab has already identified critical differences in DNA methylation pattern between subcutaneous and visceral fat. These differences have prompted us to focus more on the adipocyte regulome – the 1-2% of accessible (open) chromatin that cell type-specifically control gene regulation. The narrow window of open chromatin provides the advantage of allowing for focused analysis of the most significant part of the adipocyte genome, while cancelling out remaining genomic noise that provides minimal information about cell type identity and status. For regulome investigations, ‘Assay for Transposase-Accessible Chromatin’ combined with deep sequencing (ATAC-seq) is being performed with primary human adipocytes derived from subcutaneous and visceral depots. The technique was proven to be clinically compatible with minute sample amounts and has the advantage of robustly identifying accessible chromatin regions. Accordingly, regulome characterization of cultured subcutaneous and visceral adipocytes will be performed along with experiments carried out with corresponding biobank material obtained from morbidly obese patients undergoing bariatric surgery.
The initial work focused on optimizing culture conditions for primary preadipocyte growth and differentiation and harvest and isolation of cell fractions (e.g. protein, RNA, and exosomes) to be used for analyses parallel to ATAC-seq. Importantly, the ATAC-seq procedure has now also been established and provided us with the first genome-wide view of the regulatory landscape that poises preadipocytes for adipogenic differentiation. Our ATAC-seq data are in good agreement with publicly available data for chromatin accessibility in adipocytes (ENCODE), which were generated by a previous method detecting DNase I hypersensitivity by deep sequencing (DHS-seq). Among other genomic locations of interest, we observe multiple accessible sites in the Hox gene clusters that play an emerging role in adipose development and depot-specificity. In ongoing efforts, we are now conducting experiments with human adipocytes derived from visceral and subcutaneous adipose tissue from bariatric surgery patients. In preparation for this, several steps have been taken to obtain adipose tissue biobank material and establish our own biobank from bariatric surgery patients. Collaborations with leading obesity experts from Tønsberg and Bergen were discussed under the symposium ‘Fedmeforskningsdagene’ (2018) and at the European Association for the Study of Diabetes (EASD, 2018) with collaborators from Germany.
Collectively, our studies will provide unprecedented information about defining features that dictate depot-specific differences between subcutaneous and visceral fat in lean versus obese individuals. It is conceivable that this information will further the opportunity to identify factors in visceral fat that can be targeted to stall progression of obesity and/or its comorbidities including diabetes and cardiovascular diseases.
Nei
Obesity is a leading risk for morbidity and mortality worldwide and storing adipose tissue (AT) in visceral areas strongly increases risk to develop metabolic dysfunctions compared to storing AT subcutaneously. We aim at identifying depot-specific differences of AT contributing to co-morbidities.We aim at identifying depot-specific differences of AT contributing to co-morbidities.We are applying an array of genome-wide methods allowing scrutiny of the genetic and epigenetic features that make up the chromatin landscape of adipocytes. Previous and ongoing work in our lab has already identified critical differences in DNA methylation pattern between subcutaneous and visceral fat. These differences have now prompted us to focus more on the adipocyte regulome – the 1-2% of accessible (open) chromatin that cell type-specifically control gene regulation. The narrow window of open chromatin provides the advantage of allowing us to focus on the most significant part of the adipocyte genome, while cancelling out remaining genomic noise that provides minimal information about cell type identity and status. For regulome investigations, Assay for Transposase-Accessible Chromatin’ combined with deep sequencing (ATAC-seq) is currently being established with primary human adipocytes derived from subcutaneous and visceral depots. The technique was proven to be clinically compatible with minute sample amounts and has the advantage of robustly identifying accessible chromatin regions. Accordingly, regulome characterization of cultured subcutaneous and visceral adipocytes will be followed by experiments carried out with corresponding biobank material obtained from morbidly obese patients undergoing bariatric surgery.
The project startet in October 2017 and initial work has been focusing on optimizing culture conditions for primary pre-adipocyte growth and differentiation and harvest and isolation of cell fractions (protein, RNA, and exosomes) to be used for analyses parallel to ATAC-seq. We find that cells from both visceral and subcutaneous adipose tissue grow well in conventional growth medium (Lonza Poietics) on specialized plastic (Corning Primaria). However, they are very sensitive to physiological stresses (e.g. shear stress, inflammation, hypoxia, oxidative stress, and nutrient toxicity) and needs to be handled with gentle care to avoid myofibroblast-like activation. The preadipocytes – in particular from visceral adipose tissue – can only be propagated for few passages before they lose their proliferation potential and become senescent. Importantly, this is not a major obstacle for the project given the great sensitivity of the ATAC-seq method, which can be applied routinely to 25,000 – 50,000 cells and has been shown to provide reasonable detection in as little as 500 cells (Corces et al. 2017). In addition to our cell culture experiments, several early steps have been taken to obtain adipose tissue biobank material and establish our own biobank from bariatric surgery patients. Collaborations with leading obesity experts from Tønsberg and Bergen have been established and discussed under the symposium ‘Fedmeforskningsdagene 2017’ held in Tønsberg in the autumn. These collaborations and a longstanding flourishing collaboration with Leipzig, Germany will provide us with biobank material for ATAC-seq experiments.
Collectively, our studies will provide unprecedented information about defining features that dictate depot-specific differences between subcutaneous and visceral fat in lean versus obese individuals. It is conceivable that this information will provide the opportunity to identify factors in visceral fat that can be targeted to stall progression of obesity and/or its comorbidities including diabetes and cardiovascular diseases.
NEI
Deltagere
- Matthias Blüher Prosjektdeltaker
- Johan Fernø Prosjektdeltaker
- Simon E Nitter Dankel Prosjektdeltaker
- Gunnar Mellgren Prosjektdeltaker
- Jens Kristoffer Hertel Prosjektdeltaker
- Jøran Hjelmesæth Prosjektdeltaker
- Lars la Cour Poulsen Postdoktorstipendiat (finansiert av denne bevilgning)
- Yvonne Böttcher Forskningsgruppeleder
eRapport er utarbeidet av Sølvi Lerfald og Reidar Thorstensen, Regionalt kompetansesenter for klinisk forskning, Helse Vest RHF, og videreutvikles av de fire RHF-ene i fellesskap, med støtte fra Helse Vest IKT
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