Isolation of nuclei from mouse white adipose tissues for single-nucleus genomics.

Lipid-filled adipocytes are incompatible with droplet-based single-cell methods, such as 10x Genomics-based technology, thus restricting droplet-based single-cell analyses of adipose tissues to the stromal vascular fraction. To overcome this limitation and obtain cellular and molecular insight into adipose tissue composition and plasticity, single-nucleus sequencing-based technologies can be applied. Here, we provide an optimized protocol for nuclei isolation from mouse adipose tissues suitable for single-nucleus RNA sequencing. This allows for transcriptomic profiling of the entire adipose tissue at single-cell resolution. For complete details on the use of this protocol, please refer to Sárvári et al., 2021.

Bacteria-host transcriptional response during endothelial invasion by Staphylococcus aureus.

Staphylococcus aureus is the cause of serious vascular infections such as sepsis and endocarditis. These infections are notoriously difficult to treat, and it is believed that the ability of S. aureus to invade endothelial cells and persist intracellularly is a key mechanism for persistence despite ongoing antibiotic treatment. Here, we used dual RNA sequencing to study the simultaneous transcriptional response of S. aureus and human endothelial cells during in vitro infections. We revealed discrete and shared differentially expressed genes for both host and pathogen at the different stages of infection. While the endothelial cells upregulated genes involved in interferon signalling and antigen presentation during late infection, S. aureus downregulated toxin expression while upregulating genes related to iron scavenging. In conclusion, the presented data provide an important resource to facilitate functional investigations into host-pathogen interaction during S. aureus invasive infection and a basis for identifying novel drug target sites.

Plasticity of Epididymal Adipose Tissue in Response to Diet-Induced Obesity at Single-Nucleus Resolution.

Adipose tissues display a remarkable ability to adapt to the dietary status. Here, we have applied single-nucleus RNA-seq to map the plasticity of mouse epididymal white adipose tissue at single-nucleus resolution in response to high-fat-diet-induced obesity. The single-nucleus approach allowed us to recover all major cell types and to reveal distinct transcriptional stages along the entire adipogenic trajectory from preadipocyte commitment to mature adipocytes. We demonstrate the existence of different adipocyte subpopulations and show that obesity leads to disappearance of the lipogenic subpopulation and increased abundance of the stressed lipid-scavenging subpopulation. Moreover, obesity is associated with major changes in the abundance and gene expression of other cell populations, including a dramatic increase in lipid-handling genes in macrophages at the expense of macrophage-specific genes. The data provide a powerful resource for future hypothesis-driven investigations of the mechanisms of adipocyte differentiation and adipose tissue plasticity.

Co-Administration of Propionate or Protocatechuic Acid Does Not Affect DHA-Specific Transcriptional Effects on Lipid Metabolism in Cultured Hepatic Cells.

Long-chain n-3 polyunsaturated fatty acids (n-3 LC-PUFAs) are collectively recognized triglyceride-lowering agents, and their preventive action is likely mediated by changes in gene expression. However, as most studies employ fish oil, which contains a mixture of n-3 LC-PUFAs, the docosahexaenoic acid (DHA)-specific transcriptional effects on lipid metabolism are still unclear. The aim of the present study was to further elucidate the DHA-induced transcriptional effects on lipid metabolism in the liver, and to investigate the effects of co-administration with other bioactive compounds having effects on lipid metabolism. To this purpose, HepG2 cells were treated for 6 or 24 h with DHA, the short-chain fatty acid propionate (PRO), and protocatechuic acid (PCA), the main human metabolite of cyanidin-glucosides. Following supplementation, we mapped the global transcriptional changes. PRO and PCA alone had a very slight effect on the transcriptome; on the contrary, supplementation of DHA highly repressed the steroid and fatty acid biosynthesis pathways, this transcriptional modulation being not affected by co-supplementation. Our results confirm that DHA effect on lipid metabolism are mediated at least in part by modulation of the expression of specific genes. PRO and PCA could contribute to counteracting dyslipidemia through other mechanisms.

Multiple mechanisms regulate H3 acetylation of enhancers in response to thyroid hormone.

Hormone-dependent activation of enhancers includes histone hyperacetylation and mediator recruitment. Histone hyperacetylation is mostly explained by a bimodal switch model, where histone deacetylases (HDACs) disassociate from chromatin, and histone acetyl transferases (HATs) are recruited. This model builds on decades of research on steroid receptor regulation of transcription. Yet, the general concept of the bimodal switch model has not been rigorously tested genome wide. We have used a genomics approach to study enhancer hyperacetylation by the thyroid hormone receptor (TR), described to operate as a bimodal switch. H3 acetylation, HAT and HDAC ChIP-seq analyses of livers from hypo- and hyperthyroid wildtype, TR deficient and NCOR1 disrupted mice reveal three types of thyroid hormone (T3)-regulated enhancers. One subset of enhancers is bound by HDAC3-NCOR1 in the absence of hormone and constitutively occupy TR and HATs irrespective of T3 levels, suggesting a poised enhancer state in absence of hormone. In presence of T3, HDAC3-NCOR1 dissociates from these enhancers leading to histone hyperacetylation, suggesting a histone acetylation rheostat function of HDACs at poised enhancers. Another subset of enhancers, not occupied by HDACs, is hyperacetylated in a T3-dependent manner, where TR is recruited to chromatin together with HATs. Lastly, a subset of enhancers, is not occupied directly by TR yet requires TR for histone hyperacetylation. This indirect enhancer activation involves co-association with TR bound enhancers within super-enhancers or topological associated domains. Collectively, this demonstrates various mechanisms controlling hormone-dependent transcription and adds significant details to the otherwise simple bimodal switch model.

Transcriptional Dynamics of Hepatic Sinusoid-Associated Cells After Liver Injury.

BACKGROUND AND AIMS: Hepatic sinusoidal cells are known actors in the fibrogenic response to injury. Activated hepatic stellate cells (HSCs), liver sinusoidal endothelial cells, and Kupffer cells are responsible for sinusoidal capillarization and perisinusoidal matrix deposition, impairing vascular exchange and heightening the risk of advanced fibrosis. While the overall pathogenesis is well understood, functional relations between cellular transitions during fibrogenesis are only beginning to be resolved. At single-cell resolution, we here explored the heterogeneity of individual cell types and dissected their transitions and crosstalk during fibrogenesis. APPROACH AND RESULTS: We applied single-cell transcriptomics to map the heterogeneity of sinusoid-associated cells in healthy and injured livers and reconstructed the single-lineage HSC trajectory from pericyte to myofibroblast. Stratifying each sinusoidal cell population by activation state, we projected shifts in sinusoidal communication upon injury. Weighted gene correlation network analysis of the HSC trajectory led to the identification of core genes whose expression proved highly predictive of advanced fibrosis in patients with nonalcoholic steatohepatitis (NASH). Among the core members of the injury-repressed gene module, we identified plasmalemma vesicle-associated protein (PLVAP) as a protein amply expressed by mouse and human HSCs. PLVAP expression was suppressed in activated HSCs upon injury and may hence define hitherto unknown roles for HSCs in the regulation of microcirculatory exchange and its breakdown in chronic liver disease. CONCLUSIONS: Our study offers a single-cell resolved account of drug-induced injury of the mammalian liver and identifies key genes that may serve important roles in sinusoidal integrity and as markers of advanced fibrosis in human NASH.

Osteogenesis depends on commissioning of a network of stem cell transcription factors that act as repressors of adipogenesis.

Mesenchymal (stromal) stem cells (MSCs) constitute populations of mesodermal multipotent cells involved in tissue regeneration and homeostasis in many different organs. Here we performed comprehensive characterization of the transcriptional and epigenomic changes associated with osteoblast and adipocyte differentiation of human MSCs. We demonstrate that adipogenesis is driven by considerable remodeling of the chromatin landscape and de novo activation of enhancers, whereas osteogenesis involves activation of preestablished enhancers. Using machine learning algorithms for in silico modeling of transcriptional regulation, we identify a large and diverse transcriptional network of pro-osteogenic and antiadipogenic transcription factors. Intriguingly, binding motifs for these factors overlap with SNPs related to bone and fat formation in humans, and knockdown of single members of this network is sufficient to modulate differentiation in both directions, thus indicating that lineage determination is a delicate balance between the activities of many different transcription factors.

Author Correction: Osteogenesis depends on commissioning of a network of stem cell transcription factors that act as repressors of adipogenesis.

In the version of this article initially published, in the graph keys in Fig. 1i, the colors indicating 'Ob' and 'Ad' were red and blue, respectively, but should have been blue and red, respectively; the shapes indicating 'MUS' and 'BM' were a triangle and a square, respectively, but should have been a square and a triangle, respectively. The errors have been corrected in the HTML and PDF versions of the article.

[Screening for intracranial aneurysms].

This review states the reasons for considering screening for intracranial aneurysms in Denmark: if patients have two first-degree relatives with intracranial aneurysms, are 30-70 years old, do not have competing disorders, which could significantly shorten life expectancy, and subsequently in patients with autosomal dominant kidney disease and a family history of subarachnoid haemorrhage. MR angiography should be the imaging study of choice, unless contraindicated. Generally, the ethical consequences ought to be considered before carrying out screening.

Insulin signaling and reduced glucocorticoid receptor activity attenuate postprandial gene expression in liver.

Hepatic circadian gene transcription is tightly coupled to feeding behavior, which has a profound impact on metabolic disorders associated with diet-induced obesity. Here, we describe a genomics approach to uncover mechanisms controlling hepatic postprandial gene expression. Combined transcriptomic and cistromic analysis identified hundreds of circadian-regulated genes and enhancers controlled by feeding. Postprandial suppression of enhancer activity was associated with reduced glucocorticoid receptor (GR) and Forkhead box O1 (FOXO1) occupancy of chromatin correlating with reduced serum corticosterone levels and increased serum insulin levels. Despite substantial co-occupancy of feeding-regulated enhancers by GR and FOXO1, selective disruption of corticosteroid and/or insulin signaling resulted in dysregulation of specific postprandial regulated gene programs. In combination, these signaling pathways operate a major part of the genes suppressed by feeding. Importantly, the feeding response was disrupted in diet-induced obese animals, which was associated with dysregulation of several corticosteroid- and insulin-regulated genes, providing mechanistic insights to dysregulated circadian gene transcription associated with obesity.

Chromatin Immunoprecipitation for Identification of Protein-DNA Interactions in Human Cells.

Chromatin immunoprecipitation (ChIP) is a powerful technique allowing for investigation of protein-DNA interactions in living cells. Here, we provide a detailed step-by-step protocol for ChIP and highlight important considerations, challenges and pitfalls often encountered in the ChIP procedure. Furthermore, we present data of key quality control (QC) steps and exemplify material performance validation on transcription factor ChIP to provide a QC guide for setting up ChIP. Finally, we provide guidelines for scaling of the ChIP procedure to ChIP sequencing (ChIP-seq) and discuss important considerations associated with this.

Hierarchical role for transcription factors and chromatin structure in genome organization along adipogenesis.

The three dimensional folding of mammalian genomes is cell type specific and difficult to alter suggesting that it is an important component of gene regulation. However, given the multitude of chromatin-associating factors, the mechanisms driving the colocalization of active chromosomal domains and the role of this organization in regulating the transcription program in adipocytes are not clear. Analysis of genome-wide chromosomal associations revealed cell type-specific spatial clustering of adipogenic genes in 3T3-L1 cells. Time course analysis demonstrated that the adipogenic 'hub', sampled by PPARγ and Lpin1, undergoes orchestrated reorganization during adipogenesis. Coupling the dynamics of genome architecture with multiple chromatin datasets indicated that among all the transcription factors (TFs) tested, RXR is central to genome reorganization at the beginning of adipogenesis. Interestingly, at the end of differentiation, the adipogenic hub was shifted to an H3K27me3-repressive environment in conjunction with attenuation of gene transcription. We propose a stage-specific hierarchy for the activity of TFs contributing to the establishment of an adipogenic genome architecture that brings together the adipogenic genetic program. In addition, the repositioning of this network in a H3K27me3-rich environment at the end of differentiation may contribute to the stabilization of gene transcription levels and reduce the developmental plasticity of these specialized cells. DATABASE: All sequence data reported in this paper have been deposited at GEO (http://www.ncbi.nlm.nih.gov/geo/) (GSE92475).

Multi-omics Analyses of Starvation Responses Reveal a Central Role for Lipoprotein Metabolism in Acute Starvation Survival in C. elegans.

Starvation causes comprehensive metabolic changes, which are still not fully understood. Here, we used quantitative proteomics and RNA sequencing to examine the temporal starvation responses in wild-type Caenorhabditis elegans and animals lacking the transcription factor HLH-30. Our findings show that starvation alters the abundance of hundreds of proteins and mRNAs in a temporal manner, many of which are involved in central metabolic pathways, including lipoprotein metabolism. We demonstrate that premature death of hlh-30 animals under starvation can be prevented by knockdown of either vit-1 or vit-5, encoding two different lipoproteins. We further show that the size and number of intestinal lipid droplets under starvation are altered in hlh-30 animals, which can be rescued by knockdown of vit-1. Taken together, this indicates that survival of hlh-30 animals under starvation is closely linked to regulation of intestinal lipid stores. We provide the most detailed poly-omic analysis of starvation responses to date, which serves as a resource for further mechanistic studies of starvation.

Dynamic Rewiring of Promoter-Anchored Chromatin Loops during Adipocyte Differentiation.

Interactions between transcriptional promoters and their distal regulatory elements play an important role in transcriptional regulation; however, the extent to which these interactions are subject to rapid modulations in response to signals is unknown. Here, we use promoter capture Hi-C to demonstrate a rapid reorganization of promoter-anchored chromatin loops within 4 hr after inducing differentiation of 3T3-L1 preadipocytes. The establishment of new promoter-enhancer loops is tightly coupled to activation of poised (histone H3 lysine 4 mono- and dimethylated) enhancers, as evidenced by the acquisition of histone H3 lysine 27 acetylation and the binding of MED1, SMC1, and P300 proteins to these regions, as well as to activation of target genes. Intriguingly, formation of loops connecting activated enhancers and promoters is also associated with extensive recruitment of corepressors such as NCoR and HDACs, indicating that this class of coregulators may play a previously unrecognized role during enhancer activation.

RNA-binding protein PSPC1 promotes the differentiation-dependent nuclear export of adipocyte RNAs.

A highly orchestrated gene expression program establishes the properties that define mature adipocytes, but the contribution of posttranscriptional factors to the adipocyte phenotype is poorly understood. Here we have shown that the RNA-binding protein PSPC1, a component of the paraspeckle complex, promotes adipogenesis in vitro and is important for mature adipocyte function in vivo. Cross-linking and immunoprecipitation followed by RNA sequencing revealed that PSPC1 binds to intronic and 3'-untranslated regions of a number of adipocyte RNAs, including the RNA encoding the transcriptional regulator EBF1. Purification of the paraspeckle complex from adipocytes further showed that PSPC1 associates with the RNA export factor DDX3X in a differentiation-dependent manner. Remarkably, PSPC1 relocates from the nucleus to the cytoplasm during differentiation, coinciding with enhanced export of adipogenic RNAs. Mice lacking PSPC1 in fat displayed reduced lipid storage and adipose tissue mass and were resistant to diet-induced obesity and insulin resistance due to a compensatory increase in energy expenditure. These findings highlight a role for PSPC1-dependent RNA maturation in the posttranscriptional control of adipose development and function.

High fat diet-induced changes of mouse hepatic transcription and enhancer activity can be reversed by subsequent weight loss.

Epigenetic factors have been suggested to play an important role in metabolic memory by trapping and maintaining initial metabolic changes within the transcriptional regulatory machinery. In this study we fed mice a high fat diet (HFD) for seven weeks followed by additional five weeks of chow, to identify HFD-mediated changes to the hepatic transcriptional program that may persist after weight loss. Mice fed a HFD displayed increased fasting insulin levels, hepatosteatosis and major changes in hepatic gene transcription associated with modulation of H3K27Ac at enhancers, but no significant changes in chromatin accessibility, indicating that HFD-regulated gene transcription is primarily controlled by modulating the activity of pre-established enhancers. After return to the same body weight as chow fed control mice, the fasting insulin, glucose, and hepatic triglyceride levels were fully restored to normal levels. Moreover, HFD-regulated H3K27Ac and mRNA levels returned to similar levels as control mice. These data demonstrates that the transcription regulatory landscape in the liver induced by HFD is highly dynamic and can be reversed by weight loss. This provides hope for efficient treatment of early obesity-associated changes to hepatic complications by simple weight loss intervention without persistent reprograming of the liver transcriptome.

The KDM5 family is required for activation of pro-proliferative cell cycle genes during adipocyte differentiation.

The KDM5 family of histone demethylases removes the H3K4 tri-methylation (H3K4me3) mark frequently found at promoter regions of actively transcribed genes and is therefore generally considered to contribute to corepression. In this study, we show that knockdown (KD) of all expressed members of the KDM5 family in white and brown preadipocytes leads to deregulated gene expression and blocks differentiation to mature adipocytes. KDM5 KD leads to a considerable increase in H3K4me3 at promoter regions; however, these changes in H3K4me3 have a limited effect on gene expression per se. By contrast, genome-wide analyses demonstrate that KDM5A is strongly enriched at KDM5-activated promoters, which generally have high levels of H3K4me3 and are associated with highly expressed genes. We show that KDM5-activated genes include a large set of cell cycle regulators and that the KDM5s are necessary for mitotic clonal expansion in 3T3-L1 cells, indicating that KDM5 KD may interfere with differentiation in part by impairing proliferation. Notably, the demethylase activity of KDM5A is required for activation of at least a subset of pro-proliferative cell cycle genes. In conclusion, the KDM5 family acts as dual modulators of gene expression in preadipocytes and is required for early stage differentiation and activation of pro-proliferative cell cycle genes.

RNA-Seq and Mass-Spectrometry-Based Lipidomics Reveal Extensive Changes of Glycerolipid Pathways in Brown Adipose Tissue in Response to Cold.

Cold exposure greatly alters brown adipose tissue (BAT) gene expression and metabolism to increase thermogenic capacity. Here, we used RNA sequencing and mass-spectrometry-based lipidomics to provide a comprehensive resource describing the molecular signature of cold adaptation at the level of the transcriptome and lipidome. We show that short-term (3-day) cold exposure leads to a robust increase in expression of several brown adipocyte genes related to thermogenesis as well as the gene encoding the hormone irisin. However, pathway analysis shows that the most significantly induced genes are those involved in glycerophospholipid synthesis and fatty acid elongation. This is accompanied by significant changes in the acyl chain composition of triacylglycerols (TAGs) as well as subspecies-selective changes of acyl chains in glycerophospholipids. These results indicate that cold adaptation of BAT is associated with significant and highly species-selective remodeling of both TAGs and glycerophospholipids.

Acute TNF-induced repression of cell identity genes is mediated by NFκB-directed redistribution of cofactors from super-enhancers.

The proinflammatory cytokine tumor necrosis factor (TNF) plays a central role in low-grade adipose tissue inflammation and development of insulin resistance during obesity. In this context, nuclear factor κ-light-chain-enhancer of activated B cells (NFκB) is directly involved and required for the acute activation of the inflammatory gene program. Here, we show that the major transactivating subunit of NFκB, v-rel avian reticuloendotheliosis viral oncogene homolog A (RELA), is also required for acute TNF-induced suppression of adipocyte genes. Notably, this repression does not involve RELA binding to the associated enhancers but rather loss of cofactors and enhancer RNA (eRNA) selectively from high-occupancy sites within super-enhancers. Based on these data, we have developed models that, with high accuracy, predict which enhancers and genes are repressed by TNF in adipocytes. We show that these models are applicable to other cell types where TNF represses genes associated with super-enhancers in a highly cell-type-specific manner. Our results propose a novel paradigm for NFκB-mediated repression, whereby NFκB selectively redistributes cofactors from high-occupancy enhancers, thereby specifically repressing super-enhancer-associated cell identity genes.

Transcriptional activation by the thyroid hormone receptor through ligand-dependent receptor recruitment and chromatin remodelling.

A bimodal switch model is widely used to describe transcriptional regulation by the thyroid hormone receptor (TR). In this model, the unliganded TR forms stable, chromatin-bound complexes with transcriptional co-repressors to repress transcription. Binding of hormone dissociates co-repressors and facilitates recruitment of co-activators to activate transcription. Here we show that in addition to hormone-independent TR occupancy, ChIP-seq against endogenous TR in mouse liver tissue demonstrates considerable hormone-induced TR recruitment to chromatin associated with chromatin remodelling and activated gene transcription. Genome-wide footprinting analysis using DNase-seq provides little evidence for TR footprints both in the absence and presence of hormone, suggesting that unliganded TR engagement with repressive complexes on chromatin is, similar to activating receptor complexes, a highly dynamic process. This dynamic and ligand-dependent interaction with chromatin is likely shared by all steroid hormone receptors regardless of their capacity to repress transcription in the absence of ligand.