DNA methylation is a defining feature of mammalian cellular identity and is essential for normal development. Most cell types, except germ cells and pre-implantation embryos, display relatively ...stable DNA methylation patterns, with 70-80% of all CpGs being methylated. Despite recent advances, we still have a limited understanding of when, where and how many CpGs participate in genomic regulation. Here we report the in-depth analysis of 42 whole-genome bisulphite sequencing data sets across 30 diverse human cell and tissue types. We observe dynamic regulation for only 21.8% of autosomal CpGs within a normal developmental context, most of which are distal to transcription start sites. These dynamic CpGs co-localize with gene regulatory elements, particularly enhancers and transcription-factor-binding sites, which allow identification of key lineage-specific regulators. In addition, differentially methylated regions (DMRs) often contain single nucleotide polymorphisms associated with cell-type-related diseases as determined by genome-wide association studies. The results also highlight the general inefficiency of whole-genome bisulphite sequencing, as 70-80% of the sequencing reads across these data sets provided little or no relevant information about CpG methylation. To demonstrate further the utility of our DMR set, we use it to classify unknown samples and identify representative signature regions that recapitulate major DNA methylation dynamics. In summary, although in theory every CpG can change its methylation state, our results suggest that only a fraction does so as part of coordinated regulatory programs. Therefore, our selected DMRs can serve as a starting point to guide new, more effective reduced representation approaches to capture the most informative fraction of CpGs, as well as further pinpoint putative regulatory elements.
Beige and brown adipocytes generate heat in response to reductions in ambient temperature. When warmed, both beige and brown adipocytes exhibit morphological “whitening,” but it is unknown whether or ...to what extent this represents a true shift in cellular identity. Using cell-type-specific profiling in vivo, we uncover a unique paradigm of temperature-dependent epigenomic plasticity of beige, but not brown, adipocytes, with conversion from a brown to a white chromatin state. Despite this profound shift in cellular identity, warm whitened beige adipocytes retain an epigenomic memory of prior cold exposure defined by an array of poised enhancers that prime thermogenic genes for rapid response during a second bout of cold exposure. We further show that a transcriptional cascade involving glucocorticoid receptor and Zfp423 can drive warm-induced whitening of beige adipocytes. These studies identify the epigenomic and transcriptional bases of an extraordinary example of cellular plasticity in response to environmental signals.
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•Cell-type-specific RNA-seq identify markers for white, brown, and beige adipocytes•Beige adipocytes exhibit reprogramming between white and brown chromatin states•Warmed beige adipocytes retain an epigenomic memory of prior cold exposure•A GR-Zfp423 pathway mediates beige adipocyte whitening during warming
Both beige and brown adipocytes “whiten” upon warming. Roh et al. elegantly show that beige, but not brown, adipocytes undergo temperature-dependent reprogramming between brown- and white-like states, while retaining epigenomic memory of prior cold exposure. A transcriptional cascade underlies this plasticity response to environmental signals.
The hypothalamic arcuate-median eminence complex (Arc-ME) controls energy balance, fertility and growth through molecularly distinct cell types, many of which remain unknown. To catalog cell types in ...an unbiased way, we profiled gene expression in 20,921 individual cells in and around the adult mouse Arc-ME using Drop-seq. We identify 50 transcriptionally distinct Arc-ME cell populations, including a rare tanycyte population at the Arc-ME diffusion barrier, a new leptin-sensing neuron population, multiple agouti-related peptide (AgRP) and pro-opiomelanocortin (POMC) subtypes, and an orexigenic somatostatin neuron population. We extended Drop-seq to detect dynamic expression changes across relevant physiological perturbations, revealing cell type-specific responses to energy status, including distinct responses in AgRP and POMC neuron subtypes. Finally, integrating our data with human genome-wide association study data implicates two previously unknown neuron populations in the genetic control of obesity. This resource will accelerate biological discovery by providing insights into molecular and cell type diversity from which function can be inferred.
Insulin resistance is a sine qua non of type 2 diabetes and is associated with many other clinical conditions. Decades of research into mechanisms underlying insulin resistance have mostly focused on ...problems in insulin signal transduction and other mitochondrial and cytosolic pathways. By contrast, relatively little attention has been focused on transcriptional and epigenetic contributors to insulin resistance, despite strong evidence that such nuclear mechanisms play a major role in the etiopathogenesis of this condition. In this review, we summarize the evidence for nuclear mechanisms of insulin resistance, focusing on three transcription factors with a major impact on insulin action in liver, muscle, and fat.
Epigenomic mechanisms direct distinct gene expression programs for different cell types. Various in vivo tissues have been subjected to epigenomic analysis; however, these studies have been limited ...by cellular heterogeneity, resulting in composite gene expression and epigenomic profiles. Here, we introduce “NuTRAP,” a transgenic mouse that allows simultaneous isolation of cell-type-specific translating mRNA and chromatin from complex tissues. Using NuTRAP, we successfully characterize gene expression and epigenomic states of various adipocyte populations in vivo, revealing significant differences compared to either whole adipose tissue or in vitro adipocyte cell lines. We find that chromatin immunoprecipitation sequencing (ChIP-seq) using NuTRAP is highly efficient, scalable, and robust with even limited cell input. We further demonstrate the general utility of NuTRAP by analyzing hepatocyte-specific epigenomic states. The NuTRAP mouse is a resource that provides a powerful system for cell-type-specific gene expression and epigenomic profiling.
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•Generation of NuTRAP mice for cell-type-specific isolation of mRNA and nuclei•The NuTRAP mouse defines gene expression and chromatin states in vivo•NuTRAP mice reveal epigenomic differences between adipocytes in vivo and in vitro•ChIP-seq using NuTRAP is robust even with limited cell inputs
Roh et al. introduce a transgenic mouse model, named “NuTRAP,” for the isolation of cell-type-specific nuclei and mRNA and characterize gene expression and epigenomic states of pure populations of adipocytes in vivo. This approach is applicable to different cell types and is highly robust even with limited cell inputs.
COVID-19, which is caused by SARS-CoV-2, can result in acute respiratory distress syndrome and multiple organ failure
, but little is known about its pathophysiology. Here we generated single-cell ...atlases of 24 lung, 16 kidney, 16 liver and 19 heart autopsy tissue samples and spatial atlases of 14 lung samples from donors who died of COVID-19. Integrated computational analysis uncovered substantial remodelling in the lung epithelial, immune and stromal compartments, with evidence of multiple paths of failed tissue regeneration, including defective alveolar type 2 differentiation and expansion of fibroblasts and putative TP63
intrapulmonary basal-like progenitor cells. Viral RNAs were enriched in mononuclear phagocytic and endothelial lung cells, which induced specific host programs. Spatial analysis in lung distinguished inflammatory host responses in lung regions with and without viral RNA. Analysis of the other tissue atlases showed transcriptional alterations in multiple cell types in heart tissue from donors with COVID-19, and mapped cell types and genes implicated with disease severity based on COVID-19 genome-wide association studies. Our foundational dataset elucidates the biological effect of severe SARS-CoV-2 infection across the body, a key step towards new treatments.
The vagus nerve innervates many organs, and most, if not all, of its motor fibers are cholinergic. However, no one knows its organizing principles—whether or not there are dedicated neurons with ...restricted targets that act as “labeled lines” to perform certain functions, including two opposing ones (gastric contraction versus relaxation). By performing unbiased transcriptional profiling of DMV cholinergic neurons, we discovered seven molecularly distinct subtypes of motor neurons. Then, by using subtype-specific Cre driver mice, we show that two of these subtypes exclusively innervate the glandular domain of the stomach where, remarkably, they contact different enteric neurons releasing functionally opposing neurotransmitters (acetylcholine versus nitric oxide). Thus, the vagus motor nerve communicates via genetically defined labeled lines to control functionally unique enteric neurons within discrete subregions of the gastrointestinal tract. This discovery reveals that the parasympathetic nervous system utilizes a striking division of labor to control autonomic function.
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•Single nucleus RNA-seq reveals seven molecularly distinct vagal motor neuron subtypes•DMV-Pdyn and DMV-Cck neurons are located in different anatomical domains of the DMV•DMV-Pdyn and DMV-Cck neurons exclusively innervate the glandular stomach•DMV-Pdyn and DMV-Cck neurons target neurochemically distinct enteric neurons
The dorsal motor nucleus of the vagus is the primary source of parasympathetic input to the digestive system. Tao et al. demonstrate a labeled line model of the DMV by showing that Cck- and Pdyn-expressing DMV neurons exclusively innervate the glandular stomach, where they target functionally opposing enteric neurons.
The thiazolidinediones (TZDs) are ligands of PPARγ that improve insulin sensitivity, but their use is limited by significant side effects. Recently, we demonstrated a mechanism wherein TZDs improve ...insulin sensitivity distinct from receptor agonism and adipogenesis: reversal of obesity-linked phosphorylation of PPARγ at serine 273. However, the role of this modification hasn’t been tested genetically. Here we demonstrate that mice encoding an allele of PPARγ that cannot be phosphorylated at S273 are protected from insulin resistance, without exhibiting differences in body weight or TZD-associated side effects. Indeed, hyperinsulinemic-euglycemic clamp experiments confirm insulin sensitivity. RNA-seq in these mice reveals reduced expression of Gdf3, a BMP family member. Ectopic expression of Gdf3 is sufficient to induce insulin resistance in lean, healthy mice. We find Gdf3 inhibits BMP signaling and insulin signaling in vitro. Together, these results highlight the diabetogenic role of PPARγ S273 phosphorylation and focus attention on a putative target, Gdf3.
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•Blocking PPARγ S273 phosphorylation protects mice from insulin resistance in obesity•These mice do not show the side effects associated with TZD-based PPARγ agonism•These mice have reduced expression of Gdf3 mainly in macrophages•Gdf3 is sufficient to cause impaired glucose homeostasis in vivo and in vitro
Hall et al. show that mice lacking phosphorylation at serine 273 of PPARγ are protected from developing insulin resistance in response to high-fat-diet feeding, associated with dramatically reduced levels of Gdf3. Gdf3 in turn is sufficient to cause impaired insulin signaling both in vitro and in vivo.
Skeletal muscle and brown adipose tissue (BAT) are functionally linked, as exercise increases browning via secretion of myokines. It is unknown whether BAT affects muscle function. Here, we find that ...loss of the transcription factor IRF4 in BAT (BATI4KO) reduces exercise capacity, mitochondrial function, ribosomal protein synthesis, and mTOR signaling in muscle and causes tubular aggregate formation. Loss of IRF4 induces myogenic gene expression in BAT, including the secreted factor myostatin, a known inhibitor of muscle function. Reducing myostatin via neutralizing antibodies or soluble receptor rescues the exercise capacity of BATI4KO mice. In addition, overexpression of IRF4 in brown adipocytes reduces serum myostatin and increases exercise capacity in muscle. Finally, mice housed at thermoneutrality have reduced IRF4 in BAT, lower exercise capacity, and elevated serum myostatin; these abnormalities are corrected by excising BAT. Collectively, our data point to an unsuspected level of BAT-muscle crosstalk driven by IRF4 and myostatin.
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•Loss of IRF4 in BAT causes decreased exercise capacity and a selective myopathy•IRF4 represses myogenic genes in BAT, including the myokine myostatin•Neutralizing serum myostatin rescues the ability of BATI4KO mice to exercise normally•Warming reduces IRF4 in BAT, causing myopathy that is reversed by removal of BAT
Skeletal muscle and brown adipose tissue (BAT) are functionally interlinked. Kong et al. report on the intriguing role of the transcription factor IRF4 in BAT to mediate BAT-muscle crosstalk through the muscle function inhibitor, myostatin. Thermoneutrality or loss of IRF4 results in elevated serum myostatin levels and decreased exercise capacity.
Obesity increases the risk of mortality because of metabolic sequelae such as type 2 diabetes and cardiovascular disease
. Thermogenesis by adipocytes can counteract obesity and metabolic diseases
. ...In thermogenic fat, creatine liberates a molar excess of mitochondrial ADP-purportedly via a phosphorylation cycle
-to drive thermogenic respiration. However, the proteins that control this futile creatine cycle are unknown. Here we show that creatine kinase B (CKB) is indispensable for thermogenesis resulting from the futile creatine cycle, during which it traffics to mitochondria using an internal mitochondrial targeting sequence. CKB is powerfully induced by thermogenic stimuli in both mouse and human adipocytes. Adipocyte-selective inactivation of Ckb in mice diminishes thermogenic capacity, increases predisposition to obesity, and disrupts glucose homeostasis. CKB is therefore a key effector of the futile creatine cycle.
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