Sex-dependent modulation of T and NK cells and gut microbiome by low sodium diet in patients with primary aldosteronism.

Background: High dietary sodium intake is a major cardiovascular risk factor and adversely affects blood pressure control. Patients with primary aldosteronism (PA) are at increased cardiovascular risk, even after medical treatment, and high dietary sodium intake is common in these patients. Here, we analyze the impact of a moderate dietary sodium restriction on microbiome composition and immunophenotype in patients with PA.

An Optimized High-Resolution Mapping Method for Glucocorticoid Receptor-DNA Binding in Mouse Primary Macrophages.

ChIP-exo is a powerful tool for achieving enhanced sensitivity and single-base-pair resolution of transcription factor (TF) binding, which utilizes a combination of chromatin immunoprecipitation (ChIP) and lambda exonuclease digestion (exo) followed by high-throughput sequencing. ChIP-nexus (chromatin immunoprecipitation experiments with nucleotide resolution through exonuclease, unique barcode, and single ligation) is an updated and simplified version of the original ChIP-exo method, which has reported an efficient adapter ligation through the DNA circularization step. Building upon an established method, we present a protocol for generating NGS (next-generation sequencing) ready and high-quality ChIP-nexus library for glucocorticoid receptor (GR).

E47 as a novel glucocorticoid-dependent gene mediating lipid metabolism in patients with endogenous glucocorticoid excess.

Purpose: E47 has been identified as a modulating transcription factor of glucocorticoid receptor target genes, its loss protecting mice from metabolic adverse effects of glucocorticoids. We aimed to analyze the role of E47 in patients with endogenous glucocorticoid excess [Cushing's syndrome (CS)] and its association with disorders of lipid and glucose metabolism.

Methods: This is a prospective cohort study including 120 female patients with CS (ACTH-dependent = 79; ACTH-independent = 41) and 26 healthy female controls.

GIP receptor agonism improves dyslipidemia and atherosclerosis independently of body weight loss in preclinical mouse model for cardio-metabolic disease.

Background: Agonism at the receptor for the glucose-dependent insulinotropic polypeptide (GIPR) is a key component of the novel unimolecular GIPR:GLP-1R co-agonists, which are among the most promising drugs in clinical development for the treatment of obesity and type 2 diabetes. The therapeutic effect of chronic GIPR agonism to treat dyslipidemia and thus to reduce the cardiovascular disease risk independently of body weight loss has not been explored yet.

Methods: After 8 weeks on western diet, LDL receptor knockout (LDLR-/-) male mice were treated with daily subcutaneous injections of long-acting acylated GIP analog (acyl-GIP; 10nmol/kg body weight) for 28 days.

Integrative analysis of macrophage ribo-Seq and RNA-Seq data define glucocorticoid receptor regulated inflammatory response genes into distinct regulatory classes.

Glucocorticoids such as dexamethasone (Dex) are widely used to treat both acute and chronic inflammatory conditions. They regulate immune responses by dampening cell-mediated immunity in a glucocorticoid receptor (GR)-dependent manner, by suppressing the expression of pro-inflammatory cytokines and chemokines and by stimulating the expression of anti-inflammatory mediators. Despite its evident clinical benefit, the mechanistic underpinnings of the gene regulatory networks transcriptionally controlled by GR in a context-specific manner remain mysterious.

How to tame your genes: mechanisms of inflammatory gene repression by glucocorticoids.

Glucocorticoids (GCs) are widely used therapeutic agents to treat a broad range of inflammatory conditions. Their functional effects are elicited by binding to the glucocorticoid receptor (GR), which regulates transcription of distinct gene networks in response to ligand. However, the mechanisms governing various aspects of undesired side effects versus beneficial immunomodulation upon GR activation remain complex and incompletely understood.

Angiopoietin-like 4 governs diurnal lipoprotein lipase activity in brown adipose tissue.

Objective: Brown adipose tissue (BAT) burns fatty acids (FAs) to produce heat, and shows diurnal oscillation in glucose and triglyceride (TG)-derived FA-uptake, peaking around wakening. Here we aimed to gain insight in the diurnal regulation of metabolic BAT activity.

Methods: RNA-sequencing, chromatin immunoprecipitation (ChIP)-sequencing, and lipidomics analyses were performed on BAT samples of wild type C57BL/6J mice collected at 3-hour intervals throughout the day.

P75 neurotrophin receptor controls subventricular zone neural stem cell migration after stroke.

Stroke is the leading cause of adult disability. Endogenous neural stem/progenitor cells (NSPCs) originating from the subventricular zone (SVZ) contribute to the brain repair process. However, molecular mechanisms underlying CNS disease-induced SVZ NSPC-redirected migration to the lesion area are poorly understood.

Protocol for using heterologous spike-ins to normalize for technical variation in chromatin immunoprecipitation.

Quantifying differential genome occupancy by chromatin immunoprecipitation (ChIP) remains challenging due to variation in chromatin fragmentation, immunoprecipitation efficiencies, and intertube variability. In this protocol, we add heterologous spike-ins from chromatin as an internal control to the mice chromatin before immunoprecipitation to normalize for technical variation in ChIP-qPCR or ChIP-seq. The choice of spike-in depends on the evolutionary conservation of the protein of interest and the antibody used.

T cells armed with C-X-C chemokine receptor type 6 enhance adoptive cell therapy for pancreatic tumours.

The efficacy of adoptive cell therapy for solid tumours is hampered by the poor accumulation of the transferred T cells in tumour tissue. Here, we show that forced expression of C-X-C chemokine receptor type 6 (whose ligand is highly expressed by human and murine pancreatic cancer cells and tumour-infiltrating immune cells) in antigen-specific T cells enhanced the recognition and lysis of pancreatic cancer cells and the efficacy of adoptive cell therapy for pancreatic cancer. In mice with subcutaneous pancreatic tumours treated with T cells with either a transgenic T-cell receptor or a murine chimeric antigen receptor targeting the tumour-associated antigen epithelial cell adhesion molecule, and in mice with orthotopic pancreatic tumours or patient-derived xenografts treated with T cells expressing a chimeric antigen receptor targeting mesothelin, the T cells exhibited enhanced intratumoral accumulation, exerted sustained anti-tumoral activity and prolonged animal survival only when co-expressing C-X-C chemokine receptor type 6.

The glucocorticoid receptor recruits the COMPASS complex to regulate inflammatory transcription at macrophage enhancers.

Glucocorticoids (GCs) are effective anti-inflammatory drugs; yet, their mechanisms of action are poorly understood. GCs bind to the glucocorticoid receptor (GR), a ligand-gated transcription factor controlling gene expression in numerous cell types. Here, we characterize GR's protein interactome and find the SETD1A (SET domain containing 1A)/COMPASS (complex of proteins associated with Set1) histone H3 lysine 4 (H3K4) methyltransferase complex highly enriched in activated mouse macrophages.

Anti-inflammatory functions of the glucocorticoid receptor require DNA binding.

The glucocorticoid receptor is an important immunosuppressive drug target and metabolic regulator that acts as a ligand-gated transcription factor. Generally, GR's anti-inflammatory effects are attributed to the silencing of inflammatory genes, while its adverse effects are ascribed to the upregulation of metabolic targets. GR binding directly to DNA is proposed to activate, whereas GR tethering to pro-inflammatory transcription factors is thought to repress transcription.

Seq-ing answers: Current data integration approaches to uncover mechanisms of transcriptional regulation.

Advancements in the field of next generation sequencing lead to the generation of ever-more data, with the challenge often being how to combine and reconcile results from different OMICs studies such as genome, epigenome and transcriptome. Here we provide an overview of the standard processing pipelines for ChIP-seq and RNA-seq as well as common downstream analyses. We describe popular multi-omics data integration approaches used to identify target genes and co-factors, and we discuss how machine learning techniques may predict transcriptional regulators and gene expression.

Anti-inflammatory glucocorticoid action: genomic insights and emerging concepts.

Glucocorticoids (GCs) are widely used immunomodulators. They regulate gene expression by binding and activating the Glucocorticoid Receptor (GR), but underlying transcriptional mechanisms remain enigmatic. This review summarizes recent findings identifyingspecific GR-bound DNA sequences whose configuration may affect transcriptional output.

The rRNA mA methyltransferase METTL5 is involved in pluripotency and developmental programs.

Covalent chemical modifications of cellular RNAs directly impact all biological processes. However, our mechanistic understanding of the enzymes catalyzing these modifications, their substrates and biological functions, remains vague. Amongst RNA modifications N-methyladenosine (mA) is widespread and found in messenger (mRNA), ribosomal (rRNA), and noncoding RNAs.

Cistromic Reprogramming of the Diurnal Glucocorticoid Hormone Response by High-Fat Diet.

The glucocorticoid receptor (GR) is a potent metabolic regulator and a major drug target. While GR is known to play integral roles in circadian biology, its rhythmic genomic actions have never been characterized. Here we mapped GR's chromatin occupancy in mouse livers throughout the day and night cycle.

Fighting the Fire: Mechanisms of Inflammatory Gene Regulation by the Glucocorticoid Receptor.

For many decades, glucocorticoids have been widely used as the gold standard treatment for inflammatory conditions. Unfortunately, their clinical use is limited by severe adverse effects such as insulin resistance, cardiometabolic diseases, muscle and skin atrophies, osteoporosis, and depression. Glucocorticoids exert their effects by binding to the Glucocorticoid Receptor (GR), a ligand-activated transcription factor which both positively, and negatively regulates gene expression.

In Vivo ChIP-Seq of Nuclear Receptors: A Rough Guide to Transform Frozen Tissues into High-Confidence Genome-Wide Binding Profiles.

Chromatin immunoprecipitation coupled to next generation sequencing (ChIP-seq) is a powerful tool to map context-dependent genome-wide binding of nuclear hormone receptors and their coregulators. This information can provide important mechanistic insight into where, when and how DNA-protein interactions are linked to target gene regulation. Here we describe a simple, yet reliable ChIP-seq method, including nuclear isolation from frozen tissue samples, cross-linking DNA-protein complexes, chromatin shearing, immunoprecipitation, and purification of ChIP DNA.

Phosphoproteomics Reveals the GSK3-PDX1 Axis as a Key Pathogenic Signaling Node in Diabetic Islets.

Progressive decline of pancreatic beta cell function is central to the pathogenesis of type 2 diabetes. Protein phosphorylation regulates glucose-stimulated insulin secretion from beta cells, but how signaling networks are remodeled in diabetic islets in vivo remains unknown. Using high-sensitivity mass spectrometry-based proteomics, we quantified 6,500 proteins and 13,000 phosphopeptides in islets of obese diabetic mice and matched controls, revealing drastic remodeling of key kinase hubs and signaling pathways.

E47 modulates hepatic glucocorticoid action.

Glucocorticoids (GCs) are effective drugs, but their clinical use is compromised by severe side effects including hyperglycemia, hyperlipidemia and obesity. They bind to the Glucocorticoid Receptor (GR), which acts as a transcription factor. The activation of metabolic genes by GR is thought to underlie these adverse effects.

Organellar Proteomics and Phospho-Proteomics Reveal Subcellular Reorganization in Diet-Induced Hepatic Steatosis.

Lipid metabolism is highly compartmentalized between cellular organelles that dynamically adapt their compositions and interactions in response to metabolic challenges. Here, we investigate how diet-induced hepatic lipid accumulation, observed in non-alcoholic fatty liver disease (NAFLD), affects protein localization, organelle organization, and protein phosphorylation in vivo. We develop a mass spectrometric workflow for protein and phosphopeptide correlation profiling to monitor levels and cellular distributions of ∼6,000 liver proteins and ∼16,000 phosphopeptides during development of steatosis.

Transcriptional programming of lipid and amino acid metabolism by the skeletal muscle circadian clock.

Circadian clocks are fundamental physiological regulators of energy homeostasis, but direct transcriptional targets of the muscle clock machinery are unknown. To understand how the muscle clock directs rhythmic metabolism, we determined genome-wide binding of the master clock regulators brain and muscle ARNT-like protein 1 (BMAL1) and REV-ERBα in murine muscles. Integrating occupancy with 24-hr gene expression and metabolomics after muscle-specific loss of BMAL1 and REV-ERBα, here we unravel novel molecular mechanisms connecting muscle clock function to daily cycles of lipid and protein metabolism.

Skeletal muscle mass is controlled by the MRF4-MEF2 axis.

Purpose Of Review: The review is focused on the unexpected role of myogenic regulatory factor 4 (MRF4) in controlling muscle mass by repressing myocyte enhancer binding factor 2 (MEF2) activity in adult skeletal muscle, and on the emerging role of MEF2 in skeletal muscle growth.

Recent Findings: The MRF4s of the MyoD family (MyoD, MYF5, MRF4, myogenin) and the MEF2 factors are known to play a major role in embryonic myogenesis. However, their function in adult muscle tissue is not known.

DNA residence time is a regulatory factor of transcription repression.

Transcription comprises a highly regulated sequence of intrinsically stochastic processes, resulting in bursts of transcription intermitted by quiescence. In transcription activation or repression, a transcription factor binds dynamically to DNA, with a residence time unique to each factor. Whether the DNA residence time is important in the transcription process is unclear.

Circadian Metabolomics in Time and Space.

Circadian rhythms are widely known to govern human health and disease, but specific pathogenic mechanisms linking circadian disruption to metabolic diseases are just beginning to come to light. This is thanks in part to the development and application of various "omics"-based tools in biology and medicine. Current high-throughput technologies allow for the simultaneous monitoring of multiple dynamic cellular events over time, ranging from gene expression to metabolite abundance and sub-cellular localization.