Steroid Receptors Reprogram FoxA1 Occupancy through Dynamic Chromatin Transitions.

The estrogen receptor (ER), glucocorticoid receptor (GR), and forkhead box protein 1 (FoxA1) are significant factors in breast cancer progression. FoxA1 has been implicated in establishing ER-binding patterns though its unique ability to serve as a pioneer factor. However, the molecular interplay between ER, GR, and FoxA1 requires further investigation. Here we show that ER and GR both have the ability to alter the genomic distribution of the FoxA1 pioneer factor. Single-molecule tracking experiments in live cells reveal a highly dynamic interaction of FoxA1 with chromatin in vivo. Furthermore, the FoxA1 factor is not associated with detectable footprints at its binding sites throughout the genome. These findings support a model wherein interactions between transcription factors and pioneer factors are highly dynamic. Moreover, at a subset of genomic sites, the role of pioneer can be reversed, with the steroid receptors serving to enhance binding of FoxA1.

Interactome maps of mouse gene regulatory domains reveal basic principles of transcriptional regulation.

A key finding of the ENCODE project is that the enhancer landscape of mammalian cells undergoes marked alterations during ontogeny. However, the nature and extent of these changes are unclear. As part of the NIH Mouse Regulome Project, we here combined DNaseI hypersensitivity, ChIP-seq, and ChIA-PET technologies to map the promoter-enhancer interactomes of pluripotent ES cells and differentiated B lymphocytes. We confirm that enhancer usage varies widely across tissues. Unexpectedly, we find that this feature extends to broadly transcribed genes, including Myc and Pim1 cell-cycle regulators, which associate with an entirely different set of enhancers in ES and B cells. By means of high-resolution CpG methylomes, genome editing, and digital footprinting, we show that these enhancers recruit lineage-determining factors. Furthermore, we demonstrate that the turning on and off of enhancers during development correlates with promoter activity. We propose that organisms rely on a dynamic enhancer landscape to control basic cellular functions in a tissue-specific manner.

Single cell-resolved study of advanced murine MASH reveals a homeostatic pericyte signaling module.

BACKGROUND & AIMS: Metabolic dysfunction-associated steatohepatitis (MASH) is linked to insulin resistance and type 2 diabetes and marked by hepatic inflammation, microvascular dysfunction, and fibrosis, impairing liver function and aggravating metabolic derangements. The liver homeostatic interactions disrupted in MASH are still poorly understood. We aimed to elucidate the plasticity and changing interactions of non-parenchymal cells associated with advanced MASH. METHODS: We characterized a diet-induced mouse model of advanced MASH at single-cell resolution and validated findings by assaying chromatin accessibility, bioimaging murine and human livers, and via functional experiments in vivo and in vitro. RESULTS: The fibrogenic activation of hepatic stellate cells (HSCs) led to deterioration of a signaling module consisting of the bile acid receptor NR1H4/FXR and HSC-specific GS-protein-coupled receptors (GSPCRs) capable of preserving stellate cell quiescence. Accompanying HSC activation, we further observed the attenuation of HSC Gdf2 expression, and a MASH-associated expansion of a CD207-positive macrophage population likely derived from both incoming monocytes and Kupffer cells. CONCLUSION: We conclude that HSC-expressed NR1H4 and GSPCRs of the healthy liver integrate postprandial cues, which sustain HSC quiescence and, through paracrine signals, overall sinusoidal health. Hence HSC activation in MASH not only drives fibrogenesis but may desensitize the hepatic sinusoid to liver homeostatic signals. IMPACT AND IMPLICATIONS: Homeostatic interactions between hepatic cell types and their deterioration in metabolic dysfunction-associated steatohepatitis are poorly characterized. In our current single cell-resolved study of advanced murine metabolic dysfunction-associated steatohepatitis, we identified a quiescence-associated hepatic stellate cell-signaling module with potential to preserve normal sinusoid function. As expression levels of its constituents are conserved in the human liver, stimulation of the identified signaling module is a promising therapeutic strategy to restore sinusoid function in chronic liver disease.

Circulating TREM2 as a noninvasive diagnostic biomarker for NASH in patients with elevated liver stiffness.

BACKGROUND AND AIMS: Reliable noninvasive biomarkers are an unmet clinical need for the diagnosis of NASH. This study investigates the diagnostic accuracy of the circulating triggering receptor expressed on myeloid cells 2 (plasma TREM2) as a biomarker for NASH in patients with NAFLD and elevated liver stiffness. APPROACH AND RESULTS: We collected cross-sectional, clinical data including liver biopsies from a derivation ( n = 48) and a validation cohort ( n = 170) of patients with elevated liver stiffness measurement (LSM ≥ 8.0 kPa). Patients with NAFLD activity scores (NAS) ≥4 were defined as having NASH. Plasma TREM2 levels were significantly elevated in patients with NASH of the derivation cohort, with an area under the receiver operating characteristics curve (AUROC) of 0.92 (95% confidence interval [CI], 0.84-0.99). In the validation cohort, plasma TREM2 level increased approximately two-fold in patients with NASH, and a strong diagnostic accuracy was confirmed (AUROC, 0.83; 95% CI, 0.77-0.89; p < 0.0001). Plasma TREM2 levels were associated with the individual histologic features of NAS: steatosis, lobular inflammation, and ballooning ( p < 0.0001), but only weakly with fibrosis stages. Dual cutoffs for rule-in and rule-out were explored: a plasma TREM2 level of ≤38 ng/ml was found to be an optimal NASH rule-out cutoff (sensitivity 90%; specificity 52%), whereas a plasma TREM2 level of ≥65 ng/ml was an optimal NASH rule-in cutoff (specificity 89%; sensitivity 54%). CONCLUSIONS: Plasma TREM2 is a plausible individual biomarker that can rule-in or rule-out the presence of NASH with high accuracy and thus has the potential to reduce the need for liver biopsies and to identify patients who are eligible for clinical trials in NASH.

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.

Collagen Density Modulates the Immunosuppressive Functions of Macrophages.

Tumor-associated macrophages (TAMs) support tumor growth by suppressing the activity of tumor-infiltrating T cells. Consistently, TAMs are considered a major limitation for the efficacy of cancer immunotherapy. However, the molecular reason behind the acquisition of an immunosuppressive TAM phenotype is not fully clarified. During tumor growth, the extracellular matrix (ECM) is degraded and substituted with a tumor-specific collagen-rich ECM. The collagen density of this tumor ECM has been associated with poor patient prognosis but the reason for this is not well understood. In this study, we investigated whether the collagen density could modulate the immunosuppressive activity of TAMs. The murine macrophage cell line RAW 264.7 was three-dimensionally cultured in collagen matrices of low and high collagen densities mimicking healthy and tumor tissue, respectively. Collagen density did not affect proliferation or viability of the macrophages. However, whole-transcriptome analysis revealed a striking response to the surrounding collagen density, including the regulation of immune regulatory genes and genes encoding chemokines. These transcriptional changes were shown to be similar in murine bone marrow-derived macrophages and TAMs isolated from murine tumors. Strikingly, coculture assays with primary T cells showed that macrophages cultured in high-density collagen were less efficient at attracting cytotoxic T cells and capable of inhibiting T cell proliferation more than macrophages cultured in low-density collagen. Our study demonstrates that a high collagen density can instruct macrophages to acquire an immunosuppressive phenotype. This mechanism could reduce the efficacy of immunotherapy and explain the link between high collagen density and poor prognosis.

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.

eRNAs promote transcription by establishing chromatin accessibility at defined genomic loci.

Transcription factors and DNA regulatory binding motifs are fundamental components of the gene regulatory network. Here, by using genome-wide binding profiling, we show extensive occupancy of transcription factors of myogenesis (MyoD and Myogenin) at extragenic enhancer regions coinciding with RNA synthesis (i.e., eRNA). In particular, multiple regions were transcribed to eRNA within the regulatory region of MYOD1, including previously characterized distal regulatory regions (DRR) and core enhancer (CE). While (CE)RNA enhanced RNA polymerase II (Pol II) occupancy and transcription at MYOD1, (DRR)RNA acted to activate the downstream myogenic genes. The deployment of transcriptional machinery to appropriate loci is contingent on chromatin accessibility, a rate-limiting step preceding Pol II assembly. By nuclease sensitivity assay, we found that eRNAs regulate genomic access of the transcriptional complex to defined regulatory regions. In conclusion, our data suggest that eRNAs contribute to establishing a cell-type-specific transcriptional circuitry by directing chromatin-remodeling events.

Noncanonical thyroid hormone signaling mediates cardiometabolic effects in vivo.

Thyroid hormone (TH) and TH receptors (TRs) α and ß act by binding to TH response elements (TREs) in regulatory regions of target genes. This nuclear signaling is established as the canonical or type 1 pathway for TH action. Nevertheless, TRs also rapidly activate intracellular second-messenger signaling pathways independently of gene expression (noncanonical or type 3 TR signaling). To test the physiological relevance of noncanonical TR signaling, we generated knockin mice with a mutation in the TR DNA-binding domain that abrogates binding to DNA and leads to complete loss of canonical TH action. We show that several important physiological TH effects are preserved despite the disruption of DNA binding of TRα and TRß, most notably heart rate, body temperature, blood glucose, and triglyceride concentration, all of which were regulated by noncanonical TR signaling. Additionally, we confirm that TRE-binding-defective TRß leads to disruption of the hypothalamic-pituitary-thyroid axis with resistance to TH, while mutation of TRα causes a severe delay in skeletal development, thus demonstrating tissue- and TR isoform-specific canonical signaling. These findings provide in vivo evidence that noncanonical TR signaling exerts physiologically important cardiometabolic effects that are distinct from canonical actions. These data challenge the current paradigm that in vivo physiological TH action is mediated exclusively via regulation of gene transcription at the nuclear level.

C/EBP maintains chromatin accessibility in liver and facilitates glucocorticoid receptor recruitment to steroid response elements.

Mechanisms regulating transcription factor interaction with chromatin in intact mammalian tissues are poorly understood. Exploiting an adrenalectomized mouse model with depleted endogenous glucocorticoids, we monitor changes of the chromatin landscape in intact liver tissue following glucocorticoid injection. Upon activation of the glucocorticoid receptor (GR), proximal regions of activated and repressed genes are remodelled, and these remodelling events correlate with RNA polymerase II occupancy of regulated genes. GR is exclusively associated with accessible chromatin and 62% percent of GR-binding sites are occupied by C/EBPß. At the majority of these sites, chromatin is preaccessible suggesting a priming function of C/EBPß for GR recruitment. Disruption of C/EBPß binding to chromatin results in attenuation of pre-programmed chromatin accessibility, GR recruitment and GR-induced chromatin remodelling specifically at sites co-occupied by GR and C/EBPß. Collectively, we demonstrate a highly cooperative mechanism by which C/EBPß regulates selective GR binding to the genome in liver tissue. We suggest that selective targeting of GR in other tissues is likely mediated by the combined action of cell-specific priming proteins and chromatin remodellers.