Transcriptional programming of translation by BCL6 controls skeletal muscle proteostasis.

Skeletal muscle is dynamically controlled by the balance of protein synthesis and degradation. Here we discover an unexpected function for the transcriptional repressor B cell lymphoma 6 (BCL6) in muscle proteostasis and strength in mice. Skeletal muscle-specific Bcl6 ablation in utero or in adult mice results in over 30% decreased muscle mass and force production due to reduced protein synthesis and increased autophagy, while it promotes a shift to a slower myosin heavy chain fibre profile. Ribosome profiling reveals reduced overall translation efficiency in Bcl6-ablated muscles. Mechanistically, tandem chromatin immunoprecipitation, transcriptomic and translational analyses identify direct BCL6 repression of eukaryotic translation initiation factor 4E-binding protein 1 (Eif4ebp1) and activation of insulin-like growth factor 1 (Igf1) and androgen receptor (Ar). Together, these results uncover a bifunctional role for BCL6 in the transcriptional and translational control of muscle proteostasis.

Epigenomic tensor predicts disease subtypes and reveals constrained tumor evolution.

Understanding the epigenomic evolution and specificity of disease subtypes from complex patient data remains a major biomedical problem. We here present DeCET (decomposition and classification of epigenomic tensors), an integrative computational approach for simultaneously analyzing hierarchical heterogeneous data, to identify robust epigenomic differences among tissue types, differentiation states, and disease subtypes. Applying DeCET to our own data from 21 uterine benign tumor (leiomyoma) patients identifies distinct epigenomic features discriminating normal myometrium and leiomyoma subtypes. Leiomyomas possess preponderant alterations in distal enhancers and long-range histone modifications confined to chromatin contact domains that constrain the evolution of pathological epigenomes. Moreover, we demonstrate the power and advantage of DeCET on multiple publicly available epigenomic datasets representing different cancers and cellular states. Epigenomic features extracted by DeCET can thus help improve our understanding of disease states, cellular development, and differentiation, thereby facilitating future therapeutic, diagnostic, and prognostic strategies.

Dynamic enhancers control skeletal muscle identity and reprogramming.

Skeletal muscles consist of fibers of differing metabolic activities and contractility, which become remodeled in response to chronic exercise, but the epigenomic basis for muscle identity and adaptation remains poorly understood. Here, we used chromatin immunoprecipitation sequencing of dimethylated histone 3 lysine 4 and acetylated histone 3 lysine 27 as well as transposase-accessible chromatin profiling to dissect cis-regulatory networks across muscle groups. We demonstrate that in vivo enhancers specify muscles in accordance with myofiber composition, show little resemblance to cultured myotube enhancers, and identify glycolytic and oxidative muscle-specific regulators. Moreover, we find that voluntary wheel running and muscle-specific peroxisome proliferator-activated receptor gamma coactivator-1 alpha (Pgc1a) transgenic (mTg) overexpression, which stimulate endurance performance in mice, result in markedly different changes to the epigenome. Exercise predominantly leads to enhancer hypoacetylation, whereas mTg causes hyperacetylation at different sites. Integrative analysis of regulatory regions and gene expression revealed that exercise and mTg are each associated with myocyte enhancer factor (MEF) 2 and estrogen-related receptor (ERR) signaling and transcription of genes promoting oxidative metabolism. However, exercise was additionally associated with regulation by retinoid X receptor (RXR), jun proto-oncogene (JUN), sine oculis homeobox factor (SIX), and other factors. Overall, our work defines the unique enhancer repertoires of skeletal muscles in vivo and reveals that divergent exercise-induced or PGC1α-driven epigenomic programs direct partially convergent transcriptional networks.

Dynamic repression by BCL6 controls the genome-wide liver response to fasting and steatosis.

Transcription is tightly regulated to maintain energy homeostasis during periods of feeding or fasting, but the molecular factors that control these alternating gene programs are incompletely understood. Here, we find that the B cell lymphoma 6 (BCL6) repressor is enriched in the fed state and converges genome-wide with PPARα to potently suppress the induction of fasting transcription. Deletion of hepatocyte Bcl6 enhances lipid catabolism and ameliorates high-fat-diet-induced steatosis. In Ppara-null mice, hepatocyte Bcl6 ablation restores enhancer activity at PPARα-dependent genes and overcomes defective fasting-induced fatty acid oxidation and lipid accumulation. Together, these findings identify BCL6 as a negative regulator of oxidative metabolism and reveal that alternating recruitment of repressive and activating transcription factors to shared cis-regulatory regions dictates hepatic lipid handling.

Loss of Transcriptional Repression by BCL6 Confers Insulin Sensitivity in the Setting of Obesity.

Accumulation of visceral adiposity is directly linked to the morbidity of obesity, while subcutaneous body fat is considered more benign. We have identified an unexpected role for B cell lymphoma 6 (BCL6), a critical regulator of immunity, in the developmental expansion of subcutaneous adipose tissue. In adipocyte-specific knockout mice (Bcl6AKO), we found that Bcl6 deletion results in strikingly increased inguinal, but not perigonadal, adipocyte size and tissue mass in addition to marked insulin sensitivity. Genome-wide RNA expression and DNA binding analyses revealed that BCL6 controls gene networks involved in cell growth and fatty acid biosynthesis. Using deuterium label incorporation and comprehensive adipokine and lipid profiling, we discovered that ablation of adipocyte Bcl6 enhances subcutaneous adipocyte lipogenesis, increases levels of adiponectin and fatty acid esters of hydroxy fatty acids (FAHFAs), and prevents steatosis. Thus, our studies identify BCL6 as a negative regulator of subcutaneous adipose tissue expansion and metabolic health.

Genomic integration of ERRγ-HNF1ß regulates renal bioenergetics and prevents chronic kidney disease.

Mitochondrial dysfunction is increasingly recognized as a critical determinant of both hereditary and acquired kidney diseases. However, it remains poorly understood how mitochondrial metabolism is regulated to support normal kidney function and how its dysregulation contributes to kidney disease. Here, we show that the nuclear receptor estrogen-related receptor gamma (ERRγ) and hepatocyte nuclear factor 1 beta (HNF1ß) link renal mitochondrial and reabsorptive functions through coordinated epigenomic programs. ERRγ directly regulates mitochondrial metabolism but cooperatively controls renal reabsorption via convergent binding with HNF1ß. Deletion of ERRγ in renal epithelial cells (RECs), in which it is highly and specifically expressed, results in severe renal energetic and reabsorptive dysfunction and progressive renal failure that recapitulates phenotypes of animals and patients with HNF1ß loss-of-function gene mutations. Moreover, ERRγ expression positively correlates with renal function and is decreased in patients with chronic kidney disease (CKD). REC-ERRγ KO mice share highly overlapping renal transcriptional signatures with human patients with CKD. Together these findings reveal a role for ERRγ in directing independent and HNF1ß-integrated programs for energy production and use essential for normal renal function and the prevention of kidney disease.

A Stringent Systems Approach Uncovers Gene-Specific Mechanisms Regulating Inflammation.

Much has been learned about transcriptional cascades and networks from large-scale systems analyses of high-throughput datasets. However, analysis methods that optimize statistical power through simultaneous evaluation of thousands of ChIP-seq peaks or differentially expressed genes possess substantial limitations in their ability to uncover mechanistic principles of transcriptional control. By examining nascent transcript RNA-seq, ChIP-seq, and binding motif datasets from lipid A-stimulated macrophages with increased attention to the quantitative distribution of signals, we identified unexpected relationships between the in vivo binding properties of inducible transcription factors, motif strength, and transcription. Furthermore, rather than emphasizing common features of large clusters of co-regulated genes, our results highlight the extent to which unique mechanisms regulate individual genes with key biological functions. Our findings demonstrate the mechanistic value of stringent interrogation of well-defined sets of genes as a complement to broader systems analyses of transcriptional cascades and networks.

Dependence of hippocampal function on ERRγ-regulated mitochondrial metabolism.

Neurons utilize mitochondrial oxidative phosphorylation (OxPhos) to generate energy essential for survival, function, and behavioral output. Unlike most cells that burn both fat and sugar, neurons only burn sugar. Despite its importance, how neurons meet the increased energy demands of complex behaviors such as learning and memory is poorly understood. Here we show that the estrogen-related receptor gamma (ERRγ) orchestrates the expression of a distinct neural gene network promoting mitochondrial oxidative metabolism that reflects the extraordinary neuronal dependence on glucose. ERRγ(-/-) neurons exhibit decreased metabolic capacity. Impairment of long-term potentiation (LTP) in ERRγ(-/-) hippocampal slices can be fully rescued by the mitochondrial OxPhos substrate pyruvate, functionally linking the ERRγ knockout metabolic phenotype and memory formation. Consistent with this notion, mice lacking neuronal ERRγ in cerebral cortex and hippocampus exhibit defects in spatial learning and memory. These findings implicate neuronal ERRγ in the metabolic adaptations required for memory formation.

Macrophage polarization phenotype regulates adiponectin receptor expression and adiponectin anti-inflammatory response.

Adiponectin (APN), a pleiotropic adipokine that exerts anti-inflammatory, antidiabetic, and antiatherogenic effects through its receptors (AdipoRs), AdipoR1 and AdipoR2, is an important therapeutic target. Factors regulating AdipoR expression in monocyte/macrophages are poorly understood, and the significance of polarized macrophage activation in controlling AdipoR expression and the APN-mediated inflammatory response has not been investigated. The aim of this study was to investigate whether the macrophage polarization phenotype controls the AdipoR expression and APN-mediated inflammatory response. With the use of mouse bone marrow and peritoneal macrophages, we demonstrate that classical activation (M1) of macrophages suppressed (40-60% of control) AdipoR expression, whereas alternative activation (M2) preserved it. Remarkably, the macrophage polarization phenotypes produced contrasting inflammatory responses to APN (EC50 5 µg/ml). In M1 macrophages, APN induced proinflammatory cytokines, TNF-α, IL-6, and IL-12 (>10-fold of control) and AdipoR levels. In contrast, in M2 macrophages, APN induced the anti-inflammatory cytokine IL-10 without altering AdipoR expression. Furthermore, M1 macrophages adapt to a cytokine environment by reversing AdipoR expression. APN induced AdipoR mRNA and protein expression by up-regulating liver X receptor-α (LXRα) in macrophages. These results provide the first evidence that macrophage polarization is a key determinant regulating AdipoR expression and differential APN-mediated macrophage inflammatory responses, which can profoundly influence their pathogenic role in inflammatory and metabolic disorders.

A role for WDR5 in integrating threonine 11 phosphorylation to lysine 4 methylation on histone H3 during androgen signaling and in prostate cancer.

Upon androgen stimulation, PKN1-mediated histone H3 threonine 11 phosphorylation (H3T11P) promotes AR target gene activation. However, the underlying mechanism is not completely understood. Here, we show that WDR5, a subunit of the SET1/MLL complex, interacts with H3T11P, and this interaction facilitates the recruitment of the MLL1 complex and subsequent H3K4 tri-methylation (H3K4me3). Using ChIP-seq, we find that androgen stimulation results in a 6-fold increase in the number of H3T11P-marked regions and induces WDR5 colocalization to one third of H3T11P-enriched promoters, thus establishing a genome-wide relationship between H3T11P and recruitment of WDR5. Accordingly, PKN1 knockdown or chemical inhibition severely blocks WDR5 chromatin association and H3K4me3 on AR target genes. Finally, WDR5 is critical in prostate cancer cell proliferation and is hyperexpressed in human prostate cancers. Together, these results identify WDR5 as a critical epigenomic integrator of histone phosphorylation and methylation and as a major driver of androgen-dependent prostate cancer cell proliferation.

Adiponectin expression protects against angiotensin II-mediated inflammation and accelerated atherosclerosis.

Adiponectin (APN), an adipocytokine produced by adipose tissue, exerts pleiotropic actions regulating inflammation, metabolism and vascular homeostasis. APN levels are inversely correlated with obesity, type-2 diabetes, hypertension and cardiovascular disease. Although renin angiotensin system (RAS) activation in these interrelated metabolic syndrome components increases angiotensin II (AngII) levels leading to vascular damage, it is unknown whether APN under these conditions provides atheroprotection. We investigated whether increasing plasma APN provides atheroprotection in a hypertensive and accelerated atherosclerosis model. Using adenoviral gene transfer, sustained APN expression increased plasma levels of total and high-molecular weight APN, leading to a significant elevation of plasma HDL-cholesterol (HDL-C). Elevated APN levels were strongly atheroprotective, yet had no impact on blood pressure. Notably, gene expression analyses revealed that APN significantly inhibited the expression of pro-inflammatory and atherogenic genes while it increased the expression of the anti-inflammatory cytokine, IL-10 and the cholesterol efflux transporters, ABCA1 and ABCG1 in the artery wall. These findings suggest that increasing APN levels may be an effective therapeutic strategy to inhibit vascular inflammation and accelerated atherosclerosis associated with RAS activation in the metabolic syndrome.

Rescue of a primary myelofibrosis model by retinoid-antagonist therapy.

Molecular targeting of the two receptor interaction domains of the epigenetic repressor silencing mediator of retinoid and thyroid hormone receptors (SMRT(mRID)) produced a transplantable skeletal syndrome that reduced radial bone growth, increased numbers of bone-resorbing periosteal osteoclasts, and increased bone fracture risk. Furthermore, SMRT(mRID) mice develop spontaneous primary myelofibrosis, a chronic, usually idiopathic disorder characterized by progressive bone marrow fibrosis. Frequently linked to polycythemia vera and chronic myeloid leukemia, myelofibrosis displays high patient morbidity and mortality, and current treatment is mostly palliative. To decipher the etiology of this disease, we identified the thrombopoietin (Tpo) gene as a target of the SMRT-retinoic acid receptor signaling pathway in bone marrow stromal cells. Chronic induction of Tpo in SMRT(mRID) mice results in up-regulation of TGF-ß and PDGF in megakaryocytes, uncontrolled proliferation of bone marrow reticular cells, and fibrosis of the marrow compartment. Of therapeutic relevance, we show that this syndrome can be rescued by retinoid antagonists, demonstrating that the physical interface between SMRT and retinoic acid receptor can be a potential therapeutic target to block primary myelofibrosis disease progression.

Insights into negative regulation by the glucocorticoid receptor from genome-wide profiling of inflammatory cistromes.

How the glucocorticoid receptor (GR) activates some genes while potently repressing others remains an open question. There are three current models for suppression: transrepression via GR tethering to AP-1/NF-κB sites, direct GR association with inhibitory elements (nGREs), and GR recruitment of the corepressor GRIP1. To gain insights into GR suppression, we used genomic analyses and genome-wide profiling of GR, p65, and c-Jun in LPS-stimulated macrophages. We show that GR mediates both activation and repression at tethered sites, GREs, and GRIP1-bound elements, indicating that motif classification is insufficient to predict regulatory polarity of GR binding. Interestingly, sites of GR repression utilize GRIP1's corepressor function and display reduced histone acetylation. Together, these findings suggest that while GR occupancy confers hormone responsiveness, the receptor itself may not participate in the regulatory effects. Furthermore, transcriptional outcome is not established by sequence but is influenced by epigenetic regulators, context, and other unrecognized regulatory determinants.

The Bcl6-SMRT/NCoR cistrome represses inflammation to attenuate atherosclerosis.

Chronic inflammation is a hallmark of atherosclerosis, but its transcriptional underpinnings are poorly understood. We show that the transcriptional repressor Bcl6 is an anti-inflammatory regulator whose loss in bone marrow of Ldlr(-/-) mice results in severe atherosclerosis and xanthomatous tendonitis, a virtually pathognomonic complication in patients with familial hypercholesterolemia. Disruption of the interaction between Bcl6 and SMRT or NCoR with a peptide inhibitor in vitro recapitulated atherogenic gene changes in mice transplanted with Bcl6-deficient bone marrow, pointing to these cofactors as key mediators of Bcl6 inflammatory suppression. Using ChIP-seq, we reveal the SMRT and NCoR corepressor cistromes, each consisting of over 30,000 binding sites with a nearly 50% overlap. While the complete cistromes identify a diversity of signaling pathways, the Bcl6-bound subcistromes for each corepressor are highly enriched for NF-κB-driven inflammatory and tissue remodeling genes. These results reveal that Bcl6-SMRT/NCoR complexes constrain immune responses and contribute to the prevention of atherosclerosis.

Research resource: Comparative nuclear receptor atlas: basal and activated peritoneal B-1 and B-2 cells.

Naïve murine B cells are typically divided into three subsets based on functional and phenotypic characteristics: innate-like B-1 and marginal zone B cells vs. adaptive B-2 cells, also known as follicular or conventional B cells. B-1 cells, the innate-immune-like component of the B cell lineage are the primary source of natural antibodies and have been shown to modulate autoimmune diseases, human B-cell leukemias, and inflammatory disorders such as atherosclerosis. On the other hand, B-2 cells are the principal mediators of the adaptive humoral immune response and represent an important pharmacological target for various conditions including rheumatoid arthritis, lupus erythematosus, and lymphomas. Using the resources of the Nuclear Receptor Signaling Atlas program, we used quantitative real-time PCR to assess the complement of the 49 murine nuclear receptor superfamily expressed in quiescent and toll-like receptor (TLR)-stimulated peritoneal B-1 and B-2 cells. We report the expression of 24 nuclear receptors in basal B-1 cells and 25 nuclear receptors in basal B-2 cells, with, in some cases, dramatic changes in response to TLR 4 or TLR 2/1 stimulation. Comparative nuclear receptor profiling between B-1 and peritoneal B-2 cells reveals a highly concordant expression pattern, albeit at quantitatively dissimilar levels. We also found that splenic B cells express 23 nuclear receptors. This catalog of nuclear receptor expression in B-1 and B-2 cells provides data to be used to better understand the specific roles of nuclear receptors in B cell function, chronic inflammation, and autoimmune disease.

Bcl-6 and NF-kappaB cistromes mediate opposing regulation of the innate immune response.

In the macrophage, toll-like receptors (TLRs) are key sensors that trigger signaling cascades to activate inflammatory programs via the NF-κB gene network. However, the genomic network targeted by TLR/NF-κB activation and the molecular basis by which it is restrained to terminate activation and re-establish quiescence is poorly understood. Here, using chromatin immunoprecipitation sequencing (ChIP-seq), we define the NF-κB cistrome, which is comprised of 31,070 cis-acting binding sites responsive to lipopolysaccharide (LPS)-induced signaling. In addition, we demonstrate that the transcriptional repressor B-cell lymphoma 6 (Bcl-6) regulates nearly a third of the Tlr4-regulated transcriptome, and that 90% of the Bcl-6 cistrome is collapsed following Tlr4 activation. Bcl-6-deficient macrophages are acutely hypersensitive to LPS and, using comparative ChIP-seq analyses, we found that the Bcl-6 and NF-κB cistromes intersect, within nucleosomal distance, at nearly half of Bcl-6-binding sites in stimulated macrophages to promote opposing epigenetic modifications of the local chromatin. These results reveal a genomic strategy for controlling the innate immune response in which repressive and inductive cistromes establish a dynamic balance between macrophage quiescence and activation via epigenetically marked cis-regulatory elements.

PPARgamma activation in adipocytes is sufficient for systemic insulin sensitization.

Although peroxisome proliferator-activated receptor gamma (PPARgamma) agonists such as thiazolidinediones (TZDs) are widely used to treat type 2 diabetes, how its activation in individual tissues contributes to TZD's therapeutic action remains controversial. As TZDs are known to have receptor-independent effects, we sought to establish gain-of-function animal models to delineate the receptor's insulin-sensitizing actions. Unexpectedly, we find that selective activation of PPARgamma in adipocytes, but not in macrophages, is sufficient for whole-body insulin sensitization equivalent to systemic TZD treatment. In addition to improved adipokine, inflammatory, and lipid profiles, PPARgamma activation in mature adipocytes normalizes serum insulin without increased adipogenesis. Co-culture studies indicated that PPARgamma-activated adipocytes broadly suppress induction of inflammatory cytokines and C-X-C family chemokines in macrophages. Collectively, these data describe an "adipocentric" model in which adipose activation of PPARgamma is sufficient for complete insulin sensitization and suggest a specific application for fat selective PPARgamma modulators in diabetic therapy.

SMRT repression of nuclear receptors controls the adipogenic set point and metabolic homeostasis.

The nuclear receptor corepressor, silencing mediator of retinoid and thyroid hormone receptors (SMRT), is recruited by a plethora of transcription factors to mediate lineage and signal-dependent transcriptional repression. We generated a knockin mutation in the receptor interaction domain (RID) of SMRT (SMRT(mRID)) that solely disrupts its interaction with nuclear hormone receptors (NHRs). SMRT(mRID) mice are viable and exhibit no gross developmental abnormalities, demonstrating that the reported lethality of SMRT knockouts is determined by non-NHR transcription factors. However, SMRT(mRID) mice exhibit widespread metabolic defects including reduced respiration, altered insulin sensitivity, and 70% increased adiposity. The latter phenotype is illustrated by the observation that SMRT(mRID)-derived MEFs display a dramatically increased adipogenic capacity and accelerated differentiation rate. Collectively, our results demonstrate that SMRT-RID-dependent repression is a key determinant of the adipogenic set point as well as an integrator of glucose metabolism and whole-body metabolic homeostasis.

PPARdelta-mediated antiinflammatory mechanisms inhibit angiotensin II-accelerated atherosclerosis.

Activation of the nuclear hormone receptor peroxisome proliferator-activated receptor delta (PPARdelta) has been shown to improve insulin resistance, adiposity, and plasma HDL levels. However, its antiatherogenic role remains controversial. Here we report atheroprotective effects of PPARdelta activation in a model of angiotensin II (AngII)-accelerated atherosclerosis, characterized by increased vascular inflammation related to repression of an antiinflammatory corepressor, B cell lymphoma-6 (Bcl-6), and the regulators of G protein-coupled signaling (RGS) proteins RGS4 and RGS5. In this model, administration of the PPARdelta agonist GW0742 (1 or 10 mg/kg) substantially attenuated AngII-accelerated atherosclerosis without altering blood pressure and increased vascular expression of Bcl-6, RGS4, and RGS5, which was associated with suppression of inflammatory and atherogenic gene expression in the artery. In vitro studies demonstrated similar changes in AngII-treated macrophages: PPARdelta activation increased both total and free Bcl-6 levels and inhibited AngII activation of MAP kinases, p38, and ERK1/2. These studies uncover crucial proinflammatory mechanisms of AngII and highlight actions of PPARdelta activation to inhibit AngII signaling, which is atheroprotective.

Nuclear receptor ERR alpha and coactivator PGC-1 beta are effectors of IFN-gamma-induced host defense.

Macrophage activation by the proinflammatory cytokine interferon-gamma (IFN-gamma) is a critical component of the host innate response to bacterial pathogenesis. However, the precise nature of the IFN-gamma-induced activation pathway is not known. Here we show using genome-wide expression and chromatin-binding profiling that IFN-gamma induces the expression of many nuclear genes encoding mitochondrial respiratory chain machinery via activation of the nuclear receptor ERR alpha (estrogen-related receptor alpha, NR3B1). Studies with macrophages lacking ERR alpha demonstrate that it is required for induction of mitochondrial reactive oxygen species (ROS) production and efficient clearance of Listeria monocytogenes (LM) in response to IFN-gamma. As a result, mice lacking ERR alpha are susceptible to LM infection, a phenotype that is localized to bone marrow-derived cells. Furthermore, we found that IFN-gamma-induced activation of ERR alpha depends on coactivator PGC-1 beta (peroxisome proliferator-activated receptor gamma coactivator-1 beta), which appears to be a direct target for the IFN-gamma/STAT-1 signaling cascade. Thus, ERR alpha and PGC-1 beta act together as a key effector of IFN-gamma-induced mitochondrial ROS production and host defense.