With the recent recognition of non-coding RNAs (ncRNAs) flanking many genes1-5, a central issue is to fully understand their potential roles in regulated gene transcription programs, possibly through different mechanisms6-12. Here, we report that an RNA-binding protein, TLS, serves as a key transcriptional regulatory sensor of DNA damage signals that, based on its allosteric modulation by RNA, specifically binds to and inhibits CBP/p300 HAT activities on a repressed gene target, cyclin D1 (CCND1). Recruitment of TLS to the CCND1 promoter to cause gene-specific repression is directed by single stranded, low copy number ncRNA transcripts tethered to the 5′ regulatory regions of CCND1 that are induced in response to DNA damage signals. Our data suggest that signal-induced ncRNAs localized to regulatory regions of transcription units can act cooperatively as selective ligands, recruiting and modulating the activities of distinct classes of RNA binding co-regulators in response to specific signals, providing an unexpected ncRNA/RNA-binding protein-based strategy to integrate transcriptional programs.

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Summary Inflammation and macrophage foam cells are characteristic features of atherosclerotic lesions, but the mechanisms linking cholesterol accumulation to inflammation and LXR-dependent response pathways are poorly understood. To investigate this relationship, we utilized lipidomic and transcriptomic methods to evaluate the effect of diet and LDL receptor genotype on macrophage foam cell formation within the peritoneal cavities of mice. Foam cell formation was associated with significant changes in hundreds of lipid species and unexpected suppression, rather than activation, of inflammatory gene expression. We provide evidence that regulated accumulation of desmosterol underlies many of the homeostatic responses observed in macrophage foam cells, including activation of LXR target genes, inhibition of SREBP target genes, selective reprogramming of fatty acid metabolism and suppression of inflammatory response genes. These observations suggest that macrophage activation in atherosclerotic lesions results from extrinsic, pro-inflammatory signals generated within the artery wall that suppress homeostatic and anti-inflammatory functions of desmosterol.

PPAR gamma is required for fat cell development and is the molecular target of antidiabetic thiazolidinediones (TZDs), which exert insulin-sensitizing effects in adipose tissue, skeletal muscle, and liver. Unexpectedly, we found that inactivation of PPAR gamma in macrophages results in the development of significant glucose intolerance plus skeletal muscle and hepatic insulin resistance in lean mice fed a normal diet. This phenotype was associated with increased expression of inflammatory markers and impaired insulin signaling in adipose tissue, muscle, and liver. PPAR gamma-deficient macrophages secreted elevated levels of factors that impair insulin responsiveness in muscle cells in a manner that was enhanced by exposure to FFAs. Consistent with this, the relative degree of insulin resistance became more severe in mice lacking macrophage PPAR gamma following high-fat feeding, and these mice were only partially responsive to TZD treatment. These findings reveal an essential role of PPAR gamma in macrophages for the maintenance of whole-body insulin action and in mediating the antidiabetic actions of TZDs.

We report the lipidomic response of the murine macrophage RAW cell line to Kdo 2 -lipid A, the active component of an inflammatory lipopolysaccharide functioning as a selective TLR4 agonist and compactin, a statin inhibitor of cholesterol biosynthesis. Analyses of lipid molecular species by dynamic quantitative mass spectrometry and concomitant transcriptomic measurements define the lipidome and demonstrate immediate responses in fatty acid metabolism represented by increases in eicosanoid synthesis and delayed responses characterized by sphingolipid and sterol biosynthesis. Lipid remodeling of glycerolipids, glycerophospholipids, and prenols also take place, indicating that activation of the innate immune system by inflammatory mediators leads to alterations in a majority of mammalian lipid categories, including unanticipated effects of a statin drug. Our studies provide a systems-level view of lipid metabolism and reveal significant connections between lipid and cell signaling and biochemical pathways that contribute to innate immune responses and to pharmacological perturbations.The "omics" revolution has provided significant insight into the genes, mRNAs, and proteins of mammalian cells, biological systems, and disease (1-3). An important function of these macromolecular classes is the production of metabolites that in turn are used by cells for replication and function. Lipids comprise major structural and metabolic components of cells and have essential functions in the formation of membranes, energy production, and intracellular signaling. Despite the central role of lipids in mammalian cell function, there has been no systematic effort to define the lipid "parts list" of a mammalian cell or the changes in these lipids associated with cellular function and disease. Many biochemical pathways leading to the synthesis and degradation of major lipid categories are known, but how these pathways interact under normal and pathological conditions remains unexplored. Recent advances in mass spectrometry have made it possible to qualitatively and quantitatively analyze a majority of cellular lipids (4 -8). We report here a comprehensive systems-level analysis of a mammalian cell lipidome through temporal measurements.We characterized lipidomic responses of RAW264.7 (RAW) macrophages to a highly specific ligand for Toll-like receptor 4 (TLR4) 4 that mimics aspects of bacterial infection. This model is of particular interest because of the essential roles that alterations in macrophage lipid metabolism play in innate and adaptive immune responses and the development of chronic inflammatory and cardiovascular diseases. Recent studies further suggest that TLR signaling in macrophages is not only required for innate immunity against viral and bacterial pathogens but also contributes to the pathogenesis of atherosclerosis, diabetes, arthritis, and other inflammatory diseases (9). Although TLR4 signaling is known to exert profound effects on the macrophage transcriptome (10), proteome (11), and selected lipid species that...

The LIPID MAPS Consortium (www.lipidmaps. org) is developing comprehensive procedures for identifying all lipids of the macrophage, following activation by endotoxin. The goal is to quantify temporal and spatial changes in lipids that occur with cellular metabolism and to develop bioinformatic approaches that establish dynamic lipid networks. To achieve these aims, an endotoxin of the highest possible analytical specification is crucial. We now report a large-scale preparation of 3-deoxy-D-manno-octulosonic acid (Kdo) 2 -Lipid A, a nearly homogeneous Re lipopolysaccharide (LPS) sub-structure with endotoxin activity equal to LPS. Kdo 2 -Lipid A was extracted from 2 kg cell paste of a heptose-deficient Escherichia coli mutant. It was purified by chromatography on silica, DEAE-cellulose, and C18 reverse-phase resin. Structure and purity were evaluated by electrospray ionization/mass spectrometry, liquid chromatography/mass spectrometry and 1 H-NMR. Its bioactivity was compared with LPS in RAW 264.7 cells and bone marrow macrophages from wild-type and toll-like receptor 4 (TLR-4)-deficient mice. Cytokine and eicosanoid production, in conjunction with gene expression profiling, were employed as readouts. Kdo 2 -Lipid A is comparable to LPS by these criteria. Its activity is reduced by . The LIPID MAPS consortium is developing quantitative methods for evaluating the composition, biosynthesis, and function of all macrophage lipids (1). These amphipathic substances not only are structural components of biological membranes but also play important roles in the pathophysiology of inflammation, atherosclerosis, and growth control. Additional lipid functions should emerge from the comprehensive analysis of macrophage lipids. Electrospray ionization/mass spectrometry (ESI/MS) (2, 3), coupled with prefractionation methods like reversephase liquid chromatography (LC), is being applied systematically to set the stage for the seamless integration of lipid metabolism into the broader fields of genomics, proteomics, and systems biology. To facilitate this endeavor, LIPID MAPS has introduced a new comprehensive classification system for biological lipids, amenable to computer-based data processing and substructure comparison (4). The eight LIPID MAPS categories are 1) fatty acyls, 2) glycerolipids, 3) glycerophospholipids, 4) sphingolipids, 5) sterol lipids, 6) prenol lipids, 7) saccharolipids,

Precise control of the innate immune response is required for resistance to microbial infections and maintenance of normal tissue homeostasis. Because this response involves coordinate regulation of hundreds of genes, it provides a powerful biological system to elucidate the molecular strategies that underlie signal- and time-dependent transitions of gene expression. Comprehensive genome-wide analysis of the epigenetic and transcription status of the TLR4-induced transcriptional program in macrophages suggests that Toll-like receptor 4 (TLR4)-dependent activation of nearly all immediate/early- (I/E) and late-response genes results from a sequential process in which signal-independent factors initially establish basal levels of gene expression that are then amplified by signal-dependent transcription factors. Promoters of I/E genes are distinguished from those of late genes by encoding a distinct set of signal-dependent transcription factor elements, including TATA boxes, which lead to preferential binding of TBP and basal enrichment for RNA polymerase II immediately downstream of transcriptional start sites. Global nuclear run-on (GRO) sequencing and total RNA sequencing further indicates that TLR4 signaling markedly increases the overall rates of both transcriptional initiation and the efficiency of transcriptional elongation of nearly all I/E genes, while RNA splicing is largely unaffected. Collectively, these findings reveal broadly utilized mechanisms underlying temporally distinct patterns of TLR4-dependent gene activation required for homeostasis and effective immune responses.

The tyrosine kinase activity of the insulin receptor was examined with partially-purified insulin receptors from adipocytes obtained from 13 lean nondiabetics, 14 obese nondiabetics, and 13 obese subjects with non-misulin-dependent diabetes (NIDDM). Incubation of receptors at 4VC with h'-32PIATP and insulin resulted in a maximal 10-12-fold increase in autophosphorylation of the 92-kDa ,8-subunit of the receptor with a half maximal effect at 1-3 ng/ml free insulin. Insulin receptor kinase activity in the three experimental groups was measured by means of both autophosphorylation and phosphorylation of the exogenous substrate Glu4:Tyrl. In the absence of insulin, autophosphorylation and Glu4:Tyrl phosphorylation activities, measured with equal numbers of insulin receptors, were comparable among the three groups. In contrast, insulin-stimulated kinase activity was comparable in the control and obese subjects, but was reduced by '50% in the NIDDM group. These findings indicate that the decrease in kinase activity in NIDDM resulted from a reduction in coupling efficiency between insulin binding and activation of the receptor kinase. The insulin receptor kinase defects observed in NIDDM could be etiologically related to insulin resistance in NIDDM and the pathogenesis of the diabetic state.