L. Balint

STAT6 transcription factor is a facilitator of the nuclear receptor PPARγ-regulated gene expression in macrophages and dendritic cells.

Summary Peroxisome proliferator-activated receptor γ (PPARγ) is a lipid-activated transcription factor regulating lipid metabolism and inflammatory response in macrophages and dendritic cells (DCs). These immune cells exposed to distinct inflammatory milieu show cell type specification as a result of altered gene expression. We demonstrate here a mechanism how inflammatory molecules modulate PPARγ signaling in distinct subsets of cells. Proinflammatory molecules inhibited whereas interleukin-4 (IL-4) stimulated PPARγ activity in macrophages and DCs. Furthermore, IL-4 signaling augmented PPARγ activity through an interaction between PPARγ and signal transducer and activators of transcription 6 (STAT6) on promoters of PPARγ target genes, including FABP4. Thus, STAT6 acts as a facilitating factor for PPARγ by promoting DNA binding and consequently increasing the number of regulated genes and the magnitude of responses. This interaction, underpinning cell type-specific responses, represents a unique way of controlling nuclear receptor signaling by inflammatory molecules in immune cells.

Peroxisome Proliferator-activated Receptor γ-regulated ABCG2 Expression Confers Cytoprotection to Human Dendritic Cells

ABCG2, a member of the ATP-binding cassette transporters has been identified as a protective pump against endogenous and exogenous toxic agents. ABCG2 was shown to be expressed at high levels in stem cells and variably regulated during cell differentiation. Here we demonstrate that functional ABCG2 is expressed in human monocyte-derived dendritic cells by the activation of a nuclear hormone receptor, PPARγ. We identified and characterized a 150-base pair long conserved enhancer region, containing three functional PPAR response elements (PPARE), upstream of the human ABCG2 gene. We confirmed the binding of the PPARγ·RXR heterodimer to this enhancer region, suggesting that PPARγ directly regulates the transcription of ABCG2. Consistent with these results, elevated expression of ABCG2 mRNA was coupled to enhanced protein production, resulting in increased xenobiotic extrusion capacity via ABCG2 in PPARγ-activated cells. Furthermore PPARγ instructed dendritic cells showed increased Hoechst dye extrusion and resistance to mitoxantrone. Collectively, these results uncovered a mechanism by which up-regulation of functional ABCG2 expression can be achieved via exogenous or endogenous activation of the lipid-activated transcription factor, PPARγ. The increased expression of the promiscuous ABCG2 transporter can significantly modify the xenobiotic and drug resistance of human myeloid dendritic cells.

Transcriptional Regulation of Human CYP27 Integrates Retinoid, Peroxisome Proliferator-Activated Receptor, and Liver X Receptor Signaling in Macrophages

ABSTRACT Cholesterol uptake and efflux are key metabolic processes associated with macrophage physiology and atherosclerosis. Peroxisome proliferator-activated receptor gamma (PPARγ) and liver X receptor alpha (LXRα) have been linked to the regulation of these processes. It remains to be identified how activation of these receptors is connected and regulated by endogenous lipid molecules. We identified CYP27, a p450 enzyme, as a link between retinoid, PPARγ, and LXR signaling. We show that the human CYP27 gene is under coupled regulation by retinoids and ligands of PPARs via a PPAR-retinoic acid receptor response element in its promoter. Induction of the enzymes expression results in an increased level of 27-hydroxycholesterol and upregulation of LXR-mediated processes. Upregulated CYP27 activity also leads to LXR-independent elimination of CYP27 metabolites as an alternative means of cholesterol efflux. Moreover, human macrophage-rich atherosclerotic lesions have an increased level of retinoid-, PPARγ-, and LXR-regulated gene expression and also enhanced CYP27 levels. Our findings suggest that nuclear receptor-regulated CYP27 expression is likely to be a key integrator of retinoic acid receptor-PPARγ-LXR signaling, relying on natural ligands and contributing to lipid metabolism in macrophages.

Selective modulators of PPAR activity as new therapeutic tools in metabolic diseases.

Peroxisome Proliferator Activated Receptors (PPARs) are regulators of metabolic pathways mainly of lipid metabolism and energy balance. Their medical importance is given by the fact that they have been implicated in development of insulin resistance, obesity and atherosclerosis. In recent years, major progress has been made in understanding the molecular basis of the function of these receptors. As a result of structural studies and identification of putative natural as well as synthetic ligands and activators of PPARs a new concept emerged and new drugs are on their ways to the clinic. The concept of Selective PPAR Modulators (SPPARM) was suggested by analogy to Selective Estrogen Receptor Modulators (SERM). SPPARMs activate the receptors in distinct ways leading to differential gene expression and biological response. The key features in understanding their action is most likely at the molecular details of ligand binding and the subsequently induced conformational changes as well as cofactor binding. A key aspect of this is that unlike classical steroid hormone receptors such as the retinoic acid receptor, the PPAR receptors have a rather large ligand-binding pocket which is not filled with the ligand entirely and the ligand also stabilizes the receptors structure. The liganded receptor can have distinct conformations and this leads to different binding affinities for the various cofactors (coactivators and corepressors). In this review, we will introduce this concept, review the literature that supports it and present an overview of the receptor selective ligands including data about their mechanism of action and biological effects.

Arginine Methylation Provides Epigenetic Transcription Memory for Retinoid-Induced Differentiation in Myeloid Cells

ABSTRACT Cellular differentiation is governed by changes in gene expression, but at the same time, a cells identity needs to be maintained through multiple cell divisions during maturation. In myeloid cell lines, retinoids induce gene expression and a well-characterized two-step lineage-specific differentiation. To identify mechanisms that contribute to cellular transcriptional memory, we analyzed the epigenetic changes taking place on regulatory regions of tissue transglutaminase, a gene whose expression is tightly linked to retinoid-induced differentiation. Here we report that the induction of an intermediary or “primed” state of myeloid differentiation is associated with increased H4 arginine 3 and decreased H3 lysine 4 methylation. These modifications occur before transcription and appear to prime the chromatin for subsequent hormone-regulated transcription. Moreover, inhibition of methyltransferase activity, preacetylation, or activation of the enzyme PAD4 attenuated retinoid-regulated gene expression, while overexpression of PRMT1, a methyltransferase, enhanced retinoid responsiveness. Taken together, our results suggest that H4 arginine 3 methylation is a bona fide positive epigenetic marker and regulator of transcriptional responsiveness as well as a signal integration mechanism during cell differentiation and, as such, may provide epigenetic memory.

The active enhancer network operated by liganded RXR supports angiogenic activity in macrophages

RXR signaling is predicted to have a major impact in macrophages, but neither the biological consequence nor the genomic basis of its ligand activation is known. Comprehensive genome-wide studies were carried out to map liganded RXR-mediated transcriptional changes, active binding sites, and cistromic interactions in the context of the macrophage genome architecture. The macrophage RXR cistrome has 5200 genomic binding sites, which are not impacted by ligand. Active enhancers are characterized by PU.1 binding, an increase of enhancer RNA, and P300 recruitment. Using these features, 387 liganded RXR-bound enhancers were linked to 226 genes, which predominantly reside in CTCF/cohesin-limited functional domains. These findings were molecularly validated using chromosome conformation capture (3C) and 3C combined with sequencing (3C-seq), and we show that selected long-range enhancers communicate with promoters via stable or RXR-induced loops and that some of the enhancers interact with each other, forming an interchromosomal network. A set of angiogenic genes, including Vegfa, has liganded RXR-controlled enhancers and provides the macrophage with a novel inducible program.

A Versatile Method to Design Stem-Loop Primer-Based Quantitative PCR Assays for Detecting Small Regulatory RNA Molecules

Short regulatory RNA-s have been identified as key regulators of gene expression in eukaryotes. They have been involved in the regulation of both physiological and pathological processes such as embryonal development, immunoregulation and cancer. One of their relevant characteristics is their high stability, which makes them excellent candidates for use as biomarkers. Their number is constantly increasing as next generation sequencing methods reveal more and more details of their synthesis. These novel findings aim for new detection methods for the individual short regulatory RNA-s in order to be able to confirm the primary data and characterize newly identified subtypes in different biological conditions. We have developed a flexible method to design RT-qPCR assays that are very sensitive and robust. The newly designed assays were tested extensively in samples from plant, mouse and even human formalin fixed paraffin embedded tissues. Moreover, we have shown that these assays are able to quantify endogenously generated shRNA molecules. The assay design method is freely available for anyone who wishes to use a robust and flexible system for the quantitative analysis of matured regulatory RNA-s.

STAT2/IRF9 directs a prolonged ISGF3-like transcriptional response and antiviral activity in the absence of STAT1.

Evidence is accumulating for the existence of a signal transducer and activator of transcription 2 (STAT2)/interferon regulatory factor 9 (IRF9)-dependent, STAT1-independent interferon alpha (IFNα) signalling pathway. However, no detailed insight exists into the genome-wide transcriptional regulation and the biological implications of STAT2/IRF9-dependent IFNα signalling as compared with interferon-stimulated gene factor 3 (ISGF3). In STAT1-defeicient U3C cells stably overexpressing human STAT2 (hST2-U3C) and STAT1-deficient murine embryonic fibroblast cells stably overexpressing mouse STAT2 (mST2-MS1KO) we observed that the IFNα-induced expression of 2′-5′-oligoadenylate synthase 2 (OAS2) and interferon-induced protein with tetratricopeptide repeats 1 (Ifit1) correlated with the kinetics of STAT2 phosphorylation, and the presence of a STAT2/IRF9 complex requiring STAT2 phosphorylation and the STAT2 transactivation domain. Subsequent microarray analysis of IFNα-treated wild-type (WT) and STAT1 KO cells overexpressing STAT2 extended our observations and identified ∼120 known antiviral ISRE-containing interferon-stimulated genes (ISGs) commonly up-regulated by STAT2/IRF9 and ISGF3. The STAT2/IRF9-directed expression profile of these IFN-stimulated genes (ISGs) was prolonged as compared with the early and transient response mediated by ISGF3. In addition, we identified a group of ‘STAT2/IRF9-specific’ ISGs, whose response to IFNα was ISGF3-independent. Finally, STAT2/IRF9 was able to trigger an antiviral response upon encephalomyocarditis virus (EMCV) and vesicular stomatitis Indiana virus (VSV). Our results further prove that IFNα-activated STAT2/IRF9 induces a prolonged ISGF3-like transcriptome and generates an antiviral response in the absence of STAT1. Moreover, the existence of ‘STAT2/IRF9-specific’ target genes predicts a novel role of STAT2 in IFNα signalling.

Activation of retinoic acid receptor signaling coordinates lineage commitment of spontaneously differentiating mouse embryonic stem cells in embryoid bodies

Retinoid signaling has been implicated in embryonic stem cell differentiation. Here we present a systematic analysis of gene expression changes in mouse embryonic stem cells (mESCs), during their spontaneous differentiation into embryoid bodies and the effect of all‐trans retinoic acid (ATRA) on this process. We show that retinoic acid is present in the serum and is sufficient to activate retinoid signaling at a basal level in undifferentiated mESCs. This signal disappears during embryoid body formation. However exogenously added ATRA resets the spontaneous differentiation programs in embryoid bodies and initiates a distinct genetic program. These data suggest that retinoid signaling not only promotes a particular pathway but also acts as a context dependent general coordinator of the differentiation states in embryonic stem cells.

Ribonucleoprotein-masked nicks at 50-kbp intervals in the eukaryotic genomic DNA

By using a microscopic approach, field inversion single-cell gel electrophoresis, we show that preformed single-strand discontinuities are present in the chromatin of resting and proliferating mammalian and yeast cells. These single-strand breaks are primarily nicks positioned at ≈50-kbp intervals throughout the entire genome that could be efficiently labeled in situ by DNA polymerase I holoenzyme but not by Klenow fragment and terminal transferase unless after ribonucleolytic treatments. The RNA molecules involved appear to comprise R-loops, recognized by the S9.6 RNA/DNA hybrid-specific antibody. By using the breakpoint cluster region of the Mixed Lineage Leukemia (MLL) gene as a model, we have found that the number of manifest nicks detected by FISH performed after field inversion single-cell gel electrophoresis depends on epigenetic context, but the difference between germ-line and translocated MLL alleles is abolished by protease treatment. Our data imply that the double-stranded genomic DNA is composed of contiguous rather than continuous single strands and reveal an aspect of higher-order chromatin organization with ribonucleoprotein-associated persistent nicks defining ≈50-kbp domains.