Gergely Nagy

Research resource: transcriptome profiling of genes regulated by RXR and its permissive and nonpermissive partners in differentiating monocyte-derived dendritic cells.

Retinoid X receptors (RXRs) are heterodimerization partners for many nuclear receptors and also act as homodimers. Heterodimers formed by RXR and a nonpermissive partner, e.g. retinoic acid receptor (RAR) and vitamin D receptor (VDR), can be activated only by the agonist of the partner receptor. In contrast, heterodimers that contain permissive partners, e.g. liver X receptor (LXR) and peroxisome proliferator-activated receptor (PPAR), can be activated by agonists for either the partner receptor or RXR, raising the possibility of pleiotropic RXR signaling. However, it is not known to what extent the receptor’s activation results in triggering mechanisms dependent or independent of permissive heterodimers. In this study, we systematically and quantitatively characterized all probable RXR-signaling pathways in differentiating human monocyte-derived dendritic cells (Mo-DCs). Using pharmacological, microarray and quantitative RT-PCR techniques, we identified and characterized gene sets regulated by RXR agonists (LG100268 and 9-cis retinoic acid) and agonists for LXRs, PPARs, RARα, and VDR. Our results demonstrated that permissiveness was partially impaired in Mo-DCs, because a large number of genes regulated by PPAR or LXR agonists was not affected by RXR-specific agonists or was regulated to a lesser extent. As expected, we found that RXR agonists regulated only small portions of RARα or VDR targets. Importantly, we could identify and characterize PPAR- and LXR-independent pathways in Mo-DCs most likely mediated by RXR homodimers. These data suggested that RXR signaling in Mo-DCs was mediated via multiple permissive heterodimers and also by mechanism(s) independent of permissive heterodimers, and it was controlled in a cell-type and gene-specific manner.

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.

Macrophage PPARγ, a Lipid Activated Transcription Factor Controls the Growth Factor GDF3 and Skeletal Muscle Regeneration

Tissue regeneration requires inflammatory and reparatory activity of macrophages. Macrophages detect and eliminate the damaged tissue and subsequently promote regeneration. This dichotomy requires the switch of effector functions of macrophages coordinated with other cell types inside the injured tissue. The gene regulatory events supporting the sensory and effector functions of macrophages involved in tissue repair are not well understood. Here we show that the lipid activated transcription factor, PPARγ, is required for proper skeletal muscle regeneration, acting in repair macrophages. PPARγ controls the expression of the transforming growth factor-β (TGF-β) family member, GDF3, which in turn regulates the restoration of skeletal muscle integrity by promoting muscle progenitor cell fusion. This work establishes PPARγ as a required metabolic sensor and transcriptional regulator of repair macrophages. Moreover, this work also establishes GDF3 as a secreted extrinsic effector protein acting on myoblasts and serving as an exclusively macrophage-derived regeneration factor in tissue repair.

Highly efficient differentiation of embryonic stem cells into adipocytes by ascorbic acid

Adipocyte differentiation and function have become the major research targets due to the increasing interest in obesity and related metabolic conditions. Although, late stages of adipogenesis have been extensively studied, the early phases remain poorly understood. Here we present that supplementing ascorbic acid (AsA) to the adipogenic differentiation cocktail enables the robust and efficient differentiation of mouse embryonic stem cells (mESCs) to mature adipocytes. Such ESC-derived adipocytes mimic the gene-expression profile of subcutaneous isolated adipocytes in vivo remarkably well, much closer than 3T3-L1 derived ones. Moreover, the differentiated cells are in a monolayer, allowing a broad range of genome-wide studies in early and late stages of adipocyte differentiation to be performed.

The IL-4/STAT6 signaling axis establishes a conserved microRNA signature in human and mouse macrophages regulating cell survival via miR-342-3p

BackgroundIL-4-driven alternative macrophage activation and proliferation are characteristic features of both antihelminthic immune responses and wound healing in contrast to classical macrophage activation, which primarily occurs during inflammatory responses. The signaling pathways defining the genome-wide microRNA expression profile as well as the cellular functions controlled by microRNAs during alternative macrophage activation are largely unknown. Hence, in the current work we examined the regulation and function of IL-4-regulated microRNAs in human and mouse alternative macrophage activation.MethodsWe utilized microarray-based microRNA profiling to detect the dynamic expression changes during human monocyte–macrophage differentiation and IL-4-mediated alternative macrophage activation. The expression changes and upstream regulatory pathways of selected microRNAs were further investigated in human and mouse in vitro and in vivo models of alternative macrophage activation by integrating small RNA-seq, ChIP-seq, ChIP-quantitative PCR, and gene expression data. MicroRNA-controlled gene networks and corresponding functions were identified using a combination of transcriptomic, bioinformatic, and functional approaches.ResultsThe IL-4-controlled microRNA expression pattern was identified in models of human and mouse alternative macrophage activation. IL-4-dependent induction of miR-342-3p and repression of miR-99b along with miR-125a-5p occurred in both human and murine macrophages in vitro. In addition, a similar expression pattern was observed in peritoneal macrophages of Brugia malayi nematode-implanted mice in vivo. By using IL4Rα- and STAT6-deficient macrophages, we were able to show that IL-4-dependent regulation of miR-342-3p, miR-99b, and miR-125a-5p is mediated by the IL-4Rα–STAT6 signaling pathway. The combination of gene expression studies and chromatin immunoprecipitation experiments demonstrated that both miR-342-3p and its host gene, EVL, are coregulated directly by STAT6. Finally, we found that miR-342-3p is capable of controlling macrophage survival through targeting an anti-apoptotic gene network including Bcl2l1.ConclusionsOur findings identify a conserved IL-4/STAT6-regulated microRNA signature in alternatively activated human and mouse macrophages. Moreover, our study indicates that miR-342-3p likely plays a pro-apoptotic role in such cells, thereby providing a negative feedback arm to IL-4-dependent macrophage proliferation.

A novel method to predict regulatory regions based on histone mark landscapes in macrophages

Macrophages as phagocytes and professional antigen presenting cells play critical roles in both innate and adaptive immunity. Main transcription factors acting during their differentiation and function are known, but the behavior and co-operation of these factors still remained unexplored. We introduce a new approach to map nucleosome-free regions using exclusively active enhancer and core promoter marking histone modification ChIP-seq data. We could detect approximately 56,000 potential active enhancers/promoters showing different lengths and histone modification shapes. Beside the highly enriched PU.1 and C/EBP sites, we could also detect binding sites for RUNX and AP-1, as well as for the MiT (MITF-TFE) family and MEF2 proteins. The PU.1 and C/EBP transcription factors are known for transforming cells into macrophages. The other transcription factors found in this study can play a role in macrophages as well, since it is known that the MiT family proteins are responsible for phagocytic activity and the MEF2 proteins specify monocytic differentiation over the granulocyte direction. Our results imply that this method can provide novel information about transcription factor organization at enhancers and core promoters as well as about the histone modifications surrounding regulatory regions in any immune or other cell types.

Combined High Tibial Osteotomy Decreases Biomechanical Changes Radiologically Detectable in the Sagittal Plane Compared With Closing-Wedge Osteotomy

PURPOSE The purpose of this randomized, prospective study was to compare radiologic changes in the sagittal plane after closing-wedge and combined high tibial osteotomies (HTOs) performed in patients with medial knee arthrosis associated with a varus deformity and requiring a 10 degrees correction. METHODS The patellar height, determined by the Insall-Salvati index (ISI), and tibial slope (TS) angle, measured by the Dejour-Bonnin method, were compared after 45 closing-wedge osteotomy (CWO) and 46 combined osteotomy (CO) procedures. CO is a relatively new technique effectively being the combination of closing-wedge and opening-wedge HTOs. It involves performing a proximal osteotomy parallel to the tibial plateau, followed by a distal osteotomy extending from the lateral part of the tibia to the line of the proximal osteotomy at the center of the tibial condyle. After closure of the lateral part of the osteotomy and consequent opening of the medial part, the removed lateral bone wedge is transferred to the gap on the medial side. RESULTS We found that at the end of the study, 12 months after the operation, CO resulted in significantly smaller changes in TS angle (4.7% v 38.2%) and ISI (2.2% v 5.7%) values than CWO. CONCLUSIONS The main conclusion of this study is that based on our radiologic findings, CO results in significantly smaller changes in TS angle and ISI values than CWO; therefore, CO is better at preserving the normal or near normal condition of the knee than CWO. Altogether, when a 10 degrees correction is required during HTO, we recommend choosing CO instead of CWO. LEVEL OF EVIDENCE Level II, prospective comparative study.

Small-angle neutron scattering study of the ultrastructure of chloroplast thylakoid membranes - Periodicity and structural flexibility of the stroma lamellae

The multilamellar organization of freshly isolated spinach and pea chloroplast thylakoid membranes was studied using small-angle neutron scattering. A broad peak at ~0.02Å(-1) is ascribed to diffraction from domains of ordered, unappressed stroma lamellae, revealing a repeat distance of 294ű7Å in spinach and 345ű11Å in pea. The peak position and hence the repeat distance of stroma lamellae is strongly dependent on the osmolarity and the ionic strength of the suspension medium, as demonstrated by varying the sorbitol and the Mg(++)-concentration in the sample. For pea thylakoid membranes, we show that the repeat distance decreases when illuminating the sample with white light, in accordance with our earlier results on spinach, also regarding the observation that addition of an uncoupler prohibits the light-induced structural changes, a strong indication that these changes are driven by the transmembrane proton gradient. We show that the magnitude of the shrinkage is strongly dependent on light intensity and that the repeat distance characteristic of the dark state after illumination is different from the initial dark state. Prolonged strong illumination leads to irreversible changes and swelling as reflected in increased repeat distances. The observed reorganizations are discussed within the frames of the current structural models of the granum-stroma thylakoid membrane assembly and the regulatory mechanisms in response to variations in the environmental conditions in vivo. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial.

The Transcription Factor STAT6 Mediates Direct Repression of Inflammatory Enhancers and Limits Activation of Alternatively Polarized Macrophages

Summary The molecular basis of signal‐dependent transcriptional activation has been extensively studied in macrophage polarization, but our understanding remains limited regarding the molecular determinants of repression. Here we show that IL‐4‐activated STAT6 transcription factor is required for the direct transcriptional repression of a large number of genes during in vitro and in vivo alternative macrophage polarization. Repression results in decreased lineage‐determining transcription factor, p300, and RNA polymerase II binding followed by reduced enhancer RNA expression, H3K27 acetylation, and chromatin accessibility. The repressor function of STAT6 is HDAC3 dependent on a subset of IL‐4‐repressed genes. In addition, STAT6‐repressed enhancers show extensive overlap with the NF‐&kgr;B p65 cistrome and exhibit decreased responsiveness to lipopolysaccharide after IL‐4 stimulus on a subset of genes. As a consequence, macrophages exhibit diminished inflammasome activation, decreased IL‐1&bgr; production, and pyroptosis. Thus, the IL‐4‐STAT6 signaling pathway establishes an alternative polarization‐specific epigenenomic signature resulting in dampened macrophage responsiveness to inflammatory stimuli. Graphical Abstract Figure. No Caption available. HighlightsIL‐4‐activated STAT6 acts as a transcriptional repressor in macrophagesIL‐4‐STAT6‐repressed enhancers associate with reduced LDTF and p300 bindingInflammatory responsiveness of the IL‐4‐repressed enhancers is attenuatedIL‐4 limits the LPS‐induced inflammasome activation, IL‐1&bgr; production, and pyroptosis &NA; The molecular bases of repressive transcriptional mechanisms contributing to macrophage polarization are not well understood. Czimmerer et al. show that in alternatively polarized macrophages, IL‐4‐activated STAT6 represses a large set of enhancers modulating the transcriptional program. STAT6‐repressed enhancers are characterized by reduced chromatin accessibility, eRNA expression, LDTF, and p300 binding. IL‐4‐STAT6‐mediated repression limits the inflammatory responsiveness including inflammasome activation, IL‐1&bgr; production, and pyroptosis. Thus, the IL4‐STAT6 pathway establishes an epigenomic signature to selectively repress the macrophage inflammation program.

Inducible super-enhancers are organized based on canonical signal-specific transcription factor binding elements

Abstract Super-enhancers are established through the interactions of several enhancers and a large number of proteins, including transcription factors and co-regulators; however, the formation of these interactions is poorly understood. By re-analysing previously published estrogen receptor alpha (ERα) ChIP-seq data sets derived from the MCF-7 cell line, we observed that in the absence of stimulation, future super-enhancers are represented by one or a few transcription factor binding event(s) and these extraordinary enhancers possess a response element largely specific to the ERα dimer. Upon hormonal stimulation, these primary binding sites are surrounded by a large amount of ERα and the critical components of active enhancers, such as P300 and MED1, and together with neighbouring sites bound by newly recruited ERα, they generate the functional super-enhancers. To further validate the role of canonical elements in super-enhancer formation, we investigated some additional signal-dependent transcription factors, confirming that certain, distinguished binding elements have a general organizer function. These results suggest that certain signal-specific transcription factors guide super-enhancer formation upon binding to strong response elements. These findings may reshape the current understanding of how these regulatory units assemble, highlighting the involvement of DNA elements instead of protein–protein interactions.