Coumarins, furocoumarins and limonoids of Citrus trifoliata and their effects on human colon adenocarcinoma cell lines.

Citrus trifoliata L. (Chinese or Japanese bitter orange) is a medicinal plant with furocoumarins and limonoids as characteristic secondary metabolites. The bitter taste of the fruit limits its use as food, however, it is applied in Asian traditional medicine for its antiphlogistic effect, to treat digestive ulcers and different gastrointestinal disorders and cancer. The phytochemical composition and pharmacological characteristics of this species have not been fully discovered, nevertheless its potential antiproliferative or cytotoxic effects might be related to furocoumarins or limonoids. Our aim was to isolate and identify secondary metabolites from C. trifoliata peel and seeds and to investigate their bioactivities that might be related to the supposed anticancer effect of the plant. By using different chromatographic methods, six pure compounds (phellopterin (2), scoparone (3), myrsellin (4), triphasiol (6), umbelliferone (7) and citropten (5,7-dimethoxycoumarin (8)) were isolated from the peel and four (imperatorin (1), auraptene (5), limonin (9) and deacetyl nomilin (10)) from the seeds of C. trifoliata fruits. These compounds are furocoumarin (1, 2), coumarin (3-8), and limonoid derivatives (9, 10). Scoparone (3) has been detected in this species for the first time. The furocoumarins (1-2) showed moderate activity on the human colorectal adenocarcinona tumor cell line COLO 320 in antiproliferative assays and 2 also had remarkable P-glycoprotein inhibitory activity and synergistic effect with doxorubicin. The coumarin 5 showed significant activity on the COLO 320 cell line in antiproliferative assays and P-glycoprotein inhibitory activity in the FACS (fluorescence activated cell sorting) assay.

Inhibition of NHE-1 Increases Smoke-Induced Proliferative Activity of Barrett's Esophageal Cell Line.

Several clinical studies indicate that smoking predisposes its consumers to esophageal inflammatory and malignant diseases, but the cellular mechanism is not clear. Ion transporters protect esophageal epithelial cells by maintaining intracellular pH at normal levels. In this study, we hypothesized that smoking affects the function of ion transporters, thus playing a role in the development of smoking-induced esophageal diseases. Esophageal cell lines were treated with cigarettesmoke extract (CSE), and the viability and proliferation of the cells, as well as the activity, mRNA and protein expression of the Na+/H+ exchanger-1 (NHE-1), were studied. NHE-1 expression was also investigated in human samples. For chronic treatment, guinea pigs were exposed to tobacco smoke, and NHE-1 activity was measured. Silencing of NHE-1 was performed by using specific siRNA. CSE treatment increased the activity and protein expression of NHE-1 in the metaplastic cells and decreased the rate of proliferation in a NHE-1-dependent manner. In contrast, CSE increased the proliferation of dysplastic cells independently of NHE-1. In the normal cells, the expression and activity of NHE-1 decreased due to in vitro and in vivo smoke exposure. Smoking enhances the function of NHE-1 in Barrett's esophagus, and this is presumably a compensatory mechanism against this toxic agent.

The transcription factor EGR2 is the molecular linchpin connecting STAT6 activation to the late, stable epigenomic program of alternative macrophage polarization.

Macrophages polarize into functionally distinct subtypes while responding to microenvironmental cues. The identity of proximal transcription factors (TFs) downstream from the polarization signals are known, but their activity is typically transient, failing to explain the long-term, stable epigenomic programs developed. Here, we mapped the early and late epigenomic changes of interleukin-4 (IL-4)-induced alternative macrophage polarization. We identified the TF, early growth response 2 (EGR2), bridging the early transient and late stable gene expression program of polarization. EGR2 is a direct target of IL-4-activated STAT6, having broad action indispensable for 77% of the induced gene signature of alternative polarization, including its autoregulation and a robust, downstream TF cascade involving PPARG. Mechanistically, EGR2 binding results in chromatin opening and the recruitment of chromatin remodelers and RNA polymerase II. Egr2 induction is evolutionarily conserved during alternative polarization of mouse and human macrophages. In the context of tissue resident macrophages, Egr2 expression is most prominent in the lung of a variety of species. Thus, EGR2 is an example of an essential and evolutionarily conserved broad acting factor, linking transient polarization signals to stable epigenomic and transcriptional changes in macrophages.

Sequence-based prediction of protein binding mode landscapes.

Interactions between disordered proteins involve a wide range of changes in the structure and dynamics of the partners involved. These changes can be classified in terms of binding modes, which include disorder-to-order (DO) transitions, when proteins fold upon binding, as well as disorder-to-disorder (DD) transitions, when the conformational heterogeneity is maintained in the bound states. Furthermore, systematic studies of these interactions are revealing that proteins may exhibit different binding modes with different partners. Proteins that exhibit this context-dependent binding can be referred to as fuzzy proteins. Here we investigate amino acid code for fuzzy binding in terms of the entropy of the probability distribution of transitions towards decreasing order. We implement these entropy calculations into the FuzPred (http://protdyn-fuzpred.org) algorithm to predict the range of context-dependent binding modes of proteins from their amino acid sequences. As we illustrate through a variety of examples, this method identifies those binding sites that are sensitive to the cellular context or post-translational modifications, and may serve as regulatory points of cellular pathways.

Labelled regulatory elements are pervasive features of the macrophage genome and are dynamically utilized by classical and alternative polarization signals.

The concept of tissue-specific gene expression posits that lineage-determining transcription factors (LDTFs) determine the open chromatin profile of a cell via collaborative binding, providing molecular beacons to signal-dependent transcription factors (SDTFs). However, the guiding principles of LDTF binding, chromatin accessibility and enhancer activity have not yet been systematically evaluated. We sought to study these features of the macrophage genome by the combination of experimental (ChIP-seq, ATAC-seq and GRO-seq) and computational approaches. We show that Random Forest and Support Vector Regression machine learning methods can accurately predict chromatin accessibility using the binding patterns of the LDTF PU.1 and four other key TFs of macrophages (IRF8, JUNB, CEBPA and RUNX1). Any of these TFs alone were not sufficient to predict open chromatin, indicating that TF binding is widespread at closed or weakly opened chromatin regions. Analysis of the PU.1 cistrome revealed that two-thirds of PU.1 binding occurs at low accessible chromatin. We termed these sites labelled regulatory elements (LREs), which may represent a dormant state of a future enhancer and contribute to macrophage cellular plasticity. Collectively, our work demonstrates the existence of LREs occupied by various key TFs, regulating specific gene expression programs triggered by divergent macrophage polarizing stimuli.

The Nuclear Receptor PPARγ Controls Progressive Macrophage Polarization as a Ligand-Insensitive Epigenomic Ratchet of Transcriptional Memory.

Macrophages polarize into distinct phenotypes in response to complex environmental cues. We found that the nuclear receptor PPARγ drove robust phenotypic changes in macrophages upon repeated stimulation with interleukin (IL)-4. The functions of PPARγ on macrophage polarization in this setting were independent of ligand binding. Ligand-insensitive PPARγ bound DNA and recruited the coactivator P300 and the architectural protein RAD21. This established a permissive chromatin environment that conferred transcriptional memory by facilitating the binding of the transcriptional regulator STAT6 and RNA polymerase II, leading to robust production of enhancer and mRNAs upon IL-4 re-stimulation. Ligand-insensitive PPARγ binding controlled the expression of an extracellular matrix remodeling-related gene network in macrophages. Expression of these genes increased during muscle regeneration in a mouse model of injury, and this increase coincided with the detection of IL-4 and PPARγ in the affected tissue. Thus, a predominantly ligand-insensitive PPARγ:RXR cistrome regulates progressive and/or reinforcing macrophage polarization.

Lyophilized human cells stored at room temperature preserve multiple RNA species at excellent quality for RNA sequencing.

Biobanks operating at ambient temperatures would dramatically reduce the costs associated with standard cryogenic storage. In the present study, we used lyophilization to stabilize unfractionated human cells in a dried state at room temperature and tested the yield and integrity of the isolated RNA by microfluidic electrophoresis, RT-qPCR and RNA sequencing. RNA yields and integrity measures were not reduced for lyophilized cells (unstored, stored for two weeks or stored for two months) compared to their paired controls. The abundance of the selected mRNAs with various expression levels, as well as enhancer-associated RNAs and cancer biomarker long non-coding RNAs (MALAT1, GAS5 and TUG1), were not significantly different between the two groups as assessed by RT-qPCR. RNA sequencing data of three lyophilized samples stored for two weeks at room temperature revealed a high degree of similarity with their paired controls in terms of the RNA biotype distribution, cumulative gene diversity, gene body read coverage and per base mismatch rate. Among the 28 differentially expressed genes transcriptional regulators, as well as certain transcript properties suggestive of a residual active decay mechanism were enriched. Our study suggests that freeze-drying of human cells is a suitable alternative for the long-term stabilization of total RNA in whole human cells for routine diagnostics and high-throughput biomedical research.

Endothelial cell activation is attenuated by everolimus via transcriptional and post-transcriptional regulatory mechanisms after drug-eluting coronary stenting.

We previously found higher level of endothelial cell (EC) activation in patients who suffered from in-stent restenosis after bare-metal stenting compared to subjects who underwent drug-eluting stenting (DES) showing no complications. Here we investigated the potential transcriptional and post-transcriptional regulatory mechanisms by which everolimus attenuated EC activation after DES. We studied the effect of everolimus on E-selectin (SELE) and VCAM1 mRNA levels when human coronary artery (HCAECs) and human umbilical vein ECs were challenged with recombinant TNF-α (100 ng/mL) for 1-24 hours in the presence or absence of everolimus using 0.5 µM concentration locally maintained by DES. EC activation was evaluated via the levels of IL-1ß and IL-6 mRNAs with miR-155 expression by RT-qPCR as well as the nuclear translocation of nuclear factor kappa beta (NF-κB) detected by fluorescence microscopy. To investigate the transcriptional regulation of E-selectin and VCAM-1, TNF-α-induced enhancer RNA (eRNA) expression at p65-bound enhancers in the neighboring genomic regions of SELE and VCAM1 genes, including SELE_-11Kb and VCAM1_-10Kb, were measured in HCAECs. Mature and precursor levels of E-selectin and VCAM-1 repressor miR-181b were quantified to analyze the post-transcriptional regulation of these genes in HCAECs. Circulating miR-181b was analyzed in plasma samples of stented subjects by stem-loop RT-qPCR. TNF-α highly elevated E-selectin and VCAM-1 expression at transcriptional level in ECs. Levels of mature, pre- and pri-miR-181b were repressed in ECs by TNF-α, while everolimus acted as a negative regulator of EC activation via inhibited translocation of NF-κB p65 subunit into cell nuclei, lowered eRNA expression at SELE and VCAM1 genes-associated enhancers and modulated expression of their post-transcriptional repressor miR-181b. Significant negative correlation was observed between plasma miR-181b and soluble E-selectin and VCAM-1 in patients. In conclusion, everolimus attenuates EC activation via reduced NF-κB p65 translocation causing decreased E-selectin and VCAM-1 expression at transcriptional and post-transcriptional level after DES.

The IL-4/STAT6/PPARγ signaling axis is driving the expansion of the RXR heterodimer cistrome, providing complex ligand responsiveness in macrophages.

Retinoid X receptor (RXR) is an obligate heterodimeric partner of several nuclear receptors (NRs), and as such a central component of NR signaling regulating the immune and metabolic phenotype of macrophages. Importantly, the binding motifs of RXR heterodimers are enriched in the tissue-selective open chromatin regions of resident macrophages, suggesting roles in subtype specification. Recent genome-wide studies revealed that RXR binds to thousands of sites in the genome, but the mechanistic details how the cistrome is established and serves ligand-induced transcriptional activity remained elusive. Here we show that IL-4-mediated macrophage plasticity results in a greatly extended RXR cistrome via both direct and indirect actions of the transcription factor STAT6. Activation of STAT6 leads to chromatin remodeling and RXR recruitment to de novo enhancers. In addition, STAT6 triggers a secondary transcription factor wave, including PPARγ. PPARγ appears to be indispensable for the development of RXR-bound de novo enhancers, whose activities can be modulated by the ligands of the PPARγ:RXR heterodimer conferring ligand selective cellular responses. Collectively, these data reveal the mechanisms leading to the dynamic extension of the RXR cistrome and identify the lipid-sensing enhancer sets responsible for the appearance of ligand-preferred gene signatures in alternatively polarized macrophages.

Analytical parameters and validation of homopolymer detection in a pyrosequencing-based next generation sequencing system.

BACKGROUND: Current technologies in next-generation sequencing are offering high throughput reads at low costs, but still suffer from various sequencing errors. Although pyro- and ion semiconductor sequencing both have the advantage of delivering long and high quality reads, problems might occur when sequencing homopolymer-containing regions, since the repeating identical bases are going to incorporate during the same synthesis cycle, which leads to uncertainty in base calling. The aim of this study was to evaluate the analytical performance of a pyrosequencing-based next-generation sequencing system in detecting homopolymer sequences using homopolymer-preintegrated plasmid constructs and human DNA samples originating from patients with cystic fibrosis. RESULTS: In the plasmid system average correct genotyping was 95.8% in 4-mers, 87.4% in 5-mers and 72.1% in 6-mers. Despite the experienced low genotyping accuracy in 5- and 6-mers, it was possible to generate amplicons with more than a 90% adequate detection rate in every homopolymer tract. When homopolymers in the CFTR gene were sequenced average accuracy was 89.3%, but varied in a wide range (52.2 - 99.1%). In all but one case, an optimal amplicon-sequencing primer combination could be identified. In that single case (7A tract in exon 14 (c.2046_2052)), none of the tested primer sets produced the required analytical performance. CONCLUSIONS: Our results show that pyrosequencing is the most reliable in case of 4-mers and as homopolymer length gradually increases, accuracy deteriorates. With careful primer selection, the NGS system was able to correctly genotype all but one of the homopolymers in the CFTR gene. In conclusion, we configured a plasmid test system that can be used to assess genotyping accuracy of NGS devices and developed an accurate NGS assay for the molecular diagnosis of CF using self-designed primers for amplification and sequencing.

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

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-κB 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-1ß 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.

Dynamic transcriptional control of macrophage miRNA signature via inflammation responsive enhancers revealed using a combination of next generation sequencing-based approaches.

MicroRNAs are important components of the post-transcriptional fine-tuning of macrophage gene expression in physiological and pathological conditions. However, the mechanistic underpinnings and the cis-acting genomic factors of how macrophage polarizing signals induce miRNA expression changes are not well characterized. Therefore, we systematically evaluated the transcriptional basis underlying the inflammation-mediated regulation of macrophage microRNome using the combination of different next generation sequencing datasets. We investigated the LPS-induced expression changes at mature miRNA and pri-miRNA levels in mouse macrophages utilizing a small RNA-seq method and publicly available GRO-seq dataset, respectively. Next, we identified an enhancer set associated with LPS-responsive pri-miRNAs based on publicly available H3K4 mono-methylation-specific ChIP-seq and GRO-seq datasets. This enhancer set was further characterized by the combination of publicly available ChIP and ATAC-seq datasets. Finally, direct interactions between the miR-155-coding genomic region and its distal regulatory elements were identified using a 3C-seq approach. Our analysis revealed 15 robustly LPS-regulated miRNAs at the transcriptional level. In addition, we found that these miRNA genes are associated with an inflammation-responsive enhancer network. Based on NFκB-p65 and JunB transcription factor binding, we showed two distinct enhancer subsets associated with LPS-activated miRNAs that possess distinct epigenetic characteristics and LPS-responsiveness. Finally, our 3C-seq analysis revealed the LPS-induced extensive reorganization of the pri-miR-155-associated functional chromatin domain as well as chromatin loop formation between LPS-responsive enhancers and the promoter region. Our genomic approach successfully combines various genome-wide datasets and allows the identification of the putative regulatory elements controlling miRNA expression in classically activated macrophages.

Extensive and functional overlap of the STAT6 and RXR cistromes in the active enhancer repertoire of human CD14+ monocyte derived differentiating macrophages.

Macrophages are able to differentiate into classically polarized (M1) or alternatively polarized (M2) states upon encountering pro-inflammatory cytokines such as interferon (IFN) γ or anti-inflammatory cytokines such as interleukin (IL) -4/IL-13, respectively. Moreover, macrophages are known to regulate lipid metabolism via multiple members of the nuclear hormone receptor family, including the retinoid X receptors (RXR). It has been also documented that cytokines are able to modulate macrophage responses to lipid signals but the nature of these interactions and the underlying mechanisms of these processes especially at the level of the chromatinized genome are not well understood. Previous work from our laboratory suggested that STAT6 is a facilitator of nuclear receptor mediated transcriptional activity acting at the genome level. This prompted us to investigate genome-wide DNA binding events and the development of cistromes in human CD14+ monocyte-derived macrophages upon exposure to IL-4. We determined the impact of IL-4 on the PU.1, RXR and STAT6 cistromes within the active enhancer regions marked by H3K27-acetylation using chromatin immunoprecipitation followed by deep sequencing and integrated bioinformatics analyses. We found that about 2/3rd of the IL-4 induced STAT6 peaks co-localized with RXR peaks. These STAT6/RXR co-peaks differed at least in part from the non-overlapping RXR peaks regarding the most enriched de novo transcription factor binding motifs. Interestingly, RXR-binding was not regulated at the STAT6/RXR co-bound enhancers following IL-4 stimulation, but differential enhancer interactions were observed between the IL-4/STAT6 and RXR signaling pathways acting in a gene selective manner. Our results suggest that there is a novel, so far uncharacterized cistromic crosstalk between RXR and STAT6 that is likely to contribute to the formation of the active enhancer repertoire, transcriptome and differential signal-specific gene regulation of polarized macrophages.

Nucleosome stability measured in situ by automated quantitative imaging.

Current approaches have limitations in providing insight into the functional properties of particular nucleosomes in their native molecular environment. Here we describe a simple and powerful method involving elution of histones using intercalators or salt, to assess stability features dependent on DNA superhelicity and relying mainly on electrostatic interactions, respectively, and measurement of the fraction of histones remaining chromatin-bound in the individual nuclei using histone type- or posttranslational modification- (PTM-) specific antibodies and automated, quantitative imaging. The method has been validated in H3K4me3 ChIP-seq experiments, by the quantitative assessment of chromatin loop relaxation required for nucleosomal destabilization, and by comparative analyses of the intercalator and salt induced release from the nucleosomes of different histones. The accuracy of the assay allowed us to observe examples of strict association between nucleosome stability and PTMs across cell types, differentiation state and throughout the cell-cycle in close to native chromatin context, and resolve ambiguities regarding the destabilizing effect of H2A.X phosphorylation. The advantages of the in situ measuring scenario are demonstrated via the marked effect of DNA nicking on histone eviction that underscores the powerful potential of topological relaxation in the epigenetic regulation of DNA accessibility.

Retinoid X receptor suppresses a metastasis-promoting transcriptional program in myeloid cells via a ligand-insensitive mechanism.

Retinoid X receptor (RXR) regulates several key functions in myeloid cells, including inflammatory responses, phagocytosis, chemokine secretion, and proangiogenic activity. Its importance, however, in tumor-associated myeloid cells is unknown. In this study, we demonstrate that deletion of RXR in myeloid cells enhances lung metastasis formation while not affecting primary tumor growth. We show that RXR deficiency leads to transcriptomic changes in the lung myeloid compartment characterized by increased expression of prometastatic genes, including important determinants of premetastatic niche formation. Accordingly, RXR-deficient myeloid cells are more efficient in promoting cancer cell migration and invasion. Our results suggest that the repressive activity of RXR on prometastatic genes is mediated primarily through direct DNA binding of the receptor along with nuclear receptor corepressor (NCoR) and silencing mediator of retinoic acid and thyroid hormone receptor (SMRT) corepressors and is largely unresponsive to ligand activation. In addition, we found that expression and transcriptional activity of RXRα is down-modulated in peripheral blood mononuclear cells of patients with lung cancer, particularly in advanced and metastatic disease. Overall, our results identify RXR as a regulator in the myeloid cell-assisted metastatic process and establish lipid-sensing nuclear receptors in the microenvironmental regulation of tumor progression.

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

BACKGROUND: IL-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. METHODS: We 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. RESULTS: The 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. CONCLUSIONS: Our 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.

Highly Dynamic Transcriptional Signature of Distinct Macrophage Subsets during Sterile Inflammation, Resolution, and Tissue Repair.

Macrophage gene expression determines phagocyte responses and effector functions. Macrophage plasticity has been mainly addressed in in vitro models that do not account for the environmental complexity observed in vivo. In this study, we show that microarray gene expression profiling revealed a highly dynamic landscape of transcriptomic changes of Ly6C(pos)CX3CR1(lo) and Ly6C(neg)CX3CR1(hi) macrophage populations during skeletal muscle regeneration after a sterile damage. Systematic gene expression analysis revealed that the time elapsed, much more than Ly6C status, was correlated with the largest differential gene expression, indicating that the time course of inflammation was the predominant driving force of macrophage gene expression. Moreover, Ly6C(pos)/Ly6C(neg) subsets could not have been aligned to canonical M1/M2 profiles. Instead, a combination of analyses suggested the existence of four main features of muscle-derived macrophages specifying important steps of regeneration: 1) infiltrating Ly6C(pos) macrophages expressed acute-phase proteins and exhibited an inflammatory profile independent of IFN-γ, making them damage-associated macrophages; 2) metabolic changes of macrophages, characterized by a decreased glycolysis and an increased tricarboxylic acid cycle/oxidative pathway, preceded the switch to and sustained their anti-inflammatory profile; 3) Ly6C(neg) macrophages, originating from skewed Ly6C(pos) cells, actively proliferated; and 4) later on, restorative Ly6C(neg) macrophages were characterized by a novel profile, indicative of secretion of molecules involved in intercellular communications, notably matrix-related molecules. These results show the highly dynamic nature of the macrophage response at the molecular level after an acute tissue injury and subsequent repair, and associate a specific signature of macrophages to predictive specialized functions of macrophages at each step of tissue injury/repair.

PRMT1 and PRMT8 regulate retinoic acid-dependent neuronal differentiation with implications to neuropathology.

Retinoids are morphogens and have been implicated in cell fate commitment of embryonic stem cells (ESCs) to neurons. Their effects are mediated by RAR and RXR nuclear receptors. However, transcriptional cofactors required for cell and gene-specific retinoid signaling are not known. Here we show that protein arginine methyl transferase (PRMT) 1 and 8 have key roles in determining retinoid regulated gene expression and cellular specification in a multistage neuronal differentiation model of murine ESCs. PRMT1 acts as a selective modulator, providing the cells with a mechanism to reduce the potency of retinoid signals on regulatory "hotspots." PRMT8 is a retinoid receptor target gene itself and acts as a cell type specific transcriptional coactivator of retinoid signaling at later stages of differentiation. Lack of either of them leads to reduced nuclear arginine methylation, dysregulated neuronal gene expression, and altered neuronal activity. Importantly, depletion of PRMT8 results in altered expression of a distinct set of genes, including markers of gliomagenesis. PRMT8 is almost entirely absent in human glioblastoma tissues. We propose that PRMT1 and PRMT8 serve as a rheostat of retinoid signaling to determine neuronal cell specification in a context-dependent manner and might also be relevant in the development of human brain malignancy.

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.

Measuring expression levels of small regulatory RNA molecules from body fluids and formalin-fixed, paraffin-embedded samples.

MicroRNAs are involved in the regulation of various pathophysiological processes such as immune regulation and cancer. Next-generation sequencing methods enable us to monitor their presence in various types of samples but we need flexible methods for validating datasets generated by high-throughput methods. Here we describe the detailed protocols to be used with our MiRNA Primer Design Tool assay design system. The presented methods allow the flexible design of the oligonucleotides needed for the RT-qPCR detection of any variant of small regulatory RNA molecules from virtually any species. This method can be used to measure miRNA levels from formalin-fixed, paraffin-embedded (FFPE) samples and various body fluids. As an example, we show the results of the hsa-miR-515-3p, hsa-miR-325, and hsa-miR-155 quantification using a specific UPL probe (Universal Probe Library) and a stem-loop RT-qPCR assay. The small nucleolar RNA RNU43 is used as endogenous control for normalization of the results. Urine from healthy pregnant women and FFPE samples from patients diagnosed with colorectal cancer and treated with antibody-based anti-EGFR monotherapy were used as samples.