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.

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.

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.

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.

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.

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.

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.

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.

Prior exposure to microenvironmental signals could fundamentally change the response of macrophages to subsequent stimuli. It is believed that T helper-2 (Th2)-cell-type cytokine interleukin-4 (IL-4) and Toll-like receptor (TLR) ligand-activated transcriptional programs mutually antagonize each other, and no remarkable convergence has been identified between them. In contrast, here, we show that IL-4-polarized macrophages established a hyperinflammatory gene expression program upon lipopolysaccharide (LPS) exposure. This phenomenon, which we termed extended synergy, was supported by IL-4-directed epigenomic remodeling, LPS-activated NF-κB-p65 cistrome expansion, and increased enhancer activity. The EGR2 transcription factor contributed to the extended synergy in a macrophage-subtype-specific manner. Consequently, the previously alternatively polarized macrophages produced increased amounts of immune-modulatory factors both in vitro and in vivo in a murine Th2 cell-type airway inflammation model upon LPS exposure. Our findings establish that IL-4-induced epigenetic reprogramming is responsible for the development of inflammatory hyperresponsiveness to TLR activation and contributes to lung pathologies.

The S. pombe orthologue of the human PAXT connection, Mtl1-Red1 Core (MTREC), is an eleven-subunit complex that targets cryptic unstable transcripts (CUTs) to the nuclear RNA exosome for degradation. It encompasses the canonical poly(A) polymerase Pla1, responsible for polyadenylation of nascent RNA transcripts as part of the cleavage and polyadenylation factor (CPF/CPSF). In this study we identify and characterise the interaction between Pla1 and the MTREC complex core component Red1 and analyse the functional relevance of this interaction in vivo. Our crystal structure of the Pla1-Red1 complex shows that a 58-residue fragment in Red1 binds to the RNA recognition motif domain of Pla1 and tethers it to the MTREC complex. Structure-based Pla1-Red1 interaction mutations show that Pla1, as part of MTREC complex, hyper-adenylates CUTs for their efficient degradation. Interestingly, the Red1-Pla1 interaction is also required for the efficient assembly of the fission yeast facultative heterochromatic islands. Together, our data suggest a complex interplay between the RNA surveillance and 3'-end processing machineries.

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.

Genotyped human B-lymphoblastoid cell lines (LCLs) are widely used models in mapping quantitative trait loci for chromatin features, gene expression, and drug response. The extent of genotype-independent functional genomic variability of the LCL model, although largely overlooked, may inform association study design. In this study, we use flow cytometry, chromatin immunoprecipitation sequencing and mRNA sequencing to study surface marker patterns, quantify genome-wide chromatin changes (H3K27ac) and transcriptome variability, respectively, among five isogenic LCLs derived from the same individual. Most of the studied LCLs were non-monoclonal and had mature B cell phenotypes. Strikingly, nearly one-fourth of active gene regulatory regions showed significantly variable H3K27ac levels, especially enhancers, among which several were classified as clustered enhancers. Large, contiguous genomic regions showed signs of coordinated activity change. Regulatory differences were mirrored by mRNA expression changes, preferentially affecting hundreds of genes involved in specialized cellular processes including immune and drug response pathways. Differential expression of DPYD, an enzyme involved in 5-fluorouracil (5-FU) catabolism, was associated with variable LCL growth inhibition mediated by 5-FU. The extent of genotype-independent functional genomic variability might highlight the need to revisit study design strategies for LCLs in pharmacogenomics.

A wide range of proteins have been reported to condensate into a dense liquid phase, forming a reversible droplet state. Failure in the control of the droplet state can lead to the formation of the more stable amyloid state, which is often disease-related. These observations prompt the question of how many proteins can undergo liquid-liquid phase separation. Here, in order to address this problem, we discuss the biophysical principles underlying the droplet state of proteins by analyzing current evidence for droplet-driver and droplet-client proteins. Based on the concept that the droplet state is stabilized by the large conformational entropy associated with nonspecific side-chain interactions, we develop the FuzDrop method to predict droplet-promoting regions and proteins, which can spontaneously phase separate. We use this approach to carry out a proteome-level study to rank proteins according to their propensity to form the droplet state, spontaneously or via partner interactions. Our results lead to the conclusion that the droplet state could be, at least transiently, accessible to most proteins under conditions found in the cellular environment.

Aging contributes to cellular stress and neurodegeneration. Our understanding is limited regarding the tissue-restricted mechanisms providing protection in postmitotic cells throughout life. Here, we show that spinal cord motoneurons exhibit a high abundance of asymmetric dimethyl arginines (ADMAs) and the presence of this posttranslational modification provides protection against environmental stress. We identify protein arginine methyltransferase 8 (PRMT8) as a tissue-restricted enzyme responsible for proper ADMA level in postmitotic neurons. Male PRMT8 knock-out mice display decreased muscle strength with aging due to premature destabilization of neuromuscular junctions. Mechanistically, inhibition of methyltransferase activity or loss of PRMT8 results in accumulation of unrepaired DNA double-stranded breaks and decrease in the cAMP response-element-binding protein 1 (CREB1) level. As a consequence, the expression of CREB1-mediated prosurvival and regeneration-associated immediate early genes is dysregulated in aging PRMT8 knock-out mice. The uncovered role of PRMT8 represents a novel mechanism of stress tolerance in long-lived postmitotic neurons and identifies PRMT8 as a tissue-specific therapeutic target in the prevention of motoneuron degeneration.SIGNIFICANCE STATEMENT Although most of the cells in our body have a very short lifespan, postmitotic neurons must survive for many decades. Longevity of a cell within the organism depends on its ability to properly regulate signaling pathways that counteract perturbations, such as DNA damage, oxidative stress, or protein misfolding. Here, we provide evidence that tissue-specific regulators of stress tolerance exist in postmitotic neurons. Specifically, we identify protein arginine methyltransferase 8 (PRMT8) as a cell-type-restricted arginine methyltransferase in spinal cord motoneurons (MNs). PRMT8-dependent arginine methylation is required for neuroprotection against age-related increased of cellular stress. Tissue-restricted expression and the enzymatic activity of PRMT8 make it an attractive target for drug development to delay the onset of neurodegenerative disorders.

5-Methylcytosine (m5C) is a prevalent base modification in tRNA and rRNA but it also occurs more broadly in the transcriptome, including in mRNA, where it serves incompletely understood molecular functions. In pursuit of potential links of m5C with mRNA translation, we performed polysome profiling of human HeLa cell lysates and subjected RNA from resultant fractions to efficient bisulfite conversion followed by RNA sequencing (bsRNA-seq). Bioinformatic filters for rigorous site calling were devised to reduce technical noise.

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.

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-1β 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.

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.

IRF3, IRF5 and IRF9 are transcription factors, which play distinct roles in the regulation of antiviral and inflammatory responses. The determinants that mediate IRF-specific enhancer selection are not fully understood. To uncover regions occupied predominantly by IRF3, IRF5 or IRF9, we performed ChIP-seq experiments in activated murine dendritic cells. The identified regions were analysed with respect to the enrichment of DNA motifs, the interferon-stimulated response element (ISRE) and ISRE half-site variants, and chromatin accessibility. Using a machine learning method, we investigated the predictability of IRF-dominance. We found that IRF5-dominant regions differed fundamentally from the IRF3- and IRF9-dominant regions: ISREs were rare, while the NFKB motif and special ISRE half-sites, such as 5'-GAGA-3' and 5'-GACA-3', were enriched. IRF3- and IRF9-dominant regions were characterized by the enriched ISRE motif and lower frequency of accessible chromatin. Enrichment analysis and the machine learning method uncovered the features that favour IRF3 or IRF9 dominancy (e.g. a tripartite form of ISRE and motifs for NF-κB for IRF3, and the GAS motif and certain ISRE variants for IRF9). This study contributes to our understanding of how IRF members, which bind overlapping sets of DNA sequences, can initiate signal-dependent responses without activating superfluous or harmful programmes.

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.