AP-1 activity induced by co-stimulation is required for chromatin opening during T cell activation.

Activation of T cells is dependent on the organized and timely opening and closing of chromatin. Herein, we identify AP-1 as the transcription factor that directs most of this remodeling. Chromatin accessibility profiling showed quick opening of closed chromatin in naive T cells within 5 h of activation. These newly opened regions were strongly enriched for the AP-1 motif, and indeed, ChIP-seq demonstrated AP-1 binding at >70% of them. Broad inhibition of AP-1 activity prevented chromatin opening at AP-1 sites and reduced the expression of nearby genes. Similarly, induction of anergy in the absence of co-stimulation during activation was associated with reduced induction of AP-1 and a failure of proper chromatin remodeling. The translational relevance of these findings was highlighted by the substantial overlap of AP-1-dependent elements with risk loci for multiple immune diseases, including multiple sclerosis, inflammatory bowel disease, and allergic disease. Our findings define AP-1 as the key link between T cell activation and chromatin remodeling.

CWL-Airflow: a lightweight pipeline manager supporting Common Workflow Language.

BACKGROUND: Massive growth in the amount of research data and computational analysis has led to increased use of pipeline managers in biomedical computational research. However, each of the >100 such managers uses its own way to describe pipelines, leading to difficulty porting workflows to different environments and therefore poor reproducibility of computational studies. For this reason, the Common Workflow Language (CWL) was recently introduced as a specification for platform-independent workflow description, and work began to transition existing pipelines and workflow managers to CWL. FINDINGS: Herein, we present CWL-Airflow, a package that adds support for CWL to the Apache Airflow pipeline manager. CWL-Airflow uses CWL version 1.0 specification and can run workflows on stand-alone MacOS/Linux servers, on clusters, or on a variety of cloud platforms. A sample CWL pipeline for processing of chromatin immunoprecipitation sequencing data is provided. CONCLUSIONS: CWL-Airflow will provide users with the features of a fully fledged pipeline manager and the ability to execute CWL workflows anywhere Airflow can run-from a laptop to a cluster or cloud environment. CWL-Airflow is available under Apache License, version 2.0 (Apache-2.0), and can be downloaded from https://barski-lab.github.io/cwl-airflow, https://scicrunch.org/resolver/RRID:SCR_017196.

Analysis of ChIP-Seq and RNA-Seq Data with BioWardrobe.

The massive amount of information produced by ChIP-Seq, RNA-Seq, and other next-generation sequencing-based methods requires computational data analysis. However, biologists performing these experiments often lack training in bioinformatics. BioWardrobe aims to bridge this gap by providing a convenient user interface and by automating routine data-processing steps. This protocol details the use of BioWardrobe for identifying and visualizing ChIP-Seq peaks, calculating RPKMs, performing differential binding or gene expression analysis, and creating plots and heat maps. We specifically describe how to use BioWardrobe's quality control measures for troubleshooting NGS-based experiments.

Polycomb protein SCML2 facilitates H3K27me3 to establish bivalent domains in the male germline.

Repressive H3K27me3 and active H3K4me2/3 together form bivalent chromatin domains, molecular hallmarks of developmental potential. In the male germline, these domains are thought to persist into sperm to establish totipotency in the next generation. However, it remains unknown how H3K27me3 is established on specific targets in the male germline. Here, we demonstrate that a germline-specific Polycomb protein, SCML2, binds to H3K4me2/3-rich hypomethylated promoters in undifferentiated spermatogonia to facilitate H3K27me3. Thus, SCML2 establishes bivalent domains in the male germline of mice. SCML2 regulates two major classes of bivalent domains: Class I domains are established on developmental regulator genes that are silent throughout spermatogenesis, while class II domains are established on somatic genes silenced during late spermatogenesis. We propose that SCML2-dependent H3K27me3 in the male germline prepares the expression of developmental regulator and somatic genes in embryonic development.

TSLP signaling in CD4+ T cells programs a pathogenic T helper 2 cell state.

Pathogenic T helper 2 (TH2) cells, which produce increased amounts of the cytokines interleukin-5 (IL-5) and IL-13, promote allergic disorders, including asthma. Thymic stromal lymphopoietin (TSLP), a cytokine secreted by epithelial and innate immune cells, stimulates such pathogenic TH2 cell responses. We found that TSLP signaling in mouse CD4+ T cells initiated transcriptional changes associated with TH2 cell programming. IL-4 signaling amplified and stabilized the genomic response of T cells to TSLP, which increased the frequency of T cells producing IL-4, IL-5, and IL-13. Furthermore, the TSLP- and IL-4-programmed TH2 cells had a pathogenic phenotype, producing greater amounts of IL-5 and IL-13 and other proinflammatory cytokines than did TH2 cells stimulated with IL-4 alone. TSLP-mediated TH2 cell induction involved distinct molecular pathways, including activation of the transcription factor STAT5 through the kinase JAK2 and repression of the transcription factor BCL6. Mice that received wild-type CD4+ T cells had exacerbated pathogenic TH2 cell responses upon exposure to house dust mites compared to mice that received TSLP receptor-deficient CD4+ T cells. Transient TSLP signaling stably programmed pathogenic potential in memory TH2 cells. In human CD4+ T cells, TSLP and IL-4 promoted the generation of TH2 cells that produced greater amounts of IL-5 and IL-13. Compared to healthy controls, asthmatic children showed enhancement of such T cell responses in peripheral blood. Our data support a sequential cytokine model for pathogenic TH2 cell differentiation and provide a mechanistic basis for the therapeutic targeting of TSLP signaling in human allergic diseases.

Rapid Recall Ability of Memory T cells is Encoded in their Epigenome.

Even though T-cell receptor (TCR) stimulation together with co-stimulation is sufficient for the activation of both naïve and memory T cells, the memory cells are capable of producing lineage specific cytokines much more rapidly than the naïve cells. The mechanisms behind this rapid recall response of the memory cells are still not completely understood. Here, we performed epigenetic profiling of human resting naïve, central and effector memory T cells using ChIP-Seq and found that unlike the naïve cells, the regulatory elements of the cytokine genes in the memory T cells are marked by activating histone modifications even in the resting state. Therefore, the ability to induce expression of rapid recall genes upon activation is associated with the deposition of positive histone modifications during memory T cell differentiation. We propose a model of T cell memory, in which immunological memory state is encoded epigenetically, through poising and transcriptional memory.

Polycomb repressive complex 1 controls uterine decidualization.

Uterine stromal cell decidualization is an essential part of the reproductive process. Decidual tissue development requires a highly regulated control of the extracellular tissue remodeling; however the mechanism of this regulation remains unknown. Through systematic expression studies, we detected that Cbx4/2, Rybp, and Ring1B [components of polycomb repressive complex 1 (PRC1)] are predominantly utilized in antimesometrial decidualization with polyploidy. Immunofluorescence analyses revealed that PRC1 members are co-localized with its functional histone modifier H2AK119ub1 (mono ubiquitination of histone-H2A at lysine-119) in polyploid cell. A potent small-molecule inhibitor of Ring1A/B E3-ubiquitin ligase or siRNA-mediated suppression of Cbx4 caused inhibition of H2AK119ub1, in conjunction with perturbation of decidualization and polyploidy development, suggesting a role for Cbx4/Ring1B-containing PRC1 in these processes. Analyses of genetic signatures by RNA-seq studies showed that the inhibition of PRC1 function affects 238 genes (154 up and 84 down) during decidualization. Functional enrichment analyses identified that about 38% genes primarily involved in extracellular processes are specifically targeted by PRC1. Furthermore, ~15% of upregulated genes exhibited a significant overlap with the upregulated Bmp2 null-induced genes in mice. Overall, Cbx4/Ring1B-containing PRC1 controls decidualization via regulation of extracellular gene remodeling functions and sheds new insights into underlying molecular mechanism(s) through transcriptional repression regulation.

Nuclear Factor κB1/RelA Mediates Inflammation in Human Lung Epithelial Cells at Atmospheric Oxygen Levels.

Oxygen levels range from 2% to 9% in vivo. Atmospheric O2 levels (21%) are known to induce cell proliferation defects and cellular senescence in primary cell cultures. However, the mechanistic basis of the deleterious effects of higher O2 levels is not fully understood. On the other hand, immortalized cells including cancer cell lines, which evade cellular senescence are normally cultured at 21% O2 and the effects of higher O2 on these cells are understudied. Here, we addressed this problem by culturing immortalized human bronchial epithelial (BEAS-2B) cells at ambient atmospheric, 21% O2 and lower, 10% O2. Our results show increased inflammatory response at 21% O2 but not at 10% O2. We found higher RelA binding at the NF-κB1/RelA target gene promoters as well as upregulation of several pro-inflammatory cytokines in cells cultured at 21% O2. RelA knockdown prevented the upregulation of the pro-inflammatory cytokines at 21% O2, suggesting NF-κB1/RelA as a major mediator of inflammatory response in cells cultured at 21% O2. Interestingly, unlike the 21% O2 cultured cells, exposure of 10% O2 cultured cells to H2O2 did not elicit inflammatory response, suggesting increased ability to tolerate oxidative stress in cells cultured at lower O2 levels.

Induction of Interleukin-9-Producing Mucosal Mast Cells Promotes Susceptibility to IgE-Mediated Experimental Food Allergy.

Experimental IgE-mediated food allergy depends on intestinal anaphylaxis driven by interleukin-9 (IL-9). However, the primary cellular source of IL-9 and the mechanisms underlying the susceptibility to food-induced intestinal anaphylaxis remain unclear. Herein, we have reported the identification of multifunctional IL-9-producing mucosal mast cells (MMC9s) that can secrete prodigious amounts of IL-9 and IL-13 in response to IL-33, and mast cell protease-1 (MCPt-1) in response to antigen and IgE complex crosslinking, respectively. Repeated intragastric antigen challenge induced MMC9 development that required T cells, IL-4, and STAT6 transcription factor, but not IL-9 signals. Mice ablated of MMC9 induction failed to develop intestinal mastocytosis, which resulted in decreased food allergy symptoms that could be restored by adoptively transferred MMC9s. Finally, atopic patients that developed food allergy displayed increased intestinal expression of Il9- and MC-specific transcripts. Thus, the induction of MMC9s is a pivotal step to acquire the susceptibility to IgE-mediated food allergy.

BioWardrobe: an integrated platform for analysis of epigenomics and transcriptomics data.

High-throughput sequencing has revolutionized biology by enhancing our ability to perform genome-wide studies. However, due to lack of bioinformatics expertise, modern technologies are still beyond the capabilities of many laboratories. Herein, we present the BioWardrobe platform, which allows users to store, visualize and analyze epigenomics and transcriptomics data using a biologist-friendly web interface, without the need for programming expertise. Predefined pipelines allow users to download data, visualize results on a genome browser, calculate RPKMs (reads per kilobase per million) and identify peaks. Advanced capabilities include differential gene expression and binding analysis, and creation of average tag -density profiles and heatmaps. BioWardrobe can be found at http://biowardrobe.com .

Transcription Factor Repertoire of Homeostatic Eosinophilopoiesis.

The production of mature eosinophils (Eos) is a tightly orchestrated process with the aim to sustain normal Eos levels in tissues while also maintaining low numbers of these complex and sensitive cells in the blood. To identify regulators of homeostatic eosinophilopoiesis in mice, we took a global approach to identify genome-wide transcriptome and epigenome changes that occur during homeostasis at critical developmental stages, including Eos-lineage commitment and lineage maturation. Our analyses revealed a markedly greater number of transcriptome alterations associated with Eos maturation (1199 genes) than with Eos-lineage commitment (490 genes), highlighting the greater transcriptional investment necessary for differentiation. Eos-lineage-committed progenitors (EoPs) were noted to express high levels of granule proteins and contain granules with an ultrastructure distinct from that of mature resting Eos. Our analyses also delineated a 976-gene Eos-lineage transcriptome that included a repertoire of 56 transcription factors, many of which have never previously been associated with Eos. EoPs and Eos, but not granulocyte-monocyte progenitors or neutrophils, expressed Helios and Aiolos, members of the Ikaros family of transcription factors, which regulate gene expression via modulation of chromatin structure and DNA accessibility. Epigenetic studies revealed a distinct distribution of active chromatin marks between genes induced with lineage commitment and genes induced with cell maturation during Eos development. In addition, Aiolos and Helios binding sites were significantly enriched in genes expressed by EoPs and Eos with active chromatin, highlighting a potential novel role for Helios and Aiolos in regulating gene expression during Eos development.

Poised chromatin and bivalent domains facilitate the mitosis-to-meiosis transition in the male germline.

BACKGROUND: The male germline transcriptome changes dramatically during the mitosis-to-meiosis transition to activate late spermatogenesis genes and to transiently suppress genes commonly expressed in somatic lineages and spermatogenesis progenitor cells, termed somatic/progenitor genes. RESULTS: These changes reflect epigenetic regulation. Induction of late spermatogenesis genes during spermatogenesis is facilitated by poised chromatin established in the stem cell phases of spermatogonia, whereas silencing of somatic/progenitor genes during meiosis and postmeiosis is associated with formation of bivalent domains which also allows the recovery of the somatic/progenitor program after fertilization. Importantly, during spermatogenesis mechanisms of epigenetic regulation on sex chromosomes are different from autosomes: X-linked somatic/progenitor genes are suppressed by meiotic sex chromosome inactivation without deposition of H3K27me3. CONCLUSIONS: Our results suggest that bivalent H3K27me3 and H3K4me2/3 domains are not limited to developmental promoters (which maintain bivalent domains that are silent throughout the reproductive cycle), but also underlie reversible silencing of somatic/progenitor genes during the mitosis-to-meiosis transition in late spermatogenesis.

Functional characterization of human T cell hyporesponsiveness induced by CTLA4-Ig.

During activation, T cells integrate multiple signals from APCs and cytokine milieu. The blockade of these signals can have clinical benefits as exemplified by CTLA4-Ig, which blocks interaction of B7 co-stimulatory molecules on APCs with CD28 on T cells. Variants of CTLA4-Ig, abatacept and belatacept are FDA approved as immunosuppressive agents in arthritis and transplantation, yet murine studies suggested that CTLA4-Ig could be beneficial in a number of other diseases. However, detailed analysis of human CD4 cell hyporesponsivness induced by CTLA4-Ig has not been performed. Herein, we established a model to study the effect of CTLA4-Ig on the activation of human naïve T cells in a human mixed lymphocytes system. Comparison of human CD4 cells activated in the presence or absence of CTLA4-Ig showed that co-stimulation blockade during TCR activation does not affect NFAT signaling but results in decreased activation of NF-κB and AP-1 transcription factors followed by a profound decrease in proliferation and cytokine production. The resulting T cells become hyporesponsive to secondary activation and, although capable of receiving TCR signals, fail to proliferate or produce cytokines, demonstrating properties of anergic cells. However, unlike some models of T cell anergy, these cells did not possess increased levels of the TCR signaling inhibitor CBLB. Rather, the CTLA4-Ig-induced hyporesponsiveness was associated with an elevated level of p27kip1 cyclin-dependent kinase inhibitor.

SCML2 establishes the male germline epigenome through regulation of histone H2A ubiquitination.

Gametogenesis is dependent on the expression of germline-specific genes. However, it remains unknown how the germline epigenome is distinctly established from that of somatic lineages. Here we show that genes commonly expressed in somatic lineages and spermatogenesis-progenitor cells undergo repression in a genome-wide manner in late stages of the male germline and identify underlying mechanisms. SCML2, a germline-specific subunit of a Polycomb repressive complex 1 (PRC1), establishes the unique epigenome of the male germline through two distinct antithetical mechanisms. SCML2 works with PRC1 and promotes RNF2-dependent ubiquitination of H2A, thereby marking somatic/progenitor genes on autosomes for repression. Paradoxically, SCML2 also prevents RNF2-dependent ubiquitination of H2A on sex chromosomes during meiosis, thereby enabling unique epigenetic programming of sex chromosomes for male reproduction. Our results reveal divergent mechanisms involving a shared regulator by which the male germline epigenome is distinguished from that of the soma and progenitor cells.

RNF8 regulates active epigenetic modifications and escape gene activation from inactive sex chromosomes in post-meiotic spermatids.

Sex chromosomes are uniquely subject to chromosome-wide silencing during male meiosis, and silencing persists into post-meiotic spermatids. Against this background, a select set of sex chromosome-linked genes escapes silencing and is activated in post-meiotic spermatids. Here, we identify a novel mechanism that regulates escape gene activation in an environment of chromosome-wide silencing in murine germ cells. We show that RNF8-dependent ubiquitination of histone H2A during meiosis establishes active epigenetic modifications, including dimethylation of H3K4 on the sex chromosomes. RNF8-dependent active epigenetic memory, defined by dimethylation of H3K4, persists throughout meiotic division. Various active epigenetic modifications are subsequently established on the sex chromosomes in post-meiotic spermatids. These RNF8-dependent modifications include trimethylation of H3K4, histone lysine crotonylation (Kcr), and incorporation of the histone variant H2AFZ. RNF8-dependent epigenetic programming regulates escape gene activation from inactive sex chromosomes in post-meiotic spermatids. Kcr accumulates at transcriptional start sites of sex-linked genes activated in an RNF8-dependent manner, and a chromatin conformational change is associated with RNF8-dependent epigenetic programming. Furthermore, we demonstrate that this RNF8-dependent pathway is distinct from that which recognizes DNA double-strand breaks. Our results establish a novel connection between a DNA damage response factor (RNF8) and epigenetic programming, specifically in establishing active epigenetic modifications and gene activation.

Long-range electron transfer in recombinant peroxidases anisotropically orientated on gold electrodes.

Long-range electron transfer (ET) in horseradish peroxidase (HRP) was studied with a wild-type recombinant form of HRP, rHRP, and recombinant forms containing histidine and cysteine tags at Gln1, Asn57, Asn189, or Ser309 amino acid residues of the protein. Chemisorption of the enzyme onto the Au electrodes through the tags introduced in different positions of the protein surface provided anisotropic orientations of the rHRPs on the Au surface, which allowed a restricted "rotation" of the rHRP molecules on the electrodes. Atomic force microscopy (AFM) studies revealed the monolayer coverage of the enzyme on gold surfaces and the specific orientations of different forms of rHRP, which may be characterized by different distances between the heme active site of rHRP and the gold electrode. The efficiency of long-range ET between the electrode and the heme of rHRP was estimated from direct non-catalytic electrochemistry of rHRPs differently orientated on Au and compared with the theoretically calculated values from the protein ET model (C. C. Page, C. C. Moser, X. Chen, P. L. Dutton, Nature, 1999, 402, 47-51), under the assumption that ET occurs within the protein structure between the heme and the tag-modified amino acid residue of the protein. Comparative analysis of the long-range ET through the rHRP showed that the highest ET rates, obtained for the rHRP forms containing the tags at C- or N-termini of the enzyme, did not correlate with the shortest ET distance, but were instead consistent with the directional ET along the most favourable ET pathway within the protein matrix.