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Osteoclasts (OCs) are bone-resorbing cells formed by the serial fusion of monocytes. In mice and humans, three distinct subsets of monocytes exist; however, it is unclear if all of them exhibit osteoclastogenic potential. Here we show that in wild-type (WT) mice, Ly6Chi and Ly6Cint monocytes are the primary source of OC formation when compared to Ly6C- monocytes. Their osteoclastogenic potential is dictated by increased expression of signaling receptors and activation of preestablished transcripts, as well as de novo gain in enhancer activity and promoter changes. In the absence of interferon regulatory factor 8 (IRF8), a transcription factor important for myelopoiesis and osteoclastogenesis, all three monocyte subsets are programmed to display higher osteoclastogenic potential. Enhanced NFATc1 nuclear translocation and amplified transcriptomic and epigenetic changes initiated at early developmental stages direct the increased osteoclastogenesis in Irf8-deficient mice. Collectively, our study provides novel insights into the transcription factors and active cis-regulatory elements that regulate OC differentiation. © 2020 American Society for Bone and Mineral Research (ASBMR).

Summary: HIRA is a histone chaperone that deposits the histone variant H3.3 in transcriptionally active genes. In DiGeorge syndromes, a DNA stretch encompassing HIRA is deleted. The syndromes manifest varied abnormalities, including immunodeficiency and thrombocytopenia. HIRA is essential in mice, as total knockout (KO) results in early embryonic death. However, the role of HIRA in hematopoiesis is poorly understood. We investigate hematopoietic cell-specific Hira deletion in mice and show that it dramatically reduces bone marrow hematopoietic stem cells (HSCs), resulting in anemia, thrombocytopenia, and lymphocytopenia. In contrast, fetal hematopoiesis is normal in Hira-KO mice, although fetal HSCs lack the reconstitution capacity. Transcriptome analysis reveals that HIRA is required for expression of many transcription factors and signaling molecules critical for HSCs. ATAC-seq analysis demonstrates that HIRA establishes HSC-specific DNA accessibility, including the SPIB/PU.1 sites. Together, HIRA provides a chromatin environment essential for HSCs, thereby steering their development and survival. : Chen et al. show that the histone H3.3 chaperone HIRA directs development and survival of adult hematopoietic stem cells (HSCs). Hira deletion impairs development of all lineages of hematopoiesis. Keywords: HIRA, histone chaperone, H3.3, hematopoietic stem cells, DiGeorge syndrome

Regulation of endosomal Toll-like receptor (TLR) responses by the chemokine CXCL4 is implicated in inflammatory and fibrotic diseases, with CXCL4 proposed to potentiate TLR responses by binding to nucleic acid TLR ligands and facilitating their endosomal delivery. Here we report that in human monocytes/macrophages, CXCL4 initiates signaling cascades and downstream epigenomic reprogramming that change the profile of the TLR8 response by selectively amplifying inflammatory gene transcription and interleukin (IL)−1β production, while partially attenuating the interferon response. Mechanistically, costimulation by CXCL4 and TLR8 synergistically activates TBK1 and IKKε, repurposes these kinases towards an inflammatory response via coupling with IRF5, and activates the NLRP3 inflammasome. CXCL4 signaling, in a cooperative and synergistic manner with TLR8, induces chromatin remodeling and activates de novo enhancers associated with inflammatory genes. Our findings thus identify new regulatory mechanisms of TLR responses relevant for cytokine storm, and suggest targeting the TBK1-IKKε-IRF5 axis may be beneficial in inflammatory diseases.

CXCL4 regulates responses of immune cells to endosomal TLRs and has been implicated in the pathogenesis of inflammatory and fibrotic diseases. However, mechanisms by which CXCL4 modulates TLR responses, and its functions in monocytes/macrophages, are still unclear. Here we report that CXCL4 changes the profile of the TLR8 response in human monocytes by selectively and dramatically amplifying inflammatory gene transcription and IL-1b production while partially attenuating the IFN response. Mechanistically, costimulation by CXCL4 and TLR8 synergistically activated TBK1/IKKe and repurposed these kinases towards an inflammatory response via coupling with IRF5, and by activating the NLRP3 inflammasome without the need for a second signal. CXCL4 strongly induced chromatin remodeling in a cooperative and synergistic manner with TLR8 signaling, inducing de novo enhancers associated with inflammatory genes. These findings identify signaling and epigenomic mechanisms that underly synergistic activation of inflammatory genes by CXCL4 and TLR8, provide a new paradigm for modulation of TLR responses that is relevant for cytokine storm, and suggest targeting the TBK1/IKKe-IRF5 axis may be beneficial in inflammatory diseases.

Differences in immune responses to viruses and autoimmune diseases such as systemic lupus erythematosus (SLE) can show sexual dimorphism. Age-associated B cells (ABC) are a population of CD11c+T-bet+ B cells critical for antiviral responses and autoimmune disorders. Absence of DEF6 and SWAP-70, two homologous guanine exchange factors, in double-knock-out (DKO) mice leads to a lupus-like syndrome in females marked by accumulation of ABCs. Here we demonstrate that DKO ABCs show sex-specific differences in cell number, upregulation of an ISG signature, and further differentiation. DKO ABCs undergo oligoclonal expansion and differentiate into both CD11c+ and CD11c− effector B cell populations with pathogenic and pro-inflammatory function as demonstrated by BCR sequencing and fate-mapping experiments. Tlr7 duplication in DKO males overrides the sex-bias and further augments the dissemination and pathogenicity of ABCs, resulting in severe pulmonary inflammation and early mortality. Thus, sexual dimorphism shapes the expansion, function and differentiation of ABCs that accompanies TLR7-driven immunopathogenesis., Autoimmunity mediated by age-associated B cells (ABC) can affect males and females differently. Here, using a lupus-like mouse model that affects females more severely, the authors observe an ABC mediated and guanine nucleotide exchange factor (GEF) restrained pathogenic process involving TLR7.

Activation of macrophage proinflammatory and antimicrobial phenotypes is regulated by IFN-γ and LPS via synergistic induction of canonical, inflammatory NF-κB target genes. However, whether IFN-γ negatively regulates components of the LPS response, and how this may affect macrophage activation, is still unclear. Here we use combined transcriptomic and epigenomic approaches to find that IFN-γ selectively abrogates LPS-induced feedback and alters macrophage metabolic pathways by suppressing TLR4-mediated gene activation. In contrast to superinduction of inflammatory genes via enhancers that bind IRF1 and STAT1, IFN-γ represses target enhancers that bind STAT3. TLR4-activated but IFN-γ-suppressed enhancers comprise two subsets discernable by differential regulation of histone acetylation and recruitment of STAT3, CDK8 and cohesin. Our findings thus show that IFN-γ suppresses feedback inhibitory and metabolic components of TLR responses to enhance macrophage activation; they also provide insights for IFN-γ-mediated selective inhibition of TLR4-induced transcription. Such inhibition can contribute to severe and sustained inflammatory responses., Macrophage activation is synergistically controlled by lipopolysaccharide (LPS) and interferon-γ (IFN-γ). Here the authors show that IFN-γ promotes macrophage activation not only by activating STAT1-dependent genes, but also by suppressing STAT3-dependent negative feedback regulation downstream of LPS signaling.

The variant histone H3.3 is incorporated into the genome in a transcription-dependent manner. This histone is thus thought to play a role in epigenetic regulation. However, our understanding of how H3.3 controls gene expression and epigenome landscape has remained incomplete. This is partly because precise localization of H3.3 in the genome has been difficult to decipher particularly for cells in vivo. To circumvent this difficulty, we generated knockin mice, by homologous recombination, to replace both of the two H3.3 loci (H3f3a and H3f3b) with the hemagglutinin-tagged H3.3 cDNA cassette, which also contained a GFP gene. We show here that the hemagglutinin-tagged H3.3 and GFP are expressed in the majority of cells in all adult tissues tested. ChIP-seq data, combined with RNA-seq, revealed a striking correlation between the level of transcripts and that of H3.3 accumulation in expressed genes. Finally, we demonstrate that H3.3 deposition is markedly enhanced upon stimulation by interferon on interferon-stimulated genes, highlighting transcription-coupled H3.3 dynamics. Together, these H3.3 knockin mice serve as a useful experimental model to study epigenome regulation in development and in various adult cells in vivo.

7576 Background: Diffuse large B-cell Lymphoma (DLBCL) is a highly heterogenous disease. Gene microarrays were initially used to classify DLBCL into germinal center B-cell-like (GCB) or activated B-cell-like (ABC) Cell of Origin (COO) subtypes. ABC is associated with shorter overall survival. In newly diagnosed patients, COO classification by RNA profiling is a validated prognostic. A simpler immunohistochemical (IHC) staining of CD10, MUM1 and BCL6 is a proxy used in clinical practice in lieu of transcriptomics due to its expense, complexity and tissue requirements. Recent advances in the HTG EdgeSeq platform allow genome-scale profiling with minimal tissue. We successfully applied this novel technology to perform simultaneous COO classification, immune cell enrichment and tumor pathway analysis using a single FFPE slide. Methods: Accuracy of the HTG EdgeSeq panel (19,000 genes) was assessed in a head-to-head comparison with RNAseq using FFPE tumor samples (n = 8). DLBCL resections and core needle biopsies were commercially sourced and COO typed using Han’s algorithm into GCB or non-GCB (n = 65). Tumor locations included: lymphoid organs, gastrointestinal tract, testes, and the pleural cavity. EdgeSeq was performed on single slides with an average tissue area of 40mm2. Transcriptomic COO classification was performed using a linear combination of genes as described in Wright et al., PNAS 2003, substituting HTG platform-specific weights. Validated COO gene sets from literature and commercial diagnostic assays were tested. Immune cell gene signature enrichment analysis was performed using xCell (Aran et al., Genome Biol 2017); pathway analysis was performed with GSEA (Subramanian et al., PNAS 2005). Results: Gene expression levels estimated from whole transcriptome EdgeSeq on single slides were highly correlated to whole transcriptome RNAseq. Differential expression analysis of GCB vs non-GCB showed that key prognostic genes were detectable and enriched in the expected subtypes. Using these pre-established signatures, subtyping accuracy was ̃93% on the training set and 89% on the test set. Immune cell enrichment analysis identified class-switched memory B-cells as more prevalent in non-GCB subjects. This is consistent with emerging evidence that memory B-cells are the primary source of ABC DLBCL and not plasma cells (Venturutti & Melnick, Blood 2020). Pathway analysis identified genes regulated by the oncogene ic transcription factor MYC were enriched in non-GCB samples; MYC protein was found to be overexpressed in ABC in a large study (Hu et al., Blood 2013). Conclusions: Combined COO typing and whole transcriptome analysis from a single slide efficiently uses precious patient tissue. Longitudinal core needle sampling may yield insights into tumor evolution and therapeutic mechanisms of action across the DLBCL treatment landscape.

Sex differences characterize immune responses to viruses and autoimmune diseases like SLE. ABCs are an emerging population of CD11c+ T-bet+ B cells critical for antiviral responses and autoimmune disorders. DEF6 and SWAP70, are two homologous molecules whose combined absence in double-knock-out mice (DKOs) leads to a lupus syndrome in females marked by an accumulation of ABCs. Here we demonstrate that DKO ABCs exhibit sex-specific differences in their expansion, upregulation of an ISG signature, and further differentiation. BCR sequencing and fate mapping experiments reveal that DKO ABCs undergo oligoclonal expansion and differentiate into both CD11c+ and CD11c- effector populations with pathogenic and proinflammatory potential. Tlr7 duplication in DKO males overrides the sex-bias and further augments the dissemination and pathogenicity of ABCs resulting in severe pulmonary inflammation and early mortality. Thus, sexual dimorphism shapes the expansion, function, and differentiation of ABCs contributing to the sex-bias that accompanies TLR7-driven immunopathogenesis.

The role of the histone chaperone SPT6 in mammalian cells is not fully understood. Here, we investigated the involvement of SPT6 in type I interferon (IFN)-induced transcription in murine fibroblasts. In RNA-seq analysis, Spt6 siRNA attenuates about half of ~ 200 IFN-stimulated genes (ISGs), while not affecting housekeeping genes. ISGs with high mRNA induction are more susceptible to Spt6 siRNA than those with lower levels of induction. ChIP analysis shows that SPT6 is recruited to highly inducible, Spt6 siRNA-sensitive ISGs, but not to other siRNA-insensitive ISGs. Furthermore, SPT6 recruitment is abrogated in cells lacking the histone methyltransferase NSD2. In co-IP experiments, SPT6 interacts with NSD2. In summary, SPT6 facilitates IFN-induced transcription, highlighting its critical role in gene activation.

Viral defense at mucosal sites depends on interferons (IFN) and IFN stimulated genes (ISGs), either of which may be constitutively expressed to maintain an “antiviral state” (AVS). However, the mechanisms that govern the AVS are poorly defined. Using a BEAS-2B respiratory epithelial cell line deficient in IRF1, we demonstrate higher susceptibility to infection with vesicular stomatitis virus (VSV) and influenza virus. IRF1-mediated restriction of VSV is IFN-independent, as blockade of types I and III IFNs and JAK-STAT signaling before infection did not affect VSV infection of either parent or IRF1 KO cells. Transcriptome analysis revealed that IRF1 regulates constitutive expression of ~300 genes, including antiviral ISGs: OAS2, BST2, and RNASEL and knockdown of any of these IRF1-dependent genes increased VSV infection. Additionally, IRF1 enhances rapid expression of IFNβ and IFNλ after stimulation with poly I:C and also regulates ISG expression. Mechanistically, IRF1 enhances recruitment of BRD4 to promotor-enhancer regions of ISGs for rapid expression and maintains levels of histone H3K4me1 for optimal constitutive expression. Finally, IRF1 also regulates constitutive expression of TLR2 and TLR3 and promotes signaling through these pattern recognition receptors (PRR). These data reveal multiple roles for IRF1 toward effective anti-viral responses by maintaining IFN-independent constitutive expression of anti-viral ISGs and supporting early IFN-dependent responses to PRR stimulation.

Histone post-translational modification patterns represent epigenetic states of genomic genes and denote the state of their transcription, past history, and future potential in gene expression. Genome-wide chromatin modification patterns reported from various laboratories are assembled in the ENCODE database, providing a fertile ground for understanding epigenetic regulation of any genes of interest across many cell types. The IRF family genes critically control innate immunity as they direct expression and activities of interferons. While these genes have similar structural and functional traits, their chromatin landscapes and epigenetic features have not been systematically evaluated. Here, by mining ENCODE database using an imputational approach, we summarize chromatin modification patterns for 6 of 9 IRF genes and show characteristic features that connote their epigenetic states. BRD4 is a BET bromodomain protein that “reads and translates” epigenetic marks into transcription. We review recent findings that BRD4 controls constitutive and signal-dependent transcription of many genes, including IRF genes. BRD4 dynamically binds to various genomic genes with a spatial and temporal specificity. Of particular importance, BRD4 is shown to critically regulate IRF-dependent anti-pathogen protection, inflammatory responses triggered by NF-κB, and the growth and spread of many cancers. The advent of small molecule inhibitors that disrupt binding of BET bromdomain to acetylated histone marks has opened new therapeutic possibilities for cancer and inflammatory diseases.

Positive coactivator 4 (PC4), a human transcriptional coactivator, is involved in diverse processes like chromatin organization and transcription regulation. It is hyperphosphorylated during mitosis, with unknown significance. For the first time, we demonstrate the function of PC4 outside the nucleus upon nuclear envelope breakdown. A fraction of PC4 associates with Aurora A and Aurora B and undergoes phosphorylation, following which PC4 activates both Aurora A and B to sustain optimal kinase activity to maintain the phosphorylation gradient for the proper functioning of the mitotic machinery. This mitotic role is evident in PC4 knockdown cells where the defects are rescued only by the catalytically active Aurora kinases, but not the kinase-dead mutants. Similarly, the PC4 phosphodeficient mutant failed to rescue such defects. Hence, our observations establish a novel mitotic function of PC4 that might be dependent on Aurora kinase-mediated phosphorylation.

The CDC 2019-Novel Coronavirus (2019-nCoV) Real-Time RT-PCR Diagnostic Panel is a real-time RT-PCR test intended for the qualitative detection of nucleic acid from the 2019-nCoV in upper and lower respiratory specimens (such as nasopharyngeal or oropharyngeal swabs, sputum, lower respiratory tract aspirates, bronchoalveolar lavage, and nasopharyngeal wash/aspirate or nasal aspirate) collected from individuals who meet 2019-nCoV clinical and/or epidemiological criteria. This protocol is adapted to screen 2019-nCoV in RNA isolated from whole blood. Results are for the identification of 2019-nCoV RNA. The 2019-nCoV RNA is generally detectable in upper and lower respiratory specimens during infection and very rarely detected in whole blood. Positive results are indicative of active infection with 2019-nCoV but do not rule out bacterial infection or co-infection with other viruses. The agent detected may not be the definite cause of disease. Negative results do not preclude 2019-nCoV infection and should not be used as the sole basis of management decisions. The CDC 2019-nCoV Real-Time RT-PCR Diagnostic Panel is a molecular in vitro diagnostic test that aids in the detection and diagnosis 2019-nCoV and is based on widely used nucleic acid amplification technology. The product contains oligonucleotide primers and dual-labeled hydrolysis probes (TaqMan®) and control material used in rRT-PCR for the in vitro qualitative detection of 2019-nCoV RNA in respiratory specimens. The oligonucleotide primers and probes for detection of 2019-nCoV were selected from regions of the virus nucleocapsid (N) gene. The panel is designed for specific detection of the 2019-nCoV (two primer/probe sets). An additional primer/probe set to detect the human RNase P gene (RP) in control samples and clinical specimens is also included in the panel. RNA isolated and purified from whole blood is reverse transcribed to cDNA and subsequently amplified in the Applied Biosystems 7500 Fast Dx Real-Time PCR Instrument with SDS version 1.4 software. Detection of viral RNA in whole blood aids in the safe operation of labs working with human blood samples.

On the role of H3.3 in retroviral silencing

The highly dynamic nucleoprotein structure of eukaryotic genome is organized in an ordered fashion, the unit of which is the nucleosome. The nucleosome is composed of core histones and DNA of variable size wrapped around it. Apart from the histone proteins, several nonhistone proteins also interact with the complex consisting of the DNA, the core and linker histones conferring highly regulated fluidity on the chromatin and permitting fine tuning of its functions. The nonhistone proteins are multifunctional and accentuate diverse cellular outcomes. In spite of the technical challenges, the architectural role of the nonhistone proteins altering the topology of the chromatin has been studied extensively. To appreciate the significance of the chromatin for genome function, it is essential to examine the role of the nonhistone proteins in different physiological conditions. Here, taking the example of a highly abundant chromatin protein, PC4 (Positive coactivator 4), we describe strategies for the identification of the chromatin-associated proteins and their structural and functional characterization.

The highly dynamic nucleoprotein structure of eukaryotic genome is organized in an ordered fashion, the unit of which is the nucleosome. The nucleosome is composed of core histones and DNA of variable size wrapped around it. Apart from the histone proteins, several nonhistone proteins also interact with the complex consisting of the DNA, the core and linker histones conferring highly regulated fluidity on the chromatin and permitting fine tuning of its functions. The nonhistone proteins are multifunctional and accentuate diverse cellular outcomes. In spite of the technical challenges, the architectural role of the nonhistone proteins altering the topology of the chromatin has been studied extensively. To appreciate the significance of the chromatin for genome function, it is essential to examine the role of the nonhistone proteins in different physiological conditions. Here, taking the example of a highly abundant chromatin protein, PC4 (Positive coactivator 4), we describe strategies for the identification of the chromatin-associated proteins and their structural and functional characterization.

Cellular stresses such as starvation, infection and inflammation trigger autophagy to clear damaged self and foreign materials. Autophagy occurs in conjunction with a global redirection of gene expression that sets a new epigenetic state. Cytokines, particularly interferons (IFNs, type I and type II) strongly induce autophagy in macrophages and dendritic cells. We showed earlier that IFNs induce chromatin exchange in IFN stimulated genes (ISGs), and the core histones are replaced by the variant histone, H3.3 [1] . The H3.3 replacement takes place during ISG transcription elongation, and requires the histone methyltransferase WHSC1 and the chromatin assembly factor HIRA. The H3.3 deposited in the ISGs remains for several cell generations, and forms a lasting epigenetic mark. We recently noted that stress induced autophagy in macrophages is dependent on and orchestrated by IRF8 [2] . IRF8 led to enhanced transcription of > 17 autophagy genes (e.g. Ulk1, Becn1, Atg4d, Atg7, Map1/lc3b, Lamp2, Cst3) that encompassed all steps of autophagy, from initial autophagosome formation to lysosomal fusion to target degradation. Upon infection of bacteria (Listeria) in macrophages, IRF8 directed high, sustained de novo transcription of multiple autophagy genes. Our more recent work shows that IRF8 plays a central role in setting the epigenetic chromatin environment in macrophages and establishes innate immune responses.