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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).

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.

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.

We demonstrate that at least three different promoter variant strains of HIV-1 subtype C have been gradually expanding and replacing the standard subtype C viruses in India, and possibly in South Africa and other global regions, over the past decade. The new viral strains contain an additional NF-κB, NF-κB-like, or RBEIII site in the viral promoter. Although the acquisition of an additional RBEIII site is a property shared by all the HIV-1 subtypes, acquiring an additional NF-κB site remains an exclusive property of subtype C. The acquired κB site is genetically distinct, binds the p50-p65 heterodimer, and strengthens the viral promoter at the levels of transcription initiation and elongation. The 4-κB viruses dominate the 3-κB "isogenic" viral strains in pairwise competition assays in T-cell lines, primary cells, and the ecotropic human immunodeficiency virus mouse model. The dominance of the 4-κB viral strains is also evident in the natural context when the subjects are coinfected with κB-variant viral strains. The mean plasma viral loads, but not CD4 counts, are significantly different in 4-κB infection suggesting that these newly emerging strains are probably more infectious. It is possible that higher plasma viral loads underlie selective transmission of the 4-κB viral strains. Several publications previously reported duplication or deletion of diverse transcription factor-binding sites in the viral promoter. Unlike previous reports, our study provides experimental evidence that the new viral strains gained a potential selective advantage as a consequence of the acquired transcription factor-binding sites and importantly that these strains have been expanding at the population level.

After screening a large number of clinical samples of HIV-1 subtype C in India, a subset of viral strains containing sequence insertions upstream of the viral enhancer has been identified. The sequence insertions contained binding sites for at least two different transcription factors NF-κB and RBEIII, importantly, in a mutually exclusive fashion. Furthermore, while some of the viral strains contained insertions of κB-like sites, a few others contained dual insertions of the RBEIII and κB sites together but only one of the two was intact. NF-κB acquisition appears to be the most common phenotype unique for subtype C with nearly half of the variant strains containing such insertions. Given that subtype C already contains three functional NF-κB sites in the viral enhancer, acquisition of a fourth NF-κB motif in some variant viral strains is intriguing. Further investigation is warranted to examine the significance of the sequence insertions for the replicative fitness of the variant viral strains.

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.

Extracellular Tat (eTat) plays an important role in HIV-1 pathogenesis. The presence of anti-Tat antibodies is negatively correlated with disease progression, hence making Tat a potential vaccine candidate. The cytotoxicity and moderate immunogenicity of Tat however remain impediments for developing Tat-based vaccines. Here, we report a novel strategy to concurrently enhance the immunogenicity and safety profile of Tat. The grafting of universal helper T-lymphocyte (HTL) epitopes, Pan DR Epitope (PADRE) and Pol711 into the cysteine rich domain (CRD) and the basic domain (BD) abolished the transactivation potential of the Tat protein. The HTL-Tat proteins elicited a significantly higher titer of antibodies as compared to the wild-type Tat in BALB/c mice. While the N-terminal epitope remained immunodominant in HTL-Tat immunizations, an additional epitope in exon-2 was recognized with comparable magnitude suggesting a broader immune recognition. Additionally, the HTL-Tat proteins induced cross-reactive antibodies of high avidity that efficiently neutralized exogenous Tat, thus blocking the activation of a Tat-defective provirus. With advantages such as presentation of multiple B-cell epitopes, enhanced antibody response and importantly, transactivation-deficient Tat protein, this approach has potential application for the generation of Tat-based HIV/AIDS vaccines.

Background The HIV-1 Subtype C, among other viral subtypes, is responsible for nearly 50% of the global infections including approximately 99% of the infections in India (UNAIDS 2012). HIV-1 subtype C is characterized by the presence of three NF-B binding sites in the enhancer as opposed to one or two such elements in other viral subtypes. Additionally, NF- Bs ite proximal to the Sp1 motifs, referred to here as the C-B site, is genetically distinct from the other two canonical NF- Bs ites (H-B sites). Furthermore, the Sp1III site proximal to the C-B motif also demonstrates subtype-specific genetic variations. Here, we examined the functional significance of the two unique regulatory elements, the C-B and the Sp1III motifs, for gene expression regulation from the viral promoter (Perkins et al. 1997). Methods and results Using several panels of reporter vectors expressing luciferase under the control of variant viral promoters, we examined gene expression in different mammalian cells under diverse activation conditions. The data confirm that the proximity and orientation of two sites, the C- Ba nd Sp1III motifs, are critical for optimal gene expression from the viral promoter. We found that a functional cooperation among the three NF-B sites is necessary for maximal promoter activity. Collectively, the data appeared to suggest that in addition to conferring a quantitative gain-offunction on the viral promoter, the presence of C-B site may have catalyzed associated genetic variation in the proximal Sp1III site through a positive selection pressure. To understand the functional association between these two sites under more relevant conditions, we constructed viral promoters consisting of only the C-B and the Sp1III sites, in the absence of other NF- Ba nd Sp1 sites. The subtype C specific Sp1III site showed higher magnitude of gene expression with the H- but not with the C- Bs ite. Experiments, using infectious viruses and the chromatin immunoprecipitation analysis, have also suggested the functional association between the two sites. In gel-shift assays the C-B site recruited the p50 and p65 heteroduplexes at a level comparable to that of the H- Bs ite. Importantly, given that the C- Bs ite is a G/C-centric motif, we asked and found that the C-B site is responsive to the p52:Bcl3 complex in contrast to the A/T-centric H-B site known to be more responsive to the p50:p65 complex promoter. Conclusions Collectively our data are suggestive of co-evolution of the two unique regulatory elements, the C- Ba nd the C-Sp1III motifs, that confer qualitative gain-of-function on the subtype C promoter thus probably playing a critical role towards the global predominance of the viral subtype. We further hypothesize that the C- Bs ite being aG /Ccentric motif, in combination with the two upstream H-B sites, confers a sustained and prolonged gene expression on HIV-1 subtype C viral promoter.

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.