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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

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.

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.

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.

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.