Biology subjects

Ozato, K.

Publications and source records attributed to Ozato, K..

13 recordsLinked to original sources

bioRxiv2026

B cell-intrinsic IRF8 transcriptionally reprograms antigen presentation to sustain CD8⁺ T cell antitumor immunity

Interferon regulatory factor 8 (IRF8) is a master transcription factor of myeloid differentiation, but whether IRF8 intrinsically controls B cell function in tumors remains unknown. Using paired single-cell transcriptomic and chromatin accessibility profiling of tumors from wild-type and Irf8-deficient mice, we identify a B cell-intrinsic IRF8 axis regulating antigen presentation and sustaining anti-tumor CD8 T cell immunity. IRF8 establishes conserved chromatin accessibility programs across myeloid cells and plasmablasts centered on antigen processing and MHC class I presentation, but engages distinct motifs by lineage: myeloid cells preferentially utilize ISRE and ETS-composite elements, whereas plasmablasts are selectively enriched for EICE elements, reflecting B lineage-specific IRF8-IRF4 cooperation. Loss of IRF8 disrupts these programs, skews B cells toward plasmablast differentiation and reduces antigen presentation machinery. B cell depletion accelerated tumor growth, while CD40 agonism activated B cells, expanded T cells, and enhanced anti-tumor immunity. B cell-specific IRF8 deletion alone accelerated tumor growth, establishing a cell-intrinsic requirement independent of myeloid IRF8 function. The IRF8-regulated B cell signature was enriched in PD-1 blockade cancer patient responders, and plasmablast abundance correlated with response in pembrolizumab-treated cancer patients. These findings establish IRF8 as a lineage-adapted regulator of antigen presentation and define the IRF8-B cell axis as a determinant of anti-tumor immunity. HighlightsIRF8 establishes a conserved chromatin accessibility across tumor-infiltrating myeloid and B cells Myeloid cells engage ISRE motifs, whereas plasmablasts rely on EICE motifs as IRF8 lineage-specific cis-regulation IRF8 regulates an antigen presentation in B cells to sustain anti-tumor T cell immunity B cell-intrinsic IRF8 transcription signature predicts patient response to PD-1 blockade immunotherapy

immunology↗

bioRxiv2026

HIRA-mediated H3.3 deposition preserves hepatocyte cell identity during liver aging

Age-associated functional decline is partly driven by progressive chromatin degeneration. Maintenance of chromatin integrity preserves cell identity and promotes healthy aging, but through different mechanisms in proliferating and non-proliferating cells. However, specific mechanisms of chromatin maintenance and their compensatory capacity in proliferating and non-proliferating cells are undefined. The histone chaperone HIRA deposits the histone variant H3.3 in a DNA replication-independent manner, leading to its accumulation in aging, non-proliferating cells. Here, we show that hepatocyte-specific loss of HIRA causes loss of cell identity, metabolic dysfunction, and accelerated fibrotic pathology with age. Transcriptomic and epigenomic analyses indicate that HIRA-H3.3 preserves chromatin integrity and sustains transcription of highly expressed genes, including cell identity genes. Partial hepatectomy, associated with induced proliferation, restores identity of HIRA knockout livers with compensatory deposition of canonical histones H3.1/2. Together, these results demonstrate that HIRA-mediated H3.3 deposition is essential for safeguarding cell identity and tissue function during aging of non-proliferating cells, but this function can be rescued by tissue regeneration and associated cell proliferation. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=124 SRC="FIGDIR/small/710643v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@a2a26aorg.highwire.dtl.DTLVardef@154e5aeorg.highwire.dtl.DTLVardef@b315a7org.highwire.dtl.DTLVardef@152bca4_HPS_FORMAT_FIGEXP M_FIG C_FIG

genomics↗

bioRxiv2025

IRF8 deficiency causes anxiety-like behavior in a sex-dependent manner

Anxiety disorder is a serious psychiatric disease that affects women twice more than men and disrupts patients daily lives. It is often comorbid with major depression and other mental diseases. Various underlying mechanisms have been proposed, such as neurotransmitters and neuroanatomical disruptions, and more recently, oxidative stress; however, much remained unclear, including the role of glial cells. Here, we investigated the role of IRF8 in anxiety disorders in the mouse model. IRF8 is a transcription factor expressed primarily in microglia in the brain. A battery of behavioral tests revealed that female IRF8 knockout (IRF8KO) mice show increased anxiety relative to male IRF8KO and wild-type mice. Female IRF8KO mice also exhibited a higher tendency for obsessive-compulsive disorder. However, these behavioral abnormalities were not observed when IRF8 was deleted postnatally, indicating that it acts during the fetal stage to control anxiety. Transcriptome analysis revealed that IRF8 deficiency leads to redox dysregulation. Further, 2,7-dichlorofluorescin diacetate (DCFDA) staining for microglia demonstrated that female IRF8KO microglia produce higher levels of reactive oxygen species (ROS) compared to WT and male IRF8KO counterparts. Detailed RNA-seq analysis, however, did not reveal specific genes that cause high ROS production in female cells. In sum, this work demonstrates that IRF8 in microglia plays a major role in controlling anxiety in a sex dependent manner.

neuroscience↗

bioRxiv2025

Histone Chaperone HIRA facilitates transcription elongation to regulate insulin sensitivity and obesity-associated adipose expansion

Adipose tissue is essential for maintaining glucose and lipid homeostasis in mammals. However, epigenomic mechanisms underlying adipose tissue function remain largely unclear. Here, we identify the histone chaperon HIRA as a novel epigenomic regulator of adipose tissue function. Adipose tissue-specific knockout of Hira in mice impairs insulin sensitivity and restrains adipose tissue expansion during high-fat diet-induced obesity. Mechanistically, HIRA is required for the expression of Adipoq, encoding the adipokine adiponectin, and lipid metabolism genes in adipose tissue. Genomic mapping reveals that HIRA binds to promoters and enhancers of Adipoq and lipid metabolism genes in adipocytes. Acute HIRA depletion using the dTAG system, followed by nascent RNA-Seq and ChIP-Seq, demonstrates that while HIRA is largely dispensable for enhancer activation and coactivator binding, it promotes transcription of target genes by facilitating RNA polymerase II pause release and subsequent elongation likely independently of H3.3 deposition. Our findings uncover a novel mechanism by which HIRA regulates transcription and establish HIRAs critical role in insulin sensitivity and lipid metabolism, providing a potential therapeutic target for obesity and insulin resistance. Significance StatementDysregulated adipose tissue function leads to metabolic disorders including obesity and insulin resistance, yet the epigenomic mechanisms that maintain its normal function are incompletely understood. This study identifies the histone chaperone HIRA as a critical regulator of insulin sensitivity and obesity-associated adipose expansion. We show that HIRA directly controls adiponectin and lipid metabolism gene expression in adipocytes by promoting RNA polymerase II pause release, rather than transcription initiation or enhancer activation, and likely in a manner independent of H3.3 deposition. These findings uncover a novel mechanism of HIRA in regulating gene expression and highlight its critical role in insulin sensitivity and lipid metabolism in adipocytes.

physiology↗

bioRxiv2024

Histone H3.3 ensures cell proliferation and genomic stability during myeloid cell development

Variant histone H3.3 is thought to be critical for survival of many cells, since it is deposited in expressed genes, a feature different from core histones. For example, H3.3 deletion leads to embryonic lethality in mice. However, requirement of H3.3 in later stage of development has remained unclear. The aim of this work was to elucidate the role of H3.3 for development of myeloid lineage, important for innate immunity. We conditionally knocked out (cKO) the H3.3 genes in myeloid progenitor cells differentiating into bone marrow derived macrophages (BMDMs). Progenitor cells lacking H3.3 were defective in replication, suffered from extensive DNA damage, and underwent apoptosis. Surviving H3.3cKO cells expressed many interferon stimulated genes (ISGs) throughout differentiation. Further, H3.3cKO BMDMs possessed chromatin accessible sites, and histone posttranslational modifications consistent with the gene expression profiles, Accordingly, H3.3cKO BMDMs retained general nucleosomal structure genome wide. In summary, H3.3 is required for proliferation of myeloid progenitor cells, but is in large part dispensable for differentiation of BMDMs.

cell biology↗

bioRxiv2024

BRD4 expression in microglia supports recruitment of T cells into the CNS and exacerbates EAE

In EAE, a mouse model of multiple sclerosis, immunization with MOG autoantigen results in the generation of Th1/Th17 T cells in the periphery. MOG-specific T cells then invade into the central nervous system (CNS), resulting in neuronal demyelination. Microglia, innate immune cells in the CNS are known to regulate various neuronal diseases. However, the role of microglia in EAE has remained elusive. BRD4 is a BET protein expressed in microglia, whether BRD4 in microglia contributes to EAE has not been determined. We show that EAE pathology was markedly reduced with microglia-specific Brd4 conditional knockout (cKO). In these mice, microglia- T cell interactions were greatly reduced, leading to the lack of T cell reactivation. Microglia specific transcriptome data showed downregulation of genes required for interaction with and reactivation of T cells in Brd4 cKO samples. In summary, BRD4 plays a critical role in regulating microglia function in normal and EAE CNS. SummaryThis study demonstrates that in a EAE model, microglia-specific Brd4 conditional knockout mice were defective in expressing genes required for microglia- T cells interaction and those involved in neuroinflammation, and demyelination resulting in fewer CNS T cell invasion and display marked reduction in EAE pathology.

neuroscience↗

bioRxiv2024

Dynamic and decay kinetics of H3 variants in live cells reveal the pivotal role of HIRA/NSD2 in maintaining the distinct H3.3 specific chromatin landscape

The incorporation of variant histone H3.3 into the genome is tightly linked with transcriptional activity, yet its precise regulatory mechanisms remain elusive. Traditional methods like Chromatin Immunoprecipitation offer static views of H3.3 distribution, lacking dynamic insights. Here, using the SNAP tag system, we employed Fluorescence Recovery After Photobleaching (FRAP) and live-cell imaging to investigate H3.3 mobility and decay kinetics in live mouse embryonic fibroblast cells. Our focus on interferon-induced transcriptional activation revealed rapid H3.3 exchange, indicative of its transcriptional regulatory role. Transcription inhibition hindered H3.3 mobility, emphasizing its involvement in transcription. Additionally, we probed into turnover dynamics(decay) of H3.1-SNAP and H3.3-SNAP variants, uncovering differential decay rates influenced by transcriptional activity and histone modifiers such as NSD2 and HIRA. Live-cell imaging showed faster decay of H3.3 compared to H3.1, further exacerbated upon NSD2/HIRA loss. Notably, HIRA and NSD2, regulators of H3.3 dynamics, proved crucial for both H3.3 mobility and decay, underscoring their pivotal role. These findings deepen our understanding of epigenetic regulation, emphasizing the dynamic nature of histone turnover in cellular function and its implications for disease pathogenesis. Taken together, this study sheds light on the dynamic behavior of H3.3 and its regulatory mechanisms, providing valuable insights into epigenetic regulation in cellular processes and disease contexts.

cell biology↗

bioRxiv2023

BRD4 directs mitotic cell division by inhibiting DNA damage

BRD4 binds to acetylated histones to regulate transcription and drive cancer cell proliferation. However, the role of BRD4 in normal cell growth remains to be elucidated. Here we investigated the question by using mouse embryonic fibroblasts with conditional Brd4 knockout (KO). We found that Brd4KO cells grow more slowly than wild type cells: they do not complete replication, fail to achieve mitosis, and exhibit extensive DNA damage throughout all cell cycle stages. BRD4 was required for expression of more than 450 cell cycle genes including genes encoding core histones and centromere/kinetochore proteins that are critical for genome replication and chromosomal segregation. Moreover, we show that many genes controlling R-loop formation and DNA damage response (DDR) require BRD4 for expression. Finally, BRD4 constitutively occupied genes controlling R-loop, DDR and cell cycle progression. We suggest that BRD4 epigenetically marks those genes and serves as a master regulator of normal cell growth.

cell biology↗

bioRxiv2023

IRF8 configures enhancer landscape in postnatal microglia and directs microglia specific transcriptional programs

Microglia are innate immune cells in the brain. Transcription factor IRF8 is highly expressed in microglia. However, its role in postnatal microglia development is unknown. We demonstrate that IRF8 binds stepwise to enhancer regions of postnatal microglia along with Sall1 and PU.1, reaching a maximum after day 14. IRF8 binding correlated with a stepwise increase in chromatin accessibility, which preceded the initiation of microglia-specific transcriptome. Constitutive and postnatal Irf8 deletion led to a loss of microglia identity and gain of disease-associated microglia-like genes. Combined analysis of scRNA-seq and scATAC-seq revealed a correlation between chromatin accessibility and transcriptome at a single-cell level. IRF8 was also required for microglia-specific DNA methylation patterns. Lastly, in the 5xFAD model, constitutive and postnatal Irf8 deletion reduced the interaction of microglia with A{beta} plaques and the size of plaques, lessening neuronal loss. Together, IRF8 sets the epigenetic landscape, which is required for postnatal microglia gene expression.

immunology↗

bioRxiv2023

Mouse Homolog of Human IRF8G388S Mutation Provides Novel Insight into Osteoclastogenesis and Tooth Root Resorption

Previously, we reported a novel mutation in the Interferon Regulatory Factor 8 (IRF8) gene associated with multiple idiopathic cervical root resorption (MICRR), an aggressive form of tooth root resorption mediated by increased osteoclast activity. The IRF8 G388S variant in the highly conserved C-terminal motif is predicted to alter the protein structure, likely impairing IRF8 function. To investigate the molecular basis of MICRR and IRF8 function, we generated Irf8 knock-in (KI) mice using CRISPR/Cas9 modeling the human IRF8G388S mutation. The heterozygous (Het) and homozygous (Homo) Irf8 KI mice showed no gross morphological defects, and the development of hematopoietic cells was unaffected and similar to that in wild-type (WT) mice. The Irf8 KI Het and Homo mice showed no difference in macrophage gene signatures important for antimicrobial defenses and inflammatory cytokine production. Consistent with the phenotype observed in MICRR patients, Irf8 KI Het and Homo mice demonstrated significantly increased osteoclast formation and resorption activity in vivo and in vitro when compared to WT mice. The oral ligature inserted Irf8 KI Het and Homo mice displayed increased osteoclast-mediated alveolar bone loss and tooth root resorption compared to WT mice. The increased osteoclastogenesis noted in KI mice is due to the inability of mutant Irf8 G388S isoform to negatively inhibit NFATc1-dependent transcriptional activation and downstream osteoclast specific transcripts. This translational study delineates the IRF8 domain important for osteoclast function and provides novel insights into the IRF8 mutation associated with MICRR. Irf8G388S mutation mainly affects osteoclastogenesis while sparing immune cell development and function. The Irf8 KI mice serve as a novel translational model for studying the etiopathology of MICRR and developing targeted therapies for MICRR and other skeletal disorders mediated by increased osteoclast activity.

genetics↗

bioRxiv2021

A single-cell genomic strategy for alternative transcript start sites identification

Alternative transcription start sites (TSSs) usage plays a critical role in gene transcription regulation in mammals. However, precisely identifying alternative TSSs remains challenging at the genome-wide level. Here, we report a single-cell genomic technology for alternative TSSs annotation and cell heterogeneity detection. In the method, we utilize Fluidigm C1 system to capture individual cells of interest, SMARTer cDNA synthesis kit to recover full-length cDNAs, then dual priming oligonucleotide system to specifically enrich TSSs for genomic analysis. We apply this method to a genome-wide study of alternative TSSs identification in two different IFN-{beta} stimulated mouse embryonic fibroblasts (MEFs). We quantify the performance of our method and find it as accurate as other single cell methods for the detection of TSSs. Our data are also clearly discriminate two IFN-{beta} stimulated MEFs. Moreover, our results indicate 82% expressed genes in these two cell types containing multiple TSSs, which is much higher than previous predictions based on CAGE (58%) or empirical determination (54%) in various cell types. This indicates that alternative TSSs are more pervasive than expected and implies our strategy could position them at an unprecedented sensitivity. It would be helpful for elucidating their biological insights in future.

genomics↗

bioRxiv2020

Monocyte Subsets with High Osteoclastogenic Potential and Their Epigenetic Regulation Orchestrated by IRF8

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 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 pre-established transcripts, as well as de novo gain in enhancer activity and promoter changes. In the absence of 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.

immunology↗

bioRxiv2018

BRD4 Deficiency Selectively Affects a Unique Developmental Subpopulation in Thymocytes

The bromodomain protein BRD4 is a driver in both inflammatory diseases and cancers. It has multiple functions, contributing to chromatin structure and transcription through its intrinsic HAT and kinase activities. Despite the wide-ranging characterization of BRD4, little is known about its in vivo function. In the present study, we have examined the role of BRD4 in T cell development by conditional deletion at various stages of thymocyte differentiation. We found that BRD4 is critical for normal T cell development. Surprisingly, BRD4 selectively regulates the progression of immature CD8 single positive (ISP) thymocytes into quiescent DP thymocytes. In striking contrast, BRD4 deletion does not affect the extensive proliferation associated with the differentiation of double negative (DN) into ISP cells. Nor does it affect the maturation of double positive (DP) into conventional CD4+ and CD8+ thymocytes. These studies lead to the unexpected conclusion that BRD4 selectively regulates preselection ISP thymocytes.\n\nOn-line SummaryImmature CD8 single-positive (ISP) thymocytes are identified as a molecularly-distinct thymocyte subpopulation, dependent on BRD4 for progression to the DP stage. DN and DP are BRD4-independent. These findings provide new insights into BRD4, a therapeutic target in inflammation and cancer.

immunology↗