BRD4 directs hematopoietic stem cell development and modulates macrophage inflammatory responses.

BRD4 is a BET family protein that binds acetylated histones and regulates transcription. BET/BRD4 inhibitors block blood cancer growth and inflammation and serve as a new therapeutic strategy. However, the biological role of BRD4 in normal hematopoiesis and inflammation is not fully understood. Analysis of Brd4 conditional knockout (KO) mice showed that BRD4 is required for hematopoietic stem cell expansion and progenitor development. Nevertheless, BRD4 played limited roles in macrophage development and inflammatory response to LPS ChIP-seq analysis showed that despite its limited importance, BRD4 broadly occupied the macrophage genome and participated in super-enhancer (SE) formation. Although BRD4 is critical for SE formation in cancer, BRD4 was not required for macrophage SEs, as KO macrophages created alternate, BRD4-less SEs that compensated BRD4 loss. This and additional mechanisms led to the retention of inflammatory responses in macrophages. Our results illustrate a context-dependent role of BRD4 and plasticity of epigenetic regulation.

The transcription factor IRF8 activates integrin-mediated TGF-ß signaling and promotes neuroinflammation.

Recent epidemiological studies have identified interferon regulatory factor 8 (IRF8) as a susceptibility factor for multiple sclerosis (MS). However, how IRF8 influences the neuroinflammatory disease has remained unknown. By studying the role of IRF8 in experimental autoimmune encephalomyelitis (EAE), a mouse model of MS, we found that Irf8(-/-) mice are resistant to EAE. Furthermore, expression of IRF8 in antigen-presenting cells (APCs, such as macrophages, dendritic cells, and microglia), but not in T cells, facilitated disease onset and progression through multiple pathways. IRF8 enhanced αvß8 integrin expression in APCs and activated TGF-ß signaling leading to T helper 17 (Th17) cell differentiation. IRF8 induced a cytokine milieu that favored growth and maintenance of Th1 and Th17 cells, by stimulating interleukin-12 (IL-12) and IL-23 production, but inhibiting IL-27 during EAE. Finally, IRF8 activated microglia and exacerbated neuroinflammation. Together, this work provides mechanistic bases by which IRF8 contributes to the pathogenesis of MS.

Interferon stimulation creates chromatin marks and establishes transcriptional memory.

Epigenetic memory for signal-dependent transcription has remained elusive. So far, the concept of epigenetic memory has been largely limited to cell-autonomous, preprogrammed processes such as development and metabolism. Here we show that IFNß stimulation creates transcriptional memory in fibroblasts, conferring faster and greater transcription upon restimulation. The memory was inherited through multiple cell divisions and led to improved antiviral protection. Of ∼2,000 IFNß-stimulated genes (ISGs), about half exhibited memory, which we define as memory ISGs. The rest, designated nonmemory ISGs, did not show memory. Surprisingly, mechanistic analysis showed that IFN memory was not due to enhanced IFN signaling or retention of transcription factors on the ISGs. We demonstrated that this memory was attributed to accelerated recruitment of RNA polymerase II and transcription/chromatin factors, which coincided with acquisition of the histone H3.3 and H3K36me3 chromatin marks on memory ISGs. Similar memory was observed in bone marrow macrophages after IFNγ stimulation, suggesting that IFN stimulation modifies the shape of the innate immune response. Together, external signals can establish epigenetic memory in mammalian cells that imparts lasting adaptive performance upon various somatic cells.

Batf3 and Id2 have a synergistic effect on Irf8-directed classical CD8α+ dendritic cell development.

Dendritic cells (DCs) are heterogeneous cell populations represented by different subtypes, each varying in terms of gene expression patterns and specific functions. Recent studies identified transcription factors essential for the development of different DC subtypes, yet molecular mechanisms for the developmental program and functions remain poorly understood. In this study, we developed and characterized a mouse DC progenitor-like cell line, designated DC9, from Irf8(-/-) bone marrow cells as a model for DC development and function. Expression of Irf8 in DC9 cells led to plasmacytoid DCs and CD8α(+) DC-like cells, with a concomitant increase in plasmacytoid DC- and CD8α(+) DC-specific gene transcripts and induction of type I IFNs and IL12p40 following TLR ligand stimulation. Irf8 expression in DC9 cells led to an increase in Id2 and Batf3 transcript levels, transcription factors shown to be important for the development of CD8α(+) DCs. We show that, without Irf8, expression of Id2 and Batf3 was not sufficient for directing classical CD8α(+) DC development. When coexpressed with Irf8, Batf3 and Id2 had a synergistic effect on classical CD8α(+) DC development. We demonstrate that Irf8 is upstream of Batf3 and Id2 in the classical CD8α(+) DC developmental program and define the hierarchical relationship of transcription factors important for classical CD8α(+) DC development.

Cascade-amplified self-immolative polymeric prodrug for cancer therapy by disrupting redox homeostasis.

The amplification of reactive oxygen species (ROS) generation and glutathione (GSH) depletion in cancer cells represents a promising strategy to disrupt redox homeostasis for cancer therapy. Quinone methide and its analogs (QM) have recently been recognized as potential GSH scavengers for anticancer applications; however, an effective QM prodrug is yet to be developed. In this study, we prepare a self-immolative polymeric prodrug (SPP), which could be selectively degraded to generate large quantities of QMs in cancer cells during the spontaneous stepwise head-to-tail degradation of SPP. The amphiphilic SPP is self-assembled into nano-sized micelles, allowing for encapsulating 2-methoxy-ß-estradiol (2ME), an anticancer drug that produces a large amount of intracellular ROS. When SPP@2ME, as the cascade-amplified prodrug, is treated on the cancer cells, 2ME is rapidly released at the ROS-rich intracellular environment by degradation of SPP, thus generating more ROS that triggers the degradation of more SPP chains. Such a domino-like cascade-amplified feedback loop significantly amplifies oxidative stress and disrupts the redox homeostasis in cancer cells. This unique strategy provides synergistic anticancer therapeutic efficacy and demonstrates an important perception in innovative and precise nanomedicine.

Self-immolative polymer-based immunogenic cell death inducer for regulation of redox homeostasis.

Doxorubicin (DOX), widely used as an anticancer drug, is considered an immunogenic cell death (ICD) inducer that enhances cancer immunotherapy. However, its extended application as an ICD inducer has been limited owing to poor antigenicity and inefficient adjuvanticity. To enhance the immunogenicity of DOX, we prepare a reactive oxygen species (ROS)-responsive self-immolative polymer (R-SIP) that can efficiently destroy redox homeostasis via self-immolation-mediated glutathione depletion in cancer cells. Owing to its amphiphilic nature, R-SIP self-assemble into nano-sized particles under aqueous conditions, and DOX is efficiently encapsulated inside the nanoparticles by a simple dialysis method. Interestingly, when treated with 4T1 cancer cells, DOX-encapsulated R-SIP (DR-SIP) induces the phosphorylation of eukaryotic translation initiation factor 2α and overexpression of ecto-calreticulin, resulting in endoplasmic reticulum-associated ICD. In addition, DR-SIP contributes to the maturation of dendritic cells by promoting the release of damage-associated molecular patterns (DAMPs) from cancer cells. When intravenously administered to tumor-bearing mice, DR-SIP remarkably inhibits tumor growth compared with DOX alone. Overall, DR-SIP may have the potential to elicit an immune response as an ICD inducer.

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.

Recent progress in nitric oxide-generating nanomedicine for cancer therapy.

Nitric oxide (NO) is an endogenous, multipotent biological signaling molecule that participates in several physiological processes. Recently, exogenous supplementation of tumor tissues with NO has emerged as a potential anticancer therapy. In particular, it induces synergistic effects with other conventional therapies (such as chemo-, radio-, and photodynamic therapies) by regulating the activity of P-glycoprotein, acting as a vascular relaxant to relieve tumor hypoxia, and participating in the metabolism of reactive oxygen species. However, NO is highly reactive, and its half-life is relatively short after generation. Meanwhile, NO-induced anticancer activity is dose-dependent. Therefore, the targeted delivery of NO to the tumor is required for better therapeutic effects. In the past decade, NO-generating nanomedicines (NONs), which enable sustained and specific NO release in tumor tissues, have been developed for enhanced cancer therapy. This review describes the recent efforts and preclinical achievements in the development of NON-based cancer therapies. The chemical structures employed in the fabrication of NONs are summarized, and the strategies involved in NON-based cancer therapies are elaborated.

Polymeric Hydrogels for Controlled Drug Delivery to Treat Arthritis.

Rheumatoid arthritis (RA) and osteoarthritis (OA) are disabling musculoskeletal disorders that affect joints and cartilage and may lead to bone degeneration. Conventional delivery of anti-arthritic agents is limited due to short intra-articular half-life and toxicities. Innovations in polymer chemistry have led to advancements in hydrogel technology, offering a versatile drug delivery platform exhibiting tissue-like properties with tunable drug loading and high residence time properties This review discusses the advantages and drawbacks of polymeric materials along with their modifications as well as their applications for fabricating hydrogels loaded with therapeutic agents (small molecule drugs, immunotherapeutic agents, and cells). Emphasis is given to the biological potentialities of hydrogel hybrid systems/micro-and nanotechnology-integrated hydrogels as promising tools. Applications for facile tuning of therapeutic drug loading, maintaining long-term release, and consequently improving therapeutic outcome and patient compliance in arthritis are detailed. This review also suggests the advantages, challenges, and future perspectives of hydrogels loaded with anti-arthritic agents with high therapeutic potential that may alter the landscape of currently available arthritis treatment modalities.

Bromodomain protein BRD4 directs and sustains CD8 T cell differentiation during infection.

In response to infection, pathogen-specific CD8 T cells differentiate into functionally diverse effector and memory T cell populations critical for resolving disease and providing durable immunity. Through small-molecule inhibition, RNAi studies, and induced genetic deletion, we reveal an essential role for the chromatin modifier and BET family member BRD4 in supporting the differentiation and maintenance of terminally fated effector CD8 T cells during infection. BRD4 bound diverse regulatory regions critical to effector T cell differentiation and controlled transcriptional activity of terminal effector-specific super-enhancers in vivo. Consequentially, induced deletion of Brd4 or small molecule-mediated BET inhibition impaired maintenance of a terminal effector T cell phenotype. BRD4 was also required for terminal differentiation of CD8 T cells in the tumor microenvironment in murine models, which we show has implications for immunotherapies. Taken together, these data reveal an unappreciated requirement for BRD4 in coordinating activity of cis regulatory elements to control CD8 T cell fate and lineage stability.

HIRA, a DiGeorge Syndrome Candidate Gene, Confers Proper Chromatin Accessibility on HSCs and Supports All Stages of Hematopoiesis.

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.

Brd4 is required for recovery from antimicrotubule drug-induced mitotic arrest: preservation of acetylated chromatin.

The mammalian bromodomain protein Brd4 interacts with mitotic chromosomes by binding to acetylated histone H3 and H4 and is thought to play a role in epigenetic memory. Mitotic cells are susceptible to antimicrotubule drugs. These drugs activate multiple response pathways and arrest cells at mitosis. We found that Brd4 was rapidly released from chromosomes upon treatment with antimicrotubule drugs, including the reversible agent nocodazole. Yet, when nocodazole was withdrawn, Brd4 was reloaded onto chromosomes, and cells proceeded to complete cell division. However, cells in which a Brd4 allele was disrupted (Brd4+/-), and expressing only half of the normal Brd4 levels, were defective in reloading Brd4 onto chromosomes. Consequently, Brd4+/- cells were impaired in their ability to recover from nocodazole-induced mitotic arrest: a large fraction of +/- cells failed to reach anaphase after drug withdrawal, and those that entered anaphase showed an increased frequency of abnormal chromosomal segregation. The reloading defect observed in Brd4+/- cells coincided with selective hypoacetylation of lysine residues on H3 and H4. The histone deacetylase inhibitor trichostatin A increased global histone acetylation and perturbed nocodazole-induced Brd4 unloading. Brd4 plays an integral part in a cellular response to drug-induced mitotic stress by preserving a properly acetylated chromatin status.

Epigenetic regulation of lipoprotein lipase gene via BRD4, which is potentially associated with adipocyte differentiation and insulin resistance.

Lipoprotein lipase (LPL) is the rate-controlling enzyme for the accumulation of triacylglycerol into adipocytes, which acts by digesting it into glycerol and fatty acids. In this study, we found that treatment with (+)-JQ1, an inhibitor of the bromodomain and extra-terminal (BET) family proteins, for 4 days from the end of stimulation to induce adipocyte differentiation reduced binding of BRD4, a BET family member, within the gene body of Lpl. This eventually downregulated the expression of Lpl in 3T3-L1 adipocytes. Longer treatment for 8 days reduced the acetylation of histones H3 and H4 within the gene body of Lpl and subsequent Lpl expression. Lpl expression in mesenteric adipose tissues was lower in Brd4+/- heterozygous mice at 14 days after birth than in wild-type mice at the same age. Furthermore, treatment with an inducer of insulin resistance, tumor necrosis factor-α, reduced BRD4 binding and histone acetylation in the gene body of Lpl and its expression. These results indicate that transcriptional elongation of Lpl controlled by BRD4 may be associated with adipocyte differentiation, and that its suppression is potentially associated with insulin resistance of adipocytes.

Immature CD8 Single-Positive Thymocytes Are a Molecularly Distinct Subpopulation, Selectively Dependent on BRD4 for Their Differentiation.

T cell differentiation in the thymus proceeds in an ordered sequence of developmental events characterized by variable expression of CD4 and CD8 coreceptors. Here, we report that immature single-positive (ISP) thymocytes are molecularly distinct from all other T cell populations in the thymus in their expression of a gene profile that is dependent on the transcription factor BRD4. Conditional deletion of BRD4 at various stages of thymic differentiation reveals that BRD4 selectively regulates the further differentiation of ISPs by targeting cell cycle and metabolic pathways, but it does not affect the extensive proliferation that results in the generation of ISPs. These studies lead to the conclusion that the ISP subpopulation is not a hybrid transitional state but a molecularly distinct subpopulation that is selectively dependent on BRD4.

Global nature of dynamic protein-chromatin interactions in vivo: three-dimensional genome scanning and dynamic interaction networks of chromatin proteins.

Genome structure and gene expression depend on a multitude of chromatin-binding proteins. The binding properties of these proteins to native chromatin in intact cells are largely unknown. Here, we describe an approach based on combined in vivo photobleaching microscopy and kinetic modeling to analyze globally the dynamics of binding of chromatin-associated proteins in living cells. We have quantitatively determined basic biophysical properties, such as off rate constants, residence time, and bound fraction, of a wide range of chromatin proteins of diverse functions in vivo. We demonstrate that most chromatin proteins have a high turnover on chromatin with a residence time on the order of seconds, that the major fraction of each protein is bound to chromatin at steady state, and that transient binding is a common property of chromatin-associated proteins. Our results indicate that chromatin-binding proteins find their binding sites by three-dimensional scanning of the genome space and our data are consistent with a model in which chromatin-associated proteins form dynamic interaction networks in vivo. We suggest that these properties are crucial for generating high plasticity in genome expression.

A Mammalian bromodomain protein, brd4, interacts with replication factor C and inhibits progression to S phase.

Brd4 belongs to the BET family of nuclear proteins that carry two bromodomains implicated in the interaction with chromatin. Expression of Brd4 correlates with cell growth and is induced during early G(1) upon mitogenic stimuli. In the present study, we investigated the role of Brd4 in cell growth regulation. We found that ectopic expression of Brd4 in NIH 3T3 and HeLa cells inhibits cell cycle progression from G(1) to S. Coimmunoprecipitation experiments showed that endogenous and transfected Brd4 interacts with replication factor C (RFC), the conserved five-subunit complex essential for DNA replication. In vitro analysis showed that Brd4 binds directly to the largest subunit, RFC-140, thereby interacting with the entire RFC. In line with the inhibitory activity seen in vivo, recombinant Brd4 inhibited RFC-dependent DNA elongation reactions in vitro. Analysis of Brd4 deletion mutants indicated that both the interaction with RFC-140 and the inhibition of entry into S phase are dependent on the second bromodomain of Brd4. Lastly, supporting the functional importance of this interaction, it was found that cotransfection with RFC-140 reduced the growth-inhibitory effect of Brd4. Taken as a whole, the present study suggests that Brd4 regulates cell cycle progression in part by interacting with RFC.

BRD4 regulates adiponectin gene induction by recruiting the P-TEFb complex to the transcribed region of the gene.

We previously reported that induction of the adipocyte-specific gene adiponectin (Adipoq) during 3T3-L1 adipocyte differentiation is closely associated with epigenetic memory histone H3 acetylation on the transcribed region of the gene. We used 3T3-L1 adipocytes and Brd4 heterozygous mice to investigate whether the induction of Adipoq during adipocyte differentiation is regulated by histone acetylation and the binding protein bromodomain containing 4 (BRD4) on the transcribed region. Depletion of BRD4 by shRNA and inhibition by (+)-JQ1, an inhibitor of BET family proteins including BRD4, reduced Adipoq expression and lipid droplet accumulation in 3T3-L1 adipocytes. Additionally, the depletion and inhibition of BRD4 reduced the expression of many insulin sensitivity-related genes, including genes related to lipid droplet accumulation in adipocytes. BRD4 depletion reduced P-TEFb recruitment and histone acetylation on the transcribed region of the Adipoq gene. The expression levels of Adipoq and fatty acid synthesis-related genes and the circulating ADIPOQ protein level were lower in Brd4 heterozygous mice than in wild-type mice at 21 days after birth. These findings indicate that BRD4 regulates the Adipoq gene by recruiting P-TEFb onto acetylated histones in the transcribed region of the gene and regulates adipocyte differentiation by regulating the expression of genes related to insulin sensitivity.

Brd4 binds to active enhancers to control cell identity gene induction in adipogenesis and myogenesis.

The epigenomic reader Brd4 is an important drug target for cancers. However, its role in cell differentiation and animal development remains largely unclear. Using two conditional knockout mouse strains and derived cells, we demonstrate that Brd4 controls cell identity gene induction and is essential for adipogenesis and myogenesis. Brd4 co-localizes with lineage-determining transcription factors (LDTFs) on active enhancers during differentiation. LDTFs coordinate with H3K4 mono-methyltransferases MLL3/MLL4 (KMT2C/KMT2D) and H3K27 acetyltransferases CBP/p300 to recruit Brd4 to enhancers activated during differentiation. Brd4 deletion prevents the enrichment of Mediator and RNA polymerase II transcription machinery, but not that of LDTFs, MLL3/MLL4-mediated H3K4me1, and CBP/p300-mediated H3K27ac, on enhancers. Consequently, Brd4 deletion prevents enhancer RNA production, cell identity gene induction and cell differentiation. Interestingly, Brd4 is dispensable for maintaining cell identity genes in differentiated cells. These findings identify Brd4 as an enhancer epigenomic reader that links active enhancers with cell identity gene induction in differentiation.

Chromatin Landscape of the IRF Genes and Role of the Epigenetic Reader BRD4.

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.

Transcription elongation factor Brd4-P-TEFb accelerates intestinal differentiation-associated SLC2A5 gene expression.

BACKGROUND: Expression of the fructose transporter gene SLC2A5 and histone acetylation in the transcribed region are induced by differentiation associated-signals such as glucocorticoids and p44/42 mitogen-activated protein kinase (MAPK) inhibition in small intestinal Caco-2 cells. METHODS: We co-treated with glucocorticoid receptor agonist dexamethasone (Dex) and p44/42 MAPK inhibitor PD98059 (PD) in Caco-2 cells with or without Brd4 small hairpin (sh) RNA expression vector, and the cells were analyzed by qRT-PCR and chromatin immunoprecipitation assays. The small intestine of wild-type mice and Brd4+/- mice during weaning period were analyzed by qRT-PCR. RESULTS: Co-treatment with Dex and PD increased binding of the bromodomain-containing protein-4 (Brd4)-positive transcriptional elongation factor-b (P-TEFb)-RNA polymerase II complex to acetylated histones in the transcribed region of SLC2A5. Brd4-protein depletion by shRNA revealed that the association of these proteins on the transcribed region of SLC2A5 promoted gene expression in a Brd4-dependent manner. Expression of small-intestine Slc2a5, but not another intestinal gene sucrase-isomaltase, during weaning period, was significantly lower in Brd4+/- mice compared with wild-type mice. CONCLUSIONS: Brd4-P-TEFb plays a crucial role in differentiation-associated transcription of SLC2A5 gene in intestinal Caco-2 cells and in the small intestine of mice during weaning period. GENERAL SIGNIFICANCE: Histone acetylation and the transcription elongation factor Brd4 are important for SLC2A5 expression in the small intestine.