Transcription Factor IRF3 Research Articles

Identification of IRF8 as a potent tumor suppressor in murine acute promyelocytic leukemia

AbstractAlthough the role of promyelocytic leukemia/retinoic acid receptor α (PML/RARA) fusion protein is well recognized in acute promyelocytic leukemia (APL), its contribution to initiation and maintenance of leukemogenesis is not completely understood. Transcriptome analysis in the murine MRP8-PML/RARA APL model has demonstrated modest alterations in gene expression accompanied by expansion of the promyelocyte compartment. Of particular interest, mice expressing PML/RARA showed downregulation of the transcription factor Irf8 mRNA. Interferon regulatory factor 8 (IRF8) is a known regulator of hematopoiesis. Previous research had implicated IRF8 as a tumor suppressor for myeloid neoplasia, and mice lacking IRF8 develop a well-differentiated myeloproliferative neoplasm characterized by expansion of neutrophilic lineage cells. We hypothesized that PML/RARA-mediated downregulation of Irf8 transcript levels contributes to the initiation of APL. We observed significant downregulation of IRF8 protein levels in highly purified promyelocyte populations of PML/RARA transgenic mice. We also found that loss of IRF8 results in expansion of promyelocytes in vivo, partially phenocopying the impact of PML/RARA expression. Moreover, survival experiments showed that complete loss of IRF8 leads to acceleration of APL onset in our PML/RARA mice. Collectively, these data identify IRF8 downregulation as an important factor in APL initiation and highlight a tumor-suppressor role for IRF8 in this acute leukemia.

Mycobacterium tuberculosis Virulent Factor ESAT-6 Drives Macrophage Differentiation Toward the Pro-inflammatory M1 Phenotype and Subsequently Switches It to the Anti-inflammatory M2 Phenotype.

Tuberculosis, a human infectious disease caused by Mycobacterium tuberculosis (M.tb), is still a major cause of morbidity and mortality worldwide. The success of M.tb as a pathogen relies mainly on its ability to divert the host innate immune responses. One way by which M.tb maintains a persistent infection in a “silent” granuloma is to inhibit inflammation and induce an immunoregulatory phenotype in host macrophages (MΦs). However, M.tb effectors governing the switch of MΦs from the pro-inflammatory M1 to the anti-inflammatory M2 phenotype remain to be determined. The Early Secreted Antigenic Target 6 kDa or ESAT-6, has been implicated in the virulence and pathogenesis of tuberculosis. Here, we investigated roles of ESAT-6 in MΦ differentiation and polarization. We found that treatment of human monocytes with ESAT-6 did not interfere with differentiation of M1 MΦs. However, ESAT-6 promoted differentiation of M0 and M2 MΦs toward the M1 phenotype, as indicated by secretion of pro-inflammatory cytokines IL-6, IL-12, and TNF-α, and induction of a typical M1 transcriptional signature. Interestingly, we found that ESAT-6 switched terminal full activation of M1 polarized MΦs to the M2 phenotype. Indeed, in the pro-inflammatory M1 MΦs, ESAT-6 was able to inhibit IL-12 and TNF-α secretion and stimulate that of IL-10. Moreover, gene expression profiling of these cells showed that ESAT-6 induced downregulation of M1 MΦ cell surface molecules CD80 and CD86, transcription factors IRF5 and c-MAF, cytokines IL-12, IL-10, and IL-6, as well as chemokines CXCL10 and CXCL1. Overall, our findings suggest ESAT-6 as being one of the effectors used by M.tb to induce the pro-inflammatory M1 phenotype at the primo-infection; a prerequisite step to promote granuloma formation and subsequently drive the phenotype switch of MΦ polarization from M1 to M2 at a later stage of the infection. Our study improves current knowledge regarding mechanisms of virulence of M.tb and may be helpful to develop novel tools targeting ESAT-6 for a better and more efficient treatment of tuberculosis.

ETS1 and PAX5 transcription factors recruit AID to Igh DNA.

B lymphocytes optimize antibody responses by class switch recombination (CSR), which changes the expressed constant region exon of the immunoglobulin heavy chain (IgH), and by somatic hypermutation (SH) that introduces point mutations in the variable regions of the antibody genes. Activation-induced cytidine deaminase (AID) is the key mutagenic enzyme that initiates both these antibody diversification processes by deaminating cytosine to uracil. Here we asked the question if transcription factors can mediate the specific targeting of the antibody diversification by recruiting AID. We have recently reported that AID is together with the transcription factors E2A, PAX5 and IRF4 in a complex on key sequences of the Igh locus. Here we report that also ETS1 is together with AID in this complex on key sequences of the Igh locus in splenic B cells of mice. Furthermore, we show that both ETS1 and PAX5 can directly recruit AID to DNA sequences from the Igh locus with the specific binding site for the transcription factor. Taken together, our findings support the notion of a targeting mechanism for the selective diversification of antibody genes with limited genome wide mutagenesis by recruitment of AID by PAX5 and ETS1 in a transcription factor complex.

Inhibiting pyrimidine biosynthesis impairs Ebola virus replication through depletion of nucleoside pools and activation of innate immune responses

Specific host pathways that may be targeted therapeutically to inhibit the replication of Ebola virus (EBOV) and other emerging viruses remain incompletely defined. A screen of 200,000 compounds for inhibition of an EBOV minigenome (MG) assay that measures the function of the viral polymerase complex identified as hits several compounds with an amino-tetrahydrocarbazole scaffold. This scaffold was structurally similar to GSK983, a compound previously described as having broad-spectrum antiviral activity due to its impairing de novo pyrimidine biosynthesis through inhibition of dihydroorotate dehydrogenase (DHODH). We generated compound SW835, the racemic version of GSK983 and demonstrated that SW835 and brequinar, another DHODH inhibitor, potently inhibit the MG assay and the replication of EBOV, vesicular stomatitis virus (VSV) and Zika (ZIKV) in vitro. Nucleoside and deoxynucleoside supplementation studies demonstrated that depletion of pyrimidine pools contributes to antiviral activity of these compounds. As reported for other DHODH inhibitors, SW835 and brequinar also induced expression of interferon stimulated genes (ISGs). ISG induction was demonstrated to occur without production of IFNα/β and independently of the IFNα receptor and was not blocked by EBOV-encoded suppressors of IFN signaling pathways. Furthermore, we demonstrated that transcription factor IRF1 is required for this ISG induction, and that IRF1 induction requires the DNA damage response kinase ATM. Therefore, de novo pyrimidine biosynthesis is critical for the replication of EBOV and other RNA viruses and inhibition of this pathway activates an ATM and IRF1-dependent innate immune response that subverts EBOV immune evasion functions.

Mutations of deubiquitinase OTUD1 are associated with autoimmune disorders

Dysregulation of innate immunity accompanied by excessive interferon production contributes to autoimmune disease. However, the mechanism by which the immune response is modulated in autoimmune disorders is largely unknown. Here we identified loss-of-function mutations of OTUD1 associated with multiple autoimmune diseases. Under inflammatory conditions, inducible OTUD1 acts as an immune checkpoint and blocks RIG-I-like receptors signaling. As a deubiquitinase, OTUD1 directly interacts with transcription factor IRF3 and removes the K63-linked poly-ubiquitin chains on IRF3 Lysine 98, which inhibits IRF3 nuclear translocation and transcriptional activity. In contrast, OTUD1 mutants impair its suppressive effects on IRF3 via attenuating the OTUD1 deubiquinase activity or its association with IRF3. Moreover, we found FOXO3 signaling is required for OTUD1 induction upon antigenic stimulation. Our data demonstrate that OTUD1 is involved in maintaining immune homeostasis and loss-of-function mutations of OTUD1 enhance the immune response and are associated with autoimmunity.

Brown Adipose Tissue Controls Skeletal Muscle Function via the Secretion of Myostatin

Skeletal muscle and brown adipose tissue (BAT) are functionally linked, as exercise increases browning via secretion of myokines. It is unknown whether BAT affects muscle function. Here, we find that loss of the transcription factor IRF4 in BAT (BATI4KO) reduces exercise capacity, mitochondrial function, ribosomal protein synthesis, and mTOR signaling in muscle and causes tubular aggregate formation. Loss of IRF4 induces myogenic gene expression in BAT, including the secreted factor myostatin, a known inhibitor of muscle function. Reducing myostatin via neutralizing antibodies or soluble receptor rescues the exercise capacity of BATI4KO mice. In addition, overexpression of IRF4 in brown adipocytes reduces serum myostatin and increases exercise capacity in muscle. Finally, mice housed at thermoneutrality have reduced IRF4 in BAT, lower exercise capacity, and elevated serum myostatin; these abnormalities are corrected by excising BAT. Collectively, our data point to an unsuspected level of BAT-muscle crosstalk driven by IRF4 and myostatin.

3025 - Ebf1 as a Transcriptional Regulator of Bone Marrow Mesenchymal Stromal Cells

Mesenchymal stem and progenitor cells (MSPC), a crucial component of BM niche orchestrate maintenance, trafficking and stage specific differentiation of hematopoietic stem and progenitor cells (HSPCs). Although, it is generally accepted that MSPCs are essential for hematopoietic homeostasis, little is known about the transcriptional networks governing MSPCs biology. Here, we identified Early B-cell factor 1 (Ebf1) as new transcription regulator of MSPCs activity. MSPCs isolated from Ebf1-/- mice show diminished capacity to form fibroblastic colonies (CFU-F) indicating reduced self-renewal. Cells expanded from these colonies display impaired in vitro differentiation towards osteoblasts, chondrocytes and adipocytes. Adoptive transfers of wild type HSPCs to Ebf1+/- recipient mice showed a decrease in the absolute numbers of HSPCs in primary recipients and reduced donor chimerism within the HSCP compartment in competitive secondary transplant experiments. Additionally, Prx1-Cre-mediated deletion of Ebf1 in MSPCs in vivo leads to reduced numbers of HSPCs and myeloid cells in the bone marrow. Single cell and bulk transcriptome analysis of MSPCs lacking Ebf1 revealed differences in the niche composition and decreased expression of lineage-instructive signals for myeloid cells. Interestingly, we also observed a reduced ability of HSPCs sorted from Prx1CreEbf1 fl/fl mice to form colonies in methylcellulose, suggesting not only impaired maintenance but also hindered function of these cells. HSPCs exposed to Ebf1-deficient niche exhibit reduced chromatin accessibility for AP-1, ETS, Runx and IRF transcription factors that are crucial for myeloid differentiation, which is consistent with decreased myeloid output observed in Prx1CreEbf1 fl/fl mice. These results support the hypothesis that defective niche can cause epigenetic reprograming of HSPCs. In conclusion, our study establishes Ebf1 as a novel regulator of MSPCs playing a crucial role in the maintenance and differentiation of HSPCs.

Porcine Deltacoronavirus Accessory Protein NS6 Antagonizes Interferon Beta Production by Interfering with the Binding of RIG-I/MDA5 to Double-Stranded RNA.

Porcine deltacoronavirus (PDCoV) has recently emerged as an enteric pathogen that can cause serious vomiting and diarrhea in suckling piglets. The first outbreak of PDCoV occurred in the United States in 2014 and was followed by reports of PDCoV in South Korea, China, Thailand, Lao People's Democratic Republic, and Vietnam, leading to economic losses for pig farms and posing a considerable threat to the swine industry worldwide. Our previous studies have shown that PDCoV encodes three accessory proteins, NS6, NS7, and NS7a, but the functions of these proteins in viral replication, pathogenesis, and immune regulation remain unclear. Here, we found that ectopic expression of accessory protein NS6 significantly inhibits Sendai virus-induced interferon beta (IFN-β) production as well as the activation of transcription factors IRF3 and NF-κB. Interestingly, NS6 does not impede the IFN-β promoter activation mediated via key molecules in the RIG-I-like receptor (RLR) signaling pathway, specifically RIG-I, MDA5, and their downstream molecules MAVS, TBK1, IKKε, and IRF3. Further analyses revealed that NS6 is not an RNA-binding protein; however, it interacts with RIG-I/MDA5. This interaction attenuates the binding of double-stranded RNA by RIG-I/MDA5, resulting in the reduction of RLR-mediated IFN-β production. Taken together, our results demonstrate that ectopic expression of NS6 antagonizes IFN-β production by interfering with the binding of RIG-I/MDA5 to double-stranded RNA, revealing a new strategy employed by PDCoV accessory proteins to counteract the host innate antiviral immune response.IMPORTANCE Coronavirus accessory proteins are species specific, and they perform multiple functions in viral pathogenicity and immunity, such as acting as IFN antagonists and cell death inducers. Our previous studies have shown that PDCoV encodes three accessory proteins. Here, we demonstrated for the first time that PDCoV accessory protein NS6 antagonizes IFN-β production by interacting with RIG-I and MDA5 to impede their association with double-stranded RNA. This is an efficient strategy of antagonizing type I IFN production by disrupting the binding of host pattern recognition receptors (PRRs) and pathogen-associated molecular patterns (PAMPs). These findings deepen our understanding of the function of accessory protein NS6, and they may direct us toward novel therapeutic targets and lead to the development of more effective vaccines against PDCoV infection.

Molecular cloning and functional characterization of duck DDX41

DEAD (Asp–Glu–Ala–Asp) box polypeptide 41 (DDX41), a receptor belonging to DExD/H-box helicase family, acts as an intracellular DNA sensor and induces type I IFN production in mammals and fish. However, the function of avian DDX41 in innate immune response is still unknown. In this study, the full-length duck DDX41 (duDDX41) cDNA sequence was cloned for the first time and encoded a putative protein of 618 amino acid residues which showed the high sequence similarity with both zebra finch and chicken DDX41s. The duDDX41 mRNA was widely distributed in all tested tissues, especially the cerebrum, cerebellum, and liver. Overexpression of duDDX41 triggered the activation of transcription factors IRF1 and NF-κB, as well as IFN-β expression in DEFs. The DEADc domain of duDDX41 played an extremely vital role in duck type I IFN signaling pathway. Knockdown of duDDX41 by siRNA silencing dramatically decreased IFN-β expression stimulated by poly(dA:dT) or duck enteritis virus (DEV). In addition, the replication of DEV was significantly inhibited in duDDX41-expressed DEFs and was enhanced in DDX41 knockdown DEFs. These results suggest that DDX41 is an important cytosolic DNA sensor and plays a crucial role in duck antiviral innate immune response.

Schistosomiasis Induces Persistent DNA Methylation and Tuberculosis-Specific Immune Changes.

Epigenetic mechanisms, such as DNA methylation, determine immune cell phenotype. To understand the epigenetic alterations induced by helminth coinfections, we evaluated the longitudinal effect of ascariasis and schistosomiasis infection on CD4+ T cell DNA methylation and the downstream tuberculosis (TB)-specific and bacillus Calmette-Guérin-induced immune phenotype. All experiments were performed on human primary immune cells from a longitudinal cohort of recently TB-exposed children. Compared with age-matched uninfected controls, children with active Schistosoma haematobium and Ascaris lumbricoides infection had 751 differentially DNA-methylated genes, with 72% hypermethylated. Gene ontology pathway analysis identified inhibition of IFN-γ signaling, cellular proliferation, and the Th1 pathway. Targeted real-time quantitative PCR after methyl-specific endonuclease digestion confirmed DNA hypermethylation of the transcription factors BATF3, ID2, STAT5A, IRF5, PPARg, RUNX2, IRF4, and NFATC1 and cytokines or cytokine receptors IFNGR1, TNFS11, RELT (TNF receptor), IL12RB2, and IL12B (p < 0.001; Sidak-Bonferroni). Functional blockage of the IFN-γ signaling pathway was confirmed, with helminth-infected individuals having decreased upregulation of IFN-γ-inducible genes (Mann-Whitney p < 0.05). Hypomethylation of the IL-4 pathway and DNA hypermethylation of the Th1 pathway was confirmed by Ag-specific multidimensional flow cytometry demonstrating decreased TB-specific IFN-γ and TNF and increased IL-4 production by CD4+ T cells (Wilcoxon signed-rank p < 0.05). In S. haematobium-infected individuals, these DNA methylation and immune phenotypic changes persisted at least 6 mo after successful deworming. This work demonstrates that helminth infection induces DNA methylation and immune perturbations that inhibit TB-specific immune control and that the duration of these changes are helminth specific.

Distinct progenitor lineages contribute to the heterogeneity of plasmacytoid dendritic cells.

Plasmacytoid dendritic cells (pDCs) are an immune subset devoted to the production of high amounts of type 1 interferons in response to viral infections. Whereas conventional dendritic cells (cDCs) originate mostly from a common dendritic cell progenitor (CDP), pDCs have been shown to develop from both CDPs and common lymphoid progenitors. Here, we found that pDCs developed predominantly from IL-7R+ lymphoid progenitor cells. Expression of SiglecH and Ly6D defined pDC lineage commitment along the lymphoid branch. Transcriptional characterization of SiglecH+Ly6D+ precursors indicated that pDC development requires high expression of the transcription factor IRF8, whereas pDC identity relies on TCF4. RNA sequencing of IL-7R+ lymphoid and CDP-derived pDCs mirrored the heterogeneity of mature pDCs observed in single-cell analysis. Both mature pDC subsets are able to secrete type 1 interferons, but only myeloid-derived pDCs share with cDCs their ability to process and present antigen.

SUMO2 and SUMO3 redundantly prevent a noncanonical type I interferon response

Detection of nucleic acids by innate immune sensors triggers the production of type I interferons (IFNs). While IFNs are essential for host defense against viral infection, dysregulated production of IFNs underlies numerous autoinflammatory diseases. We have found that the loss of sumoylation results in a potent, spontaneous IFN response. Vertebrates possess three small ubiquitin-like modifiers (SUMOs) that can be conjugated onto target proteins and alter protein function in diverse but still poorly characterized ways. We demonstrate that regulation of IFN by sumoylation is redundantly mediated by both SUMO2 and SUMO3, but not SUMO1, revealing a previously unknown function of SUMO2/3. Remarkably, this IFN response is independent of all known IFN-inducing pathways and does not require either of the canonical IFN-associated transcription factors IRF3 or IRF7. Taken together, our findings demonstrate that SUMO2 and SUMO3 are specific and essential negative regulators of a noncanonical mechanism of IFN induction.

Host HDAC4 regulates the antiviral response by inhibiting the phosphorylation of IRF3.

Class II HDACs, such as HDAC4, are critical regulators of the immune response in various immune cells; however, its role in innate immunity remains largely unknown. Here, we report that the overexpression of HDAC4 suppresses the production of type I interferons triggered by pattern-recognition receptors (PRRs). HDAC4 repressed the translocation of transcription factor IRF3 to the nucleus, thereby decreasing IRF3-mediated IFN-β expression. In particular, we also determined that HDAC4 can be phosphorylated and simultaneously block the phosphorylation of IRF3 at Ser386 and Ser396 by TBK1 and IKKε, respectively, by interacting with the kinase domain of TBK1 and IKKε. Furthermore, IFN-β may stimulate the expression of HDAC4. Our findings suggest that HDAC4 acts as a regulator of PRR signaling and is a novel mechanism of negative feedback regulation for preventing an over-reactive innate immune response.

Monitoring Extracellular Vesicle Cargo Active Uptake by Imaging Flow Cytometry.

Extracellular vesicles are essential for long distance cell–cell communication. They function as carriers of different compounds, including proteins, lipids and nucleic acids. Pathogens, like malaria parasites (Plasmodium falciparum, Pf), excel in employing vesicle release to mediate cell communication in diverse processes, particularly in manipulating the host response. Establishing research tools to study the interface between pathogen-derived vesicles and their host recipient cells will greatly benefit the scientific community. Here, we present an imaging flow cytometry (IFC) method for monitoring the uptake of malaria-derived vesicles by host immune cells. By staining different cargo components, we were able to directly track the cargo’s internalization over time and measure the kinetics of its delivery. Impressively, we demonstrate that this method can be used to specifically monitor the translocation of a specific protein within the cellular milieu upon internalization of parasitic cargo; namely, we were able to visually observe how uptaken parasitic Pf-DNA cargo leads to translocation of transcription factor IRF3 from the cytosol to the nucleus within the recipient immune cell. Our findings demonstrate that our method can be used to study cellular dynamics upon vesicle uptake in different host–pathogen and pathogen–pathogen systems.

Investigation of the immunological mechanisms underlying the attenuation of vaccinia virus lacking host-range factor C7

Abstract Vaccinia virus C7 protein is an important host-range factor for the vaccinia virus life cycle in mammalian cells. Type I IFN plays an important role in host defense of viral infection. However, the role of C7 in immune modulation of the IFN pathway is unclear. Here, we find that C7 functions as a dual inhibitor of IFN production and IFN signaling by interfering with the activation of two transcription factors IRF3 and STAT2. Furthermore, vaccinia virus with deletion of C7 (VACVΔC7L) is non-pathogenic in wild-type C57BL/6J mice in an intranasal infection model. We elucidated the immunological mechanisms underlying the attenuation phenotype of VACVΔC7L, which can be summarized as follows: (i) intranasal infection of VACVΔC7L triggers the production of interferons (IFNs), proinflammatory cytokines and chemokines in bronchoalveolar lavage fluid (BAL), whereas WT VACV does not; (ii) intranasal infection of VACVΔC7L results in the recruitment of dendritic cells, inflammatory monocytes, neutrophils, CD8+, and CD4+ in the BAL, whereas WT VACV does not; and (iii) infection of primary type II lung alveolar epithelial cells (AECs) with VACVΔC7L induces IFN, proinflammatory cytokine and chemokine gene expression and protein secretion, whereas WT VACV does not. However, VACVΔC7L dramatically gained virulence in STAT2 or IFNAR1-deficient mice, with increased titers in the lungs and systemic dissemination of the viruses to the blood and distant organs. Taken together, our results provide evidence that vaccinia C7 is a key virulence factor that antagonize both IFN production and signaling and IFNAR1 and STAT2 are critical in restricting vaccinia viral replication in the lung alveolar epithelial cells.

Deconstructing T-B interactions by manipulation of IRF4 to understand regulation of T follicular helper cells.

Abstract Germinal center (GC) derived high affinity antibody is important for protecting against infectious disease. Interactions between T follicular helper (Tfh) cells and GC B cells are requisite for affinity maturation. While it is clear that bi-directional signals are important for the generation of Tfh and GC B cells, a full understanding of the signaling dynamics that lead to efficient T and B cell differentiation is not known. Studying these B cell-derived signals and their role in Tfh cell commitment, maintenance, and function during GC reactions will lead to an improved understanding of this tightly regulated system. The transcription factor IRF4 is necessary and sufficient for generating GC B and plasma cell fates through graded expression. We have shown previously through gene ontology analysis that IRF4 targets include genes involved in T and B cell interaction. This raises the possibility that beyond cell-specific control of B cell fates, IRF4-regulated genes in B cells may facilitate interactions between B and T cells. We hypothesize that IRF4 controls a gene program in B cells necessary to generate efficient T cell activation and Tfh cell differentiation. Using in-vitro co-culture, we assessed changes in T and B cell activation and functionality by flow cytometry. We found that Irf4−/− B cells processed antigen poorly and led to quantitatively and qualitatively reduced T cell activation compared to Irf4+/+ B cells. This indicates a major role for IRF4 in controlling a B cell gene program necessary for efficient T cell activation. We are currently investigating the nature of these signals through in-vitro and in-vivo approaches in order to deconstruct the molecular nature of T-B interactions during the GC program.

MHCII+ Macrophages in Peritoneal and Pleural Cavities Require IRF4 for Development from Circulating Monocytes

Abstract Resident macrophages in peritoenal and pleural cavities exist as two distinct populations: F4/80+ICAM2+macrophages and MHCII+ macrophages. F4/80+ ICAM2+macrophages are derived from CX3CR1+embryonic precursors, and maitained by the transcription factor Gata6. However, the origin and regulating factors that maintain MHCII+ macrophages remain unknown. Here, we show that the MHCII+ macrophages arise postnatally from monocytes, which continuously replenish this subset through adulthood. Gene expression analysis identified distinct surface markers such as CD226 and revealed that the transcription factor IRF4 was selectively expressed in these macrophages relative to other organs. Monocytes first enter peritoneal or pleural cavities to become MHC II+ cells that up-regulate CD226 later as they continue to mature. In the absence of IRF4 or after administration of oral antibiotics, MHCII+CD226−CD11c− monocyte-derived cells accumulated in peritoneal and pleural cavities, but CD11c+ CD226+ macrophages were lost. Thus, MHC II+ resident peritoneal and pleural macrophages are continuously replenished by blood monocytes recruited to the peritoneal and pleural cavities constitutively, starting after birth, where they require IRF4 and signals likely derived from the microbiome to fully differentiate.

GATA3, SATB1, IRF4 complex binds to the Th2 cytokine locus through cis-acting element RHS6, regulating allergic airway inflammation.

Abstract Rad50 hypersensitive site 6 (RHS6) is a cis-acting element in the Th2 locus control region which is essential for the expression of Th2 cytokine genes. However, molecular mechanisms how it regulates the genes are poorly understood. We found that RHS6 recruits transcription factors GATA3, SATB1, and IRF4 during Th2 cell differentiation. GATA3, SATB1, and IRF4 have a crucial role in regulating Th2 cytokine genes. Knock-out or knock-down of each of GATA3, SATB1, and IRF4 genes significantly decrease the expression of Th2 cytokine genes. Mechanistically RHS6 mediates the transcription factor-induced regulation of Th2 cytokine genes. GATA3, SATB1, and IRF4 physically associates with each other, and forms a protein complex at RHS6 sequence. They directly bind to the many regulatory elements throughout the Th2 cytokine locus, RHS6 dependent manner. Furthermore, deletion of RHS6 caused a dramatic resistance to airway inflammation in ovalbumin-induced allergic inflammation model. Together, RHS6 recruits GATA3, SATB1, and IRF4 coordinating their regulation of Th2 cytokine genes, so is essential for Th2-mediated allergic airway inflammation.

Inhibitor of MyoD Family A (I-mfa) regulates HSPC and myeloid differentiation

Abstract Hematopoietic stem and progenitor cell (HSPC) populations are capable of expanding in response to immunological insult or homeostatic turnover. Regulation of the fate decision between self-renewal and differentiation allows for both maintenance of blood cell populations and sustained hematopoiesis across the entire life-span. I-mfa, a small cytosolic protein, has been shown to inhibit lineage-specific transcription factors as well as regulate β-catenin/Wnt signaling. We have previously shown that I-mfa-null mice have increased myeloid cells resulting in increased osteoclasts and hypothesized that I-mfa regulates myeloid lineage differentiation and may affect HSPC populations. In order to test this, bone marrow (BM) from I-mfa−/− mice was analyzed by flow cytometry, showing increases in both long-term and short-term hematopoietic stem cells compared to WT controls, an effect which increased with age. Additionally, both common myeloid precursors (CMP) and granulocyte-macrophage precursors (GMP) were increased in I-mfa−/− mice. Conditional knockout of I-mfa in all hematopoietic lineages using Vav1-Cre lead to similar findings, while conditional knockout in myeloid cells with LysM-Cre showed only increases in myeloid progenitors. These data indicate that I-mfa has separate roles in regulating HSPC expansion and myeloid cell differentiation. Mechanistically, M-CSF-stimulated I-mfa−/− BM had significantly increased transcription of β-catenin signaling target genes Ccnd1, Axin2, and Myc, as well as myelopoietic transcription factors Spi1, Irf8, and Klf4, compared to WT. Further studies are being conducted to determine the effect of I-mfa on quiescence, Wnt ligand sensitivity, and BM reconstitution capacity.

The IRF4 gene regulatory module functions as a read-write integrator to dynamically control T helper cell fate

Abstract Transcriptional regulation during CD4+ T cell fate decisions enables their differentiation into distinct states, guiding immune responses towards antibody production via Tfh cells or inflammation by Teff cells. Tfh–Teff fate commitment is regulated by mutual antagonism between the transcription factors Bcl6 and Blimp-1. Here we examined how T cell receptor (TCR) signals establish and arbitrate Bcl6–Blimp-1 counter-antagonism. We found that the TCR-signal induced transcription factor IRF4 is essential for the differentiation of Bcl6-expressing Tfh and Blimp-1-expressing Teff cells. Increased TCR signaling raised IRF4 amounts and promoted Teff fates at the expense of Tfh ones. Importantly, orthogonal induction of IRF4 expression redirected Tfh fate trajectories towards those of Teff and this occurred independently of IL-2 signals. Mechanistically, we linked greater IRF4 abundance with its recruitment towards low affinity binding sites within Teff cis-regulatory elements, including those of Prdm1. We propose that the Irf4 locus functions as the “reader” of TCR signal strength, in turn, concentration-dependent activity of IRF4 “writes” T helper fate choice.