PU.1 cooperates with IRF4 and IRF8 to suppress pre-B-cell leukemia.
ABSTRACT: The Ets family transcription factor PU.1 and the interferon regulatory factor (IRF)4 and IRF8 regulate gene expression by binding to composite DNA sequences known as Ets/interferon consensus elements. Although all three factors are expressed from the onset of B-cell development, single deficiency of these factors in B-cell progenitors only mildly impacts on bone marrow B lymphopoiesis. Here we tested whether PU.1 cooperates with IRF factors in regulating early B-cell development. Lack of PU.1 and IRF4 resulted in a partial block in development the pre-B-cell stage. The combined deletion of PU.1 and IRF8 reduced recirculating B-cell numbers. Strikingly, all PU.1/IRF4 and ~50% of PU.1/IRF8 double deficient mice developed pre-B-cell acute lymphoblastic leukemia (B-ALL) associated with reduced expression of the established B-lineage tumor suppressor genes, Ikaros and Spi-B. These genes are directly regulated by PU.1/IRF4/IRF8, and restoration of Ikaros or Spi-B expression inhibited leukemic cell growth. In summary, we demonstrate that PU.1, IRF4 and IRF8 cooperate to regulate early B-cell development and to prevent pre-B-ALL formation.
Project description:Interferon regulatory factor 4 (IRF4) and IRF8 regulate B, T, macrophage, and dendritic cell differentiation. They are recruited to cis-regulatory Ets-IRF composite elements by PU.1 or Spi-B. How these IRFs target genes in most T cells is enigmatic given the absence of specific Ets partners. Chromatin immunoprecipitation sequencing in T helper 17 (T(H)17) cells reveals that IRF4 targets sequences enriched for activating protein 1 (AP-1)-IRF composite elements (AICEs) that are co-bound by BATF, an AP-1 factor required for T(H)17, B, and dendritic cell differentiation. IRF4 and BATF bind cooperatively to structurally divergent AICEs to promote gene activation and T(H)17 differentiation. The AICE motif directs assembly of IRF4 or IRF8 with BATF heterodimers and is also used in T(H)2, B, and dendritic cells. This genomic regulatory element and cognate factors appear to have evolved to integrate diverse immunomodulatory signals.
Project description:The transcription factor (TF) interferon regulatory factor-4 (IRF4) promotes both germinal center (GC) reactions and plasma cell (PC) differentiation by binding to alternative DNA motifs including AP-1-IRF composite elements, Ets-IRF composite elements (EICEs), and interferon sequence response elements (ISREs). Although all of these motifs mediate transcriptional activation by IRF4, it is still unknown how some of the IRF4 target genes are downregulated upon PC differentiation. Here, we revealed a molecular mechanism of IRF4-mediated gene downregulation during PC differentiation. By combining IRF4 chromatin immunoprecipitation sequence and gene expression analysis, we identified zinc finger-IRF composite elements (ZICEs) in IRF4 binding regions aligned with genes whose expression was downregulated in PCs. The zinc finger TFs Ikaros and Aiolos were identified as IRF4 binding partners in PCs, and Ikaros but not Aiolos was essential for IRF4 binding to the ZICE sequence and for PC differentiation. The Ebf1 gene, which positively controls B-cell activation and GC reactions, was identified as one of the Ikaros/IRF4 target genes. Importantly, while the ZICE embeds the ISRE motif, IRF4 bound the ZICE motif as heterodimers with Ikaros for repression of target genes, which include Ebf1 In contrast, if the zinc finger motif is juxtaposed to the EICE motif, the Ikaros/PU.1/IRF4 complex functioned to activate target gene expression. Our findings revealed a novel mode of IRF4 activity upon PC differentiation where upon forming an Ikaros/IRF4 DNA-bound complex, a subset of genes is repressed.
Project description:Interferon regulatory factor 4 (IRF4) is an IRF family transcription factor with critical roles in lymphoid development and in regulating the immune response. IRF4 binds DNA weakly owing to a carboxy-terminal auto-inhibitory domain, but cooperative binding with factors such as PU.1 or SPIB in B cells increases binding affinity, allowing IRF4 to regulate genes containing ETS-IRF composite elements (EICEs; 5'-GGAAnnGAAA-3'). Here we show that in mouse CD4(+) T cells, where PU.1/SPIB expression is low, and in B cells, where PU.1 is well expressed, IRF4 unexpectedly can cooperate with activator protein-1 (AP1) complexes to bind to AP1-IRF4 composite (5'-TGAnTCA/GAAA-3') motifs that we denote as AP1-IRF composite elements (AICEs). Moreover, BATF-JUN family protein complexes cooperate with IRF4 in binding to AICEs in pre-activated CD4(+) T cells stimulated with IL-21 and in T(H)17 differentiated cells. Importantly, BATF binding was diminished in Irf4(-/-) T cells and IRF4 binding was diminished in Batf(-/-) T cells, consistent with functional cooperation between these factors. Moreover, we show that AP1 and IRF complexes cooperatively promote transcription of the Il10 gene, which is expressed in T(H)17 cells and potently regulated by IL-21. These findings reveal that IRF4 can signal via complexes containing ETS or AP1 motifs depending on the cellular context, thus indicating new approaches for modulating IRF4-dependent transcription.
Project description:Langerhans cells (LC) can prime tolerogenic as well as immunogenic responses in skin, but the genomic states and transcription factors (TF) regulating these context-specific responses are unclear. Bulk and single-cell transcriptional profiling demonstrates that human migratory LCs are robustly programmed for MHC-I and MHC-II antigen presentation. Chromatin analysis reveals enrichment of ETS-IRF and AP1-IRF composite regulatory elements in antigen-presentation genes, coinciding with expression of the TFs, PU.1, IRF4 and BATF3 but not IRF8. Migration of LCs from the epidermis is accompanied by upregulation of IRF4, antigen processing components and co-stimulatory molecules. TNF stimulation augments LC cross-presentation while attenuating IRF4 expression. CRISPR-mediated editing reveals IRF4 to positively regulate the LC activation programme, but repress NF2EL2 and NF-kB pathway genes that promote responsiveness to oxidative stress and inflammatory cytokines. Thus, IRF4-dependent genomic programming of human migratory LCs appears to enable LC maturation while attenuating excessive inflammatory and immunogenic responses in the epidermis.
Project description:Interferon regulatory factors (IRF) have critical functions in lymphoid development and in immune response regulation. Although many studies have described the function of IRF4 in CD4+ T cells, few have focused on the IRF4 homologue, IRF8. Here, we show that IRF8 is required for Th9 differentiation in vitro and in vivo. IRF8 functions through a transcription factor complex consisting of IRF8, IRF4, PU.1 and BATF, which binds to DNA and boosts Il9 transcription. By contrast, IRF8 deficiency promotes the expression of other genes such as Il4, as IRF8 dimerises with the transcriptional repressor ETV6 and inhibits Il4 expression. In vivo, IRF8 is essential for the anti-tumour effects of Th9 cells in mouse melanoma models. Our results show that IRF8 complexes boost the Th9 program and repress Il4 expression to modulate Th9 cell differentiation, thereby implicating IRF8 as a potential therapeutic target to affect Th9 responses in cancer therapy.
Project description:Spi-B and PU.1 are highly related members of the E26-transformation-specific (ETS) family of transcription factors that have similar, but not identical, roles in B cell development. PU.1 and Spi-B are both expressed in B cells, and have been demonstrated to redundantly activate transcription of genes required for B cell differentiation and function. It was hypothesized that Spi-B and PU.1 occupy a similar set of regions within the genome of a B lymphoma cell line.To compare binding regions of Spi-B and PU.1, murine WEHI-279 lymphoma cells were infected with retroviral vectors encoding 3XFLAG-tagged PU.1 or Spi-B. Anti-FLAG chromatin immunoprecipitation followed by next generation sequencing (ChIP-seq) was performed. Analysis for high-stringency enriched genomic regions demonstrated that PU.1 occupied 4528 regions and Spi-B occupied 3360 regions. The majority of regions occupied by Spi-B were also occupied by PU.1. Regions bound by Spi-B and PU.1 were frequently located immediately upstream of genes associated with immune response and activation of B cells. Motif-finding revealed that both transcription factors were predominantly located at the ETS core domain (GGAA), however, other unique motifs were identified when examining regions associated with only one of the two factors. Motifs associated with unique PU.1 binding included POU2F2, while unique motifs in the Spi-B regions contained a combined ETS-IRF motif.Our results suggest that complementary biological functions of PU.1 and Spi-B may be explained by their interaction with a similar set of regions in the genome of B cells. However, sites uniquely occupied by PU.1 or Spi-B provide insight into their unique functions.
Project description:Epstein-Barr virus (EBV) is linked to a broad spectrum of B-cell malignancies. EBV nuclear antigen 3C (EBNA3C) is an encoded latent antigen required for growth transformation of primary human B-lymphocytes. Interferon regulatory factor 4 (IRF4) and 8 (IRF8) are transcription factors of the IRF family that regulate diverse functions in B cell development. IRF4 is an oncoprotein with anti-apoptotic properties and IRF8 functions as a regulator of apoptosis and tumor suppressor in many hematopoietic malignancies. We now demonstrate that EBNA3C can contribute to B-cell transformation by modulating the molecular interplay between cellular IRF4 and IRF8. We show that EBNA3C physically interacts with IRF4 and IRF8 with its N-terminal domain in vitro and forms a molecular complex in cells. We identified the Spi-1/B motif of IRF4 as critical for EBNA3C interaction. We also demonstrated that EBNA3C can stabilize IRF4, which leads to downregulation of IRF8 by enhancing its proteasome-mediated degradation. Further, si-RNA mediated knock-down of endogenous IRF4 results in a substantial reduction in proliferation of EBV-transformed lymphoblastoid cell lines (LCLs), as well as augmentation of DNA damage-induced apoptosis. IRF4 knockdown also showed reduced expression of its targeted downstream signalling proteins which include CDK6, Cyclin B1 and c-Myc all critical for cell proliferation. These studies provide novel insights into the contribution of EBNA3C to EBV-mediated B-cell transformation through regulation of IRF4 and IRF8 and add another molecular link to the mechanisms by which EBV dysregulates cellular activities, increasing the potential for therapeutic intervention against EBV-associated cancers.
Project description:PU.1, IKAROS, E2A, EBF, and PAX5 comprise a transcriptional network that orchestrates B-cell lineage specification, commitment, and differentiation. Here we identify interferon regulatory factor 8 (IRF8) as another component of this complex, and show that it also modulates lineage choice by hematopoietic stem cells (HSCs). IRF8 binds directly to an IRF8/Ets consensus sequence located in promoter regions of Sfpi1 and Ebf1, which encode PU.1 and EBF, respectively, and is associated with transcriptional repression of Sfpi1 and transcriptional activation of Ebf1. Bone marrows of IRF8 knockout mice (IRF8(-/-)) had significantly reduced numbers of pre-pro-B cells and increased numbers of myeloid cells. Although HSCs of IRF8(-/-) mice failed to differentiate to B220(+) B-lineage cells in vitro, the defect could be rescued by transfecting HSCs with wild-type but not with a signaling-deficient IRF8 mutant. In contrast, overexpression of IRF8 in HSC-differentiated progenitor cells resulted in growth inhibition and apoptosis. We also found that IRF8 was expressed at higher levels in pre-pro-B cells than more mature B cells in wild-type mice. Together, these results indicate that IRF8 modulates lineage choice by HSCs and is part of the transcriptional network governing B-cell lineage specification, commitment, and differentiation.
Project description:The IRF and Ets families of transcription factors regulate the expression of a range of genes involved in immune cell development and function. However, the understanding of the molecular mechanisms of each family member has been limited due to their redundancy and broad effects on multiple lineages of cells. Here, we report that double deletion of floxed Irf8 and Spi1 (encoding PU.1) by Mb1-Cre (designated DKO mice) in the B cell lineage resulted in severe defects in the development of follicular and germinal center (GC) B cells. Class-switch recombination and antibody affinity maturation were also compromised in DKO mice. RNA-seq (sequencing) and ChIP-seq analyses revealed distinct IRF8 and PU.1 target genes in follicular and activated B cells. DKO B cells had diminished expression of target genes vital for maintaining follicular B cell identity and GC development. Moreover, our findings reveal that expression of B-cell lymphoma protein 6 (BCL6), which is critical for development of germinal center B cells, is dependent on IRF8 and PU.1 in vivo, providing a mechanism for the critical role for IRF8 and PU.1 in the development of GC B cells.
Project description:Interferon regulatory factor 4 (IRF4) is an IRF family transcription factor with critical roles in lymphoid development and in regulating the immune response. IRF4 binds DNA weakly owing to a carboxy-terminal auto-inhibitory domain, but cooperative binding with factors such as PU.1 or SPIB in B cells increases binding affinity, allowing IRF4 to regulate genes containing ETS–IRF composite elements (EICEs; 5'-GGAAnnGAAA-3'). Here we show that in mouse CD4+ T cells, where PU.1/SPIB expression is low, and in B cells, where PU.1 is well expressed, IRF4 unexpectedly can cooperate with activator protein-1 (AP1) complexes to bind to AP1–IRF4 composite (5'-TGAnTCA/GAAA-3') motifs that we denote as AP1–IRF composite elements (AICEs). Moreover, BATF–JUN family protein complexes cooperate with IRF4 in binding to AICEs in pre-activated CD4+ T cells stimulated with IL-21 and in TH17 differentiated cells. Importantly, BATF binding was diminished in Irf4-/- T cells and IRF4 binding was diminished in Batf-/- T cells, consistent with functional cooperation between these factors. Moreover, we show that AP1 and IRF complexes cooperatively promote transcription of the Il10 gene, which is expressed in TH17 cells and potently regulated by IL-21. These findings reveal that IRF4 can signal via complexes containing ETS or AP1 motifs depending on the cellular context, thus indicating new approaches for modulating IRF4-dependent transcription. Overall design: Genome-wide transcription factors mapping and binding of IRF4, BATF, IRF8, STAT3, JUN etc in WT, Irf4-/- and Batf-/- mice in different cell types (B cells, CD4+ T cells and TH17 cells) cultured with or without IL-21 was conducted. RNA-Seq is conducted in mouse B cells, CD4+ T cells, TH1/TH2/TH9/TH17/Treg.