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B cell development requires the ordered rearrangement of Ig genes encoding H and L chain proteins that assemble into BCRs or Abs capable of recognizing specific Ags. Igκ rearrangement is promoted by chromatin accessibility and by relative abundance of RAG1/2 proteins. Expression of the E26 transformation-specific transcription factor Spi-C is activated in response to dsDNA double-stranded breaks in small pre-B cells to negatively regulate pre-BCR signaling and Igκ rearrangement. However, it is not clear if Spi-C regulates Igκ rearrangement through transcription or by controlling RAG expression. In this study, we investigated the mechanism of Spi-C negative regulation of Igκ L chain rearrangement. Using an inducible expression system in a pre-B cell line, we found that Spi-C negatively regulated Igκ rearrangement, Igκ transcript levels, and Rag1 transcript levels. We found that Igκ and Rag1 transcript levels were increased in small pre-B cells from Spic−/− mice. In contrast, Igκ and Rag1 transcript levels were activated by PU.1 and were decreased in small pre-B cells from PU.1-deficient mice. Using chromatin immunoprecipitation analysis, we identified an interaction site for PU.1 and Spi-C located in the Rag1 promoter region. These results suggest that Spi-C and PU.1 counterregulate Igκ transcription and Rag1 transcription to effect Igκ recombination in small pre-B cells.

SummaryIn mammalian cells expression of repetitive genomic sequences is repressed by heterochromatin, underscoring the potential threat of repeat expression to cellular homeostasis. However, the specific consequences of ectopic repeat expression remains unclear. Here we demonstrate that EZH2 inhibitors stimulate repeat misexpression and cell death in resting splenic B cells. We show that B cells are uniquely sensitive to these agents because of high levels of H3K27me3 at repeats and correspondingly low DNA methylation. We generated a pattern recognition receptor loss-of-function mouse model called RIC with mutations inRigi,Ifih1(MDA5), andCgasto specifically block the consequences of repeat misexpression. In both WT and RIC mutant B cells, EZH2 inhibition caused focused loss of H3K27me3 at repetitive elements and upregulated their expression. However, expression of inflammatory chemokines and cell death were interrupted by the RIC mutations. Furthermore, the chemokine expression patterns induced by EZH2 inhibitors resemble the B cell response to Epstein-Barr virus infection. This study demonstrates a viral mimicry effect induced by pharmacological activation of repeat expression that induces inflammation and B cell death.

B cell development requires the ordered rearrangement ofIggenes encoding H and L chain proteins that assemble into BCRs or Abs capable of recognizing specific Ags.Igκ rearrangement is promoted by chromatin accessibility and by relative abundance of RAG1/2 proteins. Expression of the E26-transformation-specific (ETS) transcription factor Spi-C is activated in response to double-stranded DNA breaks (DSBs) in small pre-B cells to negatively regulate pre-BCR signaling andIgκ rearrangement. However, it is not clear if Spi-C regulatesIgκ rearrangement through transcription or by controlling RAG expression. In this study, we investigated the mechanism of Spi-C negative regulation ofIgκ light chain rearrangement. Using an inducible expression system in a pre-B cell line, we found that Spi-C negatively regulatedIgκ rearrangement,Igκ transcript levels, andRag1transcript levels. We found thatIgκ andRag1transcript levels were increased in small pre-B cells fromSpic-/-mice. In contrast,Igκ andRag1transcript levels were activated by PU.1 and were decreased in small pre-B cells from PU.1-deficient mice. Using chromatin immunoprecipitation analysis, we identified an interaction site for PU.1 and Spi-C located in theRag1promoter region. These results suggest that Spi-C and PU.1 counterregulateIgκ transcription andRag1transcription to effectIgκ recombination in small pre-B cells.

Spi-C is an E26 transformation-specific transcription factor closely related to PU.1 and Spi-B. Spi-C has lineage-instructive functions important in B cell development, Ab-generating responses, and red pulp macrophage generation. This research examined the regulation of Spi-C expression in mouse B cells. To determine the mechanism of Spic regulation, we identified the Spic promoter and upstream regulatory elements. The Spic promoter had unidirectional activity that was reduced by mutation of an NF-κB binding site. Reverse transcription-quantitative PCR analysis revealed that Spic expression was reduced in B cells following treatment with cytokines BAFF + IL-4 + IL-5, anti-IgM Ab, or LPS. Cytochalasin treatment partially prevented downregulation of Spic. Unstimulated B cells upregulated Spic on culture. Spic was repressed by an upstream regulatory region interacting with the heme-binding regulator Bach2. Taken together, these data indicate that Spi-C is dynamically regulated by external signals in B cells and provide insight into the mechanism of regulation.

Spi-C is an E26 transformation-specific transcription factor closely related to PU.1 and Spi-B. Spi-C has lineage-instructive functions important in antibody-generating responses, B cell development, and red pulp macrophage generation. Spi-C is inducible by heme- and NF-κB-dependent pathways in macrophages. The present research aimed to examine the regulation of Spi-C expression in B cells. RT-qPCR analysis revealed that Spic expression was reduced in B cells following addition of lipopolysaccharide, anti-IgM antibodies, CD40L, or cytokines BAFF + IL-4 + IL-5. Cytochalasin treatment partially prevented downregulation of Spic. Unstimulated B cells upregulated Spic over time in culture. To determine the mechanism of Spic regulation, we examined the Spic promoter and upstream regulatory elements. The Spic promoter had unidirectional activity, which was reduced by mutation of an NF-κB binding site. Spic was repressed by an upstream regulatory region interacting with the heme-binding regulator Bach2. Taken together, these data indicate that Spi-C is dynamically regulated by external signals in B cells and provide insight into the mechanism of regulation.

Cell fate decisions during hematopoiesis are the consequence of a complex mixture of inputs from cell-intrinsic and cell-extrinsic factors. In rare cases, expression of a single transcription factor, or a few key factors, may be sufficient to dictate lineage differentiation in a precursor cell. The E26-transformation-specific-family transcription factor Spi-C has emerged as an example of a lineage-instructive factor involved in the generation of mature, specialized subsets of both myeloid and lymphoid cells. Spi-C can instruct differentiation of splenic precursors into red pulp macrophages responsible for phagocytosing senescent red blood cells. In the B cell compartment, Spi-C acts as a key regulator of cell fate decisions at the pro-B to pre-B cell stage and for plasma cell differentiation. Spi-C regulates key genes including Nfkb1, Bach2, Syk, and Blnk to regulate cell cycle entry and B cell differentiation. Here, we review the biology of the lineage-instructive transcription factor Spi-C and its contribution to mechanisms of disease in macrophages and B cells. This article is categorized under: Cancer > Molecular and Cellular Physiology Immune System Diseases > Molecular and Cellular Physiology Infectious Diseases > Genetics/Genomics/Epigenetics.

Precursor B cell acute lymphoblastic leukemia (B-ALL) is caused by genetic lesions in developing B cells that function as drivers for the accumulation of additional mutations in an evolutionary selection process. We investigated secondary drivers of leukemogenesis in a mouse model of B-ALL driven by PU.1/Spi-B deletion (Mb1-CreΔPB). Whole-exome-sequencing analysis revealed recurrent mutations in Jak3 (encoding Janus kinase 3), Jak1, and Ikzf3 (encoding Aiolos). Mutations with a high variant-allele frequency (VAF) were dominated by C→T transition mutations that were compatible with activation-induced cytidine deaminase, whereas the majority of mutations, with a low VAF, were dominated by C→A transversions associated with 8-oxoguanine DNA damage caused by reactive oxygen species (ROS). The Janus kinase (JAK) inhibitor ruxolitinib delayed leukemia onset, reduced ROS and ROS-induced gene expression signatures, and altered ROS-induced mutational signatures. These results reveal that JAK mutations can alter the course of leukemia clonal evolution through ROS-induced DNA damage.

The DNA-binding protein PU.1 is a myeloid lineage–determining and pioneering transcription factor due to its ability to bind "closed" genomic sites and maintain "open" chromatin state for myeloid lineage–specific genes. The precise mechanism of PU.1 in cell type–specific programming is yet to be elucidated. The melanoma cell line B16BL6, although it is nonmyeloid lineage, expressed Toll-like receptors and activated the transcription factor NF-κB upon stimulation by the bacterial cell wall component lipopolysaccharide. However, it did not produce cytokines, such as IL-1β mRNA. Ectopic PU.1 expression induced remodeling of a novel distal enhancer (located ∼10 kbp upstream of the IL-1β transcription start site), marked by nucleosome depletion, enhancer-promoter looping, and histone H3 lysine 27 acetylation (H3K27ac). PU.1 induced enhancer-promoter looping and H3K27ac through two distinct PU.1 regions. These PU.1-dependent events were independently required for subsequent signal-dependent and co-dependent events: NF-κB recruitment and further H3K27ac, both of which were required for enhancer RNA (eRNA) transcription. In murine macrophage RAW264.7 cells, these PU.1-dependent events were constitutively established and readily expressed eRNA and subsequently IL-1β mRNA by lipopolysaccharide stimulation. In summary, this study showed a sequence of epigenetic events in programming IL-1β transcription by the distal enhancer priming and eRNA production mediated by PU.1 and the signal-dependent transcription factor NF-κB.

Differentiation of myeloid progenitor cells into macrophages is accompanied by increased PU.1 concentration and increasing cell cycle length, culminating in cell cycle arrest. Induction of PU.1 expression in a cultured myeloid cell line expressing low PU.1 concentration results in decreased levels of mRNA encoding ATP-Citrate Lyase (ACL) and cell cycle arrest. ACL is an essential enzyme for generating acetyl-CoA, a key metabolite for the first step in fatty acid synthesis as well as for histone acetylation. We hypothesized that ACL may play a role in cell cycle regulation in the myeloid lineage. In this study, we found that acetyl-CoA or acetate supplementation was sufficient to rescue cell cycle progression in cultured BN cells treated with an ACL inhibitor or induced for PU.1 expression. Acetyl-CoA supplementation was also sufficient to rescue cell cycle progression in BN cells treated with a fatty acid synthase (FASN) inhibitor. We demonstrated that acetyl-CoA was utilized in both fatty acid synthesis and histone acetylation pathways to promote proliferation. Finally, we found thatAclymRNA transcript levels decrease during normal macrophage differentiation from bone marrow precursors. Our results suggest that regulation of ACL activity is a potentially important point of control for cell cycle regulation in the myeloid lineage.

Precursor B-cell acute lymphoblastic leukemia (B-ALL) is associated with recurrent mutations that occur in cancer-initiating cells. There is a need to understand how driver mutations influence clonal evolution of leukemia. The E26-transformation-specific (ETS) transcription factors PU.1 and Spi-B (encoded by Spi1 and Spib) execute a critical role in B-cell development and serve as complementary tumor suppressors. Here, we used a mouse model to conditionally delete Spi1 and Spib genes in developing B cells. These mice developed B-ALL with a median time to euthanasia of 18 weeks. We performed RNA and whole-exome sequencing (WES) on leukemias isolated from Mb1-CreΔPB mice and identified single nucleotide variants (SNVs) in Jak1, Jak3, and Ikzf3 genes, resulting in amino acid sequence changes. Jak3 mutations resulted in amino acid substitutions located in the pseudo-kinase (R653H, V670A) and in the kinase (T844M) domains. Introduction of Jak3 T844M into Spi1/Spib-deficient precursor B cells was sufficient to promote proliferation in response to low IL-7 concentrations in culture, and to promote proliferation and leukemia-like disease in transplanted mice. We conclude that mutations in Janus kinases represent secondary drivers of leukemogenesis that cooperate with Spi1/Spib deletion. This mouse model represents a useful tool to study clonal evolution in B-ALL.

Precursor B-cell acute lymphoblastic leukemia (B-ALL) is associated with recurrent mutations that occur in cancer-initiating cells. There is a need to understand how spontaneous driver mutations influence clonal evolution in leukemia. The ETS-transcription factors PU.1 and Spi-B (encoded bySpi1andSpib) execute a critical role in B cell development and serve as complementary tumour suppressors by opposing the proliferative events mediated by IL-7R signaling. Here, we used a mouse model to conditionally deleteSpi1andSpibgenes in developing B cells. These mice developed B-ALL with a median time to euthanasia of 18 weeks. We performed RNA and whole-exome sequencing (WES) on leukemias isolated from Mb1-CreΔPB mice and identified single-nucleotide variants (SNVs) inJak1,Jak3andIkzf3genes, resulting in amino acid changes and in the gain of early stop-codons. JAK3 mutations resulted in amino acid substitutions located in the pseudo-kinase (R653H, V670A) and in the kinase (T844M) domains. Introduction of these mutations into wild-type pro-B cells conferred survival and proliferation advantages. We conclude that mutations in Janus kinases represent secondary drivers of leukemogenesis in the absence of Spi-B and PU.1 transcription factors. This mouse model represents an useful tool to study clonal evolution and tumour heterogeneity in B-ALL.

During macrophage development, myeloid progenitor cells undergo terminal differentiation coordinated with reduced cell cycle progression. Differentiation of macrophages from myeloid progenitors is accompanied by increased expression of the E26 transformation-specific transcription factor PU.1. Reduced PU.1 expression leads to increased proliferation and impaired differentiation of myeloid progenitor cells. It is not understood how PU.1 coordinates macrophage differentiation with reduced cell cycle progression. In this study, we utilized cultured PU.1- inducible myeloid cells to perform genome-wide chromatin immunoprecipitation sequencing (ChIP-seq) analysis coupled with gene expression analysis to determine targets of PU.1 that may be involved in regulating cell cycle progression. We found that genes encoding cell cycle regulators and enzymes involved in lipid anabolism were directly and inducibly bound by PU.1 although their steady-state mRNA transcript levels were reduced. Inhibition of lipid anabolism was sufficient to reduce cell cycle progression in these cells. Induction of PU.1 reduced expression of E2f1, an important activator of genes involved in cell cycle and lipid anabolism, indirectly through microRNA 223. Next-generation sequencing identified microRNAs validated as targeting cell cycle and lipid anabolism for downregulation. These results suggest that PU.1 coordinates cell cycle progression with differentiation through induction of microRNAs targeting cell cycle regulators and lipid anabolism.

B cell development and Ig rearrangement are governed by cell type– and developmental stage–specific transcription factors. PU.1 and Spi-B are E26-transformation–specific transcription factors that are critical for B cell differentiation. To determine whether PU.1 and Spi-B are required for B cell development in the bone marrow, Spi1 (encoding PU.1) was conditionally deleted in B cells by Cre recombinase under control of the Mb1 gene in Spib (encoding Spi-B)–deficient mice. Combined deletion of Spi1 and Spib resulted in a lack of mature B cells in the spleen and a block in B cell development in the bone marrow at the small pre-B cell stage. To determine target genes of PU.1 that could explain this block, we applied a gain-of-function approach using a PU.1/Spi-B–deficient pro-B cell line in which PU.1 can be induced by doxycycline. PU.1-induced genes were identified by integration of chromatin immunoprecipitation–sequencing and RNA-sequencing data. We found that PU.1 interacted with multiple sites in the Igκ locus, including Vκ promoters and regions located downstream of Vκ second exons. Induction of PU.1 induced Igκ transcription and rearrangement. Upregulation of Igκ transcription was impaired in small pre-B cells from PU.1/Spi-B–deficient bone marrow. These studies reveal an important role for PU.1 in the regulation of Igκ transcription and rearrangement and a requirement for PU.1 and Spi-B in B cell development.

The most frequently occurring genetic abnormality in pediatric B-lymphocyte-lineage acute lymphoblastic leukemia is the t(12;21) chromosomal translocation that results in a ETV6-RUNX1 (also known as TEL-AML1) fusion gene. Expression of ETV6-RUNX1 induces a preleukemic condition leading to acquisition of secondary driver mutations, but the mechanism is poorly understood. SPI-B (encoded by SPIB) is an important transcriptional activator of B-cell development and differentiation. We hypothesized that SPIB is directly transcriptionally repressed by ETV6-RUNX1. Using chromatin immunoprecipitation, we identified a regulatory region in the first intron of SPIB that interacts with ETV6-RUNX1. Mutation of the RUNX1 binding site in SPIB intron 1 prevented transcriptional repression in transient transfection assays. Next, we sought to determine to what extent gene expression in REH cells can be altered by ectopic SPI-B expression. SPI-B expression was forced using CRISPR-mediated gene activation and also using a retroviral vector. Forced expression of SPI-B resulted in altered gene expression and, at high levels, impaired cell proliferation and induced apoptosis. Finally, we identified CARD11 and CDKN1A (encoding p21) as transcriptional targets of SPI-B involved in regulation of proliferation and apoptosis. Taken together, this study identifies SPIB as an important target of ETV6-RUNX1 in regulation of B-cell gene expression in t(12;21) leukemia.

Malignant neoplasms or cancers of the B lymphocyte lineage are commonly called leukemias and lymphomas. B cell cancers primarily involve blood, lymphatic tissues, or bone marrow and arise due to acquired driver mutations at distinct stages of development and differentiation, followed by a clonal evolution process involving acquisition of additional driver mutations. Recent advances suggest that B cell development may be an intrinsically dangerous process due to the large number of cell divisions that occur during ontogeny, in addition to off-target mutations induced by expression of the mutagenic enzymes activation-induced cytidine deaminase and recombination-activating gene (RAG)-1/RAG-2. B cell cancers that will be considered in detail in this article include acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia, Hodgkin lymphoma, non-Hodgkin lymphomas, Waldenstrom's macroglobulinemia, and multiple myeloma. For each B cell neoplasm considered in this article, characteristics described will include incidence, classification schemes, mechanisms of transformation, cell of origin, and current treatments. Mechanisms of neoplastic transformation are discussed in detail for the example of precursor B-ALL.

B cell acute lymphoblastic leukemia (B-ALL) is frequently associated with mutations or chromosomal translocations of genes encoding transcription factors. Conditional deletion of genes encoding the E26-transformation-specific transcription factors, PU.1 and Spi-B, in B cells (ΔPB mice) leads to B-ALL in mice at 100% incidence rate and with a median survival of 21 wk. We hypothesized that PU.1 and Spi-B may redundantly activate transcription of genes encoding tumor suppressors in the B cell lineage. Characterization of aging ΔPB mice showed that leukemia cells expressing IL-7R were found in enlarged thymuses. IL-7R-expressing B-ALL cells grew in culture in response to IL-7 and could be maintained as cell lines. Cultured ΔPB cells expressed reduced levels of B cell linker protein (BLNK), a known tumor suppressor gene, compared with controls. The Blnk promoter contained a predicted PU.1 and/or Spi-B binding site that was required for promoter activity and occupied by PU.1 and/or Spi-B as determined by chromatin immunoprecipitation. Restoration of BLNK expression in cultured ΔPB cells opposed IL-7-dependent proliferation and induced early apoptosis. We conclude that the tumor suppressor BLNK is a target of transcriptional activation by PU.1 and Spi-B in the B cell lineage. Copyright © 2012 by The American Association of Immunologists, Inc.

PU.1 is a master transcription factor whose levels directly influence hematopoiesis, leukemia, susceptibility to sepsis, and macrophage function. Though measurement of PU.1 levels is important to health and disease, most studies have relied on PCR or western blots to measure the expression of this transcription factor. An accessible, validated assay that could measure PU.1 protein in subpopulations of cells is needed. In this work we present an optimized flow cytometric assay to detect PU.1 in subpopulations of immune cells. Murine myeloid cells were fixed in paraformaldehyde, permeabilized, and then stained with anti PU.1 in the presence and absence of a blocking peptide containing the binding site of the antibody. The bound anti PU.1 was then visualized with a labeled second antibody. Methanol and ethanol were tested for their relative ability to permeabilize cells and detect PU.1. The effect of the procedure upon the ability to detect cellular subpopulations was examined. Relative PU.1 1evels in normal T cells, B cells, monocytes, macrophages, dendritic cells, neutrophils, and progenitors from the spleen and/or bone marrow were determined. Finally, PU.1 levels in proliferating myeloid cells from burn mice were determined. There was a dose dependent increase in the amount of PU.1 detected with increasing amounts of PU.1 antibody that was not seen when blocking peptide was used. Methanol or ethanol gave equivalent results as permeabilization agents, but the latter allowed easier detection of surface antigens when surface staining was performed prior to permeabilization. T cells had little if any PU.1, while B cells had intermediate levels of PU.1, and myeloid cells had high levels of PU.1. Monocytes had higher levels of PU.1 than did neutrophils or spleen macrophages. Plasmacytoid dendritic cells had lower levels of PU.1 than did conventional dendritic cells. Immature myeloid cells had higher levels of PU.1 than did mature myeloid cells. In addition, PU.1 levels were higher in proliferating cells than the corresponding non proliferating cells. Myeloid cells derived from burn mice tended to have higher levels of PU.1 than did unburned, but proliferating cells from burn or sham mice showed no difference in their levels of PU.1. This assay should be a useful addition to the tools used to study the function of PU.1 in health and disease.

The E26 transformation-specific (Ets) transcription factor PU.1 is required to generate lymphoid progenitor cells from hematopoietic stem cells, but it is not required to generate B cells from committed B-cell lineage progenitors. We hypothesized that PU.1 function in B-cell differentiation is complemented by the related Ets transcription factor Spi-B. To test this hypothesis, mice were generated lacking both PU.1 and Spi-B in the B-cell lineage. Unlike mice lacking PU.1 or Spi-B, mice deficient in both PU.1 and Spi-B in the B-cell lineage had reduced frequencies of B cells as well as impaired B-cell differentiation. Strikingly, all PU.1 and Spi-B–deficient mice developed pre-B cell acute lymphoblastic leukemia before 30 weeks of age. Pre-B cells accumulated in the thymus resulting in massive thymic enlargement and dyspnea. These findings demonstrate that PU.1 and Spi-B are essential transcriptional regulators of B-cell differentiation as well as novel tumor suppressors in the B-cell lineage.

Splenic B-2 cells can be divided into two major subsets: follicular (FO) and marginal zone (MZ) B cells. FO and MZ B cells are generated from immature transitional B cells. Few transcription factors have been identified that regulate FO B cell differentiation. The highly related proteins PU.1, Spi-B, and Spi-C are transcription factors of the E26-transformation-specific family and are important for B cell differentiation and function. To determine whether these proteins play a role in the differentiation of FO B cells, we performed a detailed analysis of splenic B cells in mice with inactivating mutations in the genes encoding PU.1 (Sfpi1) or Spi-B (Spib). Sfpi1+/− Spib−/− (PUB) mice had a 9-fold reduction in the frequency of CD23+ FO B cells compared with that of wild-type mice. In contrast, PUB mice had a 2-fold increase in the frequency of MZ B cells that was confirmed by immunofluorescence staining. Expression of Spi-C in Eμ-Spi-C transgenic PUB mice partially rescued frequencies of CD23+ B cells. Gene expression analysis, in vitro reporter assays, and chromatin immunoprecipitation experiments showed that transcription of the Fcer2a gene encoding CD23 is activated by PU.1, Spi-B, and Spi-C. These results demonstrate that FO B cell differentiation is regulated by the E26-transformation-specific transcription factors PU.1, Spi-B, and Spi-C.

Coexpression of PU.1 and GATA-1 is required for proper specification of the mast cell lineage; however, in the myeloid and erythroid lineages, PU.1 and GATA-1 are functionally antagonistic. In this study, we report a transcriptional network in which PU.1 positively regulates GATA-1 expression in mast cell development. We isolated a variant mRNA isoform of GATA-1 in murine mast cells that is significantly upregulated during mast cell differentiation. This isoform contains an alternatively spliced first exon (IB) that is distinct from the first exon (IE) incorporated in the major erythroid mRNA transcript. In contrast to erythroid and megakaryocyte cells, in mast cells we show that PU.1 and GATA-2 predominantly occupy potential cis-regulatory elements in the IB exon region in vivo. Using reporter assays, we identify an enhancer flanking the IB exon that is activated by PU.1. Furthermore, we observe that in PU.1 -/- fetal liver cells, low levels of the IE GATA-1 isoform is expressed, but the variant IB isoform is absent. Reintroduction of PU.1 restores variant IB isoform and upregulates total GATA-1 protein expression, which is concurrent with mast cell differentiation. Our results are consistent with a transcriptional hierarchy in which PU.1, possibly in concert with GATA-2, activates GATA-1 expression in mast cells in a pathway distinct from that seen in the erythroid and megakaryocytic lineages. Copyright © 2010 by The American Association of Immunologists, Inc.

Activated B-cell diffuse large B-cell lymphoma (ABC-DLBCL) is associated with a poor prognosis compared with other DLBCL types and therefore represents a top priority for developing novel therapies. Lenalidomide, an immunomodulatory drug in trials for treatment of ABC-DLBCL, targets the transcription factor IKAROS for degradation by the cereblon E3 ubiquitin ligase complex. In this study, we investigated whether the gene encoding the transcription factor SPI-B is a target of IKAROS. Using cultured ABC-DLBCL cell lines, we found that high levels of SPI-B expression conferred resistance to lenalidomide. Lenalidomide treatment of ABC-DLBCL cells resulted in downregulation of SPIB at the level of transcription. SPIB was regulated directly by IKAROS through a binding site located in the first intron of the gene. Inhibition of IKAROS binding using CRISPR/Cas9-mediated transcriptional repression downregulated endogenous SPIB transcription. Finally, ectopic expression of IKAROS protected SPIB from downregulation. These results show that the mechanism of action of lenalidomide in ABC-DLBCL cells involves downregulation of SPIB transcription by cereblon-induced degradation of IKAROS. These results have implications for the design of synthetic lethal therapy for the treatment of ABC-DLBCL.

Spi-C is an Ets family transcription factor closely related to PU.1 and Spi-B. Expression of Spi-C is developmentally regulated in the B-cell lineage, but its function remains unknown. To determine the function of Spi-C in B-cell development, we generated mice expressing a B-cell-specific Spi-C transgene under the control of the IgH intronic enhancer. Spi-C transgenic mice had 50% fewer B cells than wild-type littermates. Flow cytometric analyses showed that splenic transitional B cells and bone marrow pre-B or immature B cells from transgenic mice were dramatically reduced compared with those of wild type. Both nonspecific and Ag-specific serum IgM levels were significantly increased in transgenic mice, while serum IgG levels were significantly decreased compared with wild type. Spi-C transgenic B cells proliferated poorly after stimulation by anti-IgM or anti-CD40 in vitro, although they responded normally to LPS stimulation. Using real-time RT-PCR, we found that several BCR signaling-related mediators were downregulated at pre-B-cell and mature B-cell stages in transgenic mice, while an inhibitor of BCR signaling was upregulated. Taken together, these data indicate that ectopic expression of Spi-C can impair B-cell development and function by affecting genes associated with BCR signaling.

The Ets transcription factor PU.1, encoded by the gene Sfpi1, functions in a concentration-dependent manner to promote myeloid and B-cell development and has been implicated in myeloid and lymphoid leukemias. To determine the consequences of reducing PU.1 concentration during hematopoiesis, we analyzed mice with two distinct hypomorphic alleles of Sfpi1 that produce PU.1 at approximately 20% (BN) or approximately 2% (Blac) of wild-type levels. Myeloid development was impaired in these mice, but less severely than in Sfpi1 null mice. To identify the downstream target genes that respond to changes in PU.1 concentration, we analyzed ex vivo interleukin-3 dependent myeloid cell lines established from Sfpi1(BN/BN), Sfpi1(Blac/Blac) and Sfpi1(-/-) fetal liver cells. Unexpectedly, many T-cell and natural killer cell genes were expressed in Sfpi1(-/-) cells and repressed in a dose-dependent manner in Sfpi1(Blac/Blac) and Sfpi1(BN/BN) cells. This pattern of dose-dependent T/NK-cell gene repression also occurred in ex vivo interleukin-7 dependent progenitor B cell lines. These results suggest that PU.1 functions in a concentration-dependent manner to repress T-cell and natural killer cell fates while promoting myeloid and B-cell fates.

The Ets family transcription factor PU.1, encoded by the gene Sfpi1 , is essential for normal hematopoiesis. A number of studies have suggested that changes in PU.1 concentration play a role in directing cell fate decisions during hematopoiesis. However, the stages of hematopoietic development at which changes in PU.1 concentration are important have not been defined until recently. Experiments using conditional null alleles, reporter alleles, and hypomorphic alleles of the Sfpi1 gene in mice demonstrate that PU.1 concentration is uniformly high during early stages of hematopoietic development. However, reduction of PU.1 concentration is required for normal development of megakaryocyte–erythroid progenitors, B cell progenitors, and T cell progenitors. PU.1 concentration increases in granulocyte–macrophage progenitors. Furthermore, experimental reduction of PU.1 concentration in the myeloid lineages leads to failed differentiation, abnormal proliferation, and leukemia. In this review, we summarize recent studies to develop a new model of PU.1 function in hematopoiesis.

Signaling through the IL-7 receptor (IL-7R) is required for development and maintenance of the immune system. The receptor for IL-7 is heterodimeric, consisting of a common gamma chain (gammac, encoded by Il2rg) and an alpha subunit (IL-7Ralpha, encoded by Il7r). The Il7r gene is expressed specifically in the immune system in a developmental stage-specific manner. It is not known how the Il7r gene is transcriptionally regulated during B cell development. The goal of this study is to elucidate the function of the Il7r promoter region in developing B cells. Using a combination of 5' rapid amplification of cDNA ends analysis, transient transfection assays, and DNase I hypersensitivity mapping, we identified the location of the Il7r promoter. Using a combination of electrophoretic mobility shift analysis, chromatin immunoprecipitation experiments, and RNA interference experiments, we found that the Ets transcription factors PU.1 and GA-binding protein (GABP) activate the Il7r promoter by interacting with a highly conserved Ets binding site. In committed B lineage cells, GABP can promote Il7r transcription in the absence of PU.1. However, the results of retroviral gene transfer experiments suggest that PU.1 is uniquely required to initiate transcription of the Il7r locus at the earliest stages of progenitor B cell generation. In summary, these results suggest that Il7r transcription is regulated by both PU.1 and GABP in developing B cells.

Spi-C is an E26 transformation-specific family transcription factor that is highly related to PU.1 and Spi-B. Spi-C is expressed in developing B cells, but its function in B cell development and function is not well characterized. To determine whether Spi-C functions as a negative regulator of Spi-B (encoded by Spib), mice were generated that were germline knockout for Spib and heterozygous for Spic (Spib−/−Spic+/−). Interestingly, loss of one Spic allele substantially rescued B cell frequencies and absolute numbers in Spib−/− mouse spleens. Spib−/−Spic+/− B cells had restored proliferation compared with Spib−/− B cells in response to anti-IgM or LPS stimulation. Investigation of a potential mechanism for the Spib−/−Spic+/− phenotype revealed that steady-state levels of Nfkb1, encoding p50, were elevated in Spib−/−Spic+/− B cells compared with Spib−/− B cells. Spi-B was shown to directly activate the Nfkb1 gene, whereas Spi-C was shown to repress this gene. These results indicate a novel role for Spi-C as a negative regulator of B cell development and function.

Background 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. Results 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. Conclusions 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. Electronic supplementary material The online version of this article (doi:10.1186/s12864-015-1303-0) contains supplementary material, which is available to authorized users.

Generation of antibodies against T-independent and T-dependent antigens requires Toll-like receptor (TLR) engagement on B cells for efficient responses. However, the regulation of TLR expression and responses in B cells is not well understood. PU.1 and Spi-B (encoded by Sfpi1 and Spib, respectively) are transcription factors of the E26 transformation-specific (ETS) family and are important for B cell development and function. It was found that B cells from mice knocked out for Spi-B and heterozygous for PU.1 (Sfpi1(+/-) Spib(-/-) [PUB] mice) proliferated poorly in response to TLR ligands compared to wild-type (WT) B cells. The NF-κB family member p50 (encoded by Nfkb1) is required for lipopolysaccharide (LPS) responsiveness in mice. PUB B cells expressed reduced Nfkb1 mRNA transcripts and p50 protein. The Nfkb1 promoter was regulated directly by PU.1 and Spi-B, as shown by reporter assays and chromatin immunoprecipitation analysis. Occupancy of the Nfkb1 promoter by PU.1 was reduced in PUB B cells compared to that in WT B cells. Finally, infection of PUB B cells with a retroviral vector encoding p50 substantially restored proliferation in response to LPS. We conclude that Nfkb1 transcriptional activation by PU.1 and Spi-B promotes TLR-mediated B cell proliferation.

Deletion of genes encoding the E26 transformation-specific transcription factors PU.1 and Spi-B in B cells (CD19-CreΔPB mice) leads to impaired B cell development, followed by B cell acute lymphoblastic leukemia at 100% incidence and with a median survival of 21 wk. However, little is known about the target genes that explain leukemogenesis in these mice. In this study we found that immature B cells were altered in frequency in the bone marrow of preleukemic CD19-CreΔPB mice. Enriched pro–B cells from CD19-CreΔPB mice induced disease upon transplantation, suggesting that these were leukemia-initiating cells. Bone marrow cells from preleukemic CD19-CreΔPB mice had increased responsiveness to IL-7 and could proliferate indefinitely in response to this cytokine. Bruton tyrosine kinase (BTK), a negative regulator of IL-7 signaling, was reduced in preleukemic and leukemic CD19-CreΔPB cells compared with controls. Induction of PU.1 expression in cultured CD19-CreΔPB pro–B cell lines induced Btk expression, followed by reduced STAT5 phosphorylation and early apoptosis. PU.1 and Spi-B regulated Btk directly as shown by chromatin immunoprecipitation analysis. Ectopic expression of BTK was sufficient to induce apoptosis in cultured pro–B cells. In summary, these results suggest that PU.1 and Spi-B activate Btk to oppose IL-7 responsiveness in developing B cells.

The generation of B lymphocyte precursors is dependent on the combinatorial action of the transcription factors PU.1, Ikaros, E2A, EBF, and Pax-5. Loss of PU.1 results in a severe reduction in Flk2+, IL-7R+ lymphoid progenitors as well as impaired expression of EBF and Pax-5. Restoration of EBF expression facilitates rapid generation of pro-B cells from PU.1−/− progenitors. Molecular analysis suggests that PU.1 directly participates in regulation of the EBF gene. Although PU.1 is dispensable for expression of most early B lineage genes, it is required for CD45R/B220. Using EBF−/− mutant progenitors, we show that EBF induces Pax-5 and the early program of B lineage gene expression. Importantly, Pax-5 does not rescue B cell development from either PU.1−/− or EBF−/− progenitors. Pax-5 expression and function are contingent on EBF. Based on these results, we propose a hierarchical regulatory network for specification and commitment to the B cell fate.

PU.1 and GATA transcription factors appear to antagonize each other's function in the development of distinct lineages of the hematopoietic system. In contrast, we demonstrate that PU.1, like GATA-2, is essential for the generation of mast cells. PU.1−/− hematopoietic progenitors can be propagated in IL-3 and differentiate into mast cells or macrophages upon restoration of PU.1 activity. Using these progenitors and a conditionally activatable PU.1 protein, we show that PU.1 can negatively regulate expression of the GATA-2 gene. In the absence of GATA-2, PU.1 promotes macrophage but not mast cell differentiation. Reexpression of GATA-2 in such progenitors enables the generation of mast cells. We propose a developmental model in which cooperative function or antagonistic crossregulation by PU.1 of GATA-2 promotes distinct myeloid cell fates.

Development of the lymphoid system is dependent on the Ets family transcription factor PU.1. We demonstrate that PU.1(-/-) hematopoietic progenitors fail to express IL-7Ralpha transcripts. Promoter and chromatin crosslinking analyses suggest that PU.1 directly regulates transcription of the IL-7Ralpha gene. Retroviral transduction of IL-7Ralpha into PU.1(-/-) progenitors restores IL-7-dependent proliferation and induces, at low frequency, the generation of pro-B cells undergoing an apparently normal program of differentiation. Although the related factor Spi-B can substitute for PU.1 in early B cell development, it is not required. These results demonstrate that PU.1 functions to regulate early B cell development in part by controlling the expression of the IL-7Ralpha gene.

PU.1 is a unique regulatory protein required for the generation of both the innate and the adaptive immune system. It functions exclusively in a cell-intrinsic manner to control the development of granulocytes, macrophages, and B and T lymphocytes. We demonstrate that mutation of the PU.1 gene causes a severe reduction in myeloid (granulocyte/macrophage) progenitors. PU.1 -/- myeloid progenitors can proliferate in vitro in response to the multilineage cytokines interleukin-3 (IL-3), IL-6 and stem cell factor but are unresponsive to the myeloid-specific cytokines granulocyte-macrophage colony-stimulating factor (GM-CSF), G-CSF and M-CSF. The failure of PU.1 -/- progenitors to respond to G-CSF is bypassed by transient signaling with IL-3. In the presence of IL-3 and G-CSF, PU.1 -/- progenitors can differentiate into granulocytic precursors containing myeloperoxidase-positive granules. Thus PU.1 is not essential for specification of granulocytic precursors, but is required for their further differentiation. The failure of PU.1 -/- progenitors to respond to M-CSF is due to lack of c-fms gene transcription. Transduction of c-fms into PU.1 -/- myeloid progenitors bypasses the block to M-CSF-dependent proliferation but does not induce detectable macrophage differentiation. Therefore, PU. 1 appears to be essential for specification of monocytic precursors. Importantly, retroviral transduction of PU.1 into mutant progenitors restores responsiveness to myeloid-specific cytokines and development of mature granulocytes and macrophages. Thus PU.1 controls myelopoiesis by regulating both proliferation and differentiation pathways.

Bone marrow is the major site of hematopoiesis in the adult mammal. Bone marrow contains a highly organized microenvironment for the support of hematopoietic stem and progenitor cells, including the production of growth factors. Bone marrow cells also produce negative regulatory factors which may regulate hematopoiesis and inflammatory responses. In this paper we describe Reptimed, a unique bone marrow-derived factor with inhibitory activity for myelopoiesis and in vitro growth of myeloid cell lines. Reptimed was partially purified from bone marrow supernatants using a combination of solid-phase extraction and size exclusion chromatography. Reptimed is < 1000 Da MW and is water soluble. Reptimed inhibited growth of granulocyte-macrophage and macrophage colonies as well as proliferation of several myeloid leukemia cell lines. Reptimed may be part of a hemoregulatory circuit.

High titers of anti-citrullinated protein/peptide antibodies (ACPAs) have been detected in sera of rheumatoid arthritis (RA) patients, implicating citrullinating enzymes in the pathogenesis of RA. Peptidylarginine deiminase type IV (PAD4) is a member of the PAD family of citrullinating enzymes and has been linked to RA. Therefore, our aim was to determine how transcription of PAD4 is regulated in the human myeloid lineage. We located the PAD4 transcription start site and promoter and phylogenetic comparisons of the area identified a 200. bp conserved region. Bioinformatics analysis predicted the presence of a NF-κB binding site and we tested this via luciferase assays. Intriguingly, mutation of the predicted NF-κB site significantly increased biological activity. We used RT-qPCR to quantify PAD4 expression in HL-60 cells treated with TNF-α to activate the canonical NF-κB pathway and found that PAD4 mRNA was reduced in response to TNF-α treatment. Finally, we used chromatin immunoprecipitation (ChIP) to determine NF-κB enrichment at the PAD4 promoter and the p50 subunit of NF-κB was more highly enriched than p65 at the PAD4 promoter. These results suggest that the p50 subunit of NF-κB may play a role in the repression of PAD4 transcription during inflammation. © 2013 Elsevier B.V.

Peyer's patches consist of domains of specialized intestinal epithelium overlying gut-associated lymphoid tissue (GALT). Luminal antigens reach the GALT by translocation through epithelial gatekeeper cells, the so-calledMcells. We recently demonstrated that all epithelial cells required for the digestive functions of the intestine are generated from Lgr5-expressing stem cells. Here, we show thatMcells also derive from these crypt-based Lgr5 stem cells. The Ets family transcription factor SpiB, known to control effector functions of bone marrow-derived immune cells, is specifically expressed inMcells. In SpiB-/-mice, Mcells are entirely absent, which occurs in a cell-autonomous fashion. It has been shown that Tnfsf11 (RankL) can induceMcell development in vivo. We show that in intestinal organoid (" minigut" ) cultures, stimulation with RankL induces SpiB expression within 24 h and expression of otherMcell markers subsequently. We conclude that RankL-induced expression of SpiB is essential for Lgr5 stem cell-derived epithelial precursors to develop intoMcells. © 2012, American Society for Microbiology.

Transforming growth factor beta (TGF-β) signaling, mediated through the transcription factors Smad2 and Smad3 (Smad2/3), directs different responses in different cell types. Here we report that Smad3 co-occupies the genome with cell-type-specific master transcription factors. Thus, Smad3 occupies the genome with Oct4 in embryonic stem cells (ESCs), Myod1 in myotubes, and PU.1 in pro-B cells. We find that these master transcription factors are required for Smad3 occupancy and that TGF-β signaling largely affects the genes bound by the master transcription factors. Furthermore, we show that induction of Myod1 in nonmuscle cells is sufficient to redirect Smad3 to Myod1 sites. We conclude that cell-type-specific master transcription factors determine the genes bound by Smad2/3 and are thus responsible for orchestrating the cell-type-specific effects of TGF-β signaling. © 2011 Elsevier Inc.

Acute myeloid leukemia (AML) is characterized by increased proliferation and reduced differentiation of myeloid lineage cells. AML is frequently associated with mutations or chromosomal rearrangements involving transcription factors. PU.1 (encoded by Sfpi1) is an E26 transformation-specific family transcription factor that is required for myeloid differentiation. Reduced PU.1 levels, caused by either mutation or repression, are associated with human AML and are sufficient to cause AML in mice. The objective of this study was to determine whether reduced PU.1 expression induces deregulation of the cell cycle in the myeloid lineage. Our results showed that immature myeloid cells expressing reduced PU.1 levels (Sfpi1(BN/BN) myeloid cells) proliferated indefinitely in cell culture and expanded in vivo. Transplantation of Sfpi1(BN/BN) cells induced AML in recipient mice. Cultured Sfpi1(BN/BN) cells expressed elevated messenger RNA transcript and protein levels of E2F1, an important regulator of cell cycle entry. Restoration of PU.1 expression in Sfpi1(BN/BN) myeloid cells blocked proliferation, induced differentiation, and reduced E2F1 expression. Taken together, these data show that PU.1 controls cell cycle exit in the myeloid lineage associated with downregulation of E2F1 expression.

PU.1 is an E26 transformation-specific family transcription factor that is required for development of the immune system. PU.1 functions at both early and late stages of lymphoid and myeloid differentiation. At least 110 direct target genes of PU.1 have been identified since its discovery in 1988. We used the published literature to determine if aspects of PU.1 function can be inferred from the identity of target genes that are directly activated. This analysis revealed that 61% of described PU.1 target genes encode extracellular proteins or transmembrane proteins, most of which are involved in cellular communication. The genes activated by PU.1 can be grouped into pathways based on function. Specific examples of cellular communication pathways regulated by PU.1 include (1) antibodies and antibody receptors, (2) cytokines and cytokine receptors regulating leukocyte growth and development, and (3) cytokines and cytokine receptors regulating inflammation. As a consequence of mutation or repression of the gene encoding PU.1, hematopoietic progenitors may be generated but there is a “failure to thrive” because they cannot interact with their environment. The loss of cellular communication caused by reduced PU.1 levels can lead to leukemia. In summary, PU.1 is a critical regulator of cellular communication in the immune system.

Despite advances in the identification of lymphoid-restricted progenitor cells, the transcription factors essential for their generation remain to be identified. Here we describe an unexpected function for the myeloid oncogene product Mef2c in lymphoid development. Mef2c deficiency was associated with profound defects in the production of B cells, T cells, natural killer cells and common lymphoid progenitor cells and an enhanced myeloid output. In multipotent progenitors, Mef2c was required for the proper expression of several key lymphoid regulators and restriction of the myeloid fate. Our studies also show that Mef2c was a critical transcriptional target of the transcription factor PU.1 during lymphopoiesis. Thus, Mef2c is a crucial component of the transcriptional network that regulates cell fate 'choice' in multipotent progenitors.

Objective It has been demonstrated that high concentration of the transcription factor PU.1 (encoded by Sfpi1 ) promotes macrophage development, whereas low concentration induces B-cell development in vitro. This has led to the hypothesis that lower levels of PU.1 activity are required for B cell than for macrophage development in vivo. We utilized an allele of Sfpi1 (termed BN) with a mutation in the first coding exon, which resulted in a reduction of PU.1 expression in order to test this hypothesis. Materials and Methods Using gene targeting in embryonic stem cells, two ATG-start site codons of PU.1 were mutated, resulting in reduced PU.1 expression originating from a third start codon. Mice were assayed for phenotypic abnormalities using fluorescence-activated cell sorting, microscopy, and colony-forming ability. In addition, isolated cells were tested for their differentiation potential in vitro and in vivo. Results Lymphoid and myeloid cells derived from cultured Sfpi1 BN/BN fetal liver cells had reduced levels of PU.1 expression and activity. B-cell development was intrinsically blocked in cells isolated from Sfpi1 BN/BN mice. In addition, myeloid development was impaired in Sfpi1 BN/BN fetal liver. However, neonatal Sfpi1 BN/BN mice had a dramatic expansion and infiltration of immature myeloid cells. Conclusion Contrary to our original hypothesis, high levels of PU.1 activity are required to induce both myeloid and B-cell development. In addition, neonatal mice homozygous for the hypomorphic allele acquire a myeloproliferative disorder and die within 1 month of age.

Background. The E26-transformation-specific (ETS) transcription factor Spi-B is required for the survival of Activated B Cell-type Diffuse Large B Cell Lymphoma cell lines and is recurrently amplified in B cell lymphoma. The ETS transcription factor PU.1 is highly related to Spi-B. Both PU.1 and Spi-B are expressed in B lymphoma cell lines, and have been demonstrated to redundantly activate transcription of genes required for differentiation and function. We hypothesized that Spi-B and PU.1 occupy similar regions of chromatin within the genome of a B lymphoma cell line. Results. 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. 1900 of these regions exhibited at least 100 base pairs of overlap for both factors. Regions bound by Spi-B and PU.1 were frequently located within 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. Conclusions. 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 lymphoma cells. However, sites uniquely occupied by PU.1 or Spi-B provide insight into their unique functions. Citation Format: Lauren A. Solomon, Stephen K.h. Li, Jan Piskorz, Li S. Xu, Rodney P. DeKoter. Genome-wide comparison of PU.1 and Spi-B binding sites in a mouse B lymphoma cell line. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2098. doi:10.1158/1538-7445.AM2015-2098

The Ets transcription factor Spi-C, expressed in B cells and macrophages, is closely related to PU.1 and has the ability to recognize the same DNA consensus sequence. However, the function of Spi-C has yet to be determined. The purpose of this study is to further examine Spi-C activity in B cell development. First, using retroviral vectors to infect PU.1−/− fetal liver progenitors, Spi-C was found to be inefficient at inducing cytokine-dependent proliferation and differentiation of progenitor B (pro-B) cells or macrophages relative to PU.1 or Spi-B. Next, Spi-C was ectopically expressed in fetal liver-derived, IL-7-dependent pro-B cell lines. Wild-type (WT) pro-B cells ectopically expressing Spi-C (WT-Spi-C) have several phenotypic characteristics of pre-B cells such as increased CD25 and decreased c-Kit surface expression. In addition, WT-Spi-C pro-B cells express increased levels of IgH sterile transcripts and reduced levels of expression and transcription of the FcγRIIb gene. Gel-shift analysis suggests that Spi-C, ectopically expressed in pro-B cells, can bind PU.1 consensus sites in the IgH intronic enhancer and FcγRIIb promoter. Transient transfection analysis demonstrated that PU.1 functions to repress the IgH intronic enhancer and activate the FcγRIIb promoter, while Spi-C opposes these activities. WT-Spi-C pro-B cells have reduced levels of dimethylation on lysine 9 of histone H3 within the IgH 3′ regulatory region, indicating that Spi-C can contribute to removal of repressive features in the IgH locus. Overall, these studies suggest that Spi-C may promote B cell differentiation by modulating the activity of PU.1-dependent genes.

A number of presumptive target genes for the Ets-family transcription factor PU.1 have been identified in the B cell lineage. However, the precise function of PU.1 in B cells has not been studied because targeted null mutation of the PU.1 gene results in a block to lymphomyeloid development at an early developmental stage. In this study, we take advantage of recently developed PU.1−/−Spi-B−/− IL-7 and stromal cell-dependent progenitor B (pro-B) cell lines to analyze the function of PU.1 and Spi-B in B cell development. We show that contrary to previously published expectations, PU.1 and/or Spi-B are not required for Ig H chain (IgH) gene transcription in pro-B cells. In fact, PU.1−/−Spi-B−/− pro-B cells have increased levels of IgH transcription compared with wild-type pro-B cells. In addition, high levels of Igκ transcription are induced after IL-7 withdrawal of wild-type or PU.1−/−Spi-B−/− pro-B cells. In contrast, we found that Igλ transcription is reduced in PU.1−/−Spi-B−/− pro-B cells relative to wild-type pro-B cells after IL-7 withdrawal. These results suggest that Igλ, but not IgH or Igκ, transcription, is dependent on PU.1 and/or Spi-B. The PU.1−/−Spi-B−/− pro-B cells have other phenotypic changes relative to wild-type pro-B cells including increased proliferation, increased CD25 expression, decreased c-Kit expression, and decreased RAG-1 expression. Taken together, our observations suggest that reduction of PU.1 and/or Spi-B activity in pro-B cells promotes their differentiation to a stage intermediate between late pro-B cells and large pre-B cells.

PU.1 is a member of the Ets family of transcription factors required for the development of various lymphoid and myeloid cell lineages, but its role in natural killer (NK) cell development is not known. The study shows that PU.1 is expressed in NK cells and that, on cell transfer into alymphoid Rag2/γc−/−mice, hematopoietic progenitors of PU.1−/−fetal liver cells could generate functional NK cells but not B or T cells. Nevertheless, the numbers of bone marrow NK cell precursors and splenic mature NK cells were reduced compared to controls. Moreover,PU.1−/− NK cells displayed reduced expression of the receptors for stem cell factor and interleukin (IL)-7, suggesting a nonredundant role for PU.1 in regulating the expression of these cytokine receptor genes during NK cell development.PU.1−/− NK cells also showed defective expression of inhibitory and activating members of the Ly49 family and failed to proliferate in response to IL-2 and IL-12. Thus, despite the less stringent requirement for PU.1 in NK cell development compared to B and T cells, PU.1 regulates NK cell differentiation and homeostasis.

The ets family transcription factor PU.1 is required for the development of multiple lineages of the immune system. Using retroviral transduction ofPU.1complementary DNA into mutant hematopoietic progenitors, we demonstrate that differing concentrations of the protein regulate the development of B lymphocytes as compared with macrophages. A low concentration of PU.1 protein induces the B cell fate, whereas a high concentration promotes macrophage differentiation and blocks B cell development. Conversely, a transcriptionally weakened mutant protein preferentially induces B cell generation. Our results suggest that graded expression of a transcription factor can be used to specify distinct cell fates in the hematopoietic system.

The Ets transcription factor PU.1 (encoded by the gene Sfpi1) functions in a concentration-dependent manner as a hematopoietic cell fate determinant. PU.1 levels are uniform in early hematopoiesis, increase during myeloid differentiation, and decrease after erythrocyte and T cell/natural killer cell commitment. It is unknown how downstream target genes respond to changes in PU.1 concentration. To address this, we generated mice with two distinct hypomorphic alleles of Sfpi1 and analyzed interleukin-3 dependent cell lines from fetal liver cells homozygous for either allele. PU.1 was produced in these cells at ∼20% (Sfpi1BN/BN) or ∼2% (Sfpi1Blac/Blac) of wild type. These cells fail to terminally differentiate as a consequence of low PU.1 expression and can be maintained as cell lines. To determine what groups of genes are expressed in response to two distinct PU.1 concentrations, we performed whole-genome microarray analysis and compared gene expression in Sfpi1BN/BN and Sfpi1Blac/Blac cell lines to Sfpi1−/− cell lines. Groups of downstream target genes were activated or repressed in four modes in response to the two discrete concentrations of PU.1: at higher but not lower PU.1 concentration, at lower but not higher PU.1 concentration, at both lower and higher concentration, and in a gradient fashion. We decided to focus on genes regulated in a gradient manner, because dose-dependency suggests that these may be direct targets of PU.1. Genes activated in a gradient manner were mostly myeloid-specific and enriched for target genes of PU.1. Genes repressed in a gradient manner included erythroid-specific genes and, unexpectedly, T cell and natural killer cell-specific genes. T cell genes were also repressed by PU.1 in cultured progenitor-B cells. With this unique allelic system, we can study three discrete concentrations of PU.1 at 20%, 2%, and 0% to examine concentration-dependent effects of PU.1 on target genes and lineage decisions. Overall, our results suggest that PU.1 functions in a concentration-dependent manner to promote myeloid differentiation and repress T cell or natural killer cell development.

Objective The Ets family transcription factor PU.1 is essential for both myeloid and lymphoid development. PU.1 was discovered because of its involvement in murine erythroleukemia. We previously described that infection with a retroviral vector encoding PU.1 immortalizes fetal liver progenitor cells in response to granulocyte-macrophage colony-stimulating factor (GM-CSF) signaling. In this study, we sought to characterize PU.1-immortalized progenitor (PIP) cells. Methods PIP cells were characterized using microscopy, reverse-transcriptase polymerase chain reaction analysis, and flow cytometric analysis. In addition, progenitors were immortalized with a retrovirus containing a PU.1 cDNA flanked by loxP sites. The differentiation potential of immortalized progenitors was tested by Cre-mediated excision of the proviral PU.1 cDNA. Results PIP cells are blastlike in morphology and express cell surface markers indicative of myeloid development. Immortalization of progenitor cells requires both an acidic activation domain and an intact DNA-binding domain of PU.1. Gene expression analysis of PIP cells demonstrated the expression of genes of both myeloid and erythroid lineages. Proliferation of PIP cells was GM-CSF dependent and restricted. Upon Cre-mediated excision of proviral PU.1 cDNA, increased expression of myeloid and erythroid-specific genes was observed; as well as the appearance of both macrophages and erythrocytes in culture. Conclusion We demonstrate that ectopic expression of PU.1 is sufficient to immortalize a hematopoietic progenitor with myeloid and erythroid differentiation potential in response to GM-CSF. These data highlight the importance of the level of PU.1 expression at critical stages of hematopoiesis.