Absence Of PU Research Articles

ERG Functionally Overlaps with Other Ets Proteins in Promoting TH9 Cell Expression of Il9 during Allergic Lung Inflammation.

CD4+ TH cells develop into subsets that are specialized in the secretion of particular cytokines to mediate restricted types of inflammation and immune responses. Among the subsets that promote development of allergic inflammatory responses, IL-9-producing TH9 cells are regulated by a number of transcription factors. We have previously shown that the E26 transformation-specific (Ets) family members PU.1 and Ets translocation variant 5 (ETV5) function in parallel to regulate IL-9. In this study we identified a third member of the Ets family of transcription factors, Ets-related gene (ERG), that mediates IL-9 production in TH9 cells in the absence of PU.1 and ETV5. Chromatin immunoprecipitation assays revealed that ERG interaction at the Il9 promoter region is restricted to the TH9 lineage and is sustained during murine TH9 polarization. Knockdown or knockout of ERG during murine or human TH9 polarization in vitro led to a decrease in IL-9 production in TH9 cells. Deletion of ERG in vivo had modest effects on IL-9 production in vitro or in vivo. However, in the absence of PU.1 and ETV5, ERG was required for residual IL-9 production in vitro and for IL-9 production by lung-derived CD4 T cells in a mouse model of chronic allergic airway disease. Thus, ERG contributes to IL-9 regulation in TH9 cells.

Lipopolysaccharide-dependent transcriptional regulation of PU.1 in microglial cells

PU.1 is a pioneer transcription factor and a master regulator of the myeloid lineage. However, the role of PU.1 in lipopolysaccharide-dependent microglial activation has yet to be investigated. So, this study was conducted to determine the effects of PU.1 in LPS-induced activation of microglial cells. We knocked out PU.1 in murine BV-2 cells using the CRISPR-Cas9 system to investigate the role of PU.1 in the expression of immune-related genes. We performed RNA sequencing (RNA-seq) of PU.1 KO and BV-2 cells to analyze the gene expression patterns in PU.1 KO cells and compare them to those in wild-type BV-2 cells. The validation of differential expressions was achieved by qRT-PCR. To explore this regulatory role of PU.1, ChIP sequencing for PU.1 and H3K27Ac was performed. The sequencing result was further confirmed by ChIP-qPCR. RNA sequencing and subsequent bioinformatic analysis revealed that the expression of most of the immune-related genes was suppressed in the absence of PU.1. Proinflammatory chemokine genes were differentially expressed in LPS-treated PU.1 KO cells. The ChIP sequencing result followed by ChIP-qPCR revealed a LPS-mediated increase in the enrichment of PU.1 binding in pro-inflammatory chemokine gene promoters and enhancer regions in wild-type BV-2 cells. There was no enrichment of PU.1 in PU.1 KO cells. The above-mentioned results suggest that PU.1 is directly involved in regulating the immune response and that this regulation of inflammatory chemokines is LPS-dependent. We hope that PU.1 would be an option for limiting neurodegeneration in a diverse range of neurological disorders.

A Zebrafish Model of Cooperating C and N Terminal CEBPA Mutations Reveals Defects in Early Myelopoeisis and HSPCs Leading to Leukaemogenesis

Acute Myeloid Leukaemia (AML) is thought to occur due to stepwise accumulation of mutations in haematopoietic stem and progenitor cells (HSPCs) or early myeloid progenitor cells (EMPs) to either block differentiation or increase proliferation. In around 10% of sporadic AML cases and several known germline predisposition kindreds the myeloid transcription factor CEBPA is mutated. 50% of cases possess biallelic mutations in both C and N terminae (dm) and 50% monoallelic mutation (sm). Using TALENs we have created both C and N terminal mutants in zebrafish to study their cooperating effects in the development of AML.Our first observation was of striking defects in mature myelocytes and monocytes in all double mutants (cebpamut/mut) as assessed by Sudan black staining at 5 days post fertilisation (dpf) and apoeb whole mount in situ hybridisation (WISH) at 3dpf. In situ hybridisation revealed defects in myelopoeisis in all cebpamut/mut fry as early as 28hpf, with markedly decreased expression of coronin and l-plastin.We then interrogated the model further to assess when and where the differentiation block was occurring. We utilised the transcription factor pu.1 both as an in vivo transgenic marker Tg(pu.1:GFP) and WISH probe at earlier time points. In primitive haematopoiesis (until 22hpf) pu.1 expression showed no defects in dm mutants. However, pu.1 expressing cells were markedly reduced in dm mutants in establishment of myelopoeisis in the caudal haematopoietic tissue (CHT), fetal liver equivalent. Following formation of the CHT at around 30hpf, differing patterns of pu.1 expression were seen in sm mutants, with cebpaWT/Cterm having significantly increased staining, which was reduced in cebpaWT/Nterm (see figure 1).HSPC numbers in cebpamut/mut were seen to be normal by WISH for gata2b and runx1 and flow cytometry in Tg(CD41:GFP) fry during early haematopoeisis (2-5dpf). However, myb expression was markedly increased at 36hpf and 3 days but only in cebpaCterm/Nterm and cebpaNterm/Nterm dm mutants (see figure 2). This suggests an accumulation of early progenitors with myeloid potential but not commitment, as implied by absence of pu.1 expression but normal CD41lo numbers and other stem cell marker expression.Absence of MCherry expression in the transgenic LysC:MCherry, which highlights mature myeloid cells in vivo, was sufficiently reliable to genotype all cebpamut/mut fry and observe them for impaired survival compared to wild-type and heterozygous siblings and for the development of leukaemia. Similarly to the increase in myb staining, leukaemic transformation was only observed in dm mutants with at least one N terminal mutation, as assessed by development of anaemia and florid Tg(CD41:GFP) expression. Flow cytometry in juvenile fish (4-6weeks) also identifies a developing pre-leukaemic phenotype with moderate yet significant expansion of HSPCs and continuing absence LysC expression in cebpaNterm/Cterm and cebpaNTerm/NTerm. However, survival is poor in all dm fish, likely due to the metabolic role of cebpa and vulnerability to infection secondary to the severe myelomonocytic defect.Our results show that absence of WT cebpa has dramatic effects myelopoiesis from early stages of differentiation in definitive haematopoiesis. In addition, accumulation of myb expressing progenitors occurs as they arise from the aorta in dm mutants, identifying this as a sub-population of HSPC vulnerable to further leukaemogenic hits. Ongoing work will define the mechanism of these effects in sm and dm mutants and include comparative expression profiling in myb and pu.1 positive cells. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Reliable Machine Learning Based Spectrum Sensing in Cognitive Radio Networks

Spectrum sensing is of crucial importance in cognitive radio (CR) networks. In this paper, a reliable spectrum sensing scheme is proposed, which uses K‐nearest neighbor, a machine learning algorithm. In the training phase, each CR user produces a sensing report under varying conditions and, based on a global decision, either transmits or stays silent. In the training phase the local decisions of CR users are combined through a majority voting at the fusion center and a global decision is returned to each CR user. A CR user transmits or stays silent according to the global decision and at each CR user the global decision is compared to the actual primary user activity, which is ascertained through an acknowledgment signal. In the training phase enough information about the surrounding environment, i.e., the activity of PU and the behavior of each CR to that activity, is gathered and sensing classes formed. In the classification phase, each CR user compares its current sensing report to existing sensing classes and distance vectors are calculated. Based on quantitative variables, the posterior probability of each sensing class is calculated and the sensing report is classified into either representing presence or absence of PU. The quantitative variables used for calculating the posterior probability are calculated through K‐nearest neighbor algorithm. These local decisions are then combined at the fusion center using a novel decision combination scheme, which takes into account the reliability of each CR user. The CR users then transmit or stay silent according to the global decision. Simulation results show that our proposed scheme outperforms conventional spectrum sensing schemes, both in fading and in nonfading environments, where performance is evaluated using metrics such as the probability of detection, total probability of error, and the ability to exploit data transmission opportunities.

ETS transcription factor family member GABPA contributes to vitamin D receptor target gene regulation

Binding motifs of the ETS-domain transcription factor GABPA are found with high significance below the summits of the vitamin D receptor (VDR) cistrome. VDR is the nuclear receptor for the biologically most active vitamin D metabolite 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3). In this study, we determined the GABPA cistrome in THP-1 human monocytes and found that it is comprised of 3822 genomic loci, some 20% of which were modulated by 1,25(OH)2D3. The GABPA cistrome showed a high overlap rate with accessible chromatin and the pioneer transcription factor PU.1. Interestingly, 23 and 12% of persistent and transient VDR binding sites, respectively, co-localized with GABPA, which is clearly higher than the rate of secondary VDR loci (4%). Some 40% of GABPA binding sites were found at transcription start sites, nearly 100 of which are of 1,25(OH)2D3 target genes. On 593 genomic loci VDR and GABPA co-localized with PU.1, while only 175 VDR sites bound GABPA in the absence of PU.1. In total, VDR sites with GABPA co-localization may control some 450 vitamin D target genes. Those genes that are co-controlled by PU.1 preferentially participate in cellular and immune signaling processes, while the remaining genes are involved in cellular metabolism pathways. In conclusion, GABPA may contribute to differential VDR target gene regulation.

The Lineage-Specific Transcription Factor PU.1 Prevents Polycomb-Mediated Heterochromatin Formation at Macrophage-Specific Genes.

Lineage-specific transcription factors (TFs) are important determinants of cellular identity, but their exact mode of action has remained unclear. Here we show using a macrophage differentiation system that the lineage-specific TF PU.1 keeps macrophage-specific genes accessible during differentiation by preventing Polycomb repressive complex 2 (PRC2) binding to transcriptional regulatory elements. We demonstrate that the distal enhancer of a gene becomes bound by PRC2 as cells differentiate in the absence of PU.1 binding and that the gene is wrapped into heterochromatin, which is characterized by increased nucleosome occupancy and H3K27 trimethylation. This renders the gene inaccessible to the transcriptional machinery and prevents induction of the gene in response to an external signal in mature cells. In contrast, if PU.1 is bound at the transcriptional regulatory region of a gene during differentiation, PRC2 is not recruited, nucleosome occupancy is kept low, and the gene can be induced in mature macrophages. Similar results were obtained at the enhancers of other macrophage-specific genes that fail to bind PU.1 as an estrogen receptor fusion (PUER) in this system. These results show that one role of PU.1 is to exclude PRC2 and to prevent heterochromatin formation at macrophage-specific genes.

Abstract 401: The Lineage-specific Transcription Factor PU.1 Prevents Heterochromatinization of Macrophage-specific Genes During Hematopoietic Differentiation

We are using a quantitative approach to determine the fractional occupancy of nucleosomes at gene regulatory regions (i.e. enhancers and promoters) in primary mouse macrophages. This has allowed us to determine the dynamics of nucleosome removal at regulatory regions upon pro-inflammatory gene induction (Gjidoda, A. et al. 2014 Nucleosomes are stably evicted from enhancers but not promoters upon induction of certain pro-inflammatory genes in mouse macrophages. PLoS ONE 9(4): e93971). Moreover, we are interested in understanding how an accessible chromatin architecture is established at regulatory regions during macrophage differentiation, so that the transcriptional machinery can be recruited to these sites in mature cells. These studies have shown that the lineage-specific transcription factor PU.1 has to be present in hematopoietic progenitors to mark macrophage-specific genes for later induction. Using a previously established macrophage differentiation system from a PU.1-/- mouse as well as primary macrophages, we find that in the absence of PU.1 binding to the enhancer of a macrophage-specific gene, the enhancer is bound by PRC2 as cells are differentiated into mature macrophages. This leads to wrapping of the whole gene locus into heterochromatin, which is associated with tri-methylation of H3K27 and increased nucleosome occupancy. Our results show that one role of lineage-specific transcription factors is to prevent heterochromatinization of cell-type specific genes during differentiation to allow their expression in mature cells.

Langerhans cells are generated by two distinct PU.1-dependent transcriptional networks

Langerhans cells (LCs) are the unique dendritic cells found in the epidermis. While a great deal of attention has focused on defining the developmental origins of LCs, reports addressing the transcriptional network ruling their differentiation remain sparse. We addressed the function of a group of key DC transcription factors-PU.1, ID2, IRF4, and IRF8-in the establishment of the LC network. We show that although steady-state LC homeostasis depends on PU.1 and ID2, the latter is dispensable for bone marrow-derived LCs. PU.1 controls LC differentiation by regulating the expression of the critical TGF-β responsive transcription factor RUNX3. PU.1 directly binds to the Runx3 regulatory elements in a TGF-β-dependent manner, whereas ectopic expression of RUNX3 rescued LC differentiation in the absence of PU.1 and promoted LC differentiation from PU.1-sufficient progenitors. These findings highlight the dual molecular network underlying LC differentiation, and show the central role of PU.1 in these processes.

Gcn5 Is Required for PU.1-Dependent IL-9 Induction in Th9 Cells

Naive CD4+ T cells differentiate into various effector Th subsets depending on the Ags and cytokine microenvironment they encounter. IL-9-secreting Th9 cells are the most recent Th subset to be described. PU.1, one of the transcription factors required for the development of Th9 cells, binds to the Il9 gene. In this study, we show that PU.1 increases histone acetylation at the Il9 locus through direct interactions with histone acetyltransferases. In the absence of PU.1, there is decreased association of Gcn5 and p300/CBP associated factor and increased association of histone deacetylases at the Il9 locus in Th9 cells. Inhibition of histone deacetylase activity augments PU.1-dependent IL-9 production. PU.1 forms a complex with Gcn5, and inhibition of the expression of Gcn5 results in reduced IL-9 production. Moreover, the effects of Gcn5 on IL-9 production are specific as the production of IL-10 and IL-21, two additional cytokines produced by Th9 cells, is not altered after decreased Gcn5 expression. Together, these data define a PU.1-dependent mechanism for altered histone acetylation and expression of the Il9 locus in Th9 cells.

Two types of C/EBPα mutations play distinct but collaborative roles in leukemogenesis: lessons from clinical data and BMT models

AbstractTwo types of mutations of a transcription factor CCAAT-enhancer binding protein α (C/EBPα) are found in leukemic cells of 5%-14% of acute myeloid leukemia (AML) patients: N-terminal mutations expressing dominant negative p30 and C-terminal mutations in the basic leucine zipper domain. Our results showed that a mutation of C/EBPα in one allele was observed in AML after myelodysplastic syndrome, while the 2 alleles are mutated in de novo AML. Unlike an N-terminal frame-shift mutant (C/EBPα-Nm)–transduced cells, a C-terminal mutant (C/EBPα-Cm)–transduced cells alone induced AML with leukopenia in mice 4-12 months after bone marrow transplantation. Coexpression of both mutants induced AML with marked leukocytosis with shorter latencies. Interestingly, C/EBPα-Cm collaborated with an Flt3-activating mutant Flt3-ITD in inducing AML. Moreover, C/EBPα-Cm strongly blocked myeloid differentiation of 32Dcl3 cells, suggesting its class II mutation-like role in leukemogenesis. Although C/EBPα-Cm failed to inhibit transcriptional activity of wild-type C/EBPα, it suppressed the synergistic effect between C/EBPα and PU.1. On the other hand, C/EBPα-Nm inhibited C/EBPα activation in the absence of PU.1, despite low expression levels of p30 protein generated by C/EBPα-Nm. Thus, 2 types of C/EBPα mutations are implicated in leukemo-genesis, involving different and cooperating molecular mechanisms.

Differential Role of Myeloid Transcription Factors, C/EBPα and PU.1 In Leukemogenesis by MLL-Fusion Oncogenes.

AbstractAbstract 1568MLL translocations found in acute leukemia possess unique clinical characteristics. They have over 50 different fusion partners and show poor prognosis. These MLL fusion proteins lost H3K4 methyltransferase activity of wild-type MLL, but gained the ability to induce aberrant expression of HoxA cluster genes. Moreover, these proteins are able to transform hematopoietic stem/progenitor cells into leukemic stem cells (LSCs).Previous studies have shown that C/EBPα and PU.1, well-known myeloid specific transcription factors, were common molecular targets of myeloid malignancies. We and others have recently shown that C/EBPα and PU.1 are negative regulators of hematopoietic stem cells, suggesting that these transcription factors may play a role in the generation of LSCs. Because we have little knowledge on the role of C/EBPα and PU.1 in MLL-leukemia, we asked whether these key myeloid transcription factors were involved in the leukemogenesis by MLL-fusion proteins, especially in the stages of leukemia initiation and/or progression.First, we investigated the role of C/EBPα and PU.1 by in vitro self-renewal capacity and in vivo leukemia formation by MLL-fusion oncogenes. Bone marrow (BM) cells were harvested from C57BL/6J mice treated with 5-FU (150 mg/kg), and pre-stimulated with recombinant mouse (rm) SCF, rmIL-6, rhFL, rhTPO (50 ng/ml each). Cells were then transduced with pMYs-IG-MLL-ENL or pMXs-IG-MLL-Septin6, serially replated in methylcellulose, and transferred to rmIL-3 (10 ng/ml) containing liquid culture (immortalized cells), or were transplanted into lethally irradiated recipients (primary leukemic cells). MLL-ENL (or MLL-Septin6) immortalized cells or MLL-ENL primary leukemic cells were transduced with pMXs-IRES-DsRed-C/EBPα-ER or pMXs-IRES-DsRed-PU.1-ER. GFP+DsRed+ cells were sorted and serially replated in methylcellulose with or without 4-hydroxytamoxifen (4-HT) (1 mM), or were treated with or without 4-HT (1 mM) for 5 days followed by transplantion into sublethally irradiated secondary recipients. The results showed that overexpression of PU.1, but not C/EBPα, completely suppressed the serial replating capacity of MLL-ENL- and MLL-Septin6-immortalized cells. Moreover, activation of PU.1 suppressed propagation of MLL-ENL leukemic cells in the secondary recipients. In contrast, activation of C/EBPα did not eradicate leukemic cells in the same settings.To elucidate the role of PU.1 in the initiation of leukemia by MLL-ENL, we took PU.1+/− BM cells, or E14.5 fetal liver (FL) cells from PU.1-/- or +/− mice, and examined their capability to initiate leukemia when they were transduced with MLL-ENL. The result showed that leukemia did not develop in the absence of PU.1, and PU.1 haploinsufficiency prolonged survival of the recipients. A role of PU.1 in leukemia progression/maintenance by MLL-ENL was also tested using PU.1 conditional knockout mice. BM cells from PU.1flox/flox or flox/- mice were transduced with pMYs-IG-MLL-ENL, and transplanted into lethally irradiated recipients. PU.1-flox allele was conditionally deleted in primary leukemia cells by induction of Cre recombinase, whose effect was assessed by transplanting Cre-treated cells into secondary recipients. The result showed that conditional inactivation of PU.1 perturbed propagation of MLL-ENL leukemic cells, indicating that PU.1 is absolutely required not only for initiation, but also for maintenance of MLL-leukemia.Taken together, these results suggest that the dosage of PU.1 activity has profound impact on the self-renewal of LSCs and in vivo leukemia formation induced by MLL-fusion oncogenes. Therefore, PU.1 may serve as a potential therapeutic target for MLL-leukemia. Disclosures:No relevant conflicts of interest to declare.

Regulation of the Interleukin-7 Receptor α Promoter by the Ets Transcription Factors PU.1 and GA-binding Protein in Developing B Cells

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.

IgD Positive L&H Cells Identify a Unique Subset of Nodular Lymphocyte Predominant Hodgkin Lymphoma

Nodular lymphocyte predominant Hodgkin lymphoma (NLPHL) is a rare B-cell lymphoma considered to be of germinal center (GC) derivation. Studies on immunoglobulin expression have been few, and post-switch immunoglobulin (IgG) has been identified in the majority of cases examined thus far. We reviewed 180 cases of NLPHL and observed the unexpected expression of IgD in 27% of cases. IgD is usually coexpressed with IgM in naive B cells but can also be seen as IgD-only in centroblasts (CD38-positive) or memory B cells (CD27-positive). We asked whether IgD-positive NLPHL differed from cases of NLPHL negative for IgD. Clinically, the IgD-positive cases presented at a younger median age (21 vs. 44 years) and had a striking male predominance (male-to-female ratio, 23:1 vs. 1.5:1). Cervical lymph nodes were more frequently involved (56% vs. 18.2%). L&H cells were localized in a predominantly extrafollicular distribution in the majority of IgD-positive cases (69%). The IgD-positive cases did not coexpress IgM or CD27 (a marker associated with memory B cells), and nearly all (93%) were weakly positive for CD38, supporting a GC derivation. The expression of Bcl-6, BOB.1, Oct2, and SWAP-70 was similar in the two groups. However, PU.1 expression was seen in 60% of the IgD-positive cases in contrast to 86% of the IgD-negative cases. The absence of PU.1 staining correlated with more L&H cells in an extrafollicular distribution, weakening the use of this marker in the differential diagnosis with T-cell rich/histiocyte rich B-cell lymphomas. To study IgD expression in "de-novo" T-cell rich/histiocyte rich B-cell lymphomas, we analyzed 20 cases and all but one were negative. In conclusion, cases of IgD-positive NLPHL do not differ from IgD-negative cases regarding cellular derivation and most other immunophenotypic characteristics. However, IgD-positive NLPHL exhibits distinctive clinical features, and more often involves the interfollicular region in a background relatively rich in T cells. IgD positivity may represent an additional useful marker in the diagnosis of NLPHL.

The IL-7 Receptor alpha Promoter Is Activated at Distinct Stages during B Cell Development by the Ets Transcription Factors PU.1 and GABP.

The cytokine interleukin-7 (IL-7) is required for B and T lymphocyte development, and for the survival and maintenance of both naive and memory T lymphocytes. The receptor for IL-7 (IL-7R) is heterodimeric, consisting of a common gamma chain (γc) and an alpha subunit (IL-7Rα). The γc is expressed in most hematopoietic cell types, but the IL-7Rα gene is regulated in a cell type and developmental stage-specific manner. We have previously shown that the Ets-family transcription factor PU.1 is required to activate transcription of the IL-7Rα gene during fetal lymphocyte development. However, several questions remain unanswered. First, the IL-7Rα promoter is poorly characterized. Second, the IL-7Rα is expressed at high levels in the T cell lineage where PU.1 is not expressed. Third, the transcription factor early B cell factor (EBF) can activate IL-7Rα transcription in developing B cells in the absence of PU.1. To address these questions, we have characterized the IL-7Rα promoter region in detail. First, we determined that the major transcription start sites in the IL-7Rα gene are downstream of an Ets/PU.1 binding site. We found that the intact Ets site is required for IL-7Rα promoter activity, as well as to mediate enhancer action from a distance. IL-7Rα promoter activity depends on the proper orientation of the Ets site relative to functional initiator sequences. We found, using gel shift analysis, that both PU.1 and the Ets transcription factor GA binding protein (GABP) are expressed in developing B cells, and can interact with the Ets binding site in the IL-7Rα promoter. However, the function of PU.1 is distinct from GABP during B cell development. Retroviral transduction of PU.1 mutant progenitor cells with a PU.1 retrovirus robustly rescues IL-7Rα transcription and IL-7-dependent B cell development. In contrast, transduction with GABPα and GABPβ1 subunits fails to activate IL-7Rα transcription in PU.1 mutant progenitor cells. We conclude that activation of the IL-7Rα gene requires PU.1 during the earliest stages of lymphocyte development, but is alternatively utilized by PU.1 and GABP after commitment to the B cell lineage.

B Cell Development in the Absence of PU.1.

Mice deficient of the ETS-family transcription factor PU.1 lack B cells as well as macrophages. While most macrophage specific genes are known to be regulated by high levels of PU.1, the reason for the defect in B cell formation is not known. Here we analyzed a mouse strain in which a floxed version of the PU.1 gene, surrounding exon 4 and 5, which encode the DNA, binding and PEST domains (developed by C. Somoza and D. Tenen), was excised by Cre mediated recombination. As expected, this strain lacks both B cells and macrophages and die at birth. Surprisingly, however, we were able to establish lymphoid cell lines from fetal livers of these mice (day 14 to day 18), which proliferated on S17 stromal cells supplemented with IL-7 and stem cell factor. These cells expressed the B lineage cell surface markers CD19, CD43, BP-1 and CD24, but not B220. They also expressed B cell transcription factors, EBF, E47, Pax5, and their target genes, Rag1, IL7R, λ5 and v-preB, as detected by RT-PCR, exhibited DJ and VDJ immunoglobulin heavy chain rearrangements, and expressed IgM after IL-7 withdrawal. We then tested the effect of PU.1 deletion in B cells in adult animals by crossing the floxed PU.1 strain with a CD19 Cre mouse line. The spleen and peripheral blood (but not bone marrow) of these mice contained B cells that were CD19+ IgMlow, IgDhigh but B220 negative and instead expressed CD43. Thus PU.1 is not essential for immunoglobulin production and late B cell development. Although PU.1−/− fetal liver cells can give rise to cells, resembling Pre-B in vitro, the process of B cell formation was delayed by almost 12 days, compared with wt fetal liver, and the efficiency was reduced approximately 25-fold. In addition, PU.1 deficient B cells demonstrated an impaired ability to engraft into the bone marrow, when injected into irradiated SCID mice. We have found that PU.1 deficient B progenitors showed reduced or undetectable levels of the SDF1 receptor CXCR4, a receptor that has been implicated in B cell homing. Taken together, our observations suggest that PU.1 plays two different roles during B cell development: for early B cell formation and for proper migration and engraftment, which might be mediated through regulation of CXCR4 expression.

Functional Differences in GATA-1 Affinity for Double Versus Single GATA-1 Binding Sites Dictate Synergistic Versus Antagonistic Interactions of PU.1 and GATA-1 in Myeloid Gene Transcription.

Instructive roles for both GATA-1 and PU.1 have been demonstrated in hematopoiesis, and recent studies have identified both antagonistic and synergistic interactions between them in myeloid gene transcription and lineage development. In prior studies, we reported that PU.1 synergizes with rather than antagonizes GATA-1 for transactivation of a hallmark eosinophil gene, the major basic protein P2 promoter (MBP-P2), which possesses a novel dual (double) GATA-binding site, similar to the palindromic double site in the murine GATA-1 control locus that may specify eosinophil lineage-specific expression of GATA-1 and eosinophil development. To address the transcriptional mechanism for PU.1-GATA-1 synergy through the MBP-P2 dual GATA site, we investigated GATA-1 and PU.1 physical and functonal interactions via their binding sites in the MBP-P2 promoter. DNA binding affinities of GATA-1 and its C- versus N-terminal zinc fingers were assessed for single versus double GATA sites in the presence or absence of PU.1. Our results show that the dual GATA site strongly binds full length GATA-1 with higher affinity than either of the single sites, using both zinc fingers, but that mutant GATA-1 proteins with C-finger or N-finger deletions retain their ability to bind, albeit at lower affinity, to the dual site. DNA binding activities of the two zinc fingers with the dual GATA site were confirmed using peptides containing only the C-finger or N-finger region. Of note, formation of GATA-1 complexes with the dual GATA site was not inhibited by the addition of PU.1, whereas formation of binding complexes for mutants of GATA-1 containing only the C- or N-finger region could be completely inhibited in a dose-response fashion by PU.1. These unique features of PU.1/GATA-1 interactions on a dual versus single GATA-1 site were confirmed using peptides containing only the C- or N-finger regions of GATA-1. Our findings indicate that both zinc fingers of GATA-1 are involved in formation of the high-affinity GATA-1 complex with the dual site. Importantly, we show that the higher affinity dual GATA-1 site complex is not affected by the addition of PU.1, whereas formation of the binding complex with a single GATA-1 site is eliminated by PU.1, emphasizing the different mechanisms of GATA-1/PU.1 interactions on dual versus single GATA binding sites. Functional analyses by transactivation confirmed that synergistic activation of the MBP-P2 promoter by GATA-1 and PU.1 is mediated by their protein-protein interactions through this unique high affinity dual GATA-1 binding site. We suggest two possible mechanisms for PU.1/GATA-1 synergy on dual GATA sites: (1) PU.1 may change GATA-1 conformation and its high affinity for the dual site, enhancing its availability for interaction with the basal transcriptional machinery. Alternatively, (2) PU.1 could impede interactions of GATA-1 with a co-repressor, e.g. FOG-1, which we and others have shown represses GATA-1 function in the eosinophil lineage.

Spi-B can functionally replace PU.1 in myeloid but not lymphoid development.

Mature macrophages, neutrophils and lymphoid cells do not develop in PU.1(-/-) mice. In contrast, mice lacking the highly related protein Spi-B generate all hematopoietic lineages but display a B-cell receptor signaling defect. These distinct phenotypes could result from functional differences between PU.1 and Spi-B or their unique temporal and tissue-specific expression (PU.1: myeloid and B cells; Spi-B: B cells only). To address this question, we introduced the Spi-B cDNA into the murine PU.1 locus by homologous recombination. In the absence of PU.1, Spi-B rescued macrophage and granulocyte development when assayed by in vitro differentiation of embryonic stem cells. Adherent, CD11b(+)/F4/80(+) cells capable of phagocytosis were detected in PU.1(Spi-B/Spi-B) embryoid bodies, and myeloid colonies were present in hematopoietic progenitor assays. Despite its ability to rescue myeloid differentiation, Spi-B did not rescue lymphoid development in a RAG-2(-/-) complementation assay. These results demonstrate an important difference between PU.1 and Spi-B. Careful comparison of these Ets factors will delineate important functional domains of PU.1 involved in lymphocyte lineage commitment and/or maturation.

BSAP can repress enhancer activity by targeting PU.1 function.

PU.1 and BSAP are transcription factors crucial for proper B-cell development. Absence of PU.1 results in loss of B, T, and myeloid cells, while absence of BSAP results in an early block in B-cell differentiation. Both of these proteins bind to the immunoglobulin kappa chain 3' enhancer, which is developmentally regulated during B-cell differentiation. We find here that BSAP can repress 3' enhancer activity. This repression can occur in plasmacytoma lines or in a non-B-cell line in which the enhancer is activated by addition of the appropriate enhancer binding transcription factors. We show that the transcription factor PU.1 is a target of the BSAP-mediated repression. Although PU.1 and BSAP can physically interact through their respective DNA binding domains, this interaction does not affect DNA binding. When PU.1 function is assayed in isolation on a multimerized PU.1 binding site, BSAP targets a portion of the PU.1 transactivation domain (residues 7 to 30) for repression. The BSAP inhibitory domain (residues 358 to 385) is needed for this repression. Interestingly, the coactivator protein p300 can eliminate this BSAP-mediated repression. We also show that PU.1 can inhibit BSAP transactivation and that this repression requires PU.1 amino acids 7 to 30. Transfection of p300 resulted in only a partial reversal of PU.1-mediated repression of BSAP. When PU.1 function is assayed in the context of the immunoglobulin kappa chain 3' enhancer and associated binding proteins, BSAP represses PU.1 function by a distinct mechanism. This repression does not require the PU.1 transactivation or PEST domains and cannot be reversed by p300 expression. The possible roles of BSAP and PU.1 antagonistic activities in hematopoietic development are discussed.

Commitment to the Monocytic Lineage Occurs in the Absence of the Transcription Factor PU.1

AbstractMice homozygous for the disruption of the PU.1 (Spi-1) gene do not produce mature macrophages. In determining the role of PU.1 in macrophage differentiation, the present study investigated whether or not there was commitment to the monocytic lineage in the absence of PU.1. Early PU.1−/− myeloid colonies were generated from neonate liver under conditions that promote primarily macrophage and granulocyte/macrophage colonies. These PU.1−/− colonies were found to contain cells with monocytic characteristics as determined by nonspecific esterase stain and the use of monoclonal antibodies that recognize early monocyte precursors, including Moma-2, ER-MP12, ER-MP20, and ER-MP58. In addition, early myeloid cells could be grown from PU.1−/− fetal liver cultures in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF). Similar to the PU.1 null colonies, the GM-CSF–dependent cells also possessed early monocytic characteristics, including the ability to phagocytize latex beads. The ability of PU.1−/− progenitors to commit to the monocytic lineage was also verified in vivo by flow cytometry and cytochemical analysis of primary neonate liver cells. The combined data shows that PU.1 is absolutely required for macrophage development after commitment to this lineage.

Commitment to the Monocytic Lineage Occurs in the Absence of the Transcription Factor PU.1

Mice homozygous for the disruption of the PU.1 (Spi-1) gene do not produce mature macrophages. In determining the role of PU.1 in macrophage differentiation, the present study investigated whether or not there was commitment to the monocytic lineage in the absence of PU.1. Early PU.1−/− myeloid colonies were generated from neonate liver under conditions that promote primarily macrophage and granulocyte/macrophage colonies. These PU.1−/− colonies were found to contain cells with monocytic characteristics as determined by nonspecific esterase stain and the use of monoclonal antibodies that recognize early monocyte precursors, including Moma-2, ER-MP12, ER-MP20, and ER-MP58. In addition, early myeloid cells could be grown from PU.1−/− fetal liver cultures in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF). Similar to the PU.1 null colonies, the GM-CSF–dependent cells also possessed early monocytic characteristics, including the ability to phagocytize latex beads. The ability of PU.1−/− progenitors to commit to the monocytic lineage was also verified in vivo by flow cytometry and cytochemical analysis of primary neonate liver cells. The combined data shows that PU.1 is absolutely required for macrophage development after commitment to this lineage.