Expression Levels Of PU Research Articles

Positive feedback loop PU.1-IL9 in Th9 promotes rheumatoid arthritis development

ObjectivesT helper 9 (Th9) cells are recognised for their characteristic expression of the transcription factor PU.1 and production of interleukin-9 (IL-9), which has been implicated in various autoimmune diseases. However,...

Lin- PU.1dim GATA-1- defines haematopoietic stem cells with long-term multilineage reconstitution activity.

Despite extensive characterization of the state and function of haematopoietic stem cells (HSCs), the use of transcription factors to define the HSC population is still limited. We show here that the HSC population in mouse bone marrow can be defined by the distinct expression levels of Spi1 and Gata1. By using a double fluorescence knock-in mouse model, PGdKI, in which the expression levels of PU.1 and GATA-1 are indicated by the expression of GFP and mCherry, respectively, we uncover that the HSCs with lymphoid and myeloid repopulating activity are specifically enriched in a Lin- PU.1dim GATA-1- (LPG) cell subset. In vivo competitive repopulation assays demonstrate that bone marrow cells gated by LPG exhibit haematopoietic reconstitution activity which is comparable to that of classical Lin- Sca1+ c-kit+ (LSK). The integrated analysis of single-cell RNA sequence data from LPG and LSK-gated cells reveals that a transcriptional network governed by core TFs contributes to regulation of HSC multipotency. These discoveries provide new clues for HSC characterization and functional study.

PU.1 Regulates Cathepsin S Expression in Large Yellow Croaker (Larimichthys crocea) Macrophages

Different morphologies have been detected in teleost macrophages. In this study, two macrophage cell lines were sub-cloned from a large yellow croaker head kidney cell line, LYCK. One type of sub-cloned cells was fusiform but the other was round, named LYC-FM and LYC-RM cells respectively, based on their morphologies. Both types showed the characteristics of macrophages, including expression of macrophage-specific marker genes, possession of phagocytic and bactericidal activities, and production of reactive oxygen species (ROS) and nitric oxide (NO). The transcription factor PU.1, crucial for the development of macrophages in mammals, was found to exist in two transcripts, PU.1a and PU.1b, in large yellow croaker, and constitutively expressed in LYC-FM and LYC-RM cells. The expression levels of PU.1a and PU.1b could be upregulated by recombinant large yellow croaker IFN-γ protein (rLcIFN-γ). Further studies showed that both PU.1a and PU.1b increased the expression of cathepsin S (CTSS) by binding to different E26−transformation−specific (Ets) motifs of the CTSS promoter. Additionally, we demonstrated that all three domains of PU.1a and PU.1b were essential for initiating CTSS expression by truncated mutation experiments. Our results therefore provide the first evidence that teleost PU.1 has a role in regulating the expression of CTSS.

The effect of treatment with fexofenadine and fluticasone propionate on the gene expression levels of Th9 transcription factors in patients with allergic rhinitis

T helper-9 (Th9) is a new T cell subset involved in allergic rhinitis (AR) pathogenesis. Fexofenadine and fluticasone propionate are the first effective line of AR treatment. This study aimed to evaluate the effect of fexofenadine and fluticasone propionate on the gene expression levels of Interferon regulatory factor 4 (IRF4), B cell-activating transcription factor-like (BATF), and SPI1 gene-encoded protein (PU.1), essential transcription factors for Th9 cell differentiation, in AR patients. Twenty-six AR patients (aged 32.8 ± 9.1 years, 13 men and 13 women) were treated with fexofenadine and fluticasone propionate for one month. Expression levels of PU.1, IRF4, and BATF genes were measured using Real-Time PCR. Our results showed that after one month of treatment, the expression level of IRF4 and BATF genes decreased significantly (P < 0.001, P < 0.01, respectively), while PU.1 gene expression was not remarkably different. Overall, our results showed that after one month of treatment with fexofenadine and fluticasone propionate, the expression levels of IRF4 and BATF genes in AR patients decreased, which may be due to this treatment regimen. However, the exact mechanism of action of fexofenadine and fluticasone propionate needs further study.

Expression profile of PU.1 in CD4+T cells from patients with systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is a multisystem autoimmune disease with complex genetic predisposing factors involved. PU.1 is an important member of the ETS transcription factors family which has diverse functions such as regulating the proliferation, differentiation of immune cells and multiple inflammatory cytokines. Previous studies preliminary explored the relation between PU.1 and SLE. To further explain the potential role of PU.1 in the pathogenesis of SLE, 40 SLE patients and 20 age-sex matched healthy controls (HC) were recruited in this study. Flow cytometry was used to test the percentages of CD4+PU.1+T cells in peripheral blood mononuclear cells (PBMCs) from patients with SLE and HC. Expression levels of PU.1 mRNA in CD4+T cells from SLE patients and HC were analyzed by real-time transcription-polymerase chain reaction. Expression levels of plasma IL-1β, IL-9, IL-18, IL-6, IFN-α, TNF-α, IL-10 and TGF-β1 were measured by enzyme-linked immunosorbent assay. The percentage of CD4+PU.1+T cells in PBMCs from patients with SLE was significantly higher than that from HC (P < 0.001). In addition, the PU.1 mRNA expression in CD4+T cells from SLE patients was increased than that from HC (P = 0.002). In SLE patients, no significant correlation was found between the percentage of CD4+PU.1+T cells and the expression of PU.1 mRNA in CD4+T cells (P > 0.05). Associations of PU.1 mRNA expression in CD4+T cells with major clinical and laboratory parameters of SLE patients were also analyzed, but no significant correlations were found. Consistent with previous studies, SLE patients had increased IL-1β, IL-18, IL-6, IFN-α, TNF-α and IL-10 plasma concentrations than HC (P < 0.01). The expression level of plasma TGF-β1 was significantly decreased in SLE patients than in HC (P < 0.001). In SLE patients, the expression level of IL-1β was positive correlated with PU.1 mRNA expression in CD4+T cells (P = 0.001). Our study first time evaluated the expression profile of PU.1 in CD4+T cells from SLE patients confirming that PU.1 may participate in the pathogenesis of SLE.

High expression of PU.1 is associated with Her-2 and shorter survival in patients with breast cancer.

The transcription factor PU.1 was previously identified as an oncogene or a tumor suppressor in different types of leukemia. The aim of the present study was to investigate the expression of PU.1 in breast cancer and to analyze its association with clinical features and prognosis. Immunohistochemistry was used to determine PU.1 expression in breast cancer tissue microarrays and paraffin-embedded sections. The association between PU.1 expression and clinicopathological factors was assessed by using chi-square test. The survival analysis of patients was conducted by using Kaplan-Meier analysis and log-rank tests. Cox regression was utilized for univariate and multivariate analyses of prognostic factors. The results indicated that the expression level of PU.1 protein in breast cancer samples was significantly higher compared with normal breast tissues (P=2.63×10-8). Furthermore, the level of PU.1 expression was detected to be positively associated with androgen receptor (P=0.027) and human epidermal growth factor receptor 2 status (P=2.03×10-21) as well as molecular subtype (P=3.51×10-11). Furthermore, patients with negative PU.1 expression had longer OR compared with those with positive PU.1 expression (P=3.67×10-4). Multivariate Cox regression analysis revealed that PU.1 expression level and tumor-node-metastasis stage were independent prognostic factors for overall survival (P=0.034 and P=0.018, respectively). Therefore, PU.1 protein expression may contribute to breast cancer progression and may be a valuable molecular marker to predict the prognosis of patients with breast cancer.

The differentiation effect of low-dose cytosine arabinoside is disturbed in PU.1-knockdown K562 cells.

We recently demonstrated by using PU.1-knockdown K562 (K562 PU.1KD) cells stably expressing PU.1 short inhibitory RNAs and PU.1-overexpressing K562 (K562 PU.1OE) cells, that therapeutic concentrations of 5-aza-2'-deoxycytidine (5-azadC) induce erythroid differentiation of these cells and that the PU.1 expression level is closely associated with the differentiating and apoptotic effects of 5-azadC on K562 cells. In this study, we investigated whether the effects of low-dose cytosine arabinoside (Ara-C), which is another erythroid differentiation inducer in K562 cells, is associated with the expression level of PU.1 in these cells. As a result, we demonstrated that the effect of Ara-C on cell viability and differentiation, as determined by the WST-8 assay and β-globin mRNA expression analysis, respectively, was suppressed in K562 PU.1KD cells compared to their controls. Collectively, these findings suggest that sufficient expression of PU.1 is indispensable for the erythroid differentiation of K562 cells.

Expression of C/EBPα and PU.1 Gene In MDS and Their Significance Analysis

AbstractAbstract 4649 ObjectiveThis study was aimed to investigate the expression levels and significance of CCAAT/enhancer binding protein alpha gene and transcription factor PU.1 gene in myelodysplasyic syndromes (MDS). MethodsA real time quantitative reverse transcriptase polymerase chain reaction (Real-time PCR) method was used to detect the expression levels of C/EBPα mRNA in bone marrow mononuclear cells (BMMNCs) of 33 patients with MDS and 14 normal controls, Reverse transcription polymerase chain reaction (RT-PCR) was established for detecting PU.1 mRNA expression levels in BMMNCs of above-mentioned samples. ResultsThe expression of C/EBPα was significantly lower in low-risk and high-risk MDS than those of normal controls (P<0.01, P<0.001, respectively), moreover, high-risk MDS showed lower C/EBPα expression than those of low-risk MDS (P<0.05). The expression level of PU.1 significantly decreased in low-risk MDS, as compared with normal controls and high-risk MDS (P<0.001), but there was no significant difference in expression level of PU.1 between high-risk MDS and normal controls (P>0.05). Conclusionreduced C/EBPα and PU.1 expression level have closely associated with the pathogenesis of MDS, they may be important molecular biological markers of MDS; The degree of down-regulated C/EBPα has closely related to the progression of MDS, the detection of C/EBPα gene expression level may be useful for assessing disease progress and prognostic of patients with MDS. Disclosures:Ding:Anhui Provincial Natural Science Foundation (070413257X): Research Funding; Fund of Anhui Provincial “115” Industrial Innovation Program: Research Funding.

PU.1 Dose-Dependently Induces Granulocyte or Macrophage Commitment by Targeting Lineage Restricted Genes and by Regulating Transcription Factors Egr2, Nab2, Cebpa and Gfi1.

PU.1 (Sfpi1) is an ets family transcription factor required for the proper generation of both myeloid (macrophages and neutrophils) and lymphoid lineages (B and T lymphocytes)(Scott 1994, McKercher 1996). Graded expression of exogenous PU.1 in murine PU.1-deficient fetal liver hematopoietic progenitors demonstrated that increased levels of PU.1 are required to initiate development of macrophages (DeKoter, 2000). We have studied the effects of graded expression of PU.1 on its occupancy in chromatin and on the development of myeloid cells in vitro. We measured changes in gene expression, PU.1 occupancy and histone modifications in PU.1-null hematopoietic progenitor cells stably expressing PU.1 fused to the ligand-binding domain of the estrogen receptor (PU.1-ER) (Walsh 2002). The level of active PU.1-ER was regulated with graded levels of the ER inducer tamoxifen. In vitro, intermediate levels of tamoxifen produced cells with granulocyte characteristics in the suspension cell fraction and macrophage-like characteristics in the attached fraction, whereas high levels of PU.1 produced mostly attached macrophage-like cells. Expression of granulocyte-specific PU.1 target mRNAs including gelatinase B (Mmp9) and myeloperoxidase (Mpo) were observed to be expressed only with intermediate levels of tamoxifen. In contrast, expression of macrophage PU.1 target mRNAs including Cd14, F4/80 and Cd68 mRNAs were observed to be gradually upregulated upon PU.1-ER activation, with the maximum expression at the highest levels of tamoxifen. Thus, the expression levels of PU.1 target genes and phenotypic characteristics of the cells are dependent on PU.1 levels. Interestingly, macrophage-like cells can be produced from granulocytic-like cells by changing tamoxifen levels and vice versa. Chromatin immunoprecipitation analysis revealed specific PU.1 occupancy within regulatory regions of the genes predominantly expressed in macrophages including Cd14 and Cd11b after treatment with high levels of tamoxifen. Specific PU.1 occupancy within regulatory regions of the granulocyte specific genes including MMP9 was observed at intermediate levels of tamoxifen. Suprisingly, chromatin immunoprecipitation analysis revealed specific PU.1 occupancy within regulatory regions of the lymphocytic PU.1 target genes including Interleukin-7 receptor (Il-7r) and RAG1 at intermediate levels of tamoxifen even though expression of these genes was not detected. Accumulation of acetylated K9 and methylated K4 of histone H3 in gene loci of macrophage and granulocytic markers such as Cd14, Cd11b, and Mmp9 correlated with their mRNA expression. However, lymphocyte-specific regulatory regions including that of Il-7r gene were hypoacetylated in H3K9 despite a marked PU.1 recruitment suggesting additional factors may be required for PU.1 mediated transactivation. To identify these molecules we have tested PU.1-dependent transcription factors: Egr2, Nab2, Cebpa and Gfi-1 and found that upon increasing PU.1 levels, expression of Egr2/Nab2 and Gfi-1/Cebpa changed in a reciprocal manner and these changes preceded expression of the lineage specific markers. We are currently testing if PU.1 directly regulates expression of Egr2, Nab2, Cebpa and Gfi-1 during granulocytic/macrophage differentiation.

Redirecting differentiation of hematopoietic progenitors by a transcription factor, GATA-2

GATA-2 is a zinc finger transcription factor essential for differentiation of immature hematopoietic cells. We analyzed the function of GATA-2 by a combined method of tetracycline-dependent conditional gene expression and in vitro hematopoietic differentiation from mouse embryonic stem (ES) cells using OP9 stroma cells (OP9 system). In the presence of macrophage colony-stimulating factor (M-CSF), the OP9 system induced macrophage differentiation. GATA-2 expression in this system inhibited macrophage differentiation and redirected the fate of hematopoietic differentiation to other hematopoietic lineages. GATA-2 expression commencing at day 5 or day 6 induced megakaryocytic or erythroid differentiation, respectively. Expression levels of PU.1, a hematopoietic transcription factor that interferes with GATA-2, appeared to play a critical role in differentiation to megakaryocytic or erythroid lineages. Transcription of PU.1 was affected by histone acetylation induced by binding of GATA-2 to the PU.1 promoter region. This study demonstrates that the function of GATA-2 is modified in a context-dependent manner by expression of PU.1, which in turn is regulated by GATA-2.

Molecular Mechanism of Allergy-Related Gene Regulation and Hematopoietic Cell Development by Transcription Factors

Transcriptional regulation for the genes encoding alpha- and beta-chains of the high-affinity receptor for IgE (FcepsilonRI) have been analyzed in mast cells and regulatory mechanisms are beginning to be elucidated. Transcription factors GATA-1 and PU.1 cooperatively transactivate the alpha-chain gene, and three transcription factors, GATA-1, Oct-1, and MZF-1, are involved in regulation of beta-chain gene expression. No single nucleotide polymorphisms (SNPs) that are functionally related to the allergic diseases have been identified in coding regions of the alpha- and beta-chain genes in a definitive way. However, recent studies on SNPs in the promoter regions have revealed that these genes are probable candidates for new types of allergy-related genes whose transcription levels are affected by transcription factors which discriminate SNPs in the promoters. Another interesting finding on transcription factors functioning in mast cells is that the expression level of PU.1 determines cell fate between mast cells and monocytes.

The Acute Promyelocytic Leukemia-Associated Fusion Proteins PML/RAR and PLZF/RAR Interact with PU.1 and Inactivate Its Transcriptional Function

The pathogenesis of acute myeloid leukemia (AML) is strictly related to a block of terminal differentiation The block of differentiation is due to a deregulated function of differentiation-relevant transcription factors. We have recently shown that the acute promyelocytic leukemia (APL)-associated fusion proteins (AAFP) PML/RAR and PLZF/RAR, as well as the AML-M2-associated AML-1/ETO fusion protein, respectively related to t(15;17), t(11;17) and t(8;21), block the VitD3-induced monocytic differentiation by sequestering the VitD3-receptor (VDR). VDR is a member of the superfamily of hormon-inducible transcription factors. The aim of the study was to answer the question of whether the AAFP are able to interact directly and thereby to inhibit functionally other transcription factors, such as PU.1, which are indispensable for the normal hematopoiesis. PU.1 is a Ets-related transcription factor which plays an important in role in the stem cell fate decision as well as in the lineage specific monocytic differentiation. Recently it has been shown that the functional inactivation of PU.1 contributes to the leukemogenesis.Here we report that i.) PML/RAR as well as PLZF/RAR strongly interacted in vivo with PU.1 as revealed by mammalian two hybrid assays; ii.) the interaction with PML as well as with PLZF was stronger than that with RAR; iii.) immunofluorescence experiments revealed a diffuse nuclear anti-PU.1 staining pattern with few „dots“ in mock transfected U937 or KG-1 control cells which changed to a „microspeckled“ pattern in PML/RAR- as well as in PLZF/RAR-positive U937 or KG-1 cells; iv.) in the control cells the portion of PU.1 exhibiting localization in „dot“ structures co-localized with PML within the „dots“ of the PML-nuclear bodies (PML-NBs); v.) in PML/RAR- and PLZF/RAR-positive cells PU.1 completely co-localized within the „microspeckles“ of the fusion proteins; vi.) the AAFP inactivated the transactivation of the PU.1 target promotors of IL-18 and of GM-CSFR; vii.) the expression levels of PU.1 were not influenced by the presence of the AAFP; viii.) the overexpression of PU.1 in Sca1+/lin- hematopoietic stem cells (HSC) led in absence of G/GM-CSF to an increase of their „replating efficiency“ and to colonies expressing high levels of the stem cell markers c-Kit and Sca1; ix.) the co-expression of PU.1 in PML/RAR- and PLZF/RAR-positive HSC increased the number of differentiated cells in the CFU of the first plating but did not influence their overall replating efficiency.Taken together these data strongly suggest that the delocalization of PU.1 might withdraw it from its normal functional environment such as the PML-NBs and that this might be an important mechanisms for its functional inactivation by AML-associated fusion proteins. Furthermore it seems that the up-regulation of PU.1 is able to overcome the leukemic differentiation block only at the level of more mature cells but not at the early stem cell level which is in accordance to its dual effect on the hematopoiesis by promoting proliferation/self renewal in normal as well as in leukemic stem cells and by inducing differentiation in more mature hematopoietic precursors.

The myeloid master regulator transcription factor PU.1 is inactivated by AML1-ETO in t(8;21) myeloid leukemia

AbstractThe transcription factor PU.1 plays a pivotal role in normal myeloid differentiation. PU.1−/− mice exhibit a complete block in myeloid differentiation. Heterozygous PU.1 mutations were reported in some patients with acute myeloid leukemia (AML), but not in AML with translocation t(8;21), which gives rise to the fusion geneAML1-ETO. Here we report a negative functional impact of AML1-ETO on the transcriptional activity of PU.1. AML1-ETO physically binds to PU.1 in t(8;21)+ Kasumi-1 cells. AML1-ETO binds to the β3β4 region in the DNA-binding domain of PU.1 and displaces the coactivator c-Jun from PU.1, thus down-regulating the transcriptional activity of PU.1. This physical interaction of AML1-ETO and PU.1 did not abolish the DNA-binding capacity of PU.1. AML1-ETO down-regulates the transactivation capacity of PU.1 in myeloid U937 cells, and the expression levels of PU.1 target genes in AML French-American-British (FAB) subtype M2 patients with t(8;21) were lower than in patients without t(8;21). Conditional expression of AML1-ETO causes proliferation in mouse bone marrow cells and inhibits antiproliferative function of PU.1. Overexpression of PU.1, however, differentiates AML1-ETO–expressing Kasumi-1 cells to the monocytic lineage. Thus, the function of PU.1 is down-regulated by AML1-ETO in t(8;21) myeloid leukemia, whereas overexpression of PU.1 restores normal differentiation.

AKT Induces Transcriptional Activity of PU.1 through Phosphorylation-mediated Modifications within Its Transactivation Domain

Signal transduction by the antigen receptor complexes is critical for developmental progression of B-lymphocytes, which are defined by assembly and sequential expression of immunoglobulin genes, which in turn are regulated by the enhancer elements. Although proximal antigen-receptor signal transduction pathways are well defined, the precise nuclear factors targeted by these signals remained unknown. Previous studies have demonstrated that tissue-restricted transcription factors including PU.1 and PU.1 interaction partner (PIP) function synergistically with c-Fos plus c-Jun to stimulate the kappaE3'-enhancer in 3T3 cells. In this study, we demonstrate that the functional synergy between these factors is enhanced in response to mitogen-activated protein kinase kinase kinase, in 3T3 cells, where the enhancer is inactive. However in S194 plasmacytoma cells, mitogen-activated protein kinase kinase kinase was able to stimulate the activity of PU.1 but unable to induce the kappaE3'-enhancer activity. We have found that Ras-phosphoinositide 3-kinase-dependent externally regulated kinase, AKT, induces kappaE3'-enhancer activity in both pre-B and plasmacytoma cells. AKT stimulation of the kappaE3'-enhancer is primarily due to PU.1 induction and is independent of PU.1 interaction with PIP. Activation of AKT had no effect on the expression levels of PU.1 or its protein-protein interaction with PIP. Using a series of deletion constructs, we have determined that the PU.1 acid-rich (amino acids 33-74) transactivation domain is necessary for AKT-mediated induction. Substitution analyses within this region indicate that phosphorylation of Ser(41) is necessary to respond to AKT. Consistent with these studies, ligation of antigen receptors in A20 B cells mimics AKT activation of PU.1. Taken together, these results provide evidence that PU.1 is induced by AKT signal in a phosphoinositide 3-kinase-dependent manner, leading to inducible or constitutive activation of its target genes.