Expression Of PU Research Articles

Radiation-inducible promoter mediated PUMA Gene in the the treatment of Tca8113

Objective: The aim of this study is to explore the therapeutic effect of the PUMA gene mediated by ra- diation-inducible promoters in the treatment of ton- gue squamous cell carcinoma. Methods: Recombi- nant pcDNA3.1 (+)/E-PUMA was constructed, in which the PUMA gene was mediated by a synthetic radiation inducible promoter. The recombined plas-mids were transfected into the Tca8113 cell and xeno- grafts of human tongue squamous carcinoma in na- ked mice respectively. After 24 h, the tumors were tr- eated with 3 Gy of irradiation to upregulate the PU- MA gene expression. PUMA mRNA was detected by RT-PCR. Proliferating cell nuclear antigen (PCNA) and apoptosis were detected by immunohistochemi- cal method and in situ end-labeling (ISEL) respecti- vely. The data were analyzed using the SPSS11.0 soft- ware package for chi-square test. RESULTS: Com-pared with the control group, the comparative survi- val rate of Tca8113 cells in the PUMA/IR group was markedly decreased and the xenografts were signify- cantly suppressed. Up-regulation of PUMA gene ex- pression was observed in the Tca8113 cells and in the xenografts after irradiation. The apoptosis indices of the Tca8113 cells and xenograft with irradiation were markedly higher than those without irradiation. At the same time, the proliferation indices of the Tca- 8113 cells and xenografts with irradiation were mark- edly lower than those without irradiation. Conclu- sions: radiation-induc ible promoters can serve as molecular switches to improve the expression of PU- MA gene in tongue squamous cell carcinoma both in vivo and in vitro. Low-dose induction radiation can significantly improve therapeutic efficiency.

Early mammalian erythropoiesis requires the Dot1L methyltransferase

Histone methylation is an important regulator of gene expression; its coordinated activity is critical in complex developmental processes such as hematopoiesis. Disruptor of telomere silencing 1-like (DOT1L) is a unique histone methyltransferase that specifically methylates histone H3 at lysine 79. We analyzed Dot1L-mutant mice to determine influence of this enzyme on embryonic hematopoiesis. Mutant mice developed more slowly than wild-type embryos and died between embryonic days 10.5 and 13.5, displaying a striking anemia, especially apparent in small vessels of the yolk sac. Further, a severe, selective defect in erythroid, but not myeloid, differentiation was observed. Erythroid progenitors failed to develop normally, showing retarded progression through the cell cycle, accumulation during G₀/G₁ stage, and marked increase in apoptosis in response to erythroid growth factors. GATA2, a factor essential for early erythropoiesis, was significantly reduced in Dot1L-deficient cells, whereas expression of PU.1, a transcription factor that inhibits erythropoiesis and promotes myelopoiesis, was increased. These data suggest a model whereby DOT1L-dependent lysine 79 of histone H3 methylation serves as a critical regulator of a differentiation switch during early hematopoiesis, regulating steady-state levels of GATA2 and PU.1 transcription, thus controlling numbers of circulating erythroid and myeloid cells.

RAD001, An Inhibitor of mTOR, Enhances Monocytic Differentiation Induced by ATRA through Phosphorylation of C/EBPα In AML Cell Lines

AbstractAbstract 2152 Background:CCAAT/ enhancer binding protein alpha (C/EBPα) is a critical transcription factor that controls monocytic and granulocytic differentiation. Several recent studies have reported that C/EBPα expression is down-regulated in acute myeloid leukemia (AML), leading to suppression of monocytic differentiation. All-trans-retinoic acid (ATRA) induces numerous transcriptional factors, including C/EBPα; however, ATRA alone is not sufficient to induce monocytic differentiation in AML. The purpose of this study was to identify agents that increase the efficacy of ATRA. RAD001 (Everolimus; provided by Novartis), a rapamycin analog, is a relatively new drug that inhibits the Akt/ PI3K/ mTOR pathway. To assess the utility of differentiation therapy as a treatment for types of AML other than acute promyelocytic leukemia, we evaluated the effects of RAD001 and ATRA combination treatment in several AML cell lines. Methods:Three AML cell lines (U-937, THP-1, and KOCL48) and two primary AML samples were treated with 2.5–5.0 nM RAD001 and 1 μM ATRA for five days. Cell growth was analyzed by counting nuclei using a Coulter counter. Monocytic differentiation was assessed by morphological analysis and flow cytometric analysis (FCM) of CD11b expression. An Annexin V assay was carried out to measure apoptosis. Microarray analysis using an Agilent expression array was employed to determine changes in gene expression associated with ATRA and RAD001 combination treatment. Quantitative RT-PCR (qRT-PCR) analysis was performed to validate the microarray results. Western blotting was carried out to measure the phosphorylation of C/EBPα at Ser 21. Results:We determined that ATRA and RAD001 treatment induced morphological changes characteristic of monocytic differentiation. Microarray analysis of THP-1 revealed that ATRA and RAD001 induced expression of a set of genes associated with monocytic differentiation, including MPEG1, CD11b, CD115 and CD14. FCM analysis confirmed that ATRA and RAD001 intensified CD11b expression in the three cell lines tested, especially in the two ATRA-resistant cell lines (KOCL48 and U937). qRT-PCR analysis also revealed that ATRA and RAD001 treatment increased expression of C/EBPα and C/EBPε, which is involved in the terminal stages of monocytic differentiation, in all three cell lines and two primary samples compared to treatment with ATRA only. Expression of PU.1 was also increased by combination treatment in all cells tested except the U937 cell line. Western blot analysis revealed that ATRA and RAD001 decreased phosphorylation of C/EBPα at serine 21. ATRA and RAD001 combination treatment also suppressed cell growth in two ATRA-resistant cell lines (growth inhibition rate: 70–80%). The Annexin V assay demonstrated that ATRA and RAD001 combination treatment strongly induced apoptosis in the three cell lines tested. Microarray analysis revealed that FasL,FADD, and caspase 8, which are associated with apoptotic pathways, showed the greatest degree of up-regulation in THP-1 cells treated with ATRA and RAD001. qRT-PCR analysis confirmed up-regulation of these genes in all three cell lines and in both primary AML samples, indicating that ATRA and RAD001 induce apoptosis in AML cells through the extrinsic cell death signaling pathway. Conclusions:RAD001 induced monocytic differentiation through induction of a set of genes associated with monocytic differentiation and phosphorylation of C/EBPα at Ser 21 when combined with ATRA. This combination therapy also induced apoptosis in AML cells through activation of the extrinsic cell death signaling pathway. Disclosures:No relevant conflicts of interest to declare.

Expanded γδ T cell populations in absence of transcription factor PU.1 (36.49)

Abstract T cell development is characterized by a succession of developmental stages that lead to the generation of mature αβ and γδ T cells. γδ T cells are considered innate cells that critically contribute to host defense. PU.1, an Ets family transcription factor, also identified as the spleen focus forming virus proviral integration site-1 (Sfpi-1) is essential for early stages of T cell development and is down regulated during the pro T-cell stage. PU.1 is expressed in Th2 cells but not Th1 cells. Expression of PU.1 in other T cell subsets has not been studied extensively. In this study, we show that PU.1 is expressed in γδ T cells, and in mice that specifically lack PU.1 in T cells using an lck-Cre transgene with a conditional Sfpi1 allele (Sfpilck-/-) there are increased numbers of γδ T cells in spleen, thymus and in the intestine when compared to wild type mice. The increase in γδ T cell numbers in PU.1 deficient mice is reflected in several subsets examined (Vγ2T, Vγ3T and Vδ4T). In mice where PU.1 was deleted using a CD4-Cre transgene, the expansion of γδ T cell numbers was restricted to the intestinal population. PU.1-deficient γδ T cells secrete higher levels of IL-17 than wild type cells, but similar levels of IFN-γ. Thus, our data show that PU.1 is expressed in γδ T cells and that deletion of PU.1 results in γδ T cell expansion. This suggests that PU.1 has a negative influence on γδ T cell development or expansion.

Overexpression of Oncogenic KRAS G12V in Human Stem and Progenitor Cells Enhances Proliferation and Initiates Monocytic Differentiation Via Intrinsic and Extrinsic Pathways.

Abstract 3975Poster Board III-911In human hematopoietic malignancies, Ras mutations are frequently present in monocytic and T-cell leukemias. In this study we investigated KRAS G12V-induced phenotypes in human stem and progenitor cells and identified signal transduction pathways that are involved. Using a retroviral expression system, KRAS G12V was introduced to human CD34+ cord blood (CB) cells and proliferation, differentiation and stem cell/progenitor frequencies were evaluated. Overexpression of constitutively active KRAS G12V induced a strong increase in cell expansion over 5-fold in MS5 bone marrow stromal cocultures as well as in cytokine-driven liquid cultures, which coincided with increased early cobblestone formation and induction of monocytic differentiation. Erythroid progenitors were greatly reduced by introduction of KRAS G12V and Q-PCR analysis revealed that expression of PU.1 was increased in conjunction with reduced GATA1 expression in KRAS G12V cells. Progenitor frequencies were increased 6-fold in KRAS-transduced cells within 1 week after plating on MS5. By week three progenitors were exhausted and KRAS-transduced cells were terminally differentiated into monocytes/macrophages. These results were in line with the strong reduction in LTC-IC frequencies at week 5, indicating that also the stem cell pool was exhausted. Intriguingly, when KRAS G12V-transduced cells were cocultured with non-transduced CB CD34+ cells, we observed that the non-transduced cells also displayed a strong growth advantage, coinciding with enhanced early cobblestone formation. Furthermore, the addition of conditioned medium from KRAS G12V-transduced cells grown on MS5 to non-transduced CB cells induced a strong growth advantage and formation of early CAFCs. These observations indicate that, besides intrinsic pathways, secreted factor(s) play an important role in the phenotypes induced by KRAS G12V in human CB CD34+ cells. Current studies include mass-spectroscopy analysis of the secretome of KRAS G12V-transduced CB CD34+ cells to identify the factor(s) that are involved.In order to elucidate signal transduction pathways that mediate KRAS G12V-induced phenotypes, Western-blot analysis was performed. These experiments revealed an increase in phospho-ERK1/2, phospho-p38 and phospho- STAT5 (Y694) levels in KRAS-transduced cells, whereas phospho-JNK was not induced and phospho-C/EBPa (S21) levels were slightly reduced. Induction of STAT5 Y649 phosphorylation by KRAS G12V was confirmed by intracellular phosphoFACS analysis, whereby both in HSCs as well as in more committed MPPs KRAS-induced phosphorylation of STAT5 was observed. KRAS-transduced cells did not show GM-CSF hypersensitivity in any measured cell population upon activation. Inhibition of the ERK/MAPK pathway using the MEK inhibitor U0126 resulted in strongly reduced expansion in MS5 cocultures, whereby both intrinsically induced proliferation as well as proliferation induced via secreted factor(s) were impaired. KRAS G12V-induced monocytic differentiation was not significantly affected by MEK inhibition. While inhibition of the JNK pathway hardly affected proliferation and differentiation of KRAS G12V cells, inhibition of the p38 pathway using SB203580 inhibitor impaired both proliferation and differentiation. When KRAS G12V-transduced cells were cocultured with non-transduced CB CD34+ cells, inhibition of p38 predominantly affected the transduced cells but not the non-transduced cells, suggesting that the p38 pathway particularly mediates intrinsic phenotypes imposed by KRAS G12V.In conclusion, we show that overexpression of oncogenic KRAS G12V in human CD34+ cells enhances proliferation and initiates monocytic differentiation via intrinsic and extrinsic pathways. Disclosures:No relevant conflicts of interest to declare.

Genetics and Epigenetics of the PU.1 Upstream Regulatory Element: AML1 Binding Sites Are Critical for Leuekmia Induced by the AML/ETO9a Fusion Oncogene

The level of expression of the transcription factor PU.1 is a critical determinant of lineage commitment in normal hematopoiesis, and dysregulation of PU.1 leads to development of leukemia. In mice with targeted disruption of the PU.1 upstream regulatory element (URE), expression of PU.1 is decreased to 20% of wild type levels and results in development of acute myeloid leukemia (AML). These data suggests that tightly regulated PU.1 expression is important to maintain normal hematopoiesis and prevent leukemogenesis. Previously, we reported that AML1 (RUNX1) regulated PU.1 expression. Here we demonstrate that AMLl regulates PU.1 through 3 AML1 binding sites in the URE. Mice with targeted mutations in the 3 AML1 binding sites have decreased PU.1 expression in multiple hematopoietic lineages at multiple different developmental stages. Conditional targeting of AML1 in transgenic mice in which the URE homology region 2 (H2, containing all 3 AML1 binding sites) is used to drive expression of a reporter decreased reporter gene expression, suggesting that AML1 regulates PU.1 through these 3 sites in URE homology region 2. Using a second mouse model with a targeted mutation in the PU.1 binding site in the PU.1 URE (which is flanked by the 3 AML1 sites), we demonstrated that PU.1 indeed autoregulates itself through the URE. These results demonstrated that AML1 regulates PU.1 through the 3 AML1 sites in the URE. However, while low levels of PU.1 lead to leukemia, we have not observed frank leukemia development in AML1 conditional knockouts or in mice with targeted disruption of the 3 AML1 sites in the PU.1 URE. We hypothesized that this might be the case because disruption of AML1 or the AML1 sites reduces PU.1 levels to about 40% of wild type, but not as great as that found in PU.1 URE knockouts, which do progress to AML (20% of wild type). We hypothesized that downregulation of PU.1 as a result of binding of AML1/ETO fusion proteins to the URE might result in further reductions of PU.1 expression, and contribute to leukemogenesis. Therefore, we predicted that development of leukemia might be delayed in mice with mutations in the PU.1 URE AML1 DNA binding sites, and this was indeed the case in a modle using a retrovirus expressing the AML1/ETO9a form. We further explored the effect of AML1 and PU.1 binding on chromatin strucutre using chromatin immunoprecipitation (Chip) in the AML1 and PU.1 site URE knockin models, and found that AML1 is involved in H3K4me3 and H3/H4 acetylation of histone tails in the PU.1 URE, while PU.1 is involved in H3/H4 acetylation but not H3K4me3; H3K4 methylation and H3 acetylation decreased in AML1 sites mutant knockin mice and H3 acetylation decreased in PU.1 site mutant knockin mice. Mutation of the AML1 site in mice not only altered the chromatin structure of the URE region, but also interefered with the physical interaction between the URE and PU.1 promoter, as assessed by chromosome capture configuration (3C) assays. Interestingly, the AML1/ETO9a fusion oncogene has a unique role on the epigenetic status of the PU.1 URE in addition to its dominant effect on the 3 AML1 sites. AML1-ETO9 blocks the autoregulation of PU.1 through the PU.1 site in the URE. In summary, our data suggests that AML1 regulates PU.1 expression through 3 AML1 binding sites in the PU.1 URE by modifying chromatin structure in the URE region. In addition, PU.1 can autoregulate itself by facilitating similar epigenetic changes. Dysregulation of the epigenetic status by chromosome translocation products such as AML1-ETO might play an important role in leukemogenesis.First two authors contribute equally to this work.

PU.1 Epigenetically Represses the Expression of Metallothionein-1 Gene in Leukemia Cells

Recently, it was reported that reduced level of PU.1 expression developed acute myeloid leukemia (AML) in mice. To clarify the mechanisms of AML, first we established PU.1 knockdown K562 (K562PU.1KD) cells, that express reduced level of PU.1 by stably transfected PU.1 siRNA. Flow cytometric analysis demonstrated that among other lineage markers, expressions of myeloid CD13/CD33 markers are significantly decreased in K562PU.1KD cells compared to control cells. We next tried to identify PU.1 target genes to elucidate the mechanisms of AML. To solve this, microarray and real time PCR analyses were performed. We revealed that several genes including metallothionein (MT) s (MT-1G, MT-1A) were markedly induced in K562PU.1KD cells. MTs are a group of low-molecular weight, cysteine rich intracellular proteins. A number of studies have shown an increased expression of MTs in various human solid tumors. To verify the relevance, we established PU.1 over-expressed K562 (K562PU.1OE) cells and found the significant suppression of these genes. Furthermore, we revealed a negative correlation between PU.1 and MTs mRNA expression in 43 primary AML specimens (MT-1G; R=−0.50, p<0.001, MT-1A; R=−0.57, p<0.0005). These results suggest that MTs are bona fide targets of PU.1. To clarify MTs regulation by PU.1, chromatin immunoprecipitation assay was employed and demonstrated an increased association of acetylated histone H3 and H4 with the MT-1G and MT-1A promoter in K562PU.1KD cells, compared to control cells. As there are 38 CpG sites in approximately −430 to +170 bp from transcription start site in MT-1G and MT-1A promoter, we next performed bisulfite DNA sequencing analysis to examine the methylation status of these CpG sites. We revealed that the proportion of the methylated CpG sites in MT- 1G, as well as MT-1A promoter were significantly decreased in K562PU.1KD cells (MT- 1G: control cells, 29–41% vs K562PU.1KD cells, 16–29%; MT-1A: control cells, 12% vs K562PU.1KD cells, 4%). In contrast, methylations of these sites were markedly increased in K562PU.1OE cells (MT-1G: control cells, 36% vs K562PU.1OE cells, 59%; MT-1A: control cells, 12% vs K562PU.1OE cells, 18%). These suggest that downregulation of PU.1 leads to the impairment of the recruitment of DNA methyltransferase (DNMT) and histone deacetylase, results in the aberrant gene expression in leukemia cells. Therefore, we next hypothesized that there is some specific effect of epigenetic drugs on PU.1 transgenic cells. From WST assay and trypan blue dye exclusion assay, we observed that K562PU.1KD cells were resistant to DNMT inhibitor 5-aza-2′-deoxycytidine (5-Azadc). Viable cells were approximately 20% increased in K562PU.1KD cells in the presence of 5-Azadc, and 20% decreased in K562PU.1OE cells compared to control cells respectively. These suggest that expression of PU.1, MTs and proportion of methylated CpG sites in MTs' promoter may useful therapeutic marker to predict the effect of 5-Azadc on leukemia cells.

Heterogeneity of Embryonic and Adult Stem Cells

New studies suggest that stem cells of embryonic, neural, and hematopoietic origin are heterogeneous, with cells moving between two or more metastable states. These cell states show a bias in their differentiation potential and correlate with specific patterns of transcription factor expression and chromatin modifications.

PU.1 expression is restored upon treatment of chronic myeloid leukemia patients

The PU.1 transcription factor is a crucial regulator of hematopoiesis which expression is altered in various leukemic processes. Our previous work in chronic myeloid leukemia (CML) cells demonstrated that interferon-α upregulated PU.1 expression. Here we show that expression of PU.1 is severely impaired in patients with CML at diagnosis. However, the PU.1 suppression is abrogated in patients in remission, after interferon-α or imatinib treatment. These effects are not found in patients with other myeloproliferative diseases such as polycythemia vera or essential thrombocythemia. PU.1 could, therefore, be used as an additional marker of the response to the treatment of the CML.

The NHR2 Oligomerization Domain of AML1/ETO as a Novel Therapeutic Target Structure in t(8;21) Positive Leukemias.

About 12% of all de novo acute myeloid leukemias are characterized by the translocation t(8;21) which generates the oncogenic fusion protein AML1/ETO. AML1/ETO is found in high molecular weight complexes (HMWC) which are crucial for the block in myeloid differentiation of AML1/ETO transformed cells. Essential for HMWC formation is the alpha-helical nervy homology region 2 (NHR2) within ETO which serves as an interacting surface for oligomerization as well as association with members of the ETO protein family. Based on the observation that the integrity of the NHR2 domain is essential for transcriptional repressor activity of ETO, we designed a peptide (NC128) aimed to selectively interfere with AML1/ETO oligomerization. Confocal laser scanning microscopy revealed colocalization of NC128 with AML1/ETO in the nucleus. In protein-protein interaction studies we could demonstrate that the NHR2 domain in NC128 was necessary and sufficient for proper binding to AML1/ETO. Expression of NC128 in AML1/ETO positive cells led to inhibition of AML1/ETO HMWC formation as demonstrated by size-exclusion chromatography. NC128 expression in the AML1/ETO growth dependent Kasumi-1 cells restores transcription of AML1/ETO target genes. Among others, the expression of PU.1, a master regulator of myeloid differentiation, was significantly upregulated in the presence of NC128. In Kasumi-1 cells as well as in U937-AML1/ETO cells expression of NC128 almost completely overcomes the block of cytokine mediated differentiation induced by AML1/ETO. In the presence of NC128, Kasumi-1 cells lose their progenitor cell characteristics and upregulate marker of early monocytic differentiation. Likewise, NC128 expressing Kasumi-1 and SKNO-1 cells are arrested in cell cycle progression with a peak in the G1 phase of the cell cycle. The binding affinity of NHR2 peptide mutants to ETO directly correlates with the intensity of growth inhibition. A derivative of NC128 which retained all NHR2 amino acids (N89) maintained full binding capacity to ETO as well as antiproliferative effects, whereas a mutant of N89 lacking 7 C-terminal amino acids (N82) significantly lost binding capacity and its antiproliferative effect. Next a codon optimized expression construct was developed in order to increase the cellular expression levels of N89. Compared to N89, expression of this construct enhances growth arrest suggesting that the NHR2 peptides act in a dose dependent manner. To investigate the influence of NC128 on primary cells, human CD34 progenitor cells were immortalized with AML1/ETO. Expression of NC128 in these cells resulted in growth arrest, loss of CD34 expression and an increased rate of apoptosis. In order to directly deliver the peptide to AML1/ETO expressing cells, the NHR2 domain was fused to the HIV-1 Tat protein transduction domain. The bacterial expressed peptides were purified by affinity chromatography and were proven to be stable in serum-containing medium for several hours. Upon protein transduction into mammalian cells, recombinant NHR2 peptides could be detected in cellular lysates. Furthermore, we already can show by co-immunoprecipitation experiments that the transducible peptides are able to specifically interact with ETO protein. Our results propose that selective interference with NHR2-mediated oligomerization could provide a promising strategy for the inhibition of the oncogenic properties of AML1/ETO. Based on our results, we are aiming to develop screening strategies for both peptides and small molecule substances to interfere with NHR2 mediated AML1/ETO complex formation.

PU.1 is dispensable to block erythroid differentiation in Friend erythroleukemia cells

Friend murine erythroleukemia cell lines derive from erythroblasts transformed with the Friend complex where the spleen-focus forming virus integrated in the vicinity of the Sfpi-1 locus. Erythroleukemia cells do not differentiate and grow indefinitely in the absence of erythropoietin. Activation of the transcription factor PU.1, encoded by the Sfpi-1 gene, is thought to be responsible for the transformed phenotype. These cells can overcome the blockage and reinitiate their differentiation program when exposed to some chemical inducers such as hexamethylene bisacetamide. In this study, we established cell cultures that were capable to proliferate unconstrained in the presence of the inducer. Resistant cell lines restart erythroid differentiation, though, if forced to exit the cell cycle or by overexpressing the transcription factor GATA-1. Unexpectedly, expression of PU.1 was suppressed in the resistant clones albeit the spleen-focus forming virus was still integrated in the proximity of the Sfpi-1 locus. Exposure to 5-Aza-2′-deoxycytidine activates PU.1 expression suggesting that the PU.1 coding gene is highly methylated in the resistant cells. Altogether these results suggest that PU.1 is dispensable to block erythroid differentiation.

Cartography of Hematopoietic Stem Cell Commitment Dependent upon a Reporter for Transcription Factor Activation

A hierarchical hematopoietic developmental tree has been proposed based on the result of prospective purification of lineage-restricted progenitors. For more detailed mapping for hematopoietic stem cell (HSC) commitment, we tracked the expression of PU.1, a major granulocyte/monocyte (GM)- and lymphoid-related transcription factor, from the HSC to the myelolymphoid progenitor stages by using a mouse line harboring a knockin reporter for PU.1. This approach enabled us to find a new progenitor population committed to GM and lymphoid lineages within the HSC fraction. This result suggests that there should be another developmental pathway independent of the conventional one with myeloid versus lymphoid bifurcation, represented by common myeloid progenitors and common lymphoid progenitors, respectively. The utilization of the transcription factor expression as a functional marker might be useful to obtain cartography of the hematopoietic development at a higher resolution.

Expression of PU.1 and terminal differentiation of alveolar macrophages in newborn rats

PU.1, which is a transcription factor, promotes the terminal differentiation of alveolar macrophages (AMs). Its expression is regulated by granulocyte/macrophage colony-stimulating factor (GM-CSF). In this study of AMs in newborn rats, we performed immunohistochemical staining, acid phosphatase staining, reverse transcriptase polymerase chain reaction (RT-PCR), quantitative real-time PCR, cytokine assay, and electron microscopy. AMs at 3 and 7 days after birth had a large foamy appearance with an intracytoplasmic accumulation of surfactants. Weak expression of PU.1 was observed in the nuclei. AMs at 15 days after birth were smaller, and PU.1 expression had increased. Ultrastructurally, AMs at 1 day after birth had a smooth surface and abundant lamellar structures in the cytoplasm, whereas AMs at 56 days after birth were characterized by (1) abundant microvillar projections on the cell surface, and (2) well-developed lysosomes and a few lamellar structures in the cytoplasm. Acid phosphatase activity and the expression of mannose receptor, scavenger receptor, and GM-CSF receptor alpha were enhanced in AMs with time after birth. These results suggest that AMs are initially immature, and that their terminal differentiation starts after birth concomitantly with an increased expression of PU.1.

Systematic RNAi Studies on the Role of Sp/KLF Factors in Globin Gene Expression and Erythroid Differentiation

Sp/KLF family of factors regulates gene expression by binding to the CACCC/GC/GT boxes in the DNA through their highly conserved three zinc finger domains. To investigate the role of this family of factors in erythroid differentiation and globin gene expression, we first measured the expression levels of selected Sp/KLF factors in primary cells of fetal and adult stages of erythroid development. This quantitative analysis revealed that their expression levels vary significantly in cells of either stages of the erythroid development. Significant difference in their expression levels was observed between fetal and adult erythroid cells for some Sp/KLF factors. Functional studies using RNA interference revealed that the silencing of Sp1 and KLF8 resulted in elevated level of γ globin expression in K562 cells. In addition, K562 cells become visibly red after Sp1 knockdown. Benzidine staining revealed significant hemoglobinization of these cells, indicating erythroid differentiation. Moreover, the expression of PU.1, ETS1 and Notch1 is significantly down-regulated in the cells that underwent erythroid differentiation following Sp1 knockdown. Overexpression of PU.1 or ETS1 efficiently blocked the erythroid differentiation caused by Sp1 knockdown in K562 cells. The expression of c-Kit, however, was significantly up-regulated. These data indicate that Sp1 may play an important role in erythroid differentiation.

Identification of PU.1 Target Genes That Are Dependent on Specific Functional Domains of the Transcription Factor PU.1.

The expression of PU.1, a member of the ets transcription factor family, is limited to the hematopoietic lineage. Using knockout and reduced PU.1 expression and gain of function mice as a model system, it has been demonstrated that PU.1 plays a key role in early myeloid and lymphoid fate decision and at later stages of myeloid differentiation and function. In PU.1 (Sfpi1) null mice, dendritic cells and monocytes could not be identified, but early myeloid progenitors and immature neutrophils were present. In mice with reduced PU.1 expression monocyte development was hindered, immature neutrophil development ensued, and the mice developed acute myeloid leukemia. The different domains of the PU.1 transcription factors have been attributed to distinct functions in early myeloid fate decision. The PU.1 transcription factor is composed of 3 major domains: transactivation, PEST, and DNA binding. We have used an immature myeloid cell line (503) derived from the Sfpi1-null mouse to analyze the effect of lentiviral gene delivery of PU.1 mutants lacking specific PU.1 domains, on differentiation of 503 cells, and to identify PU.1 target genes that are dependent on these specific domains for their expression. We found that a PU.1 mutant lacking the acidic domain leads to differentiation of the 503 cells towards neutrophil lineage, measured by specific lineage specific surface markers. Expression of a PU.1 mutant lacking the glutamine rich region of the transactivation domain did not promote differentiation to neutrophils. Moreover, the glutamine rich and acidic motifs are not necessary for the expression of the CD11b. Despite the independence of CD11b expression from both transactivation domain motifs, its expression was dependent on PU.1 DNA binding. Using cDNA microarray analysis, we identified PU.1 target genes that are regulated by specific domains of the PU.1 transcription factor. In our Affymetrix cDNA microarray analysis we identified over 1300 gene changes that are exclusively dependent on either the acidic domain or the glutamine rich region of the PU.1 transactivation domain for regulation. In addition, we found 33 PU.1- genes that are repressed by the PU.1 mutant lacking the acidic domain, but activated by the glutamine rich region PU.1 mutant, and 38 genes that are regulated in the opposite direction, thus allowing us to begin to identify the hierarchy of the PU.1-regulated gene cascade assocated with cellular function. Currently, we are investigating the biological significance of some of these differentially expressed target genes. For example, one of the identified genes, Neutrophil Elastase (ELA2), is highly upregulated in the 503 cells expressing full length PU.1, or the PU.1 mutant lacking the acidic domain, but is profoundly downregulated in the 503 cell line expressing the glutamine rich region deletion mutant. It has been shown that ELA2 deficient mice are protected form acute promyelocytic leukemia. We are currently investigating the functional role of the PU.1-dependent regulation of ELA2 in our model system. In addition, we are identifying target genes that are regulated exclusively by other PU.1 domains, i.e. the PEST domain. Taken together, we identified and are analyzing the functional importance of specific target genes in PU.1-dependent myeloid differentiation that are regulated by different functional domains of the PU.1 transcription factor.

A Putative Role for VEGFR-1 (FLT-1) in B Cell Commitment and Differentiation.

VEGF and its receptors are expressed in the hematopoietic system. A role for FLT-1 in particular was described in monocyte-macrophage migration and lineage differentiation (Sawano A et al, 2001), megakaryocytes maturation (Casella I et al, 2003) and dendritic cell differentiation (Dikov M et al, 2005). Given that the expression of this receptor in the lymphoid lineage is not known, we to studied FLT-1 expression and a putative function in normal lymphoid progenitors. To address this question we induced in vitro CD34+ cells differentiation into the B cell lineage using a well established assay (on S17 stromal cells). With this approach, we observed that FLT-1 is expressed throughout B cell differentiation increasing along the differentiation process, and reaching its highest at the “immature B cell” stage. We also neutralized FLT-1 during B cell differentiation in vitro. Surprisingly, in the presence of the FLT-1 neutralizing antibody (6.12 monoclonal Ab, from ImClone systems), at the end of the assays (4 different experiments) a significantly higher number of CD19+ cells (mainly immature B cells) were detected. Analyzing some of the transcription factors known to be involved in the commitment and differentiation of lymphoid B cells, we observed that the expression of PU.1, Pax5 and E47 was up-regulated by FLT-1 neutralization. Next, given that FLT-1 function was mainly associated with cell migration, and since it is expressed in B cells that are ready to exit the bone marrow into secondary lymphoid organs, we reasoned that FLT-1 might have a role in B cells exit from the bone marrow. For this purpose, we treated mice with the FLT-1 neutralizing Ab for 3 days and analyzed B cells levels in bone marrow and peripheral blood. FLT-1 neutralization led to a significant decrease (p<0.05) in B cells in the bone marrow and peripheral blood. Taken together, our data supports a clear role for FLT-1 in B cell commitment. To understand if VEGF/PlGF signalling through FLT-1 promotes myeloid differentiation, suppresses B cell differentiation or simply regulates the quiescent state of hematopoietic stem cells, we differentiated in vitro CD34+/FLT-1− cells and CD34+/FLT-1+ cells (10% of CD34+ cells) using the assay described above. Interestingly, CD34+/FLT-1− differentiation in vitro largely promoted B cell differentiation, while CD34+/FLT-1+ cells originated mostly myeloid cell differentiation. We are currently exploiting the molecular basis whereby FLT-1 signalling may impair B cells commitment and possibly promotes myeloid 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.

Why does the autoantibody against granulocyte‐macrophage colony‐stimulating factor cause lesions only in the lung?

During the course of investigating the etiology of idiopathic pulmonary alveolar proteinosis (IPAP), the authors found that the autoantibody against granulocyte-macrophage colony-stimulating factor (GM-CSF) was consistently present in both sera and the lung tissue. The autoantibody completely blocked bioactivity by direct binding with high specificity and avidity. Because of the existence of patients with congenital PAP who lack GM-CSF receptors and the development of PAP in GM-CSF and GM-CSF receptor knock-out mice, the autoantibody is likely to be the causative agent for IPAP. However, this finding posed the question as to why the autoantibody against GM-CSF caused lesions only in the lung. To answer this question, the authors investigated, using immunohistochemistry, confocal laser microscopy, and immunoblotting, the expression of PU.1 in tissue macrophages. PU.1 is a critical transcription factor for terminal differentiation of alveolar macrophage (AM), as reported previously in the lung, liver, spleen, kidney, intestine and brain. PU.1 was consistently expressed in the nuclei of normal AM, but faintly or not at all in IPAP-AM. Moreover, it was not expressed in the nuclei of any other normal tissue macrophages tested. Blood monocytes expressed PU.1 in the cytosol but not in the nuclei. These results suggested that the differentiation pathway is different between AM and other tissue macrophages. PU.1 was not expressed in the nuclei of newborn rat lung AM but was gradually expressed during the first 10 days. The authors demonstrated that GM-CSF is crucial for terminal differentiation of AM, but not for that of other tissue macrophages.

PU.1 Regulates Cathepsin S Expression in Professional APCs

Cathepsin S (CTSS) is a cysteine protease that is constitutively expressed in APCs and mediates processing of MHC class II-associated invariant chain. CTSS and the Ets family transcription factor PU.1 are highly expressed in cells of both myeloid (macrophages and dendritic cells) and lymphoid (B lymphocytes) lineages. Therefore, we hypothesized that PU.1 participates in the transcriptional regulation of CTSS in these cells. In A549 cells (a human epithelial cell line that does not express either CTSS or PU.1), the expression of PU.1 enhances CTSS promoter activity approximately 5- to 10-fold. In RAW cells (a murine macrophage-like cell line that constitutively expresses both CTSS and PU.1), the expression of a dominant-negative PU.1 protein and a short-interfering RNA PU.1 construct attenuates basal CTSS promoter activity, mRNA levels, and protein expression. EMSAs show binding of PU.1 to oligonucleotides derived from the CTSS promoter at two different Ets consensus binding elements. Mutation of these sites decreases the baseline CTSS activity in RAW cells that constitutively express PU.1. Chromatin immunoprecipitation experiments show binding of PU.1 with the CTSS promoter in this same region. Finally, the expression of PU.1, in concert with several members of the IFN regulatory factor family, enhances CTSS promoter activity beyond that achieved by PU.1 alone. These data indicate that PU.1 participates in the regulation of CTSS transcription in APCs. Thus, manipulation of PU.1 expression may directly alter the endosomal proteolytic environment in these cells.

Distinctive and indispensable roles of PU.1 in maintenance of hematopoietic stem cells and their differentiation

AbstractThe PU.1 transcription factor is a key regulator of hematopoietic development, but its role at each hematopoietic stage remains unclear. In particular, the expression of PU.1 in hematopoietic stem cells (HSCs) could simply represent “priming” of genes related to downstream myelolymphoid lineages. By using a conditional PU.1 knock-out model, we here show that HSCs express PU.1, and its constitutive expression is necessary for maintenance of the HSC pool in the bone marrow. Bone marrow HSCs disrupted with PU.1 in situ could not maintain hematopoiesis and were outcompeted by normal HSCs. PU.1-deficient HSCs also failed to generate the earliest myeloid and lymphoid progenitors. PU.1 disruption in granulocyte/monocyte-committed progenitors blocked their maturation but not proliferation, resulting in myeloblast colony formation. PU.1 disruption in common lymphoid progenitors, however, did not prevent their B-cell maturation. In vivo disruption of PU.1 in mature B cells by the CD19-Cre locus did not affect B-cell maturation, and PU.1-deficient mature B cells displayed normal proliferation in response to mitogenic signals including the cross-linking of surface immunoglobulin M (IgM). Thus, PU.1 plays indispensable and distinct roles in hematopoietic development through supporting HSC self-renewal as well as commitment and maturation of myeloid and lymphoid lineages.