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1
Madrange, L., P. Ehabouryi, O. Ferrandon, M. Mazeti, and J. Rodeaud. "Étude de la formation et de la stabilité des mousses chimiques de surface de la Vienne." Revue des sciences de l'eau 6, no. 3 (April 12, 2005): 315–35. http://dx.doi.org/10.7202/705178ar.
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Le recensement de la charge polluante rejetée dans la rivière Vienne (France) par les usines et les stations d'épuration de Limoges à Confolens a été effectué. Des campagnes de prélèvement et d'observations visuelles ont permis de localiser les lieux d'apparition de mousses en aval d'usines de fabrication de pâte à papier et de cartons. L'étude du pouvoir moussant des mélanges des deux principaux rejets polluants (papeterie et cartonnerie) a permis de mettre en évidence des phénomènes de synergie entre certains mélanges se traduisant à la fois par une augmentation du pouvoir moussant et de la stabilité de la mousse dans le temps. L'étude par « HPLC » montre l'apparition de pics supplémentaires confirmant l'interaction entre les constituants des rejets; le principal effluent a pu être suivi à l'aide de ses caractéristiques chimiques dans la rivière et dans les mousses jusqu'à Confolens.
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Christians, Jean-François. "Redécouverte de Schistostega pennata (Hedwig) F. Weber et D. Mohr (Schistostegaceae, Bryophyta) dans le massif du Pilat (Loire, France)." Bulletin mensuel de la Société linnéenne de Lyon 84, no. 7 (2015): 215–25. http://dx.doi.org/10.3406/linly.2015.17768.
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La redécouverte d’anciennes localités de Schistostega pennata dans le Parc naturel régional du Pilat est consécutive à des prospections ciblées, menées dans le cadre d’une enquête participative initiée par Isabelle Charissou sur le site «Bryophytes de France », également relayée par la Société Botanique du Centre-Ouest et par l’Amicale Legendre des Botanistes du Limousin. À cette occasion, de nouvelles stations ont également pu être mises en évidence. La biologie et l’écologie de cette mousse sont présentées, puis seront précisées ses répartitions ancienne et actuelle pour le massif du Pilat.
3
Thomas, Marc, Emmanuel Discamps, Mathieu Lejay, Xavier Muth, and Jean-Guillaume Bordes. "Os qui roule n’amasse pas mousse. Une expérimentation sur le tri différentiel des vestiges lithiques et osseux dans un écoulement turbulent." Paléo 33 (2023): 146–63. http://dx.doi.org/10.4000/1296o.
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Les vestiges lithiques et fauniques constituent les principaux témoins des occupations paléolithiques. Les proportions relatives de ces matériaux au sein des assemblages sont utilisées pour définir la fonction des sites ou parties de site. Or, de nombreux processus naturels sont susceptibles d’occasionner des tris sur les ensembles de vestiges, parmi lesquels les écoulements turbulents, intervenant dans de nombreux domaines (e.g. fluviatile, ruissellement concentré). Afin d’évaluer cet impact, de nombreuses expérimentations ont déjà été réalisées, mais peu d’entre elles ont concerné à la fois les vestiges fauniques et lithiques. Ainsi, l’impact comparé des écoulements de régime turbulent sur ces deux types de vestiges reste difficile à caractériser. De plus, la plupart des expérimentations connues concernent des ossements complets, parfois encore articulés, rendant toute comparaison difficile avec le référentiel fossile où les ossements sont fracturés et brûlés lors des activités de subsistance.Dans cet article, nous présentons les résultats d’une expérimentation dont l’objectif était d’évaluer l’impact relatif d’un écoulement turbulent sur différentes catégories (silex, vestiges osseux brûlés et non brûlés, tissu compact et spongieux) et classes de taille de vestiges.Le principal résultat de cette expérimentation est la mise en évidence d’une mobilité plus forte de toutes les catégories et classes de taille de vestiges fauniques par rapport aux vestiges lithiques, et ce quelle que soit leur taille.Le tri généré est corrélé à la densité des vestiges, soit, du plus mobile au moins mobile : l’os spongieux brûlé, l’os compact brûlé, l’os spongieux non brûlé, l’os compact non brûlé et le silex. Par ailleurs, nous avons pu mesurer qu’un tri dimensionnel même léger des vestiges lithiques implique un tri conséquent des ensembles de vestiges fauniques. Dans le cas inverse, lorsque l’analyse granulométrique des vestiges lithiques ne révèle pas de tri, nos expérimentations soulignent que ce résultat ne peut être avancé comme argument pour un bon degré d’intégrité des ensembles fauniques.Ainsi, lorsque des processus sédimentaires impliquant des écoulements concentrés sont mis en évidence par les études géoarchéologiques, des précautions doivent être prises concernant les interprétations d’ordre archéozoologique.
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Hwang, Cheol Kyu, Chun Sung Kim, Hack Sun Choi, Scott R. McKercher, and Horace H. Loh. "Transcriptional Regulation of Mouse μ Opioid Receptor Gene by PU.1." Journal of Biological Chemistry 279, no. 19 (March 3, 2004): 19764–74. http://dx.doi.org/10.1074/jbc.m400755200.
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We previously reported that the 34-bp cis-acting element of the mouse μ opioid receptor (MOR) gene represses transcription of the MOR gene from the distal promoter. Using a yeast one-hybrid screen to identify potential transcription factors of the MOR promoter, we have identified PU.1 as one of the candidate genes. PU.1 is a member of theetsfamily of transcription factors, expressed predominantly in hematopoietic cells and microglia of brain. PU.1 plays an essential role in the development of both lymphoid and myeloid lineages. Opioids exert neuromodulatory as well as immunomodulatory effects, which are transduced by MOR. Moreover, MOR-deficient mice exhibit increased proliferation of hematopoietic cells, suggesting a possible link between the opioid system and hematopoietic development. The PU.1 protein binds to the 34-bp element of the MOR gene in a sequence-specific manner confirmed by electrophoretic mobility shift assay and supershift assays. We have also determined endogenous PU.1 interactions with the 34-bp element of MOR promoter by chromatin immunoprecipitation assays. In co-transfection studies PU.1 represses MOR promoter reporter constructs through its PU.1 binding site. When the PU.1 gene is disrupted as in PU.1 knock-out mice and using small interfering RNA-based strategy in RAW264.7 cells, the transcription of the endogenous target MOR gene is increased significantly. This increase is probably mediated through modification of the chromatin structure, as suggested by the reversal of the PU.1-mediated repression of MOR promoter activity after trichostatin A treatment in neuroblastoma NMB cells. Our results suggest that PU.1 may be an important regulator of the MOR gene, particularly in brain and immune cells.
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Gerasimo, P., C. Duserre, and H. Metivier. "Biological Behaviour of Pu Administered to Animals as Pu-Standard LICAM(C) Complex: Therapeutical Attempts to Decrease Pu Kidney Burden." Human Toxicology 5, no. 5 (September 1986): 309–18. http://dx.doi.org/10.1177/096032718600500503.
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The biological fate of plutonium (Pu) introduced as a Pu-standard LICAM(C) complex was investigated in male rats of two strains, in male and female mice and in the baboon. We observed that, whatever the animal species or the entry route, this complex was deposited rapidly in the kidney. In addition, more of the complex accumulated in the rat (16% of injected radioactivity) than in either the mouse (7%) or baboon (5%). This Pu deposit was cleared spontaneously with a half-life of 10 days in the rat and only 5 days in the mouse. We noted that the complex was deposited on bone during this period and that, 10 days after the introduction of Pu, the skeleton became the main organ of retention of Pu (7% of the dose in the rat, 4% in mice and 3% in the baboon). In spite of this, which would indicate that Pu-standard LICAM(C) resembles a weak complex, gut transfer was comparable with that of a strong complex 1.10-3 ( f1 = 1.10-3) . Pu deposit seemed to be pH dependent and could be modified be varying the pH balance of urine. Bicarbonate was among the most effective of the different drugs used to affect this balance, as 5 h of continuous perfusion decreased the kidney Pu burden by a factor of 4. Such efficacy was also observed with diethylenetriaminepenta-acetic acid (DTPA) perfusion. The pragmatic consequence of these experiments is the recommendation of mixed therapy: standard LICAM(C) plus bicarbonate or DTPA.
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Zhou, Jing, Bo Li, Jun Wu, Fuhong He, Qiang Li, Xiaomei Yan, Yue Zhang, et al. "Essential Role for PU.1 in MEIS1 Activation and MLL Fusion Leukemia,." Blood 118, no. 21 (November 18, 2011): 3466. http://dx.doi.org/10.1182/blood.v118.21.3466.3466.
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Abstract Abstract 3466 Down-regulation of transcription factor PU.1, a key regulator of hematopoiesis, induces myeloid leukemia in mice, demonstrating a role of PU.1 as tumor suppressor. Recent studies, however, have also suggested that PU.1 is required for repopulation/self-renewal capacity of normal hematopoietic stem cells (HSCs), and presence of PU.1 activity may be necessary to favor growth of myeloid leukemia stem cells. To explore whether PU.1 could possibly act as an oncogene in the development of certain type of myeloid leukemia, we set to look for differential up-regulation of PU.1 among AML patients with distinct cytogenetic and genetic alterations in public databases. Consistent with recent molecular studies showing suppression of PU.1 expression by AML1-ETO and PML-RARa fusion proteins, PU.1 expresses at a significant lower level in AML patients with t(8;21) and t(15;17) translocations. In contrast, PU.1 expression level in MLL leukemia patients is significantly higher than that of other subgroups of AML. In addition, we found that a set of PU.1 direct target genes, as defined by genome wide location analysis of this factor, expresses at higher level in MLL leukemia patients comparing with those with t(8;21) and t(15;17) translocations, supporting an increased PU.1 activity in this subgroup of leukemia. In our effort to characterize the functional consequence of high expression of PU.1 in AML, we found that PU.1 plays an essential role in activation of MEIS1, an oncogene essential for MLL leukemia stem cell potential, and in development of MLL fusion leukemia. MEIS1, as PU.1, is differentially up-regulated in MLL leukemia patients, and expresses at a significant lower level in AML patients with t(8;21) and t(15;17) translocations. Among AML patients with higher level MEIS1 expression, a positive correlation was observed between expression of PU.1 and that of MEIS1. Using promoter reporter assay, electro mobility shift assay (EMSA) and chromatin immunoprecipiation (ChIP) analysis, we found that PU.1 directly binds to and activates MEIS1 promoter in vitro and in vivo. Analysis of a hypomorphic PU.1 mouse model indicated that PU.1 is required to maintain Meis1 expression in murine HSCs and progenitors, and knockdown of PU.1 in patient-derived MLL leukemia cell lines resulted in lower enrichment of PU.1 protein at MEIS1 promoter, accompanied by down-regulation of MEIS1 expression and decreased proliferation and survival of these cells. We are now examining whether the ability of MLL-AF9 fusion protein to drive leukemia is compromised in PU.1-deficient mouse HSC/HPCs, and whether introduction of exogenous Meis1 can compensate for the loss of PU.1 in the development of MLL-AF9 leukemia in mouse bone marrow transplantation model. Finally, we are also testing knock-down of PU.1 as a therapeutic approach to primary AMLs isolated from MLL leukemia patients. Collectively, our data indicate that PU.1 is required for the pathogenesis of MLL associated leukemia, at least partially, through direct activation of MEIS1. In veiw of the dependency of MEIS1 in MLL leukemic transformation, targeting PU.1 mediated MEIS1 gene activation could be an alternative or synergistic approach for MLL leukemia therapies aimed at inhibition of DOT1L and HOXA9. Disclosures: No relevant conflicts of interest to declare.
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Staber, Philipp B., Pu Zhang, Min Ye, Gang Huang, Boris Bartholdy, Annalisa DiRuscio, Alexander K. Ebralidze, and Daniel G. Tenen. "Autoregulation of the Transcription Factor PU.1 Via Its Evolutionarily Conserved Upstream Regulatory Element Is Critical in Adult Mouse Hematopoiesis." Blood 114, no. 22 (November 20, 2009): 1468. http://dx.doi.org/10.1182/blood.v114.22.1468.1468.
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Abstract Abstract 1468 Poster Board I-491 Background: Levels of the Ets transcription factor PU.1 control normal hematopoietic differentiation and even modest alterations can lead to leukemia and lymphoma. Regulation of PU.1 levels at different stages of hematopoiesis requires multiple interactions between several regulatory elements and transcription factors. Our previous studies identified a potential autoregulatory mechanism of the PU.1 gene through the combined activity of the proximal promoter and an evolutionarily conserved upstream regulatory element (URE), located at –14 kb relative to the transcription start site in mice. PU.1 binds to a conserved PU.1 site in the PU.1 URE both in vitro and in vivo. Approach: To ask at which stages of hematopoietic differentiation autoregulation of PU.1 via binding to its URE might play a role, we developed a mouse model with targeted disruption of the PU.1 binding site in the PU.1 URE. Results: Targeted mutation of the PU.1 autoregulatory site in PU.1 URE abolished PU.1 binding as verified by Chromatin Immuno-precipitation (ChIP). PU.1 URE activity was manifestly reduced resulting in a variety of lineage-specific abnormalities. As shown here in adult mice, the absence of the autoregulatory PU.1 site affected PU.1 expression in a lineage dependent manner. PU.1 expression was markedly decreased in phenotypic long term hematopoietic stem cells (LT-HSC: CD150+/CD48−/ c-kit+/sca-1+/lin−) and short term HSCs (ST-HSCs: CD150−/CD48+/ c-kit+/sca-1+/lin−) and, to a lesser extent, in Common Myeloid Progenitors (CMPs: lin−/c-kit+/Sca-1−/CD34+/FcrRlow), and Megakaryocyte/Erythrocyte Progenitors (MEPs: lin−/c-kit+/Sca-1−/CD34−/FcrRhigh). Within the lymphoid linage, PU.1 levels were unchanged in Common Lymphoid Progenitors (CLPs: lin−/c-kitlow/Sca-1low /IL-7Ra+/Thy1.1−) and pre-B-cells (B220+/ CD43−), up in pro-B-cells (B220+/CD43+), and down in mature B cells. Myeloid cells appeared to be unaffected. Interestingly, while PU.1 levels were decreased in LT- and ST-HSC populations, only phenotypic LT-HSCs were reduced in number. To further analyze HSC function of PU.1 site mutated mice we performed limiting dilution transplantation assays and measured the frequency of competitive repopulation units (CRU) using the congenic Ly5.1/Ly5.2 system. Our preliminary data indicated a decrease of LT-HSC function in PU.1 site mutated mice, although their homing and engraftment functions were not affected. This was also observed in mice with targeted disruption of all three AML-1 sites that are in close proximity of the PU.1 site at the PU.1 URE. While AML-1 itself appeared not to influence LT-HSC function (M. Ichikawa, T. Asai et al. Nature Medicine, 2004), we found that the conformational changes of the URE present in mice with disrupted AML-1 binding sites, as measured by Quantitative Chromosome Conformation Capture, impede PU.1 binding to its autoregulatory site. Conclusion: PU.1 indeed autoregulates its expression via binding to the -14kb URE in a lineage specific manner in vivo. Our data point to a critical role of PU.1 autoregulation especially for long-term hematopoietic stem cell function. Disclosures: No relevant conflicts of interest to declare.
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Bonfield, Tracey L., Baisakhi Raychaudhuri, Anagha Malur, Susamma Abraham, Bruce C. Trapnell, Mani S. Kavuru, and Mary Jane Thomassen. "PU.1 regulation of human alveolar macrophage differentiation requires granulocyte-macrophage colony-stimulating factor." American Journal of Physiology-Lung Cellular and Molecular Physiology 285, no. 5 (November 2003): L1132—L1136. http://dx.doi.org/10.1152/ajplung.00216.2003.
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Granulocyte-macrophage colony-stimulating factor (GM-CSF) is critically implicated in lung homeostasis in the GM-CSF knockout mouse model. These animals develop an isolated lung lesion reminiscent of pulmonary alveolar proteinosis (PAP) seen in humans. The development of the adult form of human alveolar proteinosis is not due to the absence of a GM-CSF gene or receptor defect but to the development of an anti-GM-CSF autoimmunity. The role of GM-CSF in the development of PAP is unknown. Studies in the GM-CSF knockout mouse have shown that lack of PU.1 protein expression in alveolar macrophages is correlated with decreased maturation, differentiation, and surfactant catabolism. This study investigates PU.1 expression in vitro and in vivo in human PAP alveolar macrophages as well as the regulation of PU.1 by GM-CSF. We show for the first time that PU.1 mRNA expression in PAP bronchoalveolar lavage cells is deficient compared with healthy controls. PU.1-dependent terminal differentiation markers CD32 (FCγII), mannose receptor, and macrophage colony-stimulating factor receptor (M-CSFR) are decreased in PAP alveolar macrophages. In vitro studies demonstrate that exogenous GMCSF treatment upregulated PU.1 and M-CSFR gene expression in PAP alveolar macrophages. Finally, in vivo studies showed that PAP patients treated with GM-CSF therapy have higher levels of PU.1 and M-CSFR expression in alveolar macrophages compared with healthy control and PAP patients before GM-CSF therapy. These observations suggest that PU.1 is critical in the terminal differentiation of human alveolar macrophages.
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Mak, Ka Sin, Alister P. W. Funnell, Richard C. M. Pearson, and Merlin Crossley. "PU.1 and Haematopoietic Cell Fate: Dosage Matters." International Journal of Cell Biology 2011 (2011): 1–6. http://dx.doi.org/10.1155/2011/808524.
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The ETS family transcription factor PU.1 is a key regulator of haematopoietic differentiation. Its expression is dynamically controlled throughout haematopoiesis in order to direct appropriate lineage specification. Elucidating the biological role of PU.1 has proved challenging. This paper will discuss how a range of experiments in cell lines and mutant and transgenic mouse models have enhanced our knowledge of the mechanisms by which PU.1 drives lineage-specific differentiation during haematopoiesis.
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Ghisi, Margherita, Mark D. McKenzie, Ethan P. Oxley, Emilia Simankowicz, Cynthia Liu, Aleksandar Dakic, Stephen L. Nutt, Matthew E. Ritchie, Johannes Zuber, and Ross A. Dickins. "Uncovering Key Downstream Effectors of PU.1 Tumor Suppression in Acute Myeloid Leukemia." Blood 128, no. 22 (December 2, 2016): 2698. http://dx.doi.org/10.1182/blood.v128.22.2698.2698.
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Abstract Mutation or altered expression of key transcription factors resulting in aberrant myeloid differentiation is a critical step in the pathogenesis of acute myeloid leukemia (AML). The ETS-domain hematopoietic transcription factor PU.1 (SPI1) is an essential regulator of myeloid differentiation. While genetic mutation of PU.1 is rare, PU.1 is down-regulated or functionally repressed in about 50% of AML patients as a consequence of recurrent translocations (such as AML1-ETO and PML-RARα) or mutations (such as FLT3-ITD). While it is well-established that the inhibition of PU.1 function promotes AML development, our understanding of the downstream targets mediating its tumor-suppressor role is still incomplete. In order to study PU.1-driven differentiation in AML, our laboratory has recently developed a mouse model of AML driven by doxycycline (Dox)-regulated knockdown of the PU.1 gene (shPU.1) on a p53-deficient background. This is an aggressive AML model that mimics high risk disease in the clinic, as loss of function mutations of p53 characterize a subset of AML patients with particularly poor prognosis. In this system, Dox treatment shuts off a GFP-linked shRNA specifically targeting PU.1. Restoration of PU.1 expression in the context of established leukemias in vivo, and in culture-adapted cell lines derived from them, triggered myeloid differentiation and induction of cell death. In order to study the dynamic transcriptional changes underpinning these effects, we performed genome-wide transcriptome analysis on shPU.1/p53-/- AML cells isolated from the bone marrow of multiple leukemic mice transplanted with 2 different primary AML (246 and 410) that were either untreated or Dox-treated for 2, 4, or 6 days. Dox-induced restoration of PU.1 expression led to dramatic transcriptional changes closely resembling those associated with the maturation of normal mouse granulocyte/macrophage progenitors (GMPs) into neutrophils. Among the differentially expressed genes at day 2 of Dox-treatment we identified 12 "early response" genes commonly induced in both 246 and 410 AML. This group of genes included the macrophage colony-stimulating factor receptor (Csf1r), a well-known target of PU.1, as well as other genes previously implicated in myeloid differentiation, cytokine signalling, survival and cancer. In order to explore the contribution of these "early-response" genes to the range of anti-leukemic effects exerted by PU.1 restoration, we have generated retroviral shRNA vectors to test whether specifically silencing each of these genes alters PU.1-induced differentiation, inhibition of proliferation and/or death of p53-/- AML cells in vitro. In conclusion, in this work we have used a novel AML mouse model driven by reversible PU.1 inhibition to identify the PU.1-regulated transcriptome in AML, and to screen for key PU.1-response genes mediating its pro-differentiation and anti-leukemic effects in the context of this disease. Disclosures No relevant conflicts of interest to declare.
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Vangala, Rajani K., Marion S. Heiss-Neumann, Janki S. Rangatia, Sheo M. Singh, Claudia Schoch, Daniel G. Tenen, Wolfgang Hiddemann, and Gerhard Behre. "The myeloid master regulator transcription factor PU.1 is inactivated by AML1-ETO in t(8;21) myeloid leukemia." Blood 101, no. 1 (January 1, 2003): 270–77. http://dx.doi.org/10.1182/blood-2002-04-1288.
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Abstract The 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.
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Pospisil, Vitek, Emanuel Necas, and Tomas Stopka. "PU.1 Activity Determines Fate of Myeloid Progenitor Cells during Lineage Commitment." Blood 108, no. 11 (November 16, 2006): 4207. http://dx.doi.org/10.1182/blood.v108.11.4207.4207.
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Abstract Myeloid cell commitment is regulated by factors interacting with chromatin in a progenitor cell entering differentiation. PU.1 is an ETS family transcription factor that has been well characterized in inducing myelopoiesis and blocking erythroid differentiation. Conditionally activated PU.1-Estrogen Receptor transgene in mouse PU.1 knockout-derived hematopoietic progenitors is known to induce macrophage differentiation. We observed that manipulation of PU.1 activity by using different levels of PU.1-ER activator, tamoxifen, was capable of producing major populations of myeloid progeny including macrophages, granulocytes, lymphocytes and mastocytes. Gradual activation of PU.1 produce distinct committed populations that require presence of PU.1 activator. Withdrawal of tamoxifen from macrophages restored cycling and pluripotent potential resembling parental cell line. Furthermore, very low PU.1 activity induced frequent chromosomal aberations such as polyploidy. To determine that PU.1 directly caused these phenotypic changes we determined its occupancy at genes representing PU.1 targets by quantitative chromatin immunoprecipitation (ChIP). We demonstrate that PU.1 is enriched at regulatory regions of lineage restricted cell populations including promoters of CD11b, CD14, IL7R, PAX5, RAG1, Gelatinase, Lysozyme, and MCP. Different PU.1 levels promote distinct cell populations that have different localizations of PU.1 in chromatin. We also tested mRNA levels of PU.1 target genes in progenitor cell line induced by PU.1. Macrophage-specific target genes such as CD11b and CD14 are induced at high PU.1 levels and granulocytic PU.1 targets such as Gelatinase and Lysozyme are induced at intermediate and high PU.1 levels. B-cell specific mRNAs such as IL7-R, PAX5, and RAG1 were induced at low and high PU.1 levels. Our data suggest that PU.1 is recruited to and transactivates its lineage-restricted target genes differently at distinct PU.1 levels and such mechanism may be involved in myeloid cell commitment and leukemogenesis.
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Chavez, James S., Jennifer L. Rabe, Giovanny Hernandez, Taylor S. Mills, Katia E. Niño, Pavel Davizon-Castillo, and Eric M. Pietras. "PU.1 Expression Defines Distinct Functional Activities in the Phenotypic HSC Compartment of a Murine Inflammatory Stress Model." Cells 11, no. 4 (February 15, 2022): 680. http://dx.doi.org/10.3390/cells11040680.
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The transcription factor PU.1 is a critical regulator of lineage fate in blood-forming hematopoietic stem cells (HSC). In response to pro-inflammatory signals, such as the cytokine IL-1β, PU.1 expression is increased in HSC and is associated with myeloid lineage expansion. To address potential functional heterogeneities arising in the phenotypic HSC compartment due to changes in PU.1 expression, here, we fractionated phenotypic HSC in mice using the SLAM surface marker code in conjunction with PU.1 expression levels, using the PU.1-EYFP reporter mouse strain. While PU.1lo SLAM cells contain extensive long-term repopulating activity and a molecular signature corresponding to HSC activity at steady state, following IL-1β treatment, HSCLT induce PU.1 expression and are replaced in the PU.1lo SLAM fraction by CD41+ HSC-like megakaryocytic progenitors (SL-MkP) with limited long-term engraftment capacity. On the other hand, the PU.1hi SLAM fraction exhibits extensive myeloid lineage priming and clonogenic activity and expands rapidly in response to IL-1β. Furthermore, we show that EPCR expression, but not CD150 expression, can distinguish HSCLT and SL-MkP under inflammatory conditions. Altogether, our data provide insights into the dynamic regulation of PU.1 and identify how PU.1 levels are linked to HSC fate in steady state and inflammatory stress conditions.
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Ghani, Saeed, Pia Riemke, Jörg Schönheit, Dido Lenze, Jürgen Stumm, Maarten Hoogenkamp, Anne Lagendijk, et al. "Macrophage development from HSCs requires PU.1-coordinated microRNA expression." Blood 118, no. 8 (August 25, 2011): 2275–84. http://dx.doi.org/10.1182/blood-2011-02-335141.
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Abstract The differentiation of HSCs into myeloid lineages requires the transcription factor PU.1. Whereas PU.1-dependent induction of myeloid-specific target genes has been intensively studied, negative regulation of stem cell or alternate lineage programs remains incompletely characterized. To test for such negative regulatory events, we searched for PU.1-controlled microRNAs (miRs) by expression profiling using a PU.1-inducible myeloid progenitor cell line model. We provide evidence that PU.1 directly controls expression of at least 4 of these miRs (miR-146a, miR-342, miR-338, and miR-155) through temporally dynamic occupation of binding sites within regulatory chromatin regions adjacent to their genomic coding loci. Ectopic expression of the most robustly induced PU.1 target miR, miR-146a, directed the selective differentiation of HSCs into functional peritoneal macrophages in mouse transplantation assays. In agreement with this observation, disruption of Dicer expression or specific antagonization of miR-146a function inhibited the formation of macrophages during early zebrafish (Danio rerio) development. In the present study, we describe a PU.1-orchestrated miR program that mediates key functions of PU.1 during myeloid differentiation.
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Lin, Ligen, Weijun Pang, Keyun Chen, Fei Wang, Jon Gengler, Yuxiang Sun, and Qiang Tong. "Adipocyte expression of PU.1 transcription factor causes insulin resistance through upregulation of inflammatory cytokine gene expression and ROS production." American Journal of Physiology-Endocrinology and Metabolism 302, no. 12 (June 15, 2012): E1550—E1559. http://dx.doi.org/10.1152/ajpendo.00462.2011.
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We have reported previously that ETS family transcription factor PU.1 is expressed in mature adipocytes of white adipose tissue. PU.1 expression is increased greatly in mouse models of genetic or diet-induced obesity. Here, we show that PU.1 expression is increased only in visceral but not subcutaneous adipose tissues of obese mice, and the adipocytes are responsible for this increase in PU.1 expression. To further address PU.1's physiological function in mature adipocytes, PU.1 was knocked down in 3T3-L1 cells using retroviral-mediated expression of PU.1-targeting shRNA. Consistent with previous findings that PU.1 regulates its target genes, such as NADPH oxidase subunits and proinflammatory cytokines in myeloid cells, the mRNA levels of proinflammatory cytokines (TNFα, IL-1β, and IL-6) and cytosolic components of NADPH oxidase (p47phox and p40phox) were downregulated significantly in PU.1-silenced adipocytes. NADPH oxidase is a main source for reactive oxygen species (ROS) generation. Indeed, silencing PU.1 suppressed NADPH oxidase activity and attenuated ROS in basal or hydrogen peroxide-treated adipocytes. Silencing PU.1 in adipocytes suppressed JNK1 activation and IRS-1 phosphorylation at Ser307. Consequently, PU.1 knockdown improved insulin signaling and increased glucose uptake in basal and insulin-stimulated conditions. Furthermore, knocking down PU.1 suppressed basal lipolysis but activated stimulated lipolysis. Collectively, these findings indicate that obesity induces PU.1 expression in adipocytes to upregulate the production of ROS and proinflammatory cytokines, both of which lead to JNK1 activation, insulin resistance, and dysregulation of lipolysis. Therefore, PU.1 might be a mediator for obesity-induced adipose inflammation and insulin resistance.
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Karpurapu, Manjula, Kavita Kumari Kakarala, Sangwoon Chung, Yunjuan Nie, Amritendu Koley, Patrick Dougherty, and John W. Christman. "Epigallocatechin gallate regulates the myeloid-specific transcription factor PU.1 in macrophages." PLOS ONE 19, no. 4 (April 25, 2024): e0301904. http://dx.doi.org/10.1371/journal.pone.0301904.
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Our previous research demonstrated that PU.1 regulates expression of the genes involved in inflammation in macrophages. Selective knockdown of PU.1 in macrophages ameliorated LPS-induced acute lung injury (ALI) in bone marrow chimera mice. Inhibitors that block the transcriptional activity of PU.1 in macrophages have the potential to mitigate the pathophysiology of LPS-induced ALI. However, complete inactivation of PU.1 gene disrupts normal myelopoiesis. Although the green tea polyphenol Epigallocatechin gallate (EGCG) has been shown to regulate inflammatory genes in various cell types, it is not known if EGCG alters the transcriptional activity of PU.1 protein. Using Schrodinger Glide docking, we have identified that EGCG binds with PU.1 protein, altering its DNA-binding and self-dimerization activity. In silico analysis shows that EGCG forms Hydrogen bonds with Glutamic Acid 209, Leucine 250 in DNA binding and Lysine 196, Tryptophan 193, and Leucine 182 in the self-dimerization domain of the PU.1 protein. Experimental validation using mouse bone marrow-derived macrophages (BMDM) confirmed that EGCG inhibits both DNA binding by PU.1 and self-dimerization. Importantly, EGCG had no impact on expression of the total PU.1 protein levels but significantly reduced expression of various inflammatory genes and generation of ROS. In summary, we report that EGCG acts as an inhibitor of the PU.1 transcription factor in macrophages.
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Antony-Debre, Ileana, Ananya Paul, Joana Leite, Kelly Mitchell, Hye Mi Kim, Luis A. Carvajal, Tihomira Tidorova, et al. "Direct Pharmacological Inhibition of the Transcription Factor PU.1 in Acute Myeloid Leukemia." Blood 130, Suppl_1 (December 7, 2017): 858. http://dx.doi.org/10.1182/blood.v130.suppl_1.858.858.
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Abstract Functionally critical decreases in levels or activity of the ETS family transcription factor PU.1 are present in approximately 2/3 of patients with acute myeloid leukemia (AML), across different AML subtypes (Sive, Leukemia 2016) including at the stem cell level (Steidl, Nat Genet 2006; Will, Nat Med 2015). Thus, targeting PU.1 could be an appealing option for treatment. As complete loss of PU.1 leads to stem cell failure (Iwasaki, Blood 2005), we hypothesized that PU.1 inhibition could eradicate leukemic cells harboring already low levels of PU.1, with modest effects on normal cells. We initially tested this hypothesis using 3 different shRNAs, and found that PU.1 inhibition led to a significant decrease in proliferation and clonogenicity, and increased apoptosis of mouse and human leukemic cell lines with low PU.1 levels, as well as the majority of primary human AML cells tested. We demonstrated that these effects were indeed due to decreased PU.1 levels by retroviral add-back experiments. The direct pharmacologic targeting of transcription factors has proven challenging in the past. Besides the core ETS binding motif (GGAA) in the DNA major groove, PU.1 binding to chromatin depends on additional minor groove contacts enriched for AT nucleotides upstream of the ETS motif, which determine selectivity for PU.1. Using an integrated screening strategy utilizing biosensor surface plasmon resonance, DNA footprinting, and cell-based dual-color PU.1 reporter assays, we developed novel small molecules of the heterocyclic diamidine family acting as first-in-class PU.1 inhibitors. Targeted occupancy by our compounds in the minor groove induces perturbations in DNA conformation that are transmitted to the PU.1 site in the major groove and thus inhibits PU.1 binding via an allosteric mechanism. Consistent with this, the inhibitory effects were selective for PU.1 versus other ETS transcription factors. Treatment with 3 different compounds led to cell growth inhibitory effect with respect to PU.1 level and preferentially affects PU.1low AML cells. Similarly to what we observed with shRNAs, treatment with our novel inhibitors led to decreased proliferation and colony forming capacity, increased apoptosis, and disrupted serial replating capacity of PU.1low AML cells and a majority of primary AML cell samples. Targeted ChIP and expression analysis showed that the compounds disrupt PU.1-promoter interaction and lead to downregulation of canonical PU.1 transcriptional targets in AML cells, confirming on-target activity in AML cells. Genome-wide analysis showed highly significant enrichment of known transcriptional targets of PU.1, and selectivity over genes regulated by other ETS family members. Comparison with published transcriptomic and PU.1 ChIP-seq data sets, as well as ARACNe analysis of the PU.1 regulon in primary AML cells, demonstrated that the inhibitors antagonize PU.1-regulated pathways at a genome-wide level. ChIP-seq performed in PU.1low AML cells confirmed a genome-wide decrease of PU.1 peaks after treatment and provides novel insight into the molecular mechanisms mediating the anti-leukemic effects of pharmacological PU.1 inhibition. To test the effects of PU.1 inhibition on normal hematopoiesis, we treated normal hematopoietic stem/progenitors cells (HSPC) in colony forming assays and saw decreased production of mature granulo-monocytic cells, consistent with PU.1's known role in this lineage. However, this effect was reversible upon drug removal, and serial replating capacity was not affected suggesting no significant effects on more immature HSPC. Congenic transplantation assays of treated normal bone marrow cells led to no change in myeloid and T-cells and only a modest decrease in B-cell numbers. Lastly, in vivo treatment with PU.1 inhibitors in mouse and human AML (xeno)transplantation models significantly decreased tumor burden and increased survival. To conclude, our study provides proof-of-principle for PU.1 inhibition as a novel therapeutic strategy in AML. Furthermore, we present the development of first-in-class PU.1 inhibitors acting via an allosteric minor groove-mediated mechanism. Our work shows that the specific pharmacological targeting of the DNA interaction of transcription factors such as PU.1 is feasible in principle, and may open the way for targeting of other transcription factors through minor groove-directed approaches. Disclosures Will: Novartis Pharmaceuticals: Consultancy, Research Funding. Steidl: Celgene: Consultancy; Aileron Therapeutics: Consultancy, Research Funding; Novartis: Research Funding; GlaxoSmithKline: Research Funding; Bayer Healthcare: Consultancy.
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Lian, Ming, Yu Fang Jiang, Shi Dong Lv, Yi Long He, Jiang Sheng Zhou, and Qing Xiong Meng. "The Anti-Obesity Effect of Instant Pu-Erh Black Tea in Mice with Hydrogenated Oil Diet-Induced Obesity." Applied Mechanics and Materials 644-650 (September 2014): 5248–51. http://dx.doi.org/10.4028/www.scientific.net/amm.644-650.5248.
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Obesity is becoming a worldwide epidemic disease, and the incidence is increasing year by year. Drinking tea has been demonstrated to have multiple beneficial effects to obese patients. This article tested the effect of instant Pu-erh black tea on weight loss in diet-induced obese (DIO) mice. The results demonstrated that instant Pu-erh black tea was be able to reduce the mouse weight dose-dependently. Its effect is stronger than that of L-carnitine, a weight loss drug currently on the market. Instant Pu-erh black tea also accelerated lipid metabolism and eased the high-fat diet-induced liver injury.
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Rekhtman, Natasha, Kevin S. Choe, Igor Matushansky, Stuart Murray, Tomas Stopka, and Arthur I. Skoultchi. "PU.1 and pRB Interact and Cooperate To Repress GATA-1 and Block Erythroid Differentiation." Molecular and Cellular Biology 23, no. 21 (November 1, 2003): 7460–74. http://dx.doi.org/10.1128/mcb.23.21.7460-7474.2003.
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ABSTRACT PU.1 and GATA-1 are two hematopoietic specific transcription factors that play key roles in development of the myeloid and erythroid lineages, respectively. The two proteins bind to one another and inhibit each other's function in transcriptional activation and promotion of their respective differentiation programs. This mutual antagonism may be an important aspect of lineage commitment decisions. PU.1 can also act as an oncoprotein since deregulated expression of PU.1 in erythroid precursors causes erythroleukemias in mice. Studies of cultured mouse erythroleukemia cell lines indicate that one aspect of PU.1 function in erythroleukemogenesis is its ability to block erythroid differentiation by repressing GATA-1 (N. Rekhtman, F. Radparvar, T. Evans, and A. I. Skoultchi, Genes Dev. 13:1398-1411, 1999). We have investigated the mechanism of PU.1-mediated repression of GATA-1. We report here that PU.1 binds to GATA-1 on DNA. We localized the repression activity of PU.1 to a small acidic N-terminal domain that interacts with the C pocket of pRB, a well-known transcriptional corepressor. Repression of GATA-1 by PU.1 requires pRB, and pRB colocalizes with PU.1 and GATA-1 at repressed GATA-1 target genes. PU.1 and pRB also cooperate to block erythroid differentiation. Our results suggest that one of the mechanisms by which PU.1 antagonizes GATA-1 is by binding to it at GATA-1 target genes and tethering to these sites a corepressor that blocks transcriptional activity and thereby erythroid differentiation.
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Ye, Min, Olga Ermaermakova-Cirilli, and Thomas Graf. "B Cell Development in the Absence of PU.1." Blood 104, no. 11 (November 16, 2004): 226. http://dx.doi.org/10.1182/blood.v104.11.226.226.
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Abstract 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.
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Saunthararajah, Yogenthiran, SiJun Yang, ShriHari Kadkol, Marie Baraoidan, Vinzon Ibanez, and Fariborz Mortazavi. "GATA-1, but Not GATA-2, Antagonizes PU.1 Mediated Transcriptional Activity at the CBFA2T3 (ETO2, MTG16) Promoter through a Mechanism Dependent on GATA DNA Binding." Blood 106, no. 11 (November 16, 2005): 1749. http://dx.doi.org/10.1182/blood.v106.11.1749.1749.
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Abstract CBFA2T3 (ETO2, MTG16), a target of chromosomal translocation in acute myeloid leukemia, has its highest expression in hematopoietic cells compared to other tissues. This suggests that its expression is regulated by major hematopoietic transcription factors. The proximal promoter from −171 to −65 bp has greater than 90% identity between mouse and human and contains recognition sites for major hematopoietic transcription factors PU.1, GATA-1 and GATA-2. Using chromatin immuno-precipitation and the MPD hematopoietic cell-line, this segment was pulled down with endogenous PU.1, GATA-1 and GATA-2. In luciferase reporter gene assays, PU.1 and GATA-2, but not GATA-1, activated the promoter. As would be expected from these findings, CBFA2T3 levels declined during terminal erythroid differentiation of primary hematopoietic cells. GATA-1, but not GATA-2, antagonized PU.1 mediated activation but this effect of GATA-1 was abrogated by mutation of the GATA DNA binding sites. Both GATA-1 and GATA-2 have been reported to antagonize PU.1 transcriptional activity by antagonizing PU.1 interactions with c-Jun (Zhang et al, Proc Natl Acad Sci USA1999;96:8705–8710); however, the DNA binding dependent mechanism reported here allows GATA-2 and GATA-1 to have contrasting relationships with PU.1 and may be the basis for the co-operation of GATA proteins with PU.1 in some contexts yet antagonism of PU.1 activity in others.
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Iino, Tadafumi, Hiromi Iwasaki, Kentaro Kohno, Shin-ichi Mizuno, Yojiro Arinobu, Daniel G. Tenen, Boris Reizis, and Koichi Akashi. "Selective Disruption of PU.1 in Mature Dendritic Cells Affects Their Tissue Distribution and T Cell Homeostasis." Blood 118, no. 21 (November 18, 2011): 518. http://dx.doi.org/10.1182/blood.v118.21.518.518.
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Abstract Abstract 518 PU.1, a hematopoietic transcription factor, is indispensable for development of conventional dendritic cells (cDCs) from hematopoietic stem cells. However, the function of PU.1 in mature cDC remains unclear. To test the possible role of PU.1 in mature cDCs, we developed mice lacking PU.1 selectively in mature cDCs (DC-PU.1D/D mice) by crossing a PU.1flox mouse line with a transgenic Itgax (CD11c)-Cre strain. In these mice, cDCs were dramatically reduced in spleen, thymus, lymph node, and skin, down to <40%, <25%, <10% and <5% of DCs in control mice respectively, whereas bone marrow cDCs and common dendritic cells progenitors (CDPs) were not affected. Surprisingly, T cell numbers were significantly decreased in DC-PU.1D/D mice, whereas thymic T cell development was normal, suggesting that maintenance of mature T cell pool might be impaired, presumably by dysfunction of PU.1D/D cDCs. In fact, PU.1D/D cDCs failed to efficiently induce ovalbumin-specific T cell response and to produce inflammatory cytokines in response to Toll like receptor (TLR) stimulation both in vitro and in vivo. The intravenous transfer of spleen PU.1D/D cDCs failed to repopulate the spleen of recipient mice, suggesting their poor survival in vivo. Furthermore, the expression of critical molecules for inflammatory responses was downregulated in PU.1D/D cDCs as compared to normal cDCs. These molecules included Myd88 and NFkB that are downstream molecules of TLR signaling, CD86 that is required for T cell stimulation, and CCR7 that is required for cDC migration. These results clearly show that PU.1 is required for development of the functional cDC pool, and the cDC pool plays a critical role in T cell homeostasis. Disclosures: No relevant conflicts of interest to declare.
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Welner, Robert S., Danielle E. Tenen, Henry Yang, Deepak Bararia, Giovanni Amabile, and Daniel G. Tenen. "Relationship Between Self-Renewal and Differentiation Pathways in Stem Cells and Leukemia." Blood 124, no. 21 (December 6, 2014): 4789. http://dx.doi.org/10.1182/blood.v124.21.4789.4789.
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Abstract Hematopoietic stem cells are capable of perpetual self-renewal and multi-lineage differentiation, properties that are maintained throughout life by minimal cell cycle activity. Our work has focused on deciphering transcriptional driven differentiation versus self-renewal pathways in stem and progenitor cells. To this end, we have studied transcription factors that control the fate of hematopoietic stem cells by combining mouse models of activated self-renewal with models that can report transcription factor expression. We chose to study the Wnt pathway, activated in several types of leukemia, in combination with the ets family PU.1 transcription factor, vital to almost all myeloid and lymphoid lineages. PU.1 regulates a number of important myeloid specific genes that mediate differentiation to a specific cell fate. To understand the interaction of these pathways, we found that over-expression of Wnt signaling or beta-catenin, the downstream signaling component of the Wnt pathway, was able to inhibit PU.1-mediated differentiation in a PU.1-inducible cell line. There was little to no up-regulation of the myeloid markers Mac1 or Gr1 with activation of Wnt signaling upon induction with 4-hydroxy-tamoxifen (4-OHT). Additionally, many genes related to myeloid differentiation were not increased as compared to control-induced cultures. To understand how these interactions might function in vitro, we crossed a Cre-responsive activated beta-catenin (floxed allele Exon3) mouse to a PU.1-GFP knock-in mouse. From this model, we are able to see changes in PU.1 (GFP) expression in specific populations of hematopoietic progenitors upon activation of beta-catenin. Most importantly, in the LT-HSCs (defined by Lin- cKitHi Sca1+ CD150+ CD48-), we observed a significant increase in GFP (PU.1) intensity upon activation of active beta-catenin. Additionally, there was an increase in the total number of LT-HSCs, as defined by surface markers. LT-HSCs with active beta-catenin and GFP (PU.1) were found to be more in cycle and they express lower levels of transcription factors related to differentiation. These results demonstrate that when beta-catenin is activated, PU.1’s role is modified and the self-renewal program is enhanced at the expense of differentiation. Furthermore, activation of beta-catenin in the hematopoietic cells of mice has been shown to lead to impaired differentiation and eventual death. Even though active beta-catenin has been shown to be essential in several subtypes of myeloid leukemias using murine models, its over-expression is not sufficient to lead to leukemic development. However, heterozygous PU.1/GFP knock-in mice were crossed to the beta-catenin overexpression model, they rapidly developed leukemia post Cre induction. This is not observed in the PU.1/GFP knock-in mice in the absence of beta-catenin activation, suggesting that Wnt signaling adds to a block in differentiation needed for leukemic transformation. These mice show splenomegaly and increased myelocytic populations in the peripheral blood. The leukemia was transplantable to secondary mice and expressed high levels of GFP (PU.1) in the spleen, bone marrow and peripheral blood. These findings demonstrate that the interaction and crosstalk between these two pathways regulate hematopoietic stem cell fate. Future studies will focus on understanding how this interaction between transcription factor and self-renewal pathways becomes disrupted in leukemic stem cells. Disclosures No relevant conflicts of interest to declare.
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Wei, Fang, Kristina Zaprazna, Junwen Wang, and Michael L. Atchison. "PU.1 Can Recruit BCL6 to DNA To Repress Gene Expression in Germinal Center B Cells." Molecular and Cellular Biology 29, no. 17 (June 29, 2009): 4612–22. http://dx.doi.org/10.1128/mcb.00234-09.
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ABSTRACT BCL6 is a transcriptional repressor crucial for germinal center formation. BCL6 represses transcription by a variety of mechanisms by binding to specific DNA sequences or by recruitment to DNA by protein interactions. We found that BCL6 can inhibit activities of the immunoglobulin kappa (Igκ) intron and 3′ enhancers. At the Igκ 3′ enhancer, BCL6 repressed enhancer activity through the PU.1 binding site. We found that BCL6 physically interacted with PU.1 in vivo and in vitro, and the results of sequential chromatin immunoprecipitation assays and transient-expression assays suggested that BCL6 recruitment to the Igκ and Igλ 3′ enhancers occurred via PU.1 interaction. By computational studies, we identified genes that are repressed in germinal center cells and whose promoters contain conserved PU.1 binding sites in mouse and human. We found that many of these promoters bound to both PU.1 and BCL6 in vivo. In addition, BCL6 knockdown resulted in increased expression of a subset of these genes, demonstrating that BCL6 is involved in their repression. The recruitment of BCL6 to promoter regions by PU.1 represents a new regulatory mechanism that expands the number of genes regulated by this important transcriptional repressor.
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Yona, Simon, and Alexander Mildner. "Good things come in threes." Science Immunology 3, no. 30 (December 7, 2018): eaav5545. http://dx.doi.org/10.1126/sciimmunol.aav5545.
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Bartholdy, Boris, Yukiya Yamamoto, Erica Evans, John Crispino, and Daniel G. Tenen. "PU.1 - c-Jun Interactions Are Crucial for PU.1 Function in Myeloid Development." Blood 114, no. 22 (November 20, 2009): 3651. http://dx.doi.org/10.1182/blood.v114.22.3651.3651.
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Abstract Abstract 3651 Poster Board III-587 The Ets transcription factor PU.1 is a master regulator absolutely required for the differentiation of monocytes, macrophages, and B cells in the fetal liver and in the adult bone marrow. PU.1 drives hematopoietic differentiation partly through direct protein-protein interactions with other transcription factors, such as the AP-1 transcription factor c-Jun. We have shown that c-Jun can be recruited to promoters which do not include AP-1 binding sites, such as the MCSFR promoter, and act as a PU.1-dependent co-activator. To address the functional importance of this interaction, we identified and studied PU.1 point mutants that lost the capability to physically interact with c-Jun while retaining normal DNA binding affinity. These mutants failed to efficiently transactivate a PU.1 target reporter, and, more importantly, were unable to induce monocyte/macrophage differentiation of the PU.1-deficient immature myeloid 503 cell line. Subsequently, we have generated knock-in mouse models harboring these single point mutations by means of homologous recombination. The mutant mice phenotypically resemble PU.1-deficient mice, have an early block in hematopoiesis, and die perinatally. We show that the mutant PU.1 mRNA and protein is expressed in long-term and short-term hematopoietic stem cells, but that the maturation into lymphoid primed multipotent progenitor (LMPP) and later progenitor populations is severely blocked, leading to an almost complete loss of mature B, T and myeloid cells. Collectively, our data strongly suggest that the PU.1-c-Jun interaction is crucial for normal PU.1 function in vivo during murine fetal hematopoiesis. Disclosures: No relevant conflicts of interest to declare.
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O’Brien, Gráinne, Lourdes Cruz-Garcia, Joanna Zyla, Natalie Brown, Rosemary Finnon, Joanna Polanska, and Christophe Badie. "Kras mutations and PU.1 promoter methylation are new pathways in murine radiation-induced AML." Carcinogenesis 41, no. 8 (October 24, 2019): 1104–12. http://dx.doi.org/10.1093/carcin/bgz175.
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Abstract Therapy-related and more specifically radiotherapy-associated acute myeloid leukaemia (AML) is a well-recognized potential complication of cytotoxic therapy for the treatment of a primary cancer. The CBA mouse model is used to study radiation leukaemogenesis mechanisms with Sfpi1/PU.1 deletion and point mutation already identified as driving events during AML development. To identify new pathways, we analysed 123 mouse radiation-induced AML (rAML) samples for the presence of mutations identified previously in human AML and found three genes to be mutated; Sfpi1 R235 (68%), Flt3-ITD (4%) and Kras G12 (3%), of which G12R was previously unreported. Importantly, a significant decrease in Sfpi1 gene expression is found almost exclusively in rAML samples without an Sfpi1 R235 mutation and is specifically associated with up-regulation of mir-1983 and mir-582-5p. Moreover, this down-regulation of Sfpi1 mRNA is negatively correlated with DNA methylation levels at specific CpG sites upstream of the Sfpi1 transcriptional start site. The down regulation of Sfpi1/PU.1 has also been reported in human AML cases revealing one common pathway of myeloid disruption between mouse and human AML where dysregulation of Sfpi1/PU.1 is a necessary step in AML development.
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Ha, Soon-Duck, Woohyun Cho, Rodney P. DeKoter, and Sung Ouk Kim. "The transcription factor PU.1 mediates enhancer-promoter looping that is required for IL-1β eRNA and mRNA transcription in mouse melanoma and macrophage cell lines." Journal of Biological Chemistry 294, no. 46 (October 4, 2019): 17487–500. http://dx.doi.org/10.1074/jbc.ra119.010149.
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The DNA-binding protein PU.1 is a myeloid lineage–determining and pioneering transcription factor due to its ability to bind “closed” genomic sites and maintain “open” chromatin state for myeloid lineage–specific genes. The precise mechanism of PU.1 in cell type–specific programming is yet to be elucidated. The melanoma cell line B16BL6, although it is nonmyeloid lineage, expressed Toll-like receptors and activated the transcription factor NF-κB upon stimulation by the bacterial cell wall component lipopolysaccharide. However, it did not produce cytokines, such as IL-1β mRNA. Ectopic PU.1 expression induced remodeling of a novel distal enhancer (located ∼10 kbp upstream of the IL-1β transcription start site), marked by nucleosome depletion, enhancer-promoter looping, and histone H3 lysine 27 acetylation (H3K27ac). PU.1 induced enhancer-promoter looping and H3K27ac through two distinct PU.1 regions. These PU.1-dependent events were independently required for subsequent signal-dependent and co-dependent events: NF-κB recruitment and further H3K27ac, both of which were required for enhancer RNA (eRNA) transcription. In murine macrophage RAW264.7 cells, these PU.1-dependent events were constitutively established and readily expressed eRNA and subsequently IL-1β mRNA by lipopolysaccharide stimulation. In summary, this study showed a sequence of epigenetic events in programming IL-1β transcription by the distal enhancer priming and eRNA production mediated by PU.1 and the signal-dependent transcription factor NF-κB.
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Shin, Dong-Mi, Chang-Hoon Lee, and Herbert Morse. "The Transcriptional Network Governed by Interferon Regulatory Factor (IRF8) In Germinal Center B Cell Lymphomas." Blood 116, no. 21 (November 19, 2010): 3638. http://dx.doi.org/10.1182/blood.v116.21.3638.3638.
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Abstract Abstract 3638 The transcription factor IRF8, is highly expressed in germinal center (GC) centroblastic B cells and diffuse large B cell lymphomas (DLBCL). IRF8 is known to play roles in GC formation as well as in early B cell fate commitment, functioning both as a transcriptional activator and repressor; however, the transcriptional network regulated by IRF8 has not been elucidated yet. To identify direct targets of IRF8 on a genome-wide scale, we used ChIP-chip and expression profiling to study human and mouse lymphoma cell lines of GC origin – human Ly1, Odh1 and Val and mouse NFS201, NFS202, and NFS205. The IRF8-negative human multiple myeloma cell line, MMS1, and mouse plasmacytoma cell line, MPC11, were used as negative controls. For the human lines, 271 IRF8 target genes with common binding sites in all three cell lines, but not in negative cell line, were highly enriched for genes involved in innate immunity and adaptive immunity. Two well-established IRF8 target motifs- GAAANNGAAA and GGAANNGAAA - were significantly enriched in promoters of these genes compared to sequences 1kb upstream. Studies of the mouse lines identified 871 IRF8 targets with the lowest number found in NFS205. NFS205 expressed little PU.1, a common partner of IRF8 in DNA binding. PU.1 ChIP-chip analyses of the mouse lines allowed us to classify IRF8 targets into those that are PU.1-associated and PU.1-independent. Analyses of expression profiling of siIRF8-treated cells were used to identify transcriptionally active IRF8 targets. Gene Set Enrichment Analysis (GSEA) revealed significantly high enrichment of IRF8 ChIP targets as well as significant enrichment of genes involved in hematopoietic development. Finally 52 IRF8 targets common to human and mouse GC neoplasms were identified and characterized as contributing to antigen presentation, BCR and CD40 signaling, and interferon signaling, which can contribute differentially to the normal GC reaction and B cells transformation. These results define the contribution of IRF8 to the transcriptional network in GC B cells and provide new insights into the workings of the GC reaction in health and disease. This work was supported by the Intramural Research Program of the NIH, National Institute of Allergy and Infectious Diseases. Disclosures: No relevant conflicts of interest to declare.
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Lian, Ming, Yu Fang Jiang, Shi Dong Lv, Yi Long He, Jiang Sheng Zhou, and Qing Xiong Meng. "The Anti-Obesity Effect of Instant Pu-Erh Ripe Tea in Mice with Hydrogenated Oil Diet-Induced Obesity." Applied Mechanics and Materials 644-650 (September 2014): 5239–43. http://dx.doi.org/10.4028/www.scientific.net/amm.644-650.5239.
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Obesity has become a fast growing epidemic in developing countries, as well as in some of the developing countries. Drinking Chinese tea has been demonstrated to have multiple beneficial effects to obese patients. It showed strong effect in reducing body fat and lowering blood sugar. This article tested the effect of instant Pu-erh ripe tea on weight loss in diet-induced obese (DIO) mice. The results demonstrated that instant Pu-erh ripe tea was be able to reduce the mouse weight dose-dependently. Its effect is stronger than that of L-carnitine, a weight loss drug currently on the market. Instant Pu-erh ripe tea also accelerated lipid metabolism and eased the high-fat diet-induced liver injury.
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Koc, Hazal, Ebru Kilicay, Zeynep Karahaliloglu, Baki Hazer, and Emir B. Denkbas. "Prevention of urinary infection through the incorporation of silver–ricinoleic acid–polystyrene nanoparticles on the catheter surface." Journal of Biomaterials Applications 36, no. 3 (February 2, 2021): 385–405. http://dx.doi.org/10.1177/0885328220983552.
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Nosocominal infections associated with biofilm formation on urinary catheters cause serious complications. The aim of this study was to investigate the feasibility of the polyurethane (PU) catheter modified with tetracycline hydrochloride (TCH) attached Ag nanoparticles embedded PolyRicinoleic acid-Polystyrene Nanoparticles (PU-TCH-AgNPs-PRici-PS NPs) and the influence on antimicrobial and antibiofilm activity of urinary catheters infected by Escherichia coli and Staphylococcus aureus. For this purpose, AgNPs embedded PRici graft PS graft copolymers (AgNPs-PRici-g-PS) were synthesized via free radical polymerization and characterized by FTIR, HNMR and DSC. AgNPs-PRici-PS NPs were prepared and optimized by the different parameters and the optimized size of nanoparticle was found as about 150 ± 1 nm. The characterization of the nanoparticles and the morphological evaluation were carried out by FTIR and SEM. Short term stability of nanoparticles was realised at 4°C for 30 days. In vitro release profiles of TCH and Ag NPs were also investigated. The formation of biofilm on PU modified TCH-Ag NPs-PRici-PS NPs, was evaluated and the biocompatibility test of the nanoparticles was realized via the mouse fibroblast (L929) and mouse urinary bladder cells (G/G An1). This is the first time that TCH-AgNPs-PRici-PS NPs used in the modification of PU catheter demonstrated high antimicrobial and antibiofilm activities against the urinary tract infection.
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Musikacharoen, Tipayaratn, Asako Oguma, Yasunobu Yoshikai, Norika Chiba, Akio Masuda, and Tetsuya Matsuguchi. "Interleukin-15 induces IL-12 receptor β1 gene expression through PU.1 and IRF 3 by targeting chromatin remodeling." Blood 105, no. 2 (January 15, 2005): 711–20. http://dx.doi.org/10.1182/blood-2004-03-0842.
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AbstractInterleukin-12 receptor β1 (IL12RB1) is expressed on a variety of immune cells, including T and natural killer (NK) cells and macrophages, and is involved in innate and adaptive immune responses. Levels of IL12RB1 mRNA are dynamically regulated by various cytokines, including interferon-γ (IFN-γ) and IL-15. To reveal the regulatory mechanisms governing IL12RB1 gene expression, we analyzed the transcriptional regulatory region of the mouse IL12RB1 gene. Promoter analyses in a mouse macrophage cell line, RAW264.7, revealed that the 2508-bp region upstream of the transcriptional start site is sufficient for the full transcriptional activation of the IL12RB1 gene by IFN-γ or IL-15. Analyses of the deletion mutants revealed critical roles of IRE/ISRE and ETS/PU.1 elements, to which IRF3 and PU.1, respectively, bound. Notably, chromatin immunoprecipitation (ChIP) assays revealed IL-15 rapidly induced histone H3 acetylation at the IL12RB1 promoter. Consistently, IL-15, as a histone deacetylase inhibitor, synergistically enhanced IL12RB1 gene expression and promoter activation by IFN-γ through increased protein binding to ETS/PU.1 and IRE/ISRE sites. Additionally, IL12RB1 promoter activation by IFN-γ was enhanced by the coexpression of a coactivator protein, CBP. Thus, IL-15 induces chromatin remodeling of the IL12RB1 gene promoter, increasing IL12RB1 mRNA expression in synergy with IFN-γ through the recruitment of PU.1 and IRF3.
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Iino, Tadafumi, Yong Chong, Shin-ichi Mizuno, Kyoko Ito, Daniel G. Tenen, Boris Reizis, and Koichi Akashi. "PU.1 Plays a Critical Role in Maintenance of Mature Dendritic Cell Pool." Blood 112, no. 11 (November 16, 2008): 3554. http://dx.doi.org/10.1182/blood.v112.11.3554.3554.
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Abstract PU.1, a hematopoietic transcription factor, is absolutely required for development of myelo-lymphoid cells from hematopoietic stem cells (HSC). PU.1-deficient mice fail to develop common myeloid progenitors (CMPs) or common lymphoid progenitors (CLPs), resulting in complete loss of dendritic cells (DC) in addition to mature myeloid and lymphoid cells. In this study, by disrupting PU.1 specifically at the mature DC stage, we here show that PU.1 is necessary for maintenance of mature DC pool. By crossing PU.1 floxed/floxed mice with a mouse line harboring the Cre transgene driven by the CD11c-BAC, we disrupted PU.1 at the CD11c+ DC stage. In these mice, development of DC precursors such as Lin−c-KitloFLT3+MCSFR+ DC progenitors, FLT3+ CLP and FLT3+CMP were not affected. The number of CD11c+B220− DCs, however, significantly reduced in all lymphoid tissues including the thymus, the spleen, the lymph node and the skin, down to <30%, <10%, <10% and <5% of DC numbers in control mice, respectively. In contrast, mice possessed normal numbers of granulocytes/monocytes, B cells, and naïve, effector or regulatory T cells. These mice have not developed any significant hematological or immune disorders at least until 6 months after birth. These results clearly show that PU.1 is required not only for DC development but also for maintenance of the peripheral DC pool. We are currently trying to elucidate the underlying mechanism for PU.1 to maintain mature DC numbers in peripheral organs.
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Cristóbal, Lara, Nerea de los Reyes, Miguel A. Ortega, Melchor Álvarez-Mon, Natalio García-Honduvilla, Julia Buján, and Andrés A. Maldonado. "Local Growth Hormone Therapy for Pressure Ulcer Healing on a Human Skin Mouse Model." International Journal of Molecular Sciences 20, no. 17 (August 26, 2019): 4157. http://dx.doi.org/10.3390/ijms20174157.
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The growth hormone is involved in skin homeostasis and wound healing. We hypothesize whether it is possible to improve pressure ulcer (PU) healing by locally applying the recombinant human growth hormone (rhGH) in a human skin mouse model. Non-obese diabetic/severe combined immunodeficient mice (n = 10) were engrafted with a full-thickness human skin graft. After 60 days with stable grafts, human skin underwent three cycles of ischemia-reperfusion with a compression device to create a PU. Mice were classified into two groups: rhGH treatment group (n = 5) and control group (n = 5). In the rhGH group for local intradermal injections, each had 0.15 mg (0.5IU) applied to the PU edges, once per week for four weeks. Evaluation of the wound healing was conducted with photographic and visual assessments, and histological analysis was performed after complete wound healing. The results showed a healing rate twice as fast in the rhGH group compared to the control group (1.25 ± 0.33 mm2/day versus 0.61 ± 0.27 mm2/day; p-value < 0.05), with a faster healing rate during the first 30 days. The rhGH group showed thicker skin (1953 ± 457 µm versus 1060 ± 208 µm; p-value < 0.05) in the repaired area, with a significant decrease in collagen type I/III ratio at wound closure (62 days, range 60–70). Local administration of the rhGH accelerates PU healing in our model. The rhGH may have a clinical use in pressure ulcer treatment.
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Bach, Christian, Philipp B. Staber, Min Ye, Pu Zhang, Alan D. Friedman, Ruud Delwel, and Daniel G. Tenen. "PU.1 Is a Downstream Target of C/EBPα in Normal Hematopoiesis and Acute Myeloid Leukemia." Blood 118, no. 21 (November 18, 2011): 1353. http://dx.doi.org/10.1182/blood.v118.21.1353.1353.
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Abstract Abstract 1353 The transcription factors PU.1 and C/EBPα are key regulators of hematopoietic cell differentiation. Tight and coordinated regulation of these factors is essential for normal hematopoiesis and even moderate alterations can lead to acute myeloid leukemia (AML). Previous studies established that in PU.1 knockout mice myeloid differentiation is blocked at an earlier stage compared to C/EBPα knockouts, consistent with PU.1 acting upstream of C/EBPα during hematopoietic differentiation. Recently, however, we and others identified a PU.1 upstream regulatory element (URE) which contains potential C/EBP binding sites. C/EBPα binds to the PU.1 URE in vitro and in vivo. Furthermore, C/EBPα transactivated the PU.1 proximal promoter in a URE dependent manner. We, therefore, hypothesized that PU.1 is a target gene of C/EBPα in hematopoietic cells. To assess the role of PU.1 as a downstream target of C/EBPα in normal hematopoiesis we performed gene expression analysis in immature hematopoietic cells of conditional C/EBPα knockout mice (Mx1-Cre). Of note, we observed a strong reduction of PU.1 expression in hematopoietic stem cells (HSCs: CD150+CD48-LSK) after excision of C/EBPα, corroborating that PU.1 is a target of C/EBPα in murine HSCs in vivo. Moreover, lentiviral PU.1 expression alleviated the myeloid differentiation block of C/EBPα−/− KSL cells as evidenced by the differentiation to Gr-1 and Mac1 positive myeloid cells. Targeted deletion of the PU.1 URE reduces PU.1 expression and induces myeloid leukemia. Additionally, inactivation of C/EBPα by various mechanisms is a common observation in many AML subtypes. Therefore, we tested if dysregulation of C/EBPα is associated with decreased PU.1 expression. Gene expression studies in several human AML cell lines revealed a positive correlation between C/EBPα and PU.1 expression. Furthermore, we analyzed expression of C/EBPα and PU.1 in a well characterized cohort of 285 AML patients. Importantly, PU.1 expression was strongly reduced in cases with either C/EBPα mutations or C/EBPα promoter silencing compared to other AML subtypes. Taken together, our data support that PU.1 is a downstream target gene of C/EBPα in normal hematopoiesis as well as human leukemia. We currently develop a mouse model containing targeted mutations of three C/EBP binding sites in the PU.1 URE. This model will help to further pinpoint the functional impact of C/EBPα mediated regulation of PU.1 in different hematopoietic populations and to determine how this regulation may contribute to leukemia development in vivo. The first two authors contributed equally to this work. Disclosures: No relevant conflicts of interest to declare.
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Vlckova, Petra, Libor Stanek, Pavel Burda, Karin Vargova, Filipp Savvulidi, Martina Kapalova, Ulrich Steidl, and Tomas Stopka. "PU.1 and p53 Double Mutant Mice Develop Aggressive AML with Dysplastic Features." Blood 120, no. 21 (November 16, 2012): 769. http://dx.doi.org/10.1182/blood.v120.21.769.769.
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Abstract Abstract 769 Introduction: Downregulation of tumour suppressor transcription factor PU.1 in haematopoietic stem and progenitor cells represents primary underlying mechanism for the development of acute myeloid leukaemia (AML) in mice with homozygous deletion of the upstream regulatory element (URE) of PU.1 gene. Human AML often display differences in aggressiveness that are associated with mutations of a well known tumour suppressor p53. We produced murine model carrying mutations of p53 and URE that develops highly aggressive AML and focused on molecular mechanisms that are responsible for AML aggressiveness. Mouse models: PU.1ure/ure (Rosenbauer F, et al. 2004) and p53−/− (Jacks T, et al. 1994) mice were used. Conditional deletion of the URE leads to downregulation of PU.1 and is marked by clonal accumulation of myeloid c-Kit+Mac-1low Gr-1low blast cells within bone marrow, spleen, and peripheral blood mirrored by lower numbers of lymphoid and erythroid cells. AML development in PU.1ure/ure mice involves a preleukaemic phase (at 2–3 months) marked by proliferation of myeloid c-Kit+Gr-1+ cells and splenomegaly. Interestingly, p53−/−mice do not develop AML, instead loss of p53 predisposes mice to solid tumours, mostly lymphomas, by 6 months of age. Results: Deletion of TP53 in the PU.1ure/ure mice (PU.1ure/ure p53−/−) results in more aggressive AML with significantly shortened overall survival, prominent hepatosplenomegaly and cachexia (wasting syndrome). Mild differences in cell surface phenotype of bone marrow derived cells were observed between PU.1ure/ure and PU.1ure/ure p53−/− mice by flow cytometry (these included: blasts expansion and lymphopenia). Next, the PU.1 expression was determined in all genotypes at progenitor and stem cell levels. PU.1 mRNA level in more aggressive PU.1ure/ure p53−/− murine AML is decreased in the entire c-Kit+tumour cell population compared to AML in PU.1ure/ure mice including haematopoietic stem and progenitor cells (HSPCs). Correspondingly to RNA level, in the PU.1ure/ure progenitors the PU.1 protein was decreased compared to p53−/− progenitors and is yet further reduced in the PU.1ure/ure p53−/− c-Kit+ Mac1+progenitors. p53−/− progenitors express similar level of PU.1 as wild type progenitors indicating that despite p53 can bind DNA as a transcription factor, it does not regulate PU.1 level directly. In addition to URE deletion we searched for other mechanisms that control PU.1 levels and found that PU.1-inhibiting microRNA miR-155 gene display altered chromatin structure and expression of both pri-miR-155 as well as its spliced mature form in the AML of PU.1ure/ure and (to higher extent in) PU.1ure/ure p53−/− murine progenitors. Upregulation of miR-155 coincides with upregulation of the Mir155hg activators: Myc and Myb. Finally, upon inhibition of either Myb or miR-155 in vitro the AML progenitors restore PU.1 levels and lose leukaemic cell growth. Conclusion: In summary, PU.1 and p53 double mutant mice develop aggressive AML with dysplastic features. Defective control of PU.1 levels in PU.1ure/ure and PU.1ure/ure p53−/−AML involves miR-155. Lastly, restored PU.1 level and cell differentiation capacity are achieved by inhibiting either Myb or miR-155 in the PU.1ure/ure p53−/− progenitors. (Grant support: P305/12/1033, UNCE 204021, PRVOUK-P24/LF1/3, SVV-2012-264507, P301/12/P380. MK was sponsored by GAUK 251070 45410, 251135 82210) Disclosures: No relevant conflicts of interest to declare.
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Kucine, Nicole, Sachie Marubayashi, Neha Bhagwat, Efthymia Papalexi, Priya Koppikar, Marta Sanchez Martin, Lauren Dong, et al. "Tumor-specific HSP90 inhibition as a therapeutic approach in JAK-mutant acute lymphoblastic leukemias." Blood 126, no. 22 (November 26, 2015): 2479–83. http://dx.doi.org/10.1182/blood-2015-03-635821.
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Key Points PU-H71, a novel purine scaffold inhibitor, shows potent therapeutic efficacy in JAK-mutant ALL cells and mouse models. HSP90 inhibition retains therapeutic efficacy in ruxolitinib-persistent JAK-mutant ALL cells.
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Fisher, Robert C., Marilyn C. Olson, Jagan M. R. Pongubala, Jeffrey M. Perkel, Michael L. Atchison, Edward W. Scott, and M. Celeste Simon. "Normal Myeloid Development Requires Both the Glutamine-Rich Transactivation Domain and the PEST Region of Transcription Factor PU.1 but Not the Potent Acidic Transactivation Domain." Molecular and Cellular Biology 18, no. 7 (July 1, 1998): 4347–57. http://dx.doi.org/10.1128/mcb.18.7.4347.
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ABSTRACT Gene targeting of transcription factor PU.1 results in an early block to fetal hematopoiesis, with no detectable lymphoid or myeloid cells produced in mouse embryos. Furthermore,PU.1 −/− embryonic stem (ES) cells fail to differentiate into Mac-1+ and F4/80+macrophages in vitro. We have previously shown that a PU.1 transgene under the control of its own promoter restores the ability ofPU.1 −/− ES cells to differentiate into macrophages. In this study, we take advantage of ourPU.1 −/− ES cell rescue system to genetically test which previously identified PU.1 functional domains are necessary for the development of mature macrophages. PU.1 functional domains include multiple N-terminal acidic and glutamine-rich transactivation domains, a PEST domain, several serine phosphorylation sites, and a C-terminal Ets DNA binding domain, all delineated and characterized by using standard biochemical and transactivational assays. By using the production of mature macrophages as a functional readout in our assay system, we have established that the glutamine-rich transactivation domain, a portion of the PEST domain, and the DNA binding domain are required for myelopoiesis. Deletion of three acidic domains, which exhibit potent transactivation potential in vitro, had no effect on the ability of PU.1 to promote macrophage development. Furthermore, mutagenesis of four independent sites of serine phosphorylation also had no effect on myelopoiesis. Collectively, our results indicate that PU.1 interacts with important regulatory proteins during macrophage development via the glutamine-rich and PEST domains. ThePU.1 −/− ES cell rescue system represents a powerful, in vitro strategy to functionally map domains of PU.1 essential for normal hematopoiesis and the generation of mature macrophages.
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Steidl, Ulrich, Frank Rosenbauer, Xuesong Gu, Bronwyn Owens, Steffen Klippel, Katharina Wagner, Manuel Aivado, Towia A. Libermann, and Daniel G. Tenen. "Knockdown of the Transcription Factor PU.1 Causes Characteristic Transcriptional Changes in Hematopoietic Stem Cells Prior to Leukemic Transformation." Blood 104, no. 11 (November 16, 2004): 1112. http://dx.doi.org/10.1182/blood.v104.11.1112.1112.
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Abstract Knockdown of the expression of the transcription factor PU.1 in mice results in an initial preleukemic expansion of myeloid progenitors and subsequent transformation to an immature acute myeloid leukemia (AML). Recent reports suggest that functional inactivation of PU.1 might also play a role in human AML. However, the molecular mechanisms underlying the malignant transformation are unknown. Examining the bone marrow of leukemic PU.1 knockdown mice we found an expansion of lin−, c-kit+, Sca1+ hematopoietic stem cells (HSC) as compared to wildtype animals. This finding prompted us to examine the transcriptome of HSC of PU.1 knockdown mice in the initial phase to look for early transcriptional changes underlying the malignant transformation. After lineage-depletion and FACS sorting of lin−, c-kit+, Sca1+ HSC we performed linear amplification of RNA which was then hybridized with Affymetrix Mouse Genome 430 2.0 arrays covering ~39,000 transcripts. Unsupervised hierarchical cluster analysis clearly distinguished PU.1 knockdown and wildtype HSC, demonstrating that reduced PU.1 expression levels lead to characteristic transcriptional changes in the stem cell compartment preceding the malignant transformation. 55 transcripts were upregulated, and 143 transcripts were downregulated in PU.1 knockdown HSC (>1.5-fold, p<0.05), reflecting the role of PU.1 as a transcriptional activator. We corroborated differential expression of 16 genes by quantitative real-time RT-PCR. Among the downregulated genes were c-fes, Bruton’s tyrosine kinase, transcription factor EC, and GM-CSF receptor α, demonstrating that those in-vitro targets of PU.1 are also affected in stem cells in vivo. BAFF-R and TACI, two members of the tumor necrosis factor receptor superfamily, receptor tyrosine kinase TEK and toll-like receptor 4 were downregulated, whereas PDGF receptor was upregulated, suggesting disturbed signaling in PU.1 knockdown HSC. We are currently investigating the causal contribution of these deregulated genes to the leukemic phenotype. In summary, our data delineate a distinct transcriptional pattern that precedes the leukemic transformation in PU.1 knockdown HSC, and provide novel insight into PU.1-mediated molecular pathways in vivo.
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Foos, Gabriele E., Kimberlee M. Fischer, Jeremiah Savage, Venkat Reddy, and Bruce E. Torbett. "Identification of PU.1 Target Genes That Are Dependent on Specific Functional Domains of the Transcription Factor PU.1." Blood 108, no. 11 (November 16, 2006): 1174. http://dx.doi.org/10.1182/blood.v108.11.1174.1174.
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Abstract 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.
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Zhang, Pu, Gang Huang, Alex Ebralidze, Jun-Yan Zhang, Annalisa DiRuscio, Chris Hetherington, Elena Levantini, and Daniel G. Tenen. "Genetics and Epigenetics of the PU.1 Upstream Regulatory Element: AML1 Binding Sites Are Critical for Leuekmia Induced by the AML/ETO9a Fusion Oncogene." Blood 112, no. 11 (November 16, 2008): 594. http://dx.doi.org/10.1182/blood.v112.11.594.594.
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Abstract 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.
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Moleriu, Lavinia, Adina Octavia Duse, Florin Borcan, Codruta Soica, Ludovic Kurunczi, Mirela Nicolov, and Marius Mioc. "Formulation and Characterization of Antibacterial Hydrogels Based on Polyurethane Microstructures and 1,2,4-Triazole Derivatives." Materiale Plastice 54, no. 2 (June 30, 2017): 348–52. http://dx.doi.org/10.37358/mp.17.2.4849.
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Antibacterial gels offer an efficient hygiene in the absence of soap and water. The most part of these products are based on mixtures of different antibacterial and fungicide substances dissolved in an alcohol. Unfortunately, all cosmetic products containing alcohols wash the sebum and dry the skin. In the present study, alcohol-free antibacterial hydrogels were obtained through the synthesis of polyurethane (PU) microstructures with an increased amount of surfactant; 5-mercapto-1,2,4-triazole derivatives were used as active compounds inside the PU microstructures as antibacterial agents. There were studied: the pH of samples, the size and stability of PU microstructures, the skin irritation, sebum level and the hydration of stratum corneum using the mouse model and their efficiency against Escherichia coli. The results indicate that were obtained hydrogels with a slightly acid pH, PU microstructures with sizes between 185 and 265 nm and with a medium stability. It was found that these new antibacterial hydrogels do not produce an important modification of skin parameters (erythema, melanin, sebum and hydration of stratum corneum) and are efficient against E. coli.
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Kitajima, Kenji, Makoto Tanaka, Jie Zheng, Hilo Yen, Ayuko Sato, Daijiro Sugiyama, Hiroki Umehara, Eiko Sakai, and Toru Nakano. "Redirecting differentiation of hematopoietic progenitors by a transcription factor, GATA-2." Blood 107, no. 5 (March 1, 2006): 1857–63. http://dx.doi.org/10.1182/blood-2005-06-2527.
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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.
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Jenal, Mathias, Venkateshwar A. Reddy, Judith Laedrach, Deborah Shan, Andreas Tobler, Bruce E. Torbett, Martin F. Fey, and Mario P. Tschan. "The Anti-Apoptotic Gene BCL2A1 Is Transcriptionally Regulated by PU.1." Blood 112, no. 11 (November 16, 2008): 3579. http://dx.doi.org/10.1182/blood.v112.11.3579.3579.
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Abstract PU.1 is a hematopoietic transcriptional regulator that is necessary for the development of both myeloid and B cells. To identify new PU.1 target genes in neutrophil development PU.1 was introduced into mouse 503 PU.1-null cells using lentiviral gene transfer and microarray analyses of two independent 503 PU.1-rescued and parental 503 cells were compared. The BCL2A1 gene was found to be more than 50-fold induced in 503 PU.1- restored as compared to the parental 503-null cells. BCL2A1 (also known as BFL-1/A1) is an anti-apoptotic member of the BCL2 family. BCL2A1 was initially identified as a tissue-specific BCL2-related factor that is induced by different reagents such as granulocyte macrophage colony-stimulating factor (GM-CSF) or all-trans retinoic acid (ATRA) during myeloid differentiation. Upregulation of BCL2A1 in granulocytes may promote a time-dependent survival. To follow up on our microarray findings we evaluated loss of PU.1 function in human NB4 acute promyelocytic leukemia (APL) cells using lentivector delivered, short hairpin (sh) RNAs targeting PU.1. Knockdown efficacy upon ATRA-treatment in the two shPU.1 expressing NB4 cell lines was 67 and 30%, respectively. Silencing of PU.1 markedly reduced BCL2A1 mRNA induction upon ATRA-treatment from 167-fold in control cells to 47- and 112-fold in the two PU.1 knockdown NB4 cell lines, respectively (Figure A). Co-transfection of PU.1 with a human BCL2A1 promoter reporter resulted in a 7-fold activation, suggesting PU.1 can directly regulate BCL2A1. Co-transfection with NF-kappaB, used as positive control, induced the BCL2A1 promoter 14.5-fold. Moreover, in vivo binding of the transcription factor PU.1 to 2/8 putative PU.1 binding sites in the BCL2A1 promoter was shown by chromatin immunoprecipitation in HL60 promyelocytic cells further supporting a role for PU.1 regulation of BCL2A1. Evaluation of BCL2A1 and PU.1 mRNA expression in CD34+ hematopoietic progenitors, granulocytes, and primary acute myeloid leukemia (AML) cells was assessed using real-time quantitative RT-PCR. BCL2A1 and PU.1 mRNA levels were significantly lower in primary AML patient samples (n=80; p<0.0001) and in CD34+ progenitor cells (n=4; p=0.0095) than in granulocytes (n=6; Figure B). In line with this observation, we found that upon ATRA therapy BCL2A1 levels were increased in 5/5 APL patients and PU.1 mRNA levels in 4/5 APL cases, respectively. Altogether, these results clearly indicate that PU.1 and BCL2A1 are co-regulated during granulocyte differentiation. Lastly, we confirmed earlier data showing that ATRA-pretreatment of NB4 cells and thus induction of PU.1 and BCL2A1, rendered these cells less sensitive to arsenic trioxide (As2O3)- induced cell death. Conversely, NB4 PU.1 knockdown cells were markedly more sensitive to As2O3 -induced cell death upon ATRA-pretreatment than the parental NB4 control cells. The increase in sensitivity to As2O3 correlated with the lower BCL2A1 levels found in the PU.1 knockdown cells. In summary, we identified the anti-apoptotic BCL2A1 gene as direct, transcriptional target of PU.1 in myeloid leukemic cells. We hypothesize that PU.1-dependent induction of BCL2A1 is necessary for the survival of normal, terminally differentiated myeloid cells. Furthermore, aberrant expression of PU.1 in erythroleukemia may result in elevated BCL2A1 levels that support increased survival of erythroblasts in this particular type of leukemia. Figure Figure
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Nishimura, Nao, Shinya Endo, Niina Ueno, Shikiko Ueno, Hiromichi Yuki, Hiroyuki Hata, Hiroaki Mitsuya, and Yutaka Okuno. "Xenograft Models of Multiple Myeloma Reveal That PU.1 Serves As a Tumor Suppressor for Multiple Myeloma." Blood 124, no. 21 (December 6, 2014): 2047. http://dx.doi.org/10.1182/blood.v124.21.2047.2047.
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Abstract PU.1 is an essential transcription factor for hematopoiesis and important for differentiation of both myeloid and lymphoid lineages. In mice conditionally knocked-out of 3.4 kb length of the enhancer region located in14 kb 5’ upstream of the PU.1 gene (URE), PU.1 is down-regulated in myeloid cells and B cells by 20% of that of wild type, and such mice develop acute myeloid leukemia and CLL-like diseases. These data strongly suggest that PU.1 has tumor suppressor activity in hematopoietic cells. We previously reported that human PU.1 is down-regulated in the majority of myeloma cell lines through the methylation of the promoter and the 17 kb upstream enhancer region (URE) of the PU.1 gene that is homologous to that in 14 kb 5’ upstream of the murine PU.1 gene. Conditionally expressed PU.1 with tet-off system induced cell growth arrest and apoptosis in two myeloma cell lines, KMS12PE and U266, suggesting that the down-regulation of PU.1 is necessary for myeloma cell growth. We have also reported that PU.1 is expressed in normal plasma cells and in contrast, PU.1 is down-regulated in primary myeloma cells from a subset of myeloma patients, who appear to have poor prognosis. In the present study, to test whether PU.1 has tumor suppressor activity in vivo, we generated xenograft mouse models. 0.6 - 1 x 107 KMS12PE cells were subcutaneously injected in 16 immunodeficient mice (Rag2-/- Jak3-/- bulb/c). The mice were then administered doxycycline through drinking water. Half of the mice (N=8) stopped taking doxycycline when the tumor sizes reached 1 cm in diameter, whereas the other half (N=8) kept taking doxycycline. Although the tumors in the mice taking doxycycline continued to grow, the tumor growth in the mice not taking doxycycline significantly slowed down. Flow cytometry analysis of the tumors in the mice that stopped taking doxycycline revealed that the cells from the tumor had completely lost PU.1 expression. Moreover, when U266 cells conditionally expressing PU.1 were subcutaneously injected to another 10 mice and the same experiment was conducted, although the tumors in the mice taking doxycycline (N=5) kept growing, the tumors in the mice not taking doxycycline (N=5), did not grow any further. The present data suggest that PU.1 serves as a tumor a suppressor in the multiple myeloma cell lines as examined in vivo. Disclosures No relevant conflicts of interest to declare.
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Sive, Jonathan I., Fernando J. Calero-Nieto, Rebecca Hannah, and Berthold Göttgens. "Genome-Scale Analysis of the Pu.1 Driven Transcriptional Programme That Overcomes the Differentiation Block in a Novel Pu.1-Inducible AML Cell Line." Blood 124, no. 21 (December 6, 2014): 869. http://dx.doi.org/10.1182/blood.v124.21.869.869.
Full textAbstract:
Abstract Transcription factors (TFs) play a key role in determining normal haematopoiesis; haematological cancers often being characterised by TF dysregulation. Pu.1 is a TF that plays a major role in myeloid differentiation, with mutations and down regulation of Pu.1 activity described in human acute myeloid leukemia (AML). To investigate the role of Pu.1 in normal and leukemic myeloid fate, we developed a model of inducible Pu.1 rescue in a murine AML cell line, and utilised it to assess genome-wide Pu.1-DNA-binding profiles and the subsequent effects on histone remodelling, gene expression and binding of the cooperative TF CEBPA. We used a previously described radiation-induced murine AML cell line known to be deficient in functional Pu.1 (Cook et al, Blood 2004; 104:3437-3444) and transduced cells with 4-hydroxy-tamoxifen (OHT)-inducible Pu.1 (PuER) or empty vector (EV). Clonal populations were produced by limiting dilution, and genotype and protein phenotype confirmed by PCR and Western blotting respectively. Incubation in 100nM OHT caused cessation of proliferation, and increased expression of the maturation markers CD11b and Gr-1. To assess the effects of Pu.1 binding, cells were incubated for 2 hours in 100nM OHT and cross-linked chromatin harvested for ChIP with antibodies against Pu.1, CEBPA and H3K27Ac. ChIP samples were amplified and sequenced, reads were mapped to the mouse reference genome, binding peaks identified, and binding levels normalised for peak length and total read count. RNA from was extracted from the same samples and analysed by microarray expression profiling. Binding peak analysis revealed an increase in Pu.1 binding sites from 5283 to 15675 following Pu.1 induction, showing that the mutant Pu.1 is able to perform limited DNA-binding, but not in a manner sufficient to induce transcription of genes necessary for normal differentiation. To investigate the effect of Pu.1 binding on histone remodelling, we selected a set of 613 peaks with ≥4-fold increase in Pu.1 read count, ≥4fold increase in H3K27Ac read count, and post-induction minimal H3K27Ac read count of ≥50 reads/region. Interestingly, these highly “dynamic” Pu.1 binding peaks were restricted to non-promoter regions, suggesting increased binding at distal enhancer sites. These 613 peaks mapped to 553 genes, which showed a small but significant overexpression post-Pu.1 induction, compared to genes adjacent to peaks with no change in Pu.1 (p = 5.357e-16). These genes were intersected with the 284 genes overexpressed in the gene expression array post Pu.1-induction, producing a list of 46 genes of relevant Pu.1 targets. Using Gene Ontology and Functional Term Enrichment analysis, we discovered a significant enrichment for genes overexpressed in bone marrow macrophages post-lipopolysaccharide stimulation (z score = -2.19). Investigating the effect of Pu.1 induction on CEBPA binding, we found that increased Pu.1 binding was enriched in CEBPA peaks which were ≥4fold increased post-Pu.1 induction (p< 0.001), suggesting recruitment of CEBPA in a significant proportion of regions. Further evidence of the relationship between Pu.1 and CEBPA comes from motif analysis of the ≥4fold Pu.1 peaks which showed CEBP motifs present in 30.0% of targets. Similarly, analysis of ≥4fold CEBPA peaks showed Ets motifs in 34.5% of these peaks with ≥ 4fold Pu.1 binding, but only 7.5% in those without. In conclusion, we present a novel model system where we have examined at genome-scale the effects of Pu.1-DNA binding to overcome the differentiation block in a Pu.1-deficient AML model. Functional Pu.1 binding rapidly induces changes in H3K27Ac profile at key enhancer sites, which subsequently drives the cell into a maturation pathway. The interaction between Pu.1 and CEBPA is also confirmed, with evidence of Pu.1 recruiting CEBPA to DNA-binding sites. Disclosures No relevant conflicts of interest to declare.
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Iino, Tadafumi, Yong Jeong, Shin-ichi Mizuno, Kentaro Kohno, Kyoko Ito, Daniel G. Tenen, Boris Reizis, and Koichi Akashi. "PU.1 Is Indispensable for Development of Mature Dendritic Cells and Their Function That Plays a Critical Role in Maintenance of Normal T Cell Pool." Blood 114, no. 22 (November 20, 2009): 1471. http://dx.doi.org/10.1182/blood.v114.22.1471.1471.
Full textAbstract:
Abstract Abstract 1471 Poster Board I-494 PU.1, a hematopoietic transcription factor, is indispensable for development of myelo-lymphoid cells from hematopoietic stem cells (HSCs). PU.1-deficient mice fail to develop common myeloid progenitors (CMPs) or common lymphoid progenitors (CLPs), resulting in complete loss of dendritic cells (DC) in addition to mature myeloid and lymphoid cells. By disrupting PU.1 specifically at the mature DC stage, we here show that PU.1 is necessary for maintenance of mature DC pool and their functions. We crossed PU.1 floxed/floxed mice with a mouse line harboring the Cre transgene driven by the CD11c-BAC. In these mice, PU.1 gene was disrupted in all conventional DCs but not in other hematopoietic cells, including lymphoid cells, myeloid cells and their progenitors. Development of DC precursors such as Lin−c-KitloFLT3+MCSFR+, FLT3+ CLP and FLT3+CMP were not affected. The number of CD11c+B220− DCs, however, significantly reduced in all lymphoid tissues including the thymus, the spleen, the lymph node and the skin, down to <40%, <25%, <10% and <5% as compared with the wild-type control, respectively. Moreover, the number of mature T cells reduced to ∼60% in the spleen as compared to the control. PU.1-deficient DCs displayed impaired functions to induce antigen-driven T cell proliferation, and to produce inflammatory cytokines (TNFa, IL-6, IL-12) in response to Toll like receptor (TLR) stimulation. These results clearly show that PU.1 is required for development of the peripheral DC pool and for maintenance of their immunological functions, which might be required for maintenance of the peripheral T cell pool. Disclosures: No relevant conflicts of interest to declare.
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Wang, Hongsheng, Shweta Jain, Jiafang Sun, Alexander L. Kovalchuk, and Herbert C. Morse. "An essential role of transcription factors PU.1 and IRF8 in follicular B cell development and the germinal center response." Journal of Immunology 200, no. 1_Supplement (May 1, 2018): 171.19. http://dx.doi.org/10.4049/jimmunol.200.supp.171.19.
Full textAbstract:
Abstract The transcription factors PU.1 and IRF8 regulate an unknown number of gene programs for differentiation of many hematopoietic cells including B cells, dendritic cells and myeloid cells. Their roles in B cell development were previously studied using B cell-specific conditional deletion mouse models, such as PU.1flox/flox-CD19Cre, IRF8flox/flox-CD19Cre, IRF8−/−PU.1flox/flox-CD19Cre, or IRF8flox/flox-PU.1flox/flox-CD19Cre mice. While PU.1-deficient B cells are phenotypically normal, deletion of IRF8 in B cells caused a moderate expansion of marginal zone B cells. Double deletion of PU.1 and IRF8 caused moderate expansion of plasma cells (PCs) in vitro. In this report, we investigated IRF8 and PU.1 double deletion mice using Mb1-Cre - termed DKO mice. FACS analysis revealed normal levels of early stage B cells in the bone marrow and transitional B cells in the spleens of DKO mice. However, follicular B cells were markedly reduced and the MZ B cell compartment was modestly expanded in DKO mice. The peritoneal CD5+ B-1a cells, which are a major source of circulating IgM, were completely absent in DKO mice. Surprisingly, the serum levels of IgM were higher and the levels of IgG3 and IgA were slightly lower in DKO than control mice. While the levels of serum IgG1 were comparable between DKO and control mice, DKO mice had no serum IgG2b or IgG2c antibodies. More intriguingly, following immunization with NP-KLH/alum, although the DKO mice produced more IgM-secreting PCs early on, they failed to generate germinal centers and produced markedly reduced levels of NP-specific switched IgG antibodies. Taken together, these data revealed a critical role of IRF8 and PU.1 in differentiation of follicular and germinal center B cells.
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Hu, Tianyuan, Kiyomi Morita, Matthew C. Hill, Yajian Jiang, Ayumi Kitano, Yusuke Saito, Feng Wang, et al. "PRDM16s transforms megakaryocyte-erythroid progenitors into myeloid leukemia–initiating cells." Blood 134, no. 7 (August 15, 2019): 614–25. http://dx.doi.org/10.1182/blood.2018888255.
Full textAbstract:
Abstract Oncogenic mutations confer on cells the ability to propagate indefinitely, but whether oncogenes alter the cell fate of these cells is unknown. Here, we show that the transcriptional regulator PRDM16s causes oncogenic fate conversion by transforming cells fated to form platelets and erythrocytes into myeloid leukemia stem cells (LSCs). Prdm16s expression in megakaryocyte-erythroid progenitors (MEPs), which normally lack the potential to generate granulomonocytic cells, caused AML by converting MEPs into LSCs. Prdm16s blocked megakaryocytic/erythroid potential by interacting with super enhancers and activating myeloid master regulators, including PU.1. A CRISPR dropout screen confirmed that PU.1 is required for Prdm16s-induced leukemia. Ablating PU.1 attenuated leukemogenesis and reinstated the megakaryocytic/erythroid potential of leukemic MEPs in mouse models and human AML with PRDM16 rearrangement. Thus, oncogenic PRDM16s expression gives MEPs an LSC fate by activating myeloid gene regulatory networks.
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Pospisil, Vitek, Pavle Krsmanovic, Jan Valecka, Kamila Chramostová, Vojtech Kulvait, Jiri Zavadil, Martin Vokurka, Peter Laslo, and Tomas Stopka. "Graded PU.1 Levels Regulate Granulocyte Vs. Macrophage Genes Via Multiple Enhancer Elements." Blood 128, no. 22 (December 2, 2016): 403. http://dx.doi.org/10.1182/blood.v128.22.403.403.
Full textAbstract:
Abstract PU.1 is a transcription factor absolutely required for normal hematopoiesis. Cumulating evidence indicates that precise levels of PU.1 expression are critical for differentiation to distinct blood lineages, and if perturbed, even modest decreases in PU.1 can lead to leukemogenesis. In contrast to extensive knowledge of regulation of PU.1 gene itself, the mechanism of how target genes senses different PU.1 levels remain largely unknown. To address this, we used PU.1-/- mouse myeloid progenitors encoding inducible PU.1 transgene (PU.1ER, PUER, Walsh 2002) that allows tight control of PU.1 activity. Interestingly, intermediate PU.1 activity induced differentiation of PUER progenitors into granulocyte like cells, while high PU.1 produced macrophages, supporting the model that different PU.1 expression is not a consequence but a driver of cell fate choice. Global expression analysis using 4 different levels of PU.1 at 8 time points (2-96 hrs) revealed that granulocyte specific genes were activated exclusively by intermediate PU.1 levels in 3 distinct modes: 1. not expressed in progenitors while strongly induced at intermediate PU.1 (e.g. Gelatinase B (Mmp9) and Neutrophil collagenase (NC) 2. moderately expressed in progenitors while strongly activated at intermediate PU.1 and repressed at high PU.1 (e.g. Myeloperaxidase (Mpo) 3. highly expressed in unstimulated progenitors with expression maintained at intermediate PU.1 but strongly repressed at high PU.1 (e.g. Neutrophil elastase (NE), Proteinase 3 (primary granule proteins), Cebpe and Gfi1 (Growth factor independent1) Majority of macrophage genes (incl. CD14, Csf1R, Egr2) were regulated as early PU.1 target genes; being gradually activated by high PU.1 activity within 8hrs. However, most granulocyte genes (NE, Mmp9, Mpo, NC but not Cebpe and GFI1) were late activated PU.1 targets (48 and 96hrs) indicating that these genes are coregulated by additional factor(s), likely an early PU.1 target. Next we analyzed the regulatory sequences (+-50kb) of two genes activated exclusively by intermediate PU.1, Mpo and Mmp9, using own and public ChIP(seq) data of transcription factors (TFs) (PU.1, GFI.1), DNAseI hypersensitive sites, histone modifications (H3K4Me, H3K27Ac, H3K9Ac) and expression of enhancer specific bidirectional ncRNAs (eRNA) (CAGE). 14 Mpo and 16 Mmp9 putative enhancers, selected by above mentioned criteria, were cloned into luciferase vector containing their proximal promoter (PP) and were tested for functional activity in response to PU.1 levels. Interestingly, the PU.1 binding motifs within these regions have a low to intermediate affinity (log of score, Jaspar) and are often present in multiples and/or enriched for binding sites of other lineage determining transcription factors. Although PU.1 bound to all of these DNA regions resembling superenhancer, just a small fraction of PU.1 binding was functionally responsive. Specifically, we identified novel enhancer elements at -3.4 kb and -15kb of MPO which were activated by intermediate (but not high) PU.1 levels. Interestingly, activity of -3.4 kb enhancer required presence of PP, while the -15kb element required presence of both PP and the -3.4kb element. Similar phenomenon was observed at -5kb and +4.6kb (intronic) MMP9 enhancers. Collectively, these observations suggest that a cooperative assembly of several cell type-specific enhancers is required for optimal Mpo and Mmp9 activation. This model is supported by our Chromosome conformation capture (3C) data identifying 3D interaction of these enhancer elements at intermediate PU.1 levels suggesting that PU.1 binding mediates DNA looping that allows enhancer cooperation. In addition, activity of these enhancers at intermediate PU.1 levels was associated with expression of bidirectional noncoding enhancer RNAs, confirming functionality of these elements. In conclusion, our data support the model that PU.1 at intermediate concentration binds to low and intermediate affinity binding sites in several enhancers of granulocyte genes, causing their successive looping and interaction with proximal promoter that leads to transcription activation. The role of cooperating TFs, mechanisms of how granulocyte genes are switched off at high PU.1 concentration and deregulation of these mechanisms in AML are being further studied. Grants 16-05649S P305/12/1033 16-31586A 16-27790A 16-31586A UNCE 204021 PRVOUK P24 Disclosures No relevant conflicts of interest to declare.