Ets Transcription Factor PU Research Articles

Chromatin structure and 3D architecture define the differential functions of PU.1 regulatory elements in blood cell lineages

The precise spatiotemporal expression of the hematopoietic ETS transcription factor PU.1, a key determinant of hematopoietic cell fates, is tightly regulated at the chromatin level. However, how chromatin signatures are linked to this dynamic expression pattern across different blood cell lineages remains uncharacterized. Here, we performed an in-depth analysis of the relationships between gene expression, chromatin structure, 3D architecture, and trans-acting factors at PU.1 cis-regulatory elements (PCREs). By identifying phylogenetically conserved DNA elements within chromatin-accessible regions in primary human blood lineages, we discovered multiple novel candidate PCREs within the upstream region of the human PU.1 locus. A subset of these elements localizes within an 8-kb-wide cluster exhibiting enhancer features, including open chromatin, demethylated DNA, enriched enhancer histone marks, present enhancer RNAs, and PU.1 occupation, presumably mediating PU.1 autoregulation. Importantly, we revealed the presence of a common 35-kb-wide CTCF-flanked insulated neighborhood that contains the PCRE cluster (PCREC), forming a chromatin territory for lineage-specific and PCRE-mediated chromatin interactions. These include functional PCRE-promoter interactions in myeloid and B cells that are absent in erythroid and T cells. By correlating chromatin structure and 3D architecture with PU.1 expression in various lineages, we were able to attribute enhancer versus silencer functions to individual elements. Our findings provide mechanistic insights into the interplay between dynamic chromatin structure and 3D architecture in the chromatin regulation of PU.1 expression. This study lays crucial groundwork for additional experimental studies that validate and dissect the role of PCREs in epigenetic regulation of normal and malignant hematopoiesis.

Transcriptional Regulation of the Lineage-Determining Gene PU.1 in Normal and Malignant Hematopoiesis: Current Understanding and Therapeutic Perspective.

The ETS transcription factor PU.1 plays an essential role in blood cell development. Its precise expression pattern is governed by cis-regulatory elements (CRE) acting at the chromatin level. CREs mediate the fine-tuning of graded levels of PU.1, deviations of which can cause acute myeloid leukemia. In this review, we perform an in-depth analysis of the regulation of PU.1 expression in normal and malignant hematopoiesis. We elaborate on the role of trans-acting factors and the biomolecular interplays in mediating local chromatin dynamics. Moreover, we discuss the current understanding of CRE bifunctionality exhibiting enhancer or silencer activities in different blood cell lineages and future directions toward gene-specific chromatin-targeted therapeutic development.

PU.1-c-Jun interaction is crucial for PU.1 function in myeloid development

The Ets transcription factor PU.1 is essential for inducing the differentiation of monocytes, macrophages, and B cells in fetal liver and adult bone marrow. PU.1 controls hematopoietic differentiation through physical interactions with other transcription factors, such as C/EBPα and the AP-1 family member c-Jun. We found that PU.1 recruits c-Jun to promoters without the AP-1 binding sites. To address the functional importance of this interaction, we generated PU.1 point mutants that do not bind c-Jun while maintaining normal DNA binding affinity. These mutants lost the ability to transactivate a target reporter that requires a physical PU.1-c-Jun interaction, and did not induce monocyte/macrophage differentiation of PU.1-deficient cells. Knock-in mice carrying these point mutations displayed an almost complete block in hematopoiesis and perinatal lethality. While the PU.1 mutants were expressed in hematopoietic stem and early progenitor cells, myeloid differentiation was severely blocked, leading to an almost complete loss of mature hematopoietic cells. Differentiation into mature macrophages could be restored by expressing PU.1 mutant fused to c-Jun, demonstrating that a physical PU.1-c-Jun interaction is crucial for the transactivation of PU.1 target genes required for myeloid commitment and normal PU.1 function in vivo during macrophage differentiation.

Direct pharmacologic regulation of the ETS transcription factor PU.1

The ETS‐family transcription factor PU.1 is essential for the self‐renewal of hematopoietic stem cells (HSC) as well as their differentiation to various lineage‐committed hematopoietic progenitors. Specific to the bone marrow niche, PU.1 regulates the expression of several key cytokines (e.g. CXCR4) and their receptors (e.g. GM‐CSF receptor, FLT3) involved in HSC mobilization. Extensive studies have shown that incomplete loss of PU.1 activity, to ~20% wildtype levels, leads to lineage arrest and aggressive leukemic disease. Conversely, lentiviral overexpression of PU.1 resolves the differentiation block in leukemic blasts, and suggests the activation of PU.1 as a potential therapeutic approach. To this end, we have identified by phage display technology a panel of selective PU.1‐binding oligopeptides. Several of these peptides promote PU.1 binding to cognate DNA sites in a dose‐dependent manner and confer resistance to PU.1 against inhibition by netropsin. To determine the essential residues responsible for their activity, we screened a targeted library of truncated hit peptides and found that as few as four residues were sufficient to enhance site‐specific DNA binding by PU.1. These peptide sequences represent leads for developing direct‐acting PU.1‐specific modulators with respect to target DNA binding and interactions with major binding partners. The resulting low‐MW reagents could significantly expand the pharmacology of transcription factors and help overcome barriers in hematopoietic research and therapy that are currently imposed by genetic approaches.Support or Funding InformationSupported by NIH HL129063.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Targeting PFKFB3 sensitizes chronic myelogenous leukemia cells to tyrosine kinase inhibitor.

Resistance to the BCR-ABL tyrosine kinase inhibitor (TKI) remains a challenge for curing the disease in chronic myeloid leukemia (CML) patients as leukemia cells may survive through BCR-ABL kinase activity-independent signal pathways. To gain insight into BCR-ABL kinase activity-independent mechanisms, we performed an initial bioinformatics screen and followed by a quantitative PCR screen of genes that were elevated in CML samples. A total of 33 candidate genes were identified to be highly expressed in TKIs resistant patients. Among those genes, 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3), controlling the limiting step of glycolysis, was found to be strongly associated with TKIs resistance. PFKFB3 knockdown or pharmacological inhibition of its kinase activity markedly enhanced the sensitivity of CML cells to TKIs. Furthermore, pharmacological inhibition of PFKFB3 inhibited CML cells growth and significantly prolonged the survival of both allograft and xenograft CML mice. ChIP-seq data analysis combined with subsequent knockdown experiment showed that the Ets transcription factor PU.1 regulated the elevated expression of PFKFB3 in TKIs-resistant CML cells. Therefore, our results showed that targeting PFKFB3 sensitizes CML cells to TKIs and PFKFB3 may be a potential BCR-ABL kinase activity-independent mechanism in CML.

Reduced PU.1 expression underlies aberrant neutrophil maturation and function in β-thalassemia mice and patients

Abstractβ-Thalassemia is associated with several abnormalities of the innate immune system. Neutrophils in particular are defective, predisposing patients to life-threatening bacterial infections. The molecular and cellular mechanisms involved in impaired neutrophil function remain incompletely defined. We used the Hbbth3/+ β-thalassemia mouse and hemoglobin E (HbE)/β-thalassemia patients to investigate dysregulated neutrophil activity. Mature neutrophils from Hbbth3/+ mice displayed a significant reduction in chemotaxis, opsonophagocytosis, and production of reactive oxygen species, closely mimicking the defective immune functions observed in β-thalassemia patients. In Hbbth3/+ mice, the expression of neutrophil CXCR2, CD11b, and reduced NAD phosphate oxidase components (p22phox, p67phox, and gp91phox) were significantly reduced. Morphological analysis of Hbbth3/+ neutrophils showed that a large percentage of mature phenotype neutrophils (Ly6GhiLy6Clow) appeared as band form cells, and a striking expansion of immature (Ly6GlowLy6Clow) hyposegmented neutrophils, consisting mainly of myelocytes and metamyelocytes, was noted. Intriguingly, expression of an essential mediator of neutrophil terminal differentiation, the ets transcription factor PU.1, was significantly decreased in Hbbth3/+ neutrophils. In addition, in vivo infection with Streptococcus pneumoniae failed to induce PU.1 expression or upregulate neutrophil effector functions in Hbbth3/+ mice. Similar changes to neutrophil morphology and PU.1 expression were observed in splenectomized and nonsplenectomized HbE/β-thalassemia patients. This study provides a mechanistic insight into defective neutrophil maturation in β-thalassemia patients, which contributes to deficiencies in neutrophil effector functions.

PFKFB3 Is a Crucial Target in the Treatment of Tyrosine Kinase Inhibitor Resistant Chronic Myelogenous Leukemia

Resistance to the BCR-ABL tyrosine kinase inhibitor (TKI) imatinib mesylate (IM) remains challenge for the treatment of chronic myeloid leukemia (CML). IM resistance often results from unknown mechanisms with wild type BCR-ABL that have no effects on TKIs binding to ABL kinase domain. The basis of such BCR-ABL-independent IM resistance remains to be elucidated. To gain insight into BCR-ABL-independent IM resistance mechanisms, we performed an initial bioinformatics screen on over represented CML genes, followed by a quantitative PCR screen of genes that were elevated in TKIs resistant CML samples. We identified a total of 33 candidate genes that were highly expressed in TKIs resistant patients. Among these genes, 6-Phosphofructo-2-Kinase/Fructose-2,6-Biphosphatase 3 (PFKFB3) controlling the limiting step of glycolysis, was found to strongly associated with TKIs resistance. RNA interference of the expression of PFKFB3 and pharmacological inhibition of its kinase activity markedly increased the sensitivity of TKIs resistant CML cells to TKIs. Furthermore, pharmacological inhibition of PFKFB3 prevented CML cells growth and significantly improved the survival of both allograft and xenograft CML mice. ChIP-seq data analysis combined with subsequent knockdown experiment demonstrated that the Ets transcription factor PU.1 regulates the elevated expression of PFKFB3 in TKIs resistant CML cells. Collectively, our results identify a therapeutically targetable mechanism of BCR-ABL-independent TKIs resistant CML. DisclosuresNo relevant conflicts of interest to declare.

Pruning of the Adipocyte Peroxisome Proliferator-Activated Receptor γ Cistrome by Hematopoietic Master Regulator PU.1

"Master" transcription factors are the gatekeepers of lineage identity. As such, they have been a major focus of efforts to manipulate cell fate for therapeutic purposes. The ETS transcription factor PU.1 has a potent ability to confer macrophage phenotypes on cells already committed to a different lineage, but how it overcomes the presence of other master regulators is not known. The nuclear receptor peroxisome proliferator-activated receptor γ (PPARγ) is the master regulator of the adipose lineage, and its genomic binding pattern in adipocytes is well characterized. Here we show that, when expressed at macrophage levels in mature adipocytes, PU.1 bound a large fraction of its macrophage sites, where it induced chromatin opening and the expression of macrophage target genes. Strikingly, PU.1 markedly reduced the genomic binding of PPARγ without changing its abundance. PU.1 expression repressed genes with nearby adipocyte-specific PPARγ binding sites, while a common macrophage-adipocyte gene expression program was retained. Together, these data reveal unexpected lability within the adipocyte PPARγ cistrome and show that, even in terminally differentiated cells, PU.1 can remodel the cistrome of another master regulator.

Efficient reprogramming of adult neural stem cells to monocytes by ectopic expression of a single gene

Neural stem cells have a broad differentiation repertoire during embryonic development and can be reprogrammed to pluripotency comparatively easily. We report that adult neural stem cells can be reprogrammed at very high efficiency to monocytes, a differentiated fate of an unrelated somatic lineage, by ectopic expression of the Ets transcription factor PU.1. The reprogrammed cells display a marker profile and functional characteristics of monocytes and integrate into tissues after transplantation. The failure to reprogram lineage-committed neural cells to monocytes with PU.1 suggests that neural stem cells are uniquely amenable to reprogramming.

PU.1 - c-Jun Interactions Are Crucial for PU.1 Function in Myeloid Development.

Abstract 3651Poster Board III-587The 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.

Autoregulation of the Transcription Factor PU.1 Via Its Evolutionarily Conserved Upstream Regulatory Element Is Critical in Adult Mouse Hematopoiesis.

Abstract 1468Poster 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.

Transcriptional Regulation of Tlr11 Gene Expression in Epithelial Cells

As sensors of invading microorganisms, Toll-like receptors (TLRs) are expressed not only on macrophages and dendritic cells (DCs) but also on epithelial cells. In the TLR family, Tlr11 appears to have the unique feature in that it is expressed primarily on epithelial cells, although it is also expressed on DCs and macrophages. Here, we demonstrate that transcription of the Tlr11 gene is regulated through two cis-acting elements, one Ets-binding site and one interferon regulatory factor (IRF)-binding site. The Ets element interacts with the epithelium-specific transcription factors, ESE-1 and ESE-3, and the IRF motif interacts with IRF-8. Thus, Tlr11 expression on epithelial cells is regulated by the transcription factors that are presumably distinct from transcription factors that regulate the expression of TLRs in innate immune cells such as macrophages and DCs. Our results imply that the distinctive transcription regulatory machinery for TLRs on epithelium may represent a promising new avenue for the development of epithelia-specific therapeutic interventions.

PU.1 Regulates TCR Expression by Modulating GATA-3 Activity

The Ets transcription factor PU.1 is a master regulator for the development of multiple lineages during hematopoiesis. The expression pattern of PU.1 is dynamically regulated during early T lineage development in the thymus. We previously revealed that PU.1 delineates heterogeneity of effector Th2 populations. In this study, we further define the function of PU.1 on the Th2 phenotype using mice that specifically lack PU.1 in T cells using an lck-Cre transgene with a conditional Sfpi1 allele (Sfpi1(lck-/-)). Although deletion of PU.1 by the lck-Cre transgene does not affect T cell development, Sfpi1(lck-/-) T cells have a lower activation threshold than wild-type T cells. When TCR engagement is limiting, Sfpi1(lck-/-) T cells cultured in Th2 polarizing conditions secrete higher levels of Th2 cytokines and have greater cytokine homogeneity than wild-type cells. We show that PU.1 modulates the levels of TCR expression in CD4(+) T cells by regulating the DNA-binding activity of GATA-3 and limiting GATA-3 regulation of TCR gene expression. GATA-3-dependent regulation of TCR expression is also observed in Th1 and Th2 cells. In CD4(+) T cells, PU.1 expression segregates into subpopulations of cells that have lower levels of surface TCR, suggesting that PU.1 contributes to the heterogeneity of TCR expression. Thus, we have identified a mechanism whereby increased GATA-3 function in the absence of the antagonizing activity of PU.1 leads to increased TCR expression, a reduced activation threshold, and increased homogeneity in Th2 populations.

Dose-dependent repression of T-cell and natural killer cell genes by PU.1 enforces myeloid and B-cell identity

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

Intracellular HMGB1 transactivates the human IL1B gene promoter through association with an Ets transcription factor PU.1

High mobility group box 1 protein (HMGB1), originally described as a non-histone, DNA binding protein, was recently identified as a late mediator of inflammation via its extracellular release from activated macrophages/monocytes. In the present study, we report that intracellular HMGB1 synergizes with a macrophage/monocyte-specific E26 transformation-specific sequence (Ets) transcription factor PU.1 to transactivate the promoter of the IL1B gene coding a 31-kDa proIL-1beta protein. The -131 to +12 IL1B promoter, which possesses a PU.1 binding motif essential for its transactivation, was induced when HMGB1 expression vector was transfected into murine RAW264.7 macrophage cells. Our glutathione S-transferase-pulldown and coimmunoprecipitation assays demonstrated direct physical interaction of HMGB1 with PU.1. Deletion of the PU.1 winged helix-turn-helix DNA-binding domain inhibited the association of the two proteins. In electrophoretic mobility shift assay using recombinant PU.1 protein, a ternary complex of PU.1, HMGB1 and PU.1-binding element within the IL1B promoter was generated. The importance of PU.1 was further supported by our observation that induction of the IL1B promoter was obtained only after PU.1 expression in PU.1-deficient murine EL4 thymoma cells. Thus, our data raise the possibility of a novel mechanism which sustains and amplifies inflammatory reactions through physical interaction of PU.1 with intracellular HMGB1 in macrophages/monocytes.

PU.1 Cooperates with SOX4 in Myeloid Cells.

Mice that express 20% the normal levels of the Ets transcription factor PU.1 develop AML, unlike mice that express 50% or 80% the normal levels, indicating that PU.1 is a dosage-sensitive tumor suppressor gene. In addition, 3 of 13 AMLs induced by transplanting mice with cells transduced with a Sox4 oncogene-containing retrovirus were found to carry a Sox4 retroviral integration in one PU.1 allele, suggesting that downregulation of PU.1 may cooperate with Sox4 in AML induction. Since the other PU.1 allele remains intact in these AMLs and a 50% decrease in PU.1 expression is not sufficient to induce AML, we hypothesized that Sox4 might further downregulate PU.1 expression in these AMLs. To test this hypothesis, we transfected HL60 cells with an expression vector carrying GFP and Sox4 cDNA or a GFP vector control alone. PU.1 mRNA levels were consistently downregulated 4 to 10 fold in cells transfected with Sox4 cDNA compared to cells transfected with the vector control, confirming that overexpression of Sox4 downregulates PU.1 expression in myeloid cells. The decrease of PU.1 mRNA was observed as early as 8 hours after Sox4 transfection, further suggesting that Sox4 may directly interact with PU.1 in myeloid cells. Consistent with this, analysis of 2 published microarray databases comprising 401 de novo AML patient samples showed that SOX4 expression is significantly negatively correlated with PU.1 expression (coefficient: −0.337, P-value: 1E-07). In order to confirm that downregulation of PU.1 cooperates with Sox4 in AML induction, we infected wild type or PU.1 heterozygous knockout bone marrow cells with the Sox4 retrovirus and then monitored the time of AML development in transplanted mice. Results showed increased penetrance (95%) of myeloid leukemia in mice transplanted with Sox4-infected PU +/– bone marrow compared to mice receiving Sox4-infected wild type marrow (60%). Myeloid leukemia was confirmed by histology in all animals of the Sox4-infected PU +/ cohort while T cell lymphoma was diagnosed in 3 animals of the Sox4 wild type cohort. Together, all experiments support the hypothesis that Sox4 cooperates with the transcription factor PU.1.

Acetylation of an Ets Transcription Factor PU.1 Suppresses Its Transcriptional Activity.

PU.1, a member of the Ets family transcription factors, is expressed restrictively in hematopoietic cells including monocytes and macrophages, and plays critical roles in the inflammatory responses and the development of hematopoietic cells. CREB-binding protein (CBP) regulates transcription by acetylating not only histones but also certain transcription factors. Here, we demonstrated that a specific inhibitor of histone deacetylases, trichostatin A (TSA) inhibits PU.1 transcriptional activity in monocytes and further showed that deletion of a histone acetyltransferase (HAT) domain of CBP resulted in synergistic cooperativity between CBP and PU.1. When human monocytic cells THP-1 were treated with TSA, our immunoprecipitaion and western blot assay showed that TSA enhanced PU.1 acetylation. Next, we investigated the effect of TSA on the transcriptional regulation of PU.1-dependent gene promoters such as the human prointerleukin 1β (IL1B) gene and the human granulocyte-macrophage colony-stimulating factor receptor α (GM-CSFRα) gene in transient transfection studies. Two distinct luciferase reporter plasmids (Luc) for the IL1B gene promoter and the GM-CSFRα gene promoter, IL1B-Luc and GM-CSFRα-Luc were used. When these plasmids were transiently transfected into THP-1 cells, TSA suppressed LPS-induced activities for the IL1B promoter and the GM-CSFRα promoter in a dose-dependent manner. In contrast, when NF-κB luciferase reporter, NF-κB-Luc was transfected into THP-1 cells, TSA synergistically increased LPS-induced NF-κB activities. Moreover, when a PU.1 expression vector, pECEPU.1 was cotransfected into PU.1-deficient murine thymocytes EL4 along with either IL1B-Luc or GM-CSFRα-Luc. The PU.1-induced promoter activities were strongly suppressed through TSA treatment. FACS analysis further indicated that TSA suppressed LPS-induced expression of IL-1β and GM-CSFRα proteins. In addition, our EMSA data showed that TSA treatment did not affect DNA binding activity of PU.1 to the IL1B promoter. PU.1 has been shown to interact physically with CBP to transactivate their target genes. In our study, expression vectors for CBP wild-type or with a deletion of its HAT domain was cotransfected into EL4 cells along with IL1B-Luc and pECEPU.1. The HAT activity-deficient mutant showed synergistic transcriptional activity with PU.1 more strongly than the wild-type CBP. In this regard, our GST-pulldown assay showed that deletion of CBP HAT domain did not change binding affinity of CBP for PU.1. Our results propose a novel molecular mechanism by which PU.1-dependent genes is negatively regulated by HAT-induced acetylation in monocytes.

Myeloid Gene Activation and T Cell/Natural Killer Cell Gene Repression in Cells Expressing Two Distinct PU.1 Concentrations.

The Ets transcription factor PU.1 (encoded by the gene Sfpi1) functions in a concentration-dependent manner as a hematopoietic cell fate determinant. PU.1 levels are uniform in early hematopoiesis, increase during myeloid differentiation, and decrease after erythrocyte and T cell/natural killer cell commitment. It is unknown how downstream target genes respond to changes in PU.1 concentration. To address this, we generated mice with two distinct hypomorphic alleles of Sfpi1 and analyzed interleukin-3 dependent cell lines from fetal liver cells homozygous for either allele. PU.1 was produced in these cells at ∼20% (Sfpi1BN/BN) or ∼2% (Sfpi1Blac/Blac) of wild type. These cells fail to terminally differentiate as a consequence of low PU.1 expression and can be maintained as cell lines. To determine what groups of genes are expressed in response to two distinct PU.1 concentrations, we performed whole-genome microarray analysis and compared gene expression in Sfpi1BN/BN and Sfpi1Blac/Blac cell lines to Sfpi1−/− cell lines. Groups of downstream target genes were activated or repressed in four modes in response to the two discrete concentrations of PU.1:1.at higher but not lower PU.1 concentration,2.at lower but not higher PU.1 concentration,3.at both lower and higher concentration, and4.in a gradient fashion.We decided to focus on genes regulated in a gradient manner, because dose-dependency suggests that these may be direct targets of PU.1. Genes activated in a gradient manner were mostly myeloid-specific and enriched for target genes of PU.1. Genes repressed in a gradient manner included erythroid-specific genes and, unexpectedly, T cell and natural killer cell-specific genes. T cell genes were also repressed by PU.1 in cultured progenitor-B cells. With this unique allelic system, we can study three discrete concentrations of PU.1 at 20%, 2%, and 0% to examine concentration-dependent effects of PU.1 on target genes and lineage decisions. Overall, our results suggest that PU.1 functions in a concentration-dependent manner to promote myeloid differentiation and repress T cell or natural killer cell development.

Identification of a monopartite sequence in PU.1 essential for nuclear import, DNA‐binding and transcription of myeloid‐specific genes

The Ets transcription factor PU.1 is an essential regulator of normal hematopoiesis, especially within the myeloid lineage. As such, endogenous PU.1 predominantly localizes to the nucleus of mammalian cells to facilitate gene regulation. However, to date, little is known regarding the mechanisms of PU.1 nuclear transport. We found, using HeLa and RAW 264.7 macrophage cells, that PU.1 enters the nucleus via passive diffusion and active transport. The latter can be facilitated by: (i) the classical pathway requiring importin alpha and beta; (ii) the non-classical pathway requiring only importin beta; or (iii) direct interaction with nucleoporins. A group of six positively charged lysine or arginine residues within the Ets DNA-binding domain was determined to be crucial in active nuclear import. These residues directly interact with importin beta to facilitate a predominantly non-classical import pathway. Furthermore, luciferase reporter assays demonstrated that these same six amino acids are crucial for PU.1-mediated transcriptional activation of myeloid-specific genes. Indeed, these residues may represent a consensus sequence vital for nuclear import, DNA-binding and transcriptional activity of Ets family members. By identifying and characterizing the mechanisms of PU.1 nuclear import and the specific amino acids involved, this report may provide insights into the molecular basis of diseases.

Intracellular HMGB1 Transactivates the Human IL1B Gene Promoter through Association with an ETS Transcription Factor PU.1.

High mobility group box chromosomal protein 1 (HMGB1) was recently identified as a late proinflammatory mediator of endotoxin lethality, which amplifies and sustains the inflammatory processes via macrophage/monocyte activation by extracellular release of HMGB1. However, it was originally described as a nonhistone nuclear protein to bind and distort the structure of DNA and to regulate transcription through association with several transcription factors. Here we investigated the effect of intracellular HMGB1 on transcriptional gene regulation of an immediate early proinflammatory cytokine IL-1β in transient transfection studies using HMGB1 expression vector (pcDNA3HMGB1) and murine macrophage cells RAW264.7. When pcDNA3HMGB1 was introduced into RAW264.7 cells with pGL3HT reporter containing the -131 to +12 minimal IL-1β gene promoter, IL-1β promoter activity was significantly induced by expression of HMGB1. The gene promoter possesses two important transcription factor binding motifs, one for PU.1, a myeloid and B cell-specific transcription factor that belongs to the ETS family, and the other a binding site for NF-IL6. To verify the functional role of intracellular HMGB1 in transactivation of the IL-1β promoter, a PU.1 expression vector and/or pcDNA3HMGB1 were cotransfected into PU.1-deficient murine thymocytes EL4 cells along with pGL3HT reporter. As a result, HMGB1 synergized with PU.1 to transactivate the IL-1β promoter, but not HMGB1 alone. This argument was supported by our GST-pulldown data, which demonstrated direct physical interaction of HMGB1 with PU.1. In addition, deletion of the PU.1 winged helix-turn-helix DNA-binding domain significantly inhibited the association of PU.1 with HMGB1. To determine whether HMGB1 could affect PU.1 DNA-binding affinity, we performed electrophoretic mobility shift assay using a radiolabeled IL-1β -59 to +12 promoter element (DT), recombinant PU.1 (rPU.1) and GST-HMGB1. Two complexes with slower and faster mobilities were generated by the addition of HMGB1 to a mixture of rPU.1 and DT probe. The two complexes were abrogated by preincubation with anti-PU.1 Ab, while anti-HMGB1 Ab reacted only with the complex with a slower mobility, indicating that the complex with a slower mobility formed by addition of HMGB1 contained both HMGB1 and PU.1, while the band with a faster mobility contained only PU.1. From the present study, we propose that intracellular HMGB1 might function as a coactivator in PU.1-mediated transcriptional activation, which facilitate access of PU.1 to specific DNA targets. The fact that PU.1 is a transcription factor essential to macrophages/monocyte-specific proinflammatory cytokine genes further raise the possibility that interaction of PU.1 with HMGB1 may play an important role in the inflammation cascade in macrophages/monocytes.