Molecular basis of Stat1 and PU.1 cooperation in cytokine-induced Fc{gamma} receptor I promoter activation

Saara Aittomäki1, Jie Yang1, Edward W. Scott2, M. Celeste Simon3 and Olli Silvennoinen1,4

1 Institute of Medical Technology, University of Tampere, 33014 Tampere, Finland 2 Department of Molecular Genetics and Microbiology, Shands Cancer Center, University of Florida, Gainesville, FL 32610, USA 3 Abramson Family Cancer Research Institute, Howard Hughes Medical Institute, University of Pennsylvania Cancer Center, Philadelphia, PA 19104, USA 4 Department of Clinical Microbiology, Tampere University Hospital, 33521 Tampere, Finland

Correspondence to: O. Silvennoinen, Institute of Medical Technology, University of Tampere, 33014 Tampere, Finland. E-mail: olli.silvennoinen{at}uta.fi
Transmitting editor: W. J. Leonard


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The high-affinity receptor for IgG (Fc{gamma}RI) is a myeloid cell-specific and IFN-{gamma}-induced gene, and thereby serves as a paradigm for cytokine-induced cell type-specific gene responses. The expression of Fc{gamma}RI is regulated by PU.1 and Stat1 transcription factors. We established an experimental model to analyze the individual functions of Stat1 and PU.1 in cytokine-induced transcription of the natural Fc{gamma}RI promoter in U3A cells lacking both factors. PU.1 was required for both the basal activity and for the IFN-{gamma}-induced Fc{gamma}RI promoter activation, while Stat1 alone could not initiate transcription. In contrast, in the context of a heterologous promoter, PU.1 inhibited the Stat1-mediated transcription. Systematic analysis of Stat1 and PU.1 mutants and Fc{gamma}RI promoter elements revealed that activation of the promoter required the DNA binding, and the transactivation functions of both Stat1 and PU.1. PU.1 and Stat1 bound the promoter elements independently, and no physical interaction between the proteins was observed. The requirement of PU.1 for Fc{gamma}RI promoter activity was supported by demonstration of in vitro interaction between PU.1 and components of the basal transcription machinery TBP and RNA polymerase II. Deletion of the acidic transactivation domain of PU.1 greatly diminished both the Fc{gamma}RI promoter activity as well as the interaction with RNA polymerase II. In contrast, Stat1 did not interact with TBP or RNA polymerase II. These results define functional cooperativity between PU.1 and Stat1 in Fc{gamma}RI promoter activation where PU.1 serves as an amplifier and bridging factor with the basal transcription machinery.

Keywords: monocyte/macrophage, signal transduction, transcription factor


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
IFN-{gamma} plays an important role in the regulation of antiviral and immune responses through binding to the cell-surface IFN-{gamma} receptor (IFN-{gamma}R) (1). IFN-{gamma} binds as a homodimer to two IFN-{gamma}R1 chains and two IFN-{gamma}R2 subunits are additionally required to assemble an active receptor complex. The IFN-{gamma}R1 and IFN-{gamma}R2 chains associate with Jak1 and Jak2 tyrosine kinases respectively, and Jaks become activated via receptor oligomerization (2). Transcriptional responses to IFN-{gamma} are largely dependent on Stat1, which is phosphorylated on Tyr701 by the Jak kinases in the receptor complex. The phosphorylated Stat1 forms SH2 domain-mediated homodimers that are translocated to the nucleus where they bind to specific regions [termed {gamma}-activated sequence (GAS)] on IFN-{gamma}-responsive promoters and modulate their transcription (1).

At the cellular level, IFN-{gamma} stimulation results in activation or repression of hundreds of genes (3). The promoter regions of IFN-{gamma}-responsive genes contain response elements for various transcription factors, and the transcriptional responses involve cooperation between different signaling pathways and cell type- and promoter-specific factors. Stat1 has been shown to cooperate with Sp1 as well as with AP1 and NF-{kappa}B transcription factors (46). The mechanisms by which Stats are connected to basal transcriptional machinery and regulate transcription are still largely unknown. However, recent studies have identified some co-activator proteins in Stat signaling. The histone acetyltransferases CREB-binding protein (CBP) and the related protein p300 function as co-activators for numerous transcription factors (7), and CBP/p300 have also been shown to interact with various Stats (8,9). Stat1-mediated transcription has also been shown to involve P/CIP co-activator (10), and the C-terminal transactivation domain of Stat1 interacts with the DNA replication factor MCM5 and BRCA1 tumor suppressor (11,12).

An important question in transcriptional regulation relates to the mechanisms by which relatively limited numbers of transcription factors can provide the specificity of gene expression. The cell type-specific gene expression represents an important model to address these questions. Several transcription factors such as GATA-1, EKLF, GATA-3, Pax5, TCF-1, Ikaros, E2A and PU.1 have been shown to play cell type-specific functions for hematopoietic cells of various lineages (13). Also, some of the IFN-{gamma}-induced gene responses show strict cell-type specificity and one important cell-type-specific target for IFN-{gamma} is Fc{gamma}RI (CD64) (14). Fc{gamma}RI is the high-affinity receptor for IgG, which mediates phagocytosis, respiratory burst and antibody-mediated cytotoxic reactions. Fc{gamma}RI is constitutively expressed on myeloid cells, but the expression of Fc{gamma}RI is rapidly induced by IFN-{gamma} treatment. Characterization of the Fc{gamma}RI promoter has identified two cis-elements within the 190 nucleotides upstream of the translation initiation site, which confer the IFN-{gamma} inducibility and myeloid cell-specific expression of the gene. The IFN-{gamma} response region (GRR) binds Stat1 and the cell-type-specific expression is mediated via a PU.1-binding element (1517).

PU.1 (Spi-1) is a member of the Ets family of transcription factors which is selectively expressed in myeloid and lymphoid cells. The Ets transcription factors are characterized by a C-terminal winged helix–loop–helix DNA-binding domain (Ets domain). The Ets domain recognizes purine-rich sequences with the core 5'-GGAA/T-3' motif, but some specificity for individual Ets proteins is conferred by the flanking sequences (18). The N-terminal part of PU.1 contains both acidic and glutamine-rich transactivation domains, and the central part consists of a proline, glutamic acid, serine and threonine-rich region (PEST) domain that mediates protein–protein interactions (19). PU.1-binding elements are present in several genes that are critical for development and function of myeloid and B cells, such as CSF-1R, CD11b, CD18, GM-CSFR, G-CSFR, IL-7R{alpha}, the macrophage scavenger receptor, PU.1 itself, Fc{gamma}RI, Ig H and L chain genes, Btk [reviewed in (18,20)], and c-rel (21). The critical role of PU.1 in regulation of these genes largely accounts for the phenotypes of PU.1–/– mice that lack B cells, T cells, macrophages and neutrophils due to defects in propagation and differentiation of progenitor cells (22,23).

The function of PU.1 in cell differentiation and activation of basal transcription has been extensively studied. Transcription factors such as AML1, C/EBPß, C/EBP{delta} and Myb cooperate with PU.1 in activation of basal transcription, and PU.1 has been shown to associate with C/EBPß, C/EBP{delta}, Rb, TFIID, HMG I/Y and SSRP (2427). Interestingly, different functional domains of PU.1 are required for regulation of myeloid and B cell differentiation and basal transcription, and also the expression level of PU.1 is a critical determinant in selection between lymphoid and myeloid differentiation (28,29). For example, Ig{kappa} activation is dependent on phosphorylation of Ser148 on PU.1 and interaction with IRF-4, while induction of myeloid differentiation is independent of Ser phosphorylation and the Ets domain, but involves the glutamine-rich transactivation domain of PU.1 (30,31). The Ets domain of PU.1 also mediates the interaction with GATA-1, which results in abrogation of PU.1 function and allows erythroid differentiation (32). The function of PU.1 in cytokine-induced transcriptional activation and its cooperation with Stat factors has been poorly understood.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Plasmids
The IRF-GAS-luc reporter has been previously characterized (33). The other reporters were constructed by inserting fragments from Fc{gamma}RI promoter, made by PCR using human genomic DNA as a template, into NotI–BamHI sites of promoterless pLuc vector (34). 189/66-{Delta}GAS-luc was constructed by mutating CAG (from –142 to –140) to ACT to disrupt the Stat1-binding site. TCC sequence from the PU.1-binding site (from –94 to –92) was mutated to GAA to give 189/66-{Delta}PU.1-luc. 189/66-{Delta}GAS{Delta}PU.1-luc harbors both CAG to ACT (from –142 to –92) and TCC to GAA (from –94 to –92) mutations. The Stat1 expression constructs were made by subcloning the Stat1 cDNAs (kindly provided by Dr J. E. Darnell Jr) (35,36) into EF-BOS vector (37) containing an HA epitope tag. PU.1 expression plasmids (31) were made by subcloning the PU.1 mutants into pCI-neo vector (Promega, Madison, WI). TBP-glutathione-S-transferase (GST) plasmid was kindly provided by Dr L. Tora.

Cell culture and transfections
Human HeLa cells, monkey COS7 cells and mouse macrophage RAW 264.7 cells were maintained in DMEM, supplemented with 10% FBS. Human monocytic HL-60 cells were grown in RPMI 1640 medium with 10% FBS, and human fibrosarcoma U3A cells, kindly provided by Dr I. Kerr (38), were cultured in DMEM with 10% Cosmic Calf Serum (Hyclone, Logan, UT).

Transfections of HeLa and U3A cells were performed using the calcium phosphate co-precipitation method. COS7 cells and RAW264.7 cells were transfected by electroporation with a GenePulser (Bio-Rad, Hercules, CA), 260 and 250 V respectively, both 960 µF.

Reporter gene assays
RAW 264.7 cells (6 x 106) were transfected with 6 µg of luciferase reporter plasmid and 4 µg of pCMV-ß-galactosidase plasmid. After electroporation, cells were divided on four 3.5-cm plates per transfection and incubated for 24 h. Cells were starved overnight in DMEM with 1% FBS and treated or left untreated for 6 h with 20 ng/ml murine IFN-{gamma}, and lysed in Promega’s Reporter Lysis Buffer. Luciferase activity was measured with 1254 Luminova luminometer (ThermoLab systems, Vantaa, Finland) and normalized against ß-galactosidase activity of the lysates.

U3A cells were transfected on six-well plates with 1 µg of reporter plasmid, 500 ng of pCMV-ß-galactosidase plasmid and different expression plasmids as indicated in the figures. One day after transfection, cells were starved overnight in DMEM with 1% FBS and treated or left untreated for 6 h with 10 ng/ml human IFN-{gamma}. Assays were performed as above.

Electrophoretic mobility shift assay (EMSA)
COS7 cells (3 x 106) were transfected with 1 µg of Stat1 or with Stat1 and 0.5 µg of PU.1 plasmid, and divided onto two plates. Cells were incubated for 2 days and starved overnight. HL-60 cells, RAW264.7 cells and transfected COS7 cells were treated with 100 ng/ml IFN-{gamma} for 15 min or left untreated. Nuclear extracts were prepared as previously described (39). Binding reactions were performed as described (40) with 6 µg of COS7 nuclear extracts or 10 µg of HL-60 or RAW264.7 extracts and oligonucleotide probe representing Fc{gamma}RI promoter region from –189 to +1, prepared by digesting the fragment from Fc{gamma}RI-luc plasmid.

GST pull-down assay
GST and GST-TBP proteins were produced in BL21 bacteria and purified with glutathione–Sepharose 4B (Amersham Biosciences, Little Chalfont, UK) according to the manufacturer’s instructions. COS7 cells (2 x 106) were transfected with 2 µg of PU.1 expression vector and after 2 days in culture nuclei were extracted as in the EMSA protocol (39). Then, 100 µg protein from COS7 extracts or 200 µg protein from HL-60 nuclear extracts in 80 µl of lysis buffer was incubated overnight at 4°C with the GST fusion proteins bound to the glutathione–Sepharose beads in 240 µl of binding buffer (12.5 mM HEPES, pH 7.4, 0.1 mM EDTA, 0.05% Nonidet P-40, 0.5% BSA, 1 mM DTT, 1 mM PMSF and 3 µg/ml aprotinin). The beads were washed 5 times with binding buffer containing 70 mM NaCl. Proteins were boiled in SDS–PAGE sample buffer, separated by 10% SDS–PAGE and analyzed by immunoblot assay with anti-PU.1 or anti-Stat1 antibody (Santa Cruz Biotechnology, Santa Cruz, CA and Transduction Laboratories, Lexington, KY respectively).

In vitro co-immunoprecipitation
COS7 whole-cell extracts were prepared by lysing the cells in a buffer containing 0.5% Nonidet P-40, 50 mM Tris–HCl, pH 8.0, 300 mM NaCl, 1 mM PMSF and 3 µg/ml aprotinin. HeLa nuclear extracts were prepared as in EMSA protocol. COS7 or HeLa extracts were immunoprecipitated with anti-RNA polymerase II antibody (Santa Cruz Biotechnology) in lysis buffer in which NaCl concentration had been adjusted to 100 mM and, after extensive washing, incubated with in vitro translated [35S]methionine-labeled PU.1 wild-type or deletion mutant {Delta}33–74 or Stat1. The bound proteins were subjected to SDS–PAGE and visualized by autoradiography.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Stat1 and PU.1 are required to activate IFN-{gamma}-induced transcription of Fc{gamma}RI in a heterologous cell system
To examine the functions of Stat1 and PU.1 in Fc{gamma}RI promoter activation, the immediate Fc{gamma}RI promoter (–189 to +1) (17) containing both Stat1 and PU.1 binding elements was cloned into a promoterless luciferase reporter gene vector (Fc{gamma}RI-luc). The basal luciferase reporter gene lacks detectable activity in all of the studied cell lines (RAW264.7, THP-1, HL-60, U3A, HeLa and HepG2). Transfection of Fc{gamma}RI-luc into murine macrophage RAW264.7 cells resulted in modest basal activation of the reporter gene and IFN-{gamma} stimulation resulted in 4-fold induction of transcription (Fig. 1B). The regulation of the Fc{gamma}RI-luc is thus reminiscent of the expression pattern of Fc{gamma}RI on human monocytes (14,40). To establish a cellular system that allows investigation of the functional roles of PU.1 and Stat1, regulation of Fc{gamma}RI-luc was next examined in the U3A fibrosarcoma cell line that lacks both PU.1 and Stat1 proteins (38). U3A cells were transfected with Fc{gamma}RI-luc in the presence or absence of Stat1{alpha} and PU.1. Fc{gamma}RI-luc alone did not show either basal or induced activity in U3A cells. Transfection of Stat1{alpha} alone did not induce significant reporter activation, but co-transfection of PU.1 and Stat1{alpha} resulted in a basal and IFN-{gamma}-induced Fc{gamma}RI-luc response that mimics the regulation of reporter gene in myeloid cells. To confirm the individual roles of Stat1 and PU.1 in the activation of Fc{gamma}RI, we constructed a promoter construct of Fc{gamma}RI containing GRR and PU.1 sites (–189 to –66). U3A cells were transfected with Stat1 and 189/66-luc constructs in which the binding site for Stat1 or PU.1, or both sites, had been mutated (189/66-{Delta}GAS-luc, 189/66-{Delta}PU.1-luc and 189/66-{Delta}GAS{Delta}PU.1-luc respectively). As shown in Fig. 1(C), 189/66-luc was activated and further induced with IFN-{gamma} in the presence of co-transfected PU.1, but mutation of either Stat1 or PU.1 elements abrogated the activity. In RAW264.7 cells, mutation of the Stat1 binding site resulted in the loss of IFN-{gamma} inducibility and mutation of the PU.1 element decreased the basal activity of the construct (Fig. 1D). Thus, the presence of both PU.1 and Stat1 is required for the optimal activation of the Fc{gamma}RI promoter, confirming that the model system reflects the regulation of the natural Fc{gamma}RI gene in myeloid cells.



View larger version (16K):
[in this window]
[in a new window]
 
Fig. 1. Stat1 and PU.1 are required for IFN-{gamma}-induced activation of Fc{gamma}RI promoter. (A) Schematic diagram of the reporter constructs. (B) RAW264.7 cells were transfected with Fc{gamma}RI-luc reporter construct and pCMV-ß-gal plasmid, and U3A cells with Fc{gamma}RI-luc, pCMV-ß-gal and empty vector or Stat1 or PU.1 plus Stat1, and treated with 10 ng/ml of IFN-{gamma} for 6 h. The luciferase values were normalized to ß-galactosidase activities and are presented as relative luciferase units. The values represent means ± SD from three experiments. (C) Effect of mutation of Stat1 and PU.1 binding sites on activation of the 189/66-luc construct. U3A cells were transfected with reporter plasmids as indicated together with Stat1 alone or together with PU.1 and treated as in (B). Representative data from one experiment is shown. (D) Effect of mutation of Stat1 and PU.1 binding sites on activation of the 189/66-luc construct in RAW264.7 cells. The cells were transfected with reporter plasmids as indicated and treated as in (B). The data represent means ± SD from three experiments.

 
DNA binding and transactivation domains of Stat1 are required for cooperation with PU.1
To determine the structural requirements of Stat1 for transcriptional activation of the Fc{gamma}RI gene, we compared different Stat1 constructs for their ability to activate transcription of the natural Fc{gamma}RI promoter and the construct consisting of Stat1 binding site from IRF-1 gene in the context of a heterologous thymidine kinase (TK) promoter (GAS-luc) (33). Equimolar amounts of Stat1 mutants (Fig. 2A), determined by western blotting (data not shown), were expressed in U3A cells together with Fc{gamma}RI-luc (Fig. 2B) or GAS-luc (Fig. 2C) reporter. Expression of PU.1 resulted in basal activation of Fc{gamma}RI-luc, but both PU.1 and wild-type Stat1{alpha} were required for IFN-{gamma} responsiveness of the reporter gene (Fig. 2B). The basal activity of Fc{gamma}RI-luc was not significantly affected by expression or IFN-{gamma}-induced activation of Stat1{alpha}. However, expression of Stat1{alpha} alone was able to mediate a strong IFN-{gamma} response in GAS-luc (Fig. 2C). The naturally occurring splice variant Stat1ß that lacks the 38 C-terminal residues of Stat1{alpha} constituting the transactivation domain was not able to stimulate either GAS-luc or Fc{gamma}RI-luc transcription after IFN-{gamma} treatment. The DNA binding of Stat1 was required for activation of both promoters, since the DNA-binding-deficient mutant of Stat1 (Glu428Ala and Glu429Ala) that still undergoes tyrosine phosphorylation and nuclear translocation (41) was unable to activate IFN-{gamma}-dependent transcription. Phosphorylation of Stat1 on serine residue 727 has been shown to selectively modulate Stat1-dependent gene responses (42), and regulate the interaction of Stat1 with MCM5 and BRCA1 proteins (11,12). Transfection of U3A cells with Stat1 (Ser727Ala) mutant expression plasmid resulted in diminished IFN-{gamma} response of both reporter genes. These results demonstrate that the DNA-binding and the transactivation functions of Stat1 are required for IFN-{gamma}-induced activation of both Fc{gamma}RI-luc and GAS-luc reporter genes. Stat1 binding to the promoter was not sufficient for transcriptional activation, which requires either the presence of PU.1 or a heterologous promoter. To rule out the possibility that PU.1 could have a general activation effect on transcription, U3A cells were transfected with the GAS-luc construct together with Stat1{alpha} and different amounts of PU.1 (Fig. 2D). Co-transfection of the same amount of PU.1 plasmid (50 ng) that supports Fc{gamma}RI-luc activation resulted in clear inhibition of the IFN-{gamma}-induced transcription of GAS-luc and the inhibition was further substantiated by increasing the amount of transfected PU.1. With Fc{gamma}RI-luc, increasing the amount of transfected PU.1 slightly enhanced the activation (data not shown).



View larger version (21K):
[in this window]
[in a new window]
 
Fig. 2. Transactivation domain and DNA-binding domain of Stat1 are required for cooperation with PU.1 in U3A cells. Protein expression levels of different Stat1 variants were checked by western blotting, and equimolar amounts were transfected into U3A cells together with PU.1 and reporter plasmids as indicated. (A) Schematic diagram of the Stat1 constructs. Effect of the various Stat1 proteins on transcription of Fc{gamma}RI-luc (B) and GAS-luc (C). (D) Effect of PU.1 on GAS-luc reporter, the DNA amounts used for transfection are indicated. Mean ± SD values from three independent experiments are shown.

 
Transactivation domains and DNA binding of PU.1 are required for the cooperation with Stat1
The experiments in Figs 1 and 2 indicate that PU.1 is required for Stat1-dependent Fc{gamma}RI promoter activation. Previous studies have shown that depending on the promoter context, different domains of PU.1 are involved in regulation of gene expression. To delineate the functional domains of PU.1 that were required for cooperation with Stat1 in IFN-{gamma} induction of the Fc{gamma}RI gene, we analyzed PU.1 mutants harboring deletions or mutations of different functional domains.

U3A cells were transfected with the Fc{gamma}RI-luc and Stat1{alpha} together with equimolar amounts (determined by western blotting) of different PU.1 constructs (31) (Fig. 3A). Phosphorylation of PU.1 on Ser148 in response to lipopolysaccharide treatment has been shown to result in enhanced transcriptional activity of PU.1 and to promote interaction with IRF-4 (43). PU.1 expression plasmid in which Ser148 had been mutated into alanine was as efficient as wild-type PU.1 in activating both basal and IFN-{gamma}-induced transcription of Fc{gamma}RI promoter (Fig. 3B). The PEST domain of PU.1 has been shown to be required for protein–protein interactions between PU.1 and IRF-4 in B cells, and also to modulate myeloid differentiation (30). Deletion of the PEST domain ({Delta}118–167) resulted in a slight decrease in the ability of PU.1 to cooperate with Stat1 and to activate basal transcription of Fc{gamma}RI. The N-terminal half of PU.1 (~100 amino acids) has been shown to harbor multiple transactivation regions (28). Deletion of the glutamine-rich domain, {Delta}75–100, which is required for myeloid differentiation (31), significantly reduced both basal and IFN-{gamma}-induced transcription of Fc{gamma}RI-luc (50% basal and 34% induced activity compared to wild-type PU.1). Deletion of a region rich in acidic amino acids, {Delta}33–74, resulted in a protein with less than a third (29%) of the basal activity of wild-type PU.1 and greatly reduced ability (14%) to cooperate with Stat1 in IFN-{gamma}-induced transcriptional activation. A mutant containing only the DNA-binding domain of PU.1 ({Delta}2–167) failed to support significant basal or IFN-{gamma}-induced transcription. Mutant {Delta}245–272 that lacks 10 carboxyl amino acids of the DNA-binding domain, and is unable to bind to DNA, was also inactive. These results indicate that the DNA-binding activity of PU.1 is indispensable, but not sufficient, for Fc{gamma}RI-luc induction, and both the basal and IFN-{gamma}-induced transcription of Fc{gamma}RI is regulated by glutamine-rich and acidic transactivation regions, and to a lesser extent also by the PEST domain.



View larger version (20K):
[in this window]
[in a new window]
 
Fig. 3. Transactivation domains and DNA binding of PU.1 are required for Fc{gamma}RI-luc activation. (A) Schematic diagram of the PU.1 constructs. (B) Different PU.1 constructs were transfected into U3A cells along with Fc{gamma}RI-luc and Stat1 as described in Fig. 2. Shown are the mean normalized values of three experiments with SD.

 
Stat1 and PU.1 bind independently to Fc{gamma}RI promoter
Next, we wanted to gain more insight into the functional cooperation between Stat1 and PU.1 in Fc{gamma}RI activation. Both Stat1 and PU.1 have been shown to interact with other transcription factors or transcriptional co-regulators (46,8,2427,44), and we analyzed whether their cooperation would involve protein–protein interaction with each other. Co-immunoprecipitation experiments in Stat1- and PU.1-transfected COS7 cells, and with in vitro translated proteins, as well as pull-down experiments with GST-PU.1 failed to demonstrate physical interaction between Stat1 and PU.1 (data not shown). Furthermore, co-expression of PU.1 was not able to enhance the activity of a GAL4–Stat1 transactivation domain fusion construct (data not shown).

To examine whether Stat1 and PU.1 would show cooperative DNA binding, EMSA was performed with the Fc{gamma}RI probe (nucleotides –189 to +1). COS7 cells, which lack endogenous PU.1, were transfected with either Stat1 or with Stat1 and PU.1. Nuclear extracts from COS7 cells and from myeloid HL-60 and RAW264.7 cells all showed similar IFN-{gamma}-stimulated DNA-binding activity (Fig. 4) which was supershifted with an antibody against the N-terminus of Stat1 (data not shown). Expression of PU.1 did not affect the Stat1-binding complex. We also investigated the possibility that IFN-{gamma} stimulation and/or Stat1 promoter binding would regulate the DNA binding of PU.1. In COS7 cells the PU.1-binding complex was detected only in PU.1-transfected cells, suggesting that in non-myeloid cells other Ets proteins are not able to substitute for PU.1 in binding to Fc{gamma}RI. IFN-{gamma} treatment did not affect the PU.1-binding complex in either COS7 or in HL-60 or RAW264.7 cells. The slight increase in PU.1-binding complex in transfected COS7 cells after IFN-{gamma} stimulation was not observed in other experiments. Furthermore, treatment of HL-60 cells with IFN-{gamma} for 30 min, and 1, 3, 6 and 24 h did not affect PU.1 binding to a probe corresponding to the PU.1 element in Fc{gamma}RI promoter (–104 to –79) (data not shown). These results indicate that PU.1 binding to the promoter is constitutive and not regulated by IFN-{gamma} or Stat1. The differences in the mobility of the PU.1 complexes are likely to represent differences in proteolytic cleavage between species and cell lines as previously noted (15). Taken together, the EMSA results indicate that Stat1 and PU.1 are binding independently to their respective response elements in Fc{gamma}RI promoter. In line with these results, supershifting the DNA-binding complexes with anti-Stat1 and anti-PU.1 antibodies failed to demonstrate Stat1 and PU.1 in the same complexes (data not shown).



View larger version (77K):
[in this window]
[in a new window]
 
Fig. 4. Stat1 and PU.1 bind to Fc{gamma}RI as separate complexes. COS7 cells were transfected with Stat1 and PU.1 as indicated, and treated with vehicle or 100 ng/ml of IFN-{gamma} for 15 min. Nuclear lysates were prepared from untreated and IFN-{gamma} treated COS7 cells, HL-60 cells and RAW264.7 cells, and analysed by EMSA with 32P-labeled Fc{gamma}RI probe. The mobilities of Stat1 and the different PU.1 complexes are indicated by arrows.

 
PU.1, but not Stat1, physically associates with TBP and RNA polymerase II
Fc{gamma}RI is a TATA-less promoter and next we addressed the question whether Stat1 or PU.1 could function as bridging molecules with general transcription factors (GTF). TBP plays a critical role in the assembly of the pre-initiation complex and previously PU.1 has been shown to interact with TBP in vitro (25). Also, in our experiments, purified recombinant TBP-GST was found to interact with transfected PU.1 in COS7 cells and with endogenous PU.1 in HL-60 cells (Fig. 5A and B). The differences in the mobility of PU.1 are likely due to species differences in proteolytic cleavage. In contrast, TBP-GST was not able to interact with Stat1 in IFN-{gamma}-treated cells (Fig. 5C). Also, in unstimulated cells where Stat1 is in a monomeric form, TBP-GST failed to interact with Stat1 (data not shown).



View larger version (48K):
[in this window]
[in a new window]
 
Fig. 5. PU.1, but not Stat1, associates with TBP and RNA polymerase II. (A) COS7 cells were transfected with PU.1 and treated with IFN-{gamma} for 15 min. Nuclear extracts were prepared and incubated with glutathione–Sepharose-bound GST or GST-TBP. Bound proteins were resolved by SDS–PAGE and immunoblotted with anti-PU.1 antibody. (B) Nuclear extracts from HL-60 cells were incubated with glutathione–Sepharose-bound GST or GST-TBP and processed as in (A). (C) The same lysates as in (A) were immunoblotted with anti-Stat1 antibody. (D) COS7 whole-cell extract was incubated with anti-RNA polymerase II antibody and the immunocomplex was purified with Protein A–Sepharose. Extract that had been incubated with Protein A–Sepharose alone was used as a control. PU.1 wild-type and Stat1 were labeled with [35S]methionine by in vitro translation, incubated with the immunoprecipitated RNA polymerase II and the control, washed extensively, eluted by SDS–PAGE sample buffer, resolved by SDS–PAGE, and autoradiographed. (E) In vitro translated wild-type PU.1 and PU.1 deletion mutant {Delta}33–74 were incubated with either control (pre-immune rabbit sera) or RNA polymerase II immunoprecipitates (PolII IP) from HeLa cell nuclear extracts. The reaction conditions and detection were carried out as in (D).

 
To investigate further the functional roles of Stat1 and PU.1 in transcriptional activation, we examined the possibility that these factors could mediate interaction with RNA polymerase II. To this end, endogenous RNA polymerase II was immunoprecipitated from COS7 cells. The RNA polymerase II immunoprecipitates were washed stringently, and then incubated with in vitro translated Stat1 and PU.1 proteins. After extensive washes the RNA polymerase II-associated proteins were analyzed by autoradiography. PU.1 was found to interact with RNA polymerase II, while no interaction between Stat1 and RNA polymerase II was detected (Fig. 5D). Deletion of the acidic domain of PU.1 resulted in decreased interaction with RNA polymerase II (Fig. 5E).


    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
We have investigated the mechanisms that underlie cell-type-specific gene regulation in IFN-{gamma} signaling in the context of the natural Fc{gamma}RI promoter. We have previously examined the function of co-activator CBP/p300 in Fc{gamma}RI activation and shown that the function of Stat1 critically requires CBP/p300 (45). In the present study, we investigated the functional cooperation between Stat1 and PU.1 in more detail, and show that Fc{gamma}RI promoter activation requires the DNA-binding as well as the transactivation functions of both PU.1 and Stat1. However, the functions of these proteins appear to differ significantly in Fc{gamma}RI activation. PU.1 mediated the basal activation of the promoter and was found to interact with components of the basal transcription machinery, whereas the function of Stat1 was restricted to IFN-{gamma}-stimulated transcription.

Fc{gamma}RI can be considered as a paradigm for a cell-type-specific IFN-{gamma} response, where Stat1 and PU.1 cooperatively regulate the promoter activation. The IFN-{gamma}-induced optimal Fc{gamma}RI promoter activation in myeloid and in non-myeloid cells demonstrated an absolute requirement for simultaneous binding of Stat1 and PU.1 to the promoter. PU.1 and Stat1 bound the promoter independently, and no evidence for physical interaction or cooperative DNA binding between PU.1 and Stat1 was detected. These results suggest that the cooperation between Stat1 and PU.1 must rely on modulation of transcriptional activation.

PU.1 is a multifaceted transcription factor that plays a critical role in regulation of numerous genes (18). The promoter context appears to determine which functional domains of PU.1 are required for transcriptional regulation. We examined the function of PU.1 in cytokine-regulated gene activation, and the results showed that both the DNA-binding Ets domain and the transactivation domains were required for the basal activity of Fc{gamma}RI promoter as well as for cooperation with Stat1. The DNA-binding domain of PU.1 is a winged helix–turn–helix structure and deletion of the amino acids 245–272 abolishes the DNA interacting wing structure between ß sheets 3 and 4 (46). The requirement for the Ets domain for Fc{gamma}RI activity was consistent with the EMSA results that showed constitutive DNA binding of PU.1. However, the Ets domain has also been shown to interact with transcriptional regulators such as GATA-1, c-Jun, cytomegalovirus IE2, C/EBPß and C/EBP{delta}, thus the Ets domain deletion could also affect critical interaction with other transcription factors (24,26,32,47,48). However, the construct encompassing only the Ets domain of PU.1 did not mediate transcriptional activation, thus the putative interaction with other transcription factors is at least not sufficient for promoter activation. The cooperation between PU.1 and c-Jun in M-CSF receptor expression also requires the transactivation domains of PU.1, while the cooperation between PU.1 and IE2 on IL-1ß gene transcription is independent of the transactivation function of PU.1 (47,48). However, in vitro macrophage development requires the glutamine-rich and PEST domains, but not the acidic transactivation domain or the Ets domain of PU.1 (31). The function of PU.1 on the Ig{kappa} 3' enhancer represents still another variation to the functional requirements of PU.1, as it is dependent only on the DNA-binding activity of PU.1 (49). Our results indicated that the acidic transactivation domain was absolutely required for Fc{gamma}RI promoter function and deletion of the glutamine-rich domain also significantly reduced the promoter activity. The differences in requirements for distinct PU.1 functional domains are likely to represent the complexity of gene regulation, and rely on differences in the composition of promoter-bound transcription factors and their interaction with GTF on various PU.1 target genes.

A typical feature for almost all myeloid-specific genes including Fc{gamma}RI is the lack of the TATA element in the promoter region. Instead, these promoters contain an initiator sequence, and PU.1 might function by recruiting TBP and thus allowing assembly of the pre-initiation complex on the promoter. Our results support the role for PU.1 in mediating basal activation through interaction with basal transcription machinery. Consistent with previous results (25), recombinant TBP-GST interacted with PU.1. However, previously we have shown that a heterologous TATA-containing promoter could not substitute for the function of PU.1 in Fc{gamma}RI activation (45). Alternatively, PU.1 may be mediating other critical functions in Fc{gamma}RI promoter activation. This hypothesis was supported by the findings that immunoprecipitated RNA polymerase II interacted with in vitro translated PU.1. The acidic transactivation domains have been shown to mediate interaction with transcriptional regulators and with basal transcription factors (50). In line with these findings, deletion of the acidic transactivation region of PU.1, which was critical for Fc{gamma}RI promoter activity, compromised the ability of PU.1 to interact with RNA polymerase II. The activation function of PU.1 was promoter dependent since PU.1 inhibited the IFN-{gamma} response of the GAS-luc reporter containing a TK promoter. The inhibitory mechanism of PU.1 in the TK promoter is currently unknown, but could involve competition of basal transcription factors on TK promoter, recruitment of histone deacetylases to the promoter or direct repression of CBP acetyltransferase activity by PU.1. PU.1 has been shown to form a complex with histone deacetylase HDAC1 and to repress the activity of c-myc (51). Hong et al., on the other hand, showed that PU.1 inhibits CBP-mediated acetylation of several histone/non-histone proteins and CBP-dependent transcriptional events, and blocks erythroid differentiation by directly binding to CBP (52). Thus, the role and function of PU.1 in transcriptional regulation is determined by the promoter context.

The molecular mechanisms leading to Stat activation and DNA binding in cytokine signaling are well established. However, most of the studies related to Stat-mediated transcriptional activation have used artificial promoters in the context of heterologous promoters thus leaving open the mechanisms by which Stats are connected to basal transcription machinery. Stat1 alone was not able to induce significant promoter activation, which is consistent with the function of PU.1 as a bridging factor for GTF. In accordance with this concept is the lack of any association between Stat1 and either TBP or RNA polymerase II, and the very poor activity of the isolated transactivation domain of Stat1 in a heterologous GAL4 transcription system (data not shown) (53). However, the transactivation domain of Stat1 was strictly required for Fc{gamma}RI promoter activation. The transactivation domain of Stat1 has been shown to mediate interaction with CBP (8,54). PU.1 has also been shown to interact with CBP, and co-transfection of CBP has been shown to stimulate both PU.1- and Stat1-dependent reporter constructs (8,44). By using well-characterized mutants of E1A (55), we previously showed that the function of Stat1 in transcriptional activation was dependent on its ability to recruit CBP/p300 (45). Recruitment of CBP/p300 was also involved in, but was not absolutely required for, the function of PU.1. The CBP interaction domain in PU.1 has been mapped to a central region encompassing the glutamine-rich domain and part of the PEST domain (44). Deletion of these regions reduced, but did not abolish, the transactivation function of PU.1 in the Fc{gamma}RI promoter, thus also supporting the view that the critical function of PU.1 in the Fc{gamma}RI promoter is to mediate the interaction with GTF. As previously shown, distinct functional domains of PU.1 are required for activation of different promoters (19). For Stat1-dependent Fc{gamma}RI gene activation, the DNA binding and transactivation functions of PU.1 were required since the transactivation domain of Stat1 fails to interact with GTF. However, the transactivation and bridging functions of PU.1 may not be required in promoters where other transcription factors can provide the interaction with GTF, and in these instances the function of PU.1 would be to regulate the holoenzyme assembly.


    Acknowledgements
 
We thank Paula Kosonen for excellent technical assistance, and Drs J. E. Darnell, L. Tora and I. Kerr for kindly providing reagents. This work was supported by the Medical Research Council (Academy of Finland), Medical Research Fund of Tampere University Hospital, Finnish Foundation for Cancer Research, Sigrid Juselius Foundation and Tuberculosis Foundation of Tampere.


    Abbreviations
 
CBP—CREB-binding protein

EMSA—electrophoretic mobility shift assay

GAS—{gamma}-activated sequence

GRR—IFN-{gamma} response region

GST—glutathione-S-transferase

IFN-{gamma}R—IFN-{gamma} receptor

PEST—proline, glutamic acid, serine and threonine-rich region

TK—thymidine kinase


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

  1. Darnell, J. E., Jr, Kerr, I. M. and Stark, G. R. 1994. Jak–STAT pathways and transcriptional activation in response to IFNs and other extracellular signaling proteins. Science 264:1415.[ISI][Medline]
  2. Silvennoinen, O., Saharinen, P., Paukku, K., Takaluoma, K. and Kovanen, P. 1997. Cytokine receptor signal transduction through Jak tyrosine kinases and Stat transcription factors. Apmis 105:497.[ISI][Medline]
  3. Ehrt, S., Schnappinger, D., Bekiranov, S., Drenkow, J., Shi, S., Gingeras, T. R., Gaasterland, T., Schoolnik, G. and Nathan, C. 2001. Reprogramming of the macrophage transcriptome in response to interferon-gamma and Mycobacterium tuberculosis. Signaling roles of nitric oxide synthase-2 and phagocyte oxidase. J. Exp. Med. 194:1123.[Abstract/Free Full Text]
  4. Look, D. C., Pelletier, M. R., Tidwell, R. M., Roswit, W. T. and Holtzman, M. J. 1995. Stat1 depends on transcriptional synergy with Sp1. J. Biol. Chem. 270:30264.[Abstract/Free Full Text]
  5. Horvai, A. E., Xu, L., Korzus, E., Brard, G., Kalafus, D., Mullen, T. M., Rose, D. W., Rosenfeld, M. G. and Glass, C. K. 1997. Nuclear integration of JAK/STAT and Ras/AP-1 signaling by CBP and p300. Proc. Natl Acad. Sci. USA 94:1074.[Abstract/Free Full Text]
  6. Ohmori, Y., Schreiber, R. D. and Hamilton, T. A. 1997. Synergy between interferon-gamma and tumor necrosis factor-alpha in transcriptional activation is mediated by cooperation between signal transducer and activator of transcription 1 and nuclear factor kappaB. J. Biol. Chem. 272:14899.[Abstract/Free Full Text]
  7. Blobel, G. A. 2000. CREB-binding protein and p300: molecular integrators of hematopoietic transcription. Blood 95:745.[Free Full Text]
  8. Zhang, J. J., Vinkemeier, U., Gu, W., Chakravarti, D., Horvath, C. M. and Darnell, J. E., Jr. 1996. Two contact regions between Stat1 and CBP/p300 in interferon gamma signaling. Proc. Natl Acad. Sci. USA 93:15092.[Abstract/Free Full Text]
  9. Shuai, K. 2000. Modulation of STAT signaling by STAT-interacting proteins. Oncogene 19:2638.[CrossRef][ISI][Medline]
  10. Torchia, J., Rose, D. W., Inostroza, J., Kamei, Y., Westin, S., Glass, C. K. and Rosenfeld, M. G. 1997. The transcriptional co-activator p/CIP binds CBP and mediates nuclear-receptor function. Nature 387:677.[CrossRef][ISI][Medline]
  11. Zhang, J. J., Zhao, Y., Chait, B. T., Lathem, W. W., Ritzi, M., Knippers, R. and Darnell, J. E., Jr. 1998. Ser727-dependent recruitment of MCM5 by Stat1alpha in IFN-gamma-induced transcriptional activation. EMBO J. 17:6963.[Abstract/Free Full Text]
  12. Ouchi, T., Lee, S. W., Ouchi, M., Aaronson, S. A. and Horvath, C. M. 2000. Collaboration of signal transducer and activator of transcription 1 (STAT1) and BRCA1 in differential regulation of IFN-gamma target genes. Proc. Natl Acad. Sci. USA 97:5208.[Abstract/Free Full Text]
  13. Orkin, S. H. 1995. Transcription factors and hematopoietic development. J. Biol. Chem. 270:4955.[Free Full Text]
  14. Guyre, P. M., Morganelli, P. M. and Miller, R. 1983. Recombinant immune interferon increases immunoglobulin G Fc receptors on cultured human mononuclear phagocytes. J. Clin. Invest. 72:393.[ISI][Medline]
  15. Perez, C., Coeffier, E., Moreau-Gachelin, F., Wietzerbin, J. and Benech, P. D. 1994. Involvement of the transcription factor PU.1/Spi-1 in myeloid cell-restricted expression of an interferon-inducible gene encoding the human high-affinity Fc gamma receptor. Mol. Cell. Biol. 14:5023.[Abstract]
  16. Pearse, R. N., Feinman, R., Shuai, K., Darnell, J. E., Jr and Ravetch, J. V. 1993. Interferon gamma-induced transcription of the high-affinity Fc receptor for IgG requires assembly of a complex that includes the 91-kDa subunit of transcription factor ISGF3. Proc. Natl Acad. Sci. USA 90:4314.[Abstract]
  17. Eichbaum, Q. G., Iyer, R., Raveh, D. P., Mathieu, C. and Ezekowitz, R. A. 1994. Restriction of interferon gamma responsiveness and basal expression of the myeloid human Fc gamma R1b gene is mediated by a functional PU.1 site and a transcription initiator consensus. J. Exp. Med. 179:1985.[Abstract]
  18. Lloberas, J., Soler, C. and Celada, A. 1999. The key role of PU.1/SPI-1 in B cells, myeloid cells and macrophages. Immunol. Today 20:184.[CrossRef][ISI][Medline]
  19. Fisher, R. C. and Scott, E. W. 1998. Role of PU.1 in hematopoiesis. Stem Cells 16:25.[Abstract/Free Full Text]
  20. Simon, M. C. 1998. PU.1 and hematopoiesis: lessons learned from gene targeting experiments. Semin. Immunol. 10:111.[CrossRef][ISI][Medline]
  21. Colucci, F., Samson, S. I., DeKoter, R. P., Lantz, O., Singh, H. and Di Santo, J. P. 2001. Differential requirement for the transcription factor PU.1 in the generation of natural killer cells versus B and T cells. Blood 97:2625.[Abstract/Free Full Text]
  22. Olson, M. C., Scott, E. W., Hack, A. A., Su, G. H., Tenen, D. G., Singh, H. and Simon, M. C. 1995. PU.1 is not essential for early myeloid gene expression but is required for terminal myeloid differentiation. Immunity 3:703.[ISI][Medline]
  23. McKercher, S. R., Torbett, B. E., Anderson, K. L., Henkel, G. W., Vestal, D. J., Baribault, H., Klemsz, M., Feeney, A. J., Wu, G. E., Paige, C. J. and Maki, R. A. 1996. Targeted disruption of the PU.1 gene results in multiple hematopoietic abnormalities. EMBO J. 15:5647.[Abstract]
  24. Yang, Z., Wara-Aswapati, N., Chen, C., Tsukada, J. and Auron, P. E. 2000. NF-IL6 (C/EBPbeta) vigorously activates il1b gene expression via a Spi-1 (PU.1) protein–protein tether. J. Biol. Chem. 275:21272.[Abstract/Free Full Text]
  25. Hagemeier, C., Bannister, A. J., Cook, A. and Kouzarides, T. 1993. The activation domain of transcription factor PU.1 binds the retinoblastoma (RB) protein and the transcription factor TFIID in vitro: RB shows sequence similarity to TFIID and TFIIB. Proc. Natl Acad. Sci. USA 90:1580.[Abstract]
  26. Nagulapalli, S., Pongubala, J. M. and Atchison, M. L. 1995. Multiple proteins physically interact with PU.1. Transcriptional synergy with NF-IL6 beta (C/EBP delta, CRP3). J. Immunol. 155:4330.[Abstract]
  27. Petrovick, M. S., Hiebert, S. W., Friedman, A. D., Hetherington, C. J., Tenen, D. G. and Zhang, D. E. 1998. Multiple functional domains of AML1: PU.1 and C/EBPalpha synergize with different regions of AML1. Mol. Cell. Biol. 18:3915.[Abstract/Free Full Text]
  28. Klemsz, M. J. and Maki, R. A. 1996. Activation of transcription by PU.1 requires both acidic and glutamine domains. Mol. Cell. Biol. 16:390.[Abstract]
  29. DeKoter, R. P. and Singh, H. 2000. Regulation of B lymphocyte and macrophage development by graded expression of PU.1. Science 288:1439.[Abstract/Free Full Text]
  30. Pongubala, J. M., Van Beveren, C., Nagulapalli, S., Klemsz, M. J., McKercher, S. R., Maki, R. A. and Atchison, M. L. 1993. Effect of PU.1 phosphorylation on interaction with NF-EM5 and transcriptional activation. Science 259:1622.[ISI][Medline]
  31. Fisher, R. C., Olson, M. C., Pongubala, J. M., Perkel, J. M., Atchison, M. L., Scott, E. W. and Simon, M. C. 1998. 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. Mol. Cell. Biol. 18:4347.[Abstract/Free Full Text]
  32. Zhang, P., Behre, G., Pan, J., Iwama, A., Wara-Aswapati, N., Radomska, H. S., Auron, P. E., Tenen, D. G. and Sun, Z. 1999. Negative cross-talk between hematopoietic regulators: GATA proteins repress PU.1. Proc. Natl Acad. Sci. USA 96:8705.[Abstract/Free Full Text]
  33. Pine, R., Canova, A. and Schindler, C. 1994. Tyrosine phosphorylated p91 binds to a single element in the ISGF2/IRF-1 promoter to mediate induction by IFN alpha and IFN gamma, and is likely to autoregulate the p91 gene. EMBO J. 13:158.[Abstract]
  34. Saksela, K. and Baltimore, D. 1993. Negative regulation of immunoglobulin kappa light-chain gene transcription by a short sequence homologous to the murine B1 repetitive element. Mol. Cell. Biol. 13:3698.[Abstract]
  35. Bromberg, J. F., Horvath, C. M., Wen, Z., Schreiber, R. D. and Darnell, J. E., Jr. 1996. Transcriptionally active Stat1 is required for the antiproliferative effects of both interferon alpha and interferon gamma. Proc. Natl Acad. Sci. USA 93:7673.[Abstract/Free Full Text]
  36. Wen, Z., Zhong, Z. and Darnell, J. E., Jr. 1995. Maximal activation of transcription by Stat1 and Stat3 requires both tyrosine and serine phosphorylation. Cell 82:241.[ISI][Medline]
  37. Mizushima, S. and Nagata, S. 1990. pEF-BOS, a powerful mammalian expression vector. Nucleic Acids Res. 18:5322.[ISI][Medline]
  38. Muller, M., Laxton, C., Briscoe, J., Schindler, C., Improta, T., Darnell, J. E., Jr, Stark, G. R. and Kerr, I. M. 1993. Complementation of a mutant cell line: central role of the 91 kDa polypeptide of ISGF3 in the interferon-alpha and -gamma signal transduction pathways. EMBO J. 12:4221.[Abstract]
  39. Saharinen, P., Ekman, N., Sarvas, K., Parker, P., Alitalo, K. and Silvennoinen, O. 1997. The Bmx tyrosine kinase induces activation of the Stat signaling pathway, which is specifically inhibited by protein kinase Cdelta. Blood 90:4341.[Abstract/Free Full Text]
  40. Aittomaki, S., Pesu, M., Groner, B., Janne, O. A., Palvimo, J. J. and Silvennoinen, O. 2000. Cooperation among Stat1, glucocorticoid receptor, and PU.1 in transcriptional activation of the high-affinity Fc gamma receptor I in monocytes. J. Immunol. 164:5689.[Abstract/Free Full Text]
  41. Horvath, C. M., Wen, Z. and Darnell, J. E., Jr. 1995. A STAT protein domain that determines DNA sequence recognition suggests a novel DNA-binding domain. Genes Dev. 9:984.[Abstract]
  42. Kovarik, P., Mangold, M., Ramsauer, K., Heidari, H., Steinborn, R., Zotter, A., Levy, D. E., Muller, M. and Decker, T. 2001. Specificity of signaling by STAT1 depends on SH2 and C-terminal domains that regulate Ser727 phosphorylation, differentially affecting specific target gene expression. EMBO J. 20:91.[Abstract/Free Full Text]
  43. Lodie, T. A., Savedra, R., Jr, Golenbock, D. T., Van Beveren, C. P., Maki, R. A. and Fenton, M. J. 1997. Stimulation of macrophages by lipopolysaccharide alters the phosphorylation state, conformation, and function of PU.1 via activation of casein kinase II. J. Immunol. 158:1848.[Abstract]
  44. Yamamoto, H., Kihara-Negishi, F., Yamada, T., Hashimoto, Y. and Oikawa, T. 1999. Physical and functional interactions between the transcription factor PU.1 and the coactivator CBP. Oncogene 18:1495.[CrossRef][ISI][Medline]
  45. Aittomaki, S., Yang, J., Scott, E. W., Simon, M. C. and Silvennoinen, O. 2002. Distinct functions for signal transducer and activator of transcription 1 and PU.1 in transcriptional activation of Fc gamma receptor I promoter. Blood 100:1078.[Abstract/Free Full Text]
  46. Kodandapani, R., Pio, F., Ni, C. Z., Piccialli, G., Klemsz, M., McKercher, S., Maki, R. A. and Ely, K. R. 1996. A new pattern for helix–turn–helix recognition revealed by the PU.1 ETS-domain–DNA complex. Nature 380:456.[CrossRef][ISI][Medline]
  47. Behre, G., Whitmarsh, A. J., Coghlan, M. P., Hoang, T., Carpenter, C. L., Zhang, D. E., Davis, R. J. and Tenen, D. G. 1999. c-Jun is a JNK-independent coactivator of the PU.1 transcription factor. J. Biol. Chem. 274:4939.[Abstract/Free Full Text]
  48. Wara-aswapati, N., Yang, Z., Waterman, W. R., Koyama, Y., Tetradis, S., Choy, B. K., Webb, A. C. and Auron, P. E. 1999. Cytomegalovirus IE2 protein stimulates interleukin 1beta gene transcription via tethering to Spi-1/PU.1. Mol. Cell. Biol. 19:6803.[Abstract/Free Full Text]
  49. Pongubala, J. M. and Atchison, M. L. 1997. PU.1 can participate in an active enhancer complex without its transcriptional activation domain. Proc. Natl Acad. Sci. USA 94:127.[Abstract/Free Full Text]
  50. Roeder, R. G. 1996. The role of general initiation factors in transcription by RNA polymerase II. Trends Biochem. Sci. 21:327.[CrossRef][ISI][Medline]
  51. Kihara-Negishi, F., Yamamoto, H., Suzuki, M., Yamada, T., Sakurai, T., Tamura, T. and Oikawa, T. 2001. In vivo complex formation of PU.1 with HDAC1 associated with PU.1-mediated transcriptional repression. Oncogene 20:6039.[CrossRef][ISI][Medline]
  52. Hong, W., Kim, A. Y., Ky, S., Rakowski, C., Seo, S. B., Chakravarti, D., Atchison, M. and Blobel, G. A. 2002. Inhibition of CBP-mediated protein acetylation by the Ets family oncoprotein PU.1. Mol. Cell. Biol. 22:3729.[Abstract/Free Full Text]
  53. Shen, Y. and Darnell, J. E., Jr. 2001. Antiviral response in cells containing Stat1 with heterologous transactivation domains. J. Virol. 75:2627.[Abstract/Free Full Text]
  54. Kamei, Y., Xu, L., Heinzel, T., Torchia, J., Kurokawa, R., Gloss, B., Lin, S. C., Heyman, R. A., Rose, D. W., Glass, C. K. and Rosenfeld, M. G. 1996. A CBP integrator complex mediates transcriptional activation and AP-1 inhibition by nuclear receptors. Cell 85:403.[ISI][Medline]
  55. Bannister, A. J. and Kouzarides, T. 1995. CBP-induced stimulation of c-Fos activity is abrogated by E1A. EMBO J. 14:4758.[Abstract]