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¹ Department of Medical Chemistry, Graduate School of Medicine, Kyoto University, Kyoto, Japan
² Solution-Oriented Research for Science and Technology, Japan Science and Technology Corporation, Yoshida-Konoe, Sakyo-ku, Kyoto 606-8501, Japan

	Abstract

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Granulocyte colony-stimulating factor (G-CSF) regulates the proliferation and differentiation of neutrophilic progenitor cells. Here, we investigated the roles of CCAAT/enhancer-binding protein (C/EBP) in the G-CSF-induced transcriptional activation and chromatin modification of the CCR2 and myeloperoxidase (MPO) genes in IL-3-dependent myeloid FDN1.1 cells. Chromatin immunoprecipitation (ChIP) and electrophoretic mobility shift assays revealed that G-CSF activates C/EBP to bind target promoters. ChIP mapping experiments across the CCR2 and MPO genes showed that G-CSF induces histone H3 modifications: the acetylation of Lys9, trimethylation of Lys4 and trimethylation of Lys9. The distribution profile of the trimethylated Lys9 was distinct from that of the two other modifications. All the G-CSF-induced C/EBP recruitment, transcriptional activation and histone modifications were reversed by re-stimulation with IL-3, and were abolished by short hairpin RNA (shRNA)-mediated knockdown of C/EBP. These results indicate that C/EBP is activated by G-CSF to bind target promoters, and plays critical roles in the transcriptional activation and dynamic chromatin modification of target genes during neutrophil differentiation.

	Introduction

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The multistep development of blood cells from hematopoietic stem cells is regulated by extracellular signals such as cytokines and microenvironmental cell–cell interactions, and the cell-fate determination of multipotential or committed progenitors is executed by the epigenetic regulation of lineage-specific gene expression (Fisher 2002; Kluger et al. 2004). Granulocyte colony-stimulating factor (G-CSF) is a cytokine that controls granulopoiesis by regulating the proliferation of myeloid progenitor cells and their differentiation into mature neutrophils. Studies using knockout mice lacking G-CSF or G-CSF receptor (G-CSFR) demonstrated that G-CSF is crucial for both steady-state granulopoiesis and emergency granulopoiesis during infection (Lieschke et al. 1994; Liu et al. 1996). G-CSF stimulates the activation of multiple signaling pathways in myeloid progenitor cells; including the JAK–STAT (Coffer et al. 2000; Ihle 2001) and Ras–MAPK pathways (Bashey et al. 1994), which then induce the short-term activation of early-response genes, such as the SOCS family, and the long-term activation of differentiation-associated genes, such as myeloperoxidase (MPO), neutrophil elastase (NE) and NADPH oxidase (Ward et al. 2000). Although STAT3 was shown to be responsible for the induction of various G-CSF-responsive immediate-early genes (Coffer et al. 2000; Alexander & Hilton 2004), it remains unclear how G-CSF-triggered signals activate the late, neutrophil-specific genes.

Previous studies have demonstrated that multiple transcription factors, such as CCAAT/enhancer-binding protein (C/EBP) family members, PU.1, Gfi1 and AML1 are involved in the differentiation of myeloid progenitors (Friedman 2002). Of these, the C/EBP family members, especially C/EBP and C/EBP, were shown to be indispensable for the production of neutrophils. The myeloid progenitors of C/EBP-null mice are blocked at an early stage of myeloid differentiation (Zhang et al. 1997), and C/EBP-null mice fail to generate terminally differentiated mature neutrophils (Yamanaka et al. 1997), indicating these factors play critical roles in the early and mid-late stages, respectively, of neutrophilic differentiation (Friedman 2002). Furthermore, recent studies have revealed a C/EBP function as a lineage-determining transcription factor that activates the myeloid differentiation program in hematopoietic progenitors (Dahl et al. 2003; Xie et al. 2004; Fukuchi et al. 2006; Suh et al. 2006). In accordance with this function, C/EBP is important for the expression of various myeloid-specific genes, including MPO, NE, G-CSFR and C/EBP (Ford et al. 1996; Oelgeschlager et al. 1996; Smith et al. 1996; Zhang et al. 1997; Friedman 2002; Wang & Friedman 2002). Studies using C/EBP(–/–) cells, G-CSFR mutants and dominant-negative C/EBP mutants have indicated that the G-CSF-triggered intracellular signals cooperate with C/EBP and other factors to activate these neutrophil-specific genes (Ward et al. 1999, 2000; Wang et al. 2001), but the molecular mechanisms by which G-CSF signaling leads to differentiation-associated gene expression and cell-fate determination remain largely unknown.

Accumulating evidence indicates that, in addition to the lineage-specific transcription factors, histone modifications and ATP-dependent chromatin remodeling play pivotal roles in the expression of lineage-specific genes and cell-fate determination during development (Fisher 2002; Narlikar et al. 2002; Margueron et al. 2005). Covalent histone modifications, such as phosphorylation, acetylation, methylation and monoubiquitylation appear to dictate dynamic transitions between transcriptionally active or silent chromatin states, and to maintain the chromatin status upon cell division (Margueron et al. 2005; Nightingale et al. 2006). For example, the acetylation of lysine residues in histones H3 and H4, and the methylation of lysines 4, 36 and 79 of histone H3 are linked to active transcription. In contrast, methylation of lysines 9 and 27 of histone H3 is strongly associated with heterochromatin formation, transcriptional repression and long-term gene silencing (Margueron et al. 2005; Martin & Zhang 2005; Nightingale et al. 2006). With regard to hematopoietic development, silencing of an immature-stage-specific gene during thymocyte development was shown to be accompanied by dynamic changes in the acetylation and methylation status of specific lysine residues of histone H3 across the entire gene locus (Su et al. 2004). Thus, the cytokine-triggered expression of lineage-specific genes is probably regulated by changes in chromatin modifications, but there is little information about the roles of cytokine signaling in chromatin dynamics during differentiation.

To explore the molecular mechanism of the G-CSF-induced expression of neutrophil-specific genes during differentiation, we previously carried out a DNA microarray analysis of G-CSF-stimulated myeloid progenitor FDC-P1 cells, and identified new G-CSF targets, including the chemokine receptor CCR2 gene (Iida et al. 2005). For this report, we identified C/EBP-binding elements in the CCR2 promoter and investigated whether C/EBP is involved in the G-CSF-induced activation of target genes. We found that G-CSF activated the promoter-binding ability of C/EBP and that the activated C/EBP was indispensable for the transcriptional activation and dynamic chromatin modification of target genes.

	Results

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Identification of C/EBP consensus sites in the CCR2 promoter

To investigate the mechanism of G-CSF-dependent transcriptional activation and the chromatin dynamics of the CCR2 gene, we used an IL-3-dependent myeloid cell line, FDN1.1, which was established by the forced expression of G-CSF receptor in myeloid precursor FDC-P1 cells (Fukunaga et al. 1991, 1993). As shown in Fig. 1A, the stimulation of FDN1.1 cells with G-CSF induced strong expression of the CCR2 gene. This expression was repressed when the cells were re-cultured in medium containing IL-3, indicating that expression of the CCR2 gene was reversibly regulated by distinct signaling pathways from IL-3 and G-CSF, as we previously showed for the MPO gene (Fukunaga et al. 1993). To characterize the promoter of the CCR2 gene, we determined the transcriptional start sites. The mouse CCR2 gene consists of three exons: exons 1 and 2 are 5'-noncoding exons, and exon 3 contains the entire coding region for CCR2 (Fig. 1B). Primer extension analysis of the CCR2 mRNA prepared from G-CSF-stimulated FDN1.1 cells revealed multiple transcription initiation sites including three major and many minor sites (Fig. 1B and Supplementary Fig. S1). We designated the start site of the most intense band as the +1 nucleotide. No consensus TATA box was found upstream of these initiation sites. We used the ALIBABA software for predicting transcription factor binding sites <http://www.gene-regulation.com/pub/programs/alibaba2/index.html> to scan the 5'-flanking region of the CCR2 gene, and found two putative C/EBP-binding sites (ATTGCATAAG and ATTTCAAAAT) between –47 and –24 (Fig. 1B). This region showed high homology with the 5'-untranslated region (+52–+75) of the human CCR2 gene (Fig. 1B), which was previously shown to be important for the constitutive expression of the CCR2 gene in human monocytic THP-1 cells (Yamamoto et al. 1999). Since the C/EBP family of proteins plays critical roles in myeloid cell differentiation (Ward et al. 2000; Friedman 2002), we investigated whether C/EBP proteins are involved in the G-CSF-dependent activation of the CCR2 gene.

View larger version (24K): [in this window] [in a new window]   Figure 1  G-CSF-induced expression of the CCR2 gene in myeloid progenitor FDN1.1 cells. (A) FDN1.1 cells were stimulated with G-CSF and cultured for 6 days (closed circles). On day 3, half the cells were washed and re-cultured with IL-3 for 3 more days (open circles). At the indicated time, the RNA was prepared and the expression levels of CCR2 mRNA (left) and MPO mRNA (right) were measured by real-time RT-PCR as described in the Experimental procedures. PCR was performed in triplicate. (B) Schematic structure and transcription start sites of the mouse CCR2 gene. The nucleotide sequence from –49 to –22 of the mouse CCR2 gene shows homology with a 5'-untranslated region of the human CCR2 gene. Two regions related to the C/EBP consensus sequence are shown in bold, and nucleotides that are identical to the consensus sequence are indicated in upper case.

Specific binding of C/EBP to the CCR2 promoter element

To examine whether C/EBP family transcription factors bind to the putative C/EBP elements of the CCR2 gene, we performed an electrophoretic mobility shift assay (EMSA) of the CCR2 promoter element, using recombinant C/EBP, C/EBPβ, C/EBP and C/EBP proteins that were expressed in COS7 cells. A well-characterized C/EBP-binding element (–83 to –44) of the mouse G-CSF receptor (G-CSFR) gene (Ito et al. 1994; Smith et al. 1996) was used as the positive control. As shown in Fig. 2A, all of the C/EBP proteins showed retarded bands when incubated with these probes. The retarded bands were supershifted by adding their specific antibodies to the reaction. (We could not perform the supershift assay for C/EBP since no antibody useful for C/EBP was available.) These results indicate that C/EBP, β, and have a potential to bind to the CCR2 promoter element as well as G-CSFR promoter element.

View larger version (69K): [in this window] [in a new window]   Figure 2  In vitro binding of CEBP to the promoter element of the CCR2 gene. (A) COS7 cells were transfected with an expression plasmid for C/EBP, β, or , or control vector (V), and their nuclear extracts (NE) were subjected to EMSA using the CCR2 probe (42 bp, –56 to approximately –15) and G-CSFR probe (41 bp, –84 to approximately –44). For supershift assay, anti-C/EBP, β and antibodies were added to the reaction mixtures as described in the Experimental procedures. (B) Nuclear extracts prepared from FDN1.1 cells cultured for 3 days in the presence of IL-3 (I) or G-CSF (G) were analyzed by EMSA using the wild-type (WT) and mutant probes of the CCR2 or G-CSFR promoter elements. Shifted bands seen with the wild-type CCR2 probe are indicated by arrowheads (C1–C4). (C) A supershift assay was performed using nuclear extracts from FDN1.1 cells cultured with IL-3 (I) or G-CSF (G) and anti-C/EBP, β and antibodies. A supershifted band is indicated by the C3' arrowhead. (D) Left panel: FDN1.1 nuclear extracts used for EMSA in (B) were analyzed by immunoblotting with an anti-C/EBP antibody. Then, the membrane was reblotted with an anti-lamin B antibody as a loading control. The band indicated by an asterisk is a degradation product of p42, which is often produced during the nuclear extract preparation from the G-CSF-treated FDN1.1 cells that contain a strong proteolytic activity (Orita et al. 1997). Right panel: The C/EBP mRNA level of IL-3- or G-CSF-cultured FDN1.1 cells was measured by real-time RT-PCR analysis.

G-CSF induces C/EBP binding to the promoter elements and dynamic histone modifications across the CCR2 and MPO genes

To determine whether C/EBP actually binds to the promoter element of the CCR2 gene in vivo, we performed a chromatin immunoprecipitation (ChIP) analysis in which cross-linked chromatin was immunoprecipitated with the anti-C/EBP antibody, and the co-precipitated DNA fragments spanning the C/EBP site were quantified by real-time PCR. Specific binding of C/EBP to the CCR2 promoter element was undetectable in IL-3-cultured FDN1.1 cells, but was strongly induced after stimulation with G-CSF (Fig. 3A). C/EBP binding to the promoter continued to increase for up to 6 days in the presence of G-CSF, but swiftly dropped when the culture medium was changed back to the IL-3-containing one. These binding profiles were closely correlated with the time courses of CCR2 expression (Fig. 1A). Since C/EBP is implicated in the neutrophil-specific expression of the MPO and G-CSFR genes (Ford et al. 1996; Smith et al. 1996), we also performed C/EBP ChIP assays of these genes, and found similar C/EBP binding kinetics for the C/EBP-binding element in their promoter regions (Fig. 3A). These results indicate that G-CSF induces C/EBP binding to the C/EBP sites of the CCR2, MPO and G-CSFR genes, and suggest that the recruited C/EBP plays an important role in their expression.

View larger version (32K): [in this window] [in a new window]   Figure 3  Induction of C/EBP binding and histone modification by G-CSF stimulation. (A) ChIP assay of the promoter of the CCR2, MPO, and G-CSFR genes with an anti-C/EBP antibody. FDN1.1 cells were cultured with G-CSF for 3 days (closed circles), and then with IL-3 (open circles) or G-CSF for an additional 3 days. At the indicated time, C/EBP binding to the promoter element was analyzed by ChIP assay for the CCR2 (–0.1 kb), MPO (–3.4 kb) and G-CSFR (–0.1 kb) amplicons with an anti-C/EBP antibody. Control ChIPs without antibody (beads) are shown by open (IL-3) and closed (G-CSF) triangles. Results shown are representative of three independent experiments and are expressed as percent recovery from the input. (B and C) ChIP mapping across the CCR2 (B) and MPO (C) genes. FDN1.1 cells were cultured with IL-3 (open circles) or G-CSF (closed circles) for 3 days. The G-CSF-cultured cells were then washed and re-cultured with IL-3 (open triangles) or G-CSF (closed triangles) for an additional 3 days. ChIP analyses were performed with anti-C/EBP, anti-H3K9ac, anti-H3K4me3, anti-H3K9me2 and anti-H3K9me3 antibodies. The positions of the amplicons are indicated below the gene structure. Exons are indicated by boxes, of which open and shaded parts represent untranslated and translated regions, respectively. The results are representative of two independent experiments.

C-terminal regions of G-CSFR are required for C/EBP activation and histone modifications

We previously demonstrated that the N-terminal region of the G-CSFR cytoplasmic domain is sufficient to transduce the proliferative signal, whereas the C-terminal region, including the four tyrosine residues, Y703, Y728, Y743 and Y763, is essential for transducing the differentiation signal (Fukunaga et al. 1991, 1993; Yoshikawa et al. 1995). To examine the role of the C-terminal region in the G-CSF-triggered C/EBP activation and histone modifications, we analyzed FDC-P1 cells expressing various truncation mutants of G-CSFR, named A, E and T (Fig. 4A). An immunoblot analysis confirmed that expression levels of the wild-type and mutant receptors are comparable among the transfectant cell lines (Fig. 4B). Consistent with our previous observation on MPO expression, the G-CSF-induced expression of the CCR2 gene was attenuated in the A and E mutant cells, and was scarcely detected in the T mutant cells (Fig. 4C). ChIP analysis with the anti-C/EBP antibody revealed that the G-CSF-induced recruitment of C/EBP to the CCR2 and MPO promoters was severely reduced in the A and E mutant cells, and was mostly abolished in the T mutant cells (Fig. 4D). Furthermore, the G-CSF-induced acetylation of H3K9 in the body of the CCR2 and MPO genes was severely reduced in all the mutant cells (Fig. 4E). These results indicate that the C-terminal region including Y763 (754–812) and the region surrounding Y703 (701–724) play important roles in G-CSF signaling to activate C/EBP binding and histone modifications of the target genes.

View larger version (37K): [in this window] [in a new window]   Figure 4  Effects of C-terminal deletion of the G-CSF receptor on G-CSF-triggered gene activation. (A) Schematic representation of the wild-type (WT) and deletion mutants with remaining tyrosine residues in the cytoplasmic domain of the G-CSFR. (B) Whole cell lysates prepared from parental cells (FDC-P1) and transformants were analyzed by immunoblotting with an anti-mouse G-CSFR antiserum MR9. The positions of the mature and less glycosylated G-CSFRs are indicated by open and solid arrowheads, respectively. The asterisks indicate nonspecific signals. (C) FDC-P1 cells expressing the wild-type (WT) or mutated G-CSFR (A, E or T) were cultured with IL-3 (open bars) or G-CSF (closed bars) for 3 days. The expression of each mRNA was determined by real-time RT-PCR analysis performed in triplicate. (D) Effect of G-CSFR deletions on G-CSF-induced C/EBP recruitment. Cells were cultured as in (B), and ChIP assays for the CCR2 (–0.1 kb) and MPO (–3.4 kb) amplicons covering the C/EBP-binding site were performed using an anti-C/EBP antibody. (E) Effect of the G-CSFR deletion on G-CSF-induced histone acetylation. Cells were cultured as in (B), and ChIP assays for the CCR2 (2.1 kb) and MPO (1.5 kb) amplicons were performed using an anti-H3K9ac antibody. The ChIP assays were performed in duplicate.

C/EBP is essential for the G-CSF-induced activation and histone modification of the CCR2 and MPO genes

Although the above results showed that the G-CSF-induced recruitment of C/EBP to the promoter is closely correlated with the chromatin modification as well as the transcriptional activation of target genes, it was not clear how C/EBP contributes to these dynamic alterations. To address this issue, we knocked down the C/EBP expression with short interfering RNA (siRNA). We constructed four short hairpin RNA (shRNA) expression plasmids (named A–D), and established stable FDN1.1 transfectants expressing these shRNAs, of which two constructs, B and D, effectively knocked down the expression of C/EBP (Fig. 5A and B). To average out the clone-to-clone deviation, a mixture of six independent transfectant clones of each construct was used. In the B and D transfectants, both the protein and the mRNA levels of C/EBP were reduced to about 15%–20% of the levels in the parental FDN1.1 cells and in a control transfectant clone expressing an shRNA for green fluorescent protein (GFP) (Fig. 5A and B). Although G-CSF stimulation up-regulated C/EBP expression, not only in the parental cells but also, to some extent, in the shRNA transfectants, the protein and mRNA levels of C/EBP in the G-CSF-stimulated B and D cells were still lower than in the unstimulated FDN1.1 or GFP cells. In both C/EBP-knockdown cell lines, the G-CSF-induced expression of the CCR2 gene was significantly attenuated and that of the MPO gene was suppressed almost completely (Fig. 5C), indicting that C/EBP is critically involved in the G-CSF-induced expression of these genes. The decreased expression of C/EBP and the relatively weak induction of CCR2 mRNA in the GFP-shRNA cells (Fig. 5B and C) are likely due to a clonal deviation of this control transfectant.

View larger version (41K): [in this window] [in a new window]   Figure 5  shRNA-mediated knockdown of C/EBP expression. (A) Immunoblot analysis of C/EBP protein levels. Parental FDN1.1 cells and transformants expressing the shRNA for GFP (control) or C/EBP (constructs B and D) were cultured with IL-3 (I) or G-CSF (G) for 3 days, and the total cell lysates were analyzed by immunoblotting with the anti-C/EBP antibody (top panel). The membrane was reblotted with an anti-tubulin antibody as a loading control (lower panel). (B–E) The control (FDN and GFP) cells and C/EBP-knockdown cells (constructs B and D) were treated with IL-3 (open bars) or G-CSF (closed bars) for 3 days, and were subjected to the following assays. (B) The C/EBP mRNA level was measured by real-time RT-PCR. (C) Expression of the CCR2 and MPO mRNAs was measured by real-time RT-PCR. (D) A ChIP analysis for the CCR2 (–0.1 kb) and MPO (–3.4 kb) amplicons was performed using an anti-C/EBP antibody. (E) ChIP analyses using anti-H3K9ac, anti-H3K4me3 and anti-H3K9me3 antibodies were performed for the indicated amplicons, which corresponded to the peak regions in the ChIP mapping results shown in Fig. 3. All of the quantitative PCR analyses were performed in duplicate.

	Discussion

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G-CSF activates C/EBP to bind the promoter element of lineage-specific genes

We initially tried luciferase reporter assay to characterize promoter elements responsible for G-CSF-induced expression of the MPO and CCR2 genes, but various reporter constructs containing not only the 5'-upstream region but also the transcribed exon–intron region did not show G-CSF-dependent reporter expression in either transient transfection assay or stable transfectant system. The difficulty of reporter assays may reflect the importance of endogenous chromatin structure of the gene loci for their regulated expression. We thus searched the CCR2 promoter region for transcription factor binding sites by a computer algorithm, and investigated a predicted candidate, C/EBP, in this study. C/EBP has been shown to be involved in the expression of various genes, not only in hematopoietic cells but also in cells of other lineages, such as hepatocytes and adipocytes (Ramji & Foka 2002), but the regulation of C/EBP activity by cytokine signaling has remained elusive. By ChIP experiments, we showed that G-CSF induced the specific binding of C/EBP to the promoter elements of the CCR2, MPO and G-CSFR genes in vivo. Although G-CSF stimulation led to an increased level of C/EBP expression, the drastic augmentation of C/EBP's occupancy of the binding sites (Fig. 3) suggests that the binding ability of C/EBP is regulated not only by its protein level but also by other mechanisms, such as the activation or de-repression of C/EBP function. Although many transcription factors are regulated by nucleo-cytoplasmic localization, this mechanism is unlikely to be operative in this case, since the C/EBP protein was present at a significant level even in the unstimulated nuclear extract, but was incapable of binding to the promoter element in the EMSA experiment (Fig. 2). These results suggest instead that the G-CSF signal acts to convert a latent form of C/EBP into a binding-capable, active form by post-transcriptional modification and/or interaction with other proteins. C/EBP is reported to be regulated by post-translational modifications, such as phosphorylation (Ross et al. 1999, 2004; Behre et al. 2002) and sumoylation (Kim et al. 2002). We examined whether G-CSF stimulation alters the phosphorylation of C/EBP, but did not detect changes in its phosphorylation status at the reported sites, including Ser21, Thr222 and Thr226 (Ross et al. 1999, 2004) (S. Iida and R. Fukunaga, unpublished observation). Alternatively, C/EBP may be activated by changes in its dimerization partners among the C/EBP family members, such as C/EBPβ, C/EBP or CHOP (Ramji & Foka 2002), or by its interaction with other transcription factors.

Previous studies have indicated that signaling pathways from phosphorylated tyrosine residues of G-CSFR as well as tyrosine-independent pathways cooperatively contribute to the proliferation and differentiation of myeloid precursor cells (Ward et al. 2000). G-CSF signals for myeloid-specific gene expression were shown to be mediated not only by Tyr703, the major docking site for STAT3, but also by the membrane-distal region that includes Tyr763 (Yoshikawa et al. 1995; Ward et al. 1999; Nakajima & Ihle 2001; Wang et al. 2001). In the present study, the G-CSF-induced C/EBP recruitment and subsequent expression and histone modification of target genes were strongly diminished, yet slightly retained, in the A and E mutant cells, and were lost almost completely in the T mutant cells (Fig. 4). This result indicates that the membrane-distal region containing Tyr763 plays a critical role for the C/EBP activation and chromatin modification, and also Tyr703 plays a minor role. Tyr763 is the major docking site for adaptor proteins such as Shc and Grb2 (Ward et al. 2000), suggesting an important role of the downstream Ras–MAPK pathway for the C/EBP activation.

Essential role of C/EBP in the G-CSF-dependent activation of lineage-specific genes

A germ-line knockout of the C/EBP gene has provided information about its physiological roles in neutrophil development, but the detailed analysis of its function in neutrophilic gene expression was hampered by the absence of neutrophil progenitors in the knockout mice (Zhang et al. 1997). Studies using the forced expression of other C/EBP members or a dominant-inhibitory C/EBP mutant in various myeloid progenitor cells suggested that C/EBP family members can play redundant functions in lineage-specific gene expression (Friedman 2002; Wang & Friedman 2002; Zhang et al. 2002), but it has been unclear how direct C/EBP's contribution is to the G-CSF-dependent gene expression.

In this study, we knocked down C/EBP by the stable expression of specific shRNAs in myeloid progenitor cells, which unequivocally demonstrated that both the transcriptional activation and histone modifications of the G-CSF-responsive genes depend critically on the presence of C/EBP. The knockdown of C/EBP suppressed the expression and histone modifications of the MPO gene almost completely, but resulted in only partial inhibition of the CCR2 expression, indicating that the G-CSF-stimulated activation of the MPO gene depends absolutely on C/EBP, whereas the CCR2 gene is regulated not only by C/EBP but also by other factors. This difference in requirement for C/EBP may be related to the distinct expression pattern of these two genes in hematopoietic cells: that is, the expression of MPO is restricted to the promyelocyte–metamyelocyte stages in neutrophil differentiation, but CCR2 is expressed in other lineages, including the monocytes/macrophages and T cells (Murphy et al. 2000).

G-CSF evokes dynamic chromatin modification across target genes

The results presented here demonstrate that histone modifications of myeloid-specific genes are dynamically regulated by cytokine signaling. In the proliferative state with IL-3 signaling, the CCR2 and MPO genes were silent and lacked detectable levels of acetyl H3K9 or trimethyl H3K4. The induction of neutrophilic differentiation by G-CSF stimulation resulted in the strong activation of these genes with a concomitant increase in the H3K9ac and H3K4me3 levels, but all of these changes were swiftly reversed by re-stimulation with IL-3. Knockdown of C/EBP attenuated the G-CSF-induced histone modifications, indicating that these modifications are regulated by specific machineries recruited by C/EBP or by general transcription machineries including RNA polymerase II. Although C/EBP was shown to interact functionally and physically with p300 histone acetyltransferase in differentiating adipocytes (Erickson et al. 2001), we could not detect significant association of C/EBP with either p300 or CREB-binding protein, a related acetyltransferase, in the G-CSF-stimulated FDN1.1 cells. Although the role of H3K4 methylation in transcriptional regulation remains elusive (Sims & Reinberg 2006), the distributions of H3K9 acetylation and H3K4 trimethylation were quite similar, suggesting that these two modifications are introduced by intimately linked machineries.

In contrast to the link between H3K4 methylation and gene activation, the methylation of H3K9 has been tightly associated with heterochromatin formation, transcriptional repression, and gene silencing in fission yeast, fruit fly and mammals (Margueron et al. 2005; Martin & Zhang 2005; Nightingale et al. 2006). Indeed, an inverse correlation between transcriptional activity and H3K9 methylation is reported for lipopolysaccharide-inducible inflammatory genes in monocytes (Saccani & Natoli 2002). Furthermore, silencing of the terminal deoxynucleotidyl transferase gene during thymocyte maturation is accompanied by increased H3K9 methylation throughout the gene locus (Su et al. 2004). We observed that H3K9 trimethylation was also induced by G-CSF concomitantly with transcriptional activation, and was reversibly down-regulated by the IL-3 signal. Although this result appears to be inconsistent with the conventional scenario, other recent studies have demonstrated an association of H3K9 methylation with actively transcribed genes in mammalian euchromatin (Vakoc et al. 2005, 2006; Brinkman et al. 2006). Together with these reports, our results indicate that H3K9 methylation may be associated with active transcription in euchromatic regions. The modification profile of the CCR2 and MPO genes revealed that H3K9ac and H3K4me3 were enriched between the promoter and the 1.5–2 kb downstream region, whereas H3K9me3 was scarcely present in this region but was distributed from the middle to the 3' end of the transcribed region (Fig. 3B and C). This H3K9me3 profile rather resembles that of H3K36 trimethylation at active genes in mammalian cells (Bannister et al. 2005; Vakoc et al. 2006) and in yeast (Pokholok et al. 2005). Recent studies indicate that H3K36 methylation functions to suppress intragenic transcription initiation during transcriptional elongation in yeast (Carrozza et al. 2005; Joshi & Struhl 2005; Keogh et al. 2005); thus, H3K9 trimethylation may also play a role in the elongation and/or termination steps, as hypothesized recently (Vakoc et al. 2005, 2006; Eissenberg & Shilatifard 2006).

In summary, this study showed that G-CSF signaling activates the promoter-binding ability of C/EBP, and that the activated C/EBP plays essential roles in the transcriptional activation and histone modifications of target genes. Further studies on the signaling machineries responsible for the C/EBP activation and subsequent chromatin modification would provide insight into the molecular mechanisms for the lineage-specific gene expression in neutrophil differentiation.

	Experimental procedures

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Cell culture, RNA preparation and transfection

Recombinant mouse interleukin-3 (IL-3) was produced in mouse C127 cells as described previously (Fukunaga et al. 1990). Recombinant human G-CSF was kindly provided by Chugai Pharmaceutical Company (Tokyo, Japan). The mouse myeloid cell line FDC-P1 and its derivatives, FDN-1.1, -A, -E and -T, were established and maintained as described (Fukunaga et al. 1991). For stimulation by G-CSF, the cells were washed twice with RPMI1640 medium containing 10% fetal calf serum (FCS, Invitrogen, Carlsbad, CA) to remove IL-3 and then cultured at a density less than 1 x 10⁶ cells/mL in the same medium containing 200 units/mL human G-CSF at 37 °C, as described previously (Iida et al. 2005). Total RNA was prepared from 1 x 10⁷ cells using the RNeasy kit with the RNase-Free DNase set (Qiagen, Venlo, the Netherlands). COS7 cells were maintained in Dulbecco's modified Eagle's medium (DMEM, Invitrogen) supplemented with 10% FCS, 100 µg/mL streptomycin and 100 µg/mL kanamycin. Transfection of COS cells with the C/EBP expression plasmids pEF-C/EBP, β and (gifts from Dr S. Akira), and pEF-C/EBP (cloned by RT-PCR) was carried out by the calcium phosphate coprecipitation method using BES-buffered saline.

Real-time, reverse-transcriptase-primed polymerase chain reaction (RT-PCR)

Quantitative analysis of mRNA by real-time RT-PCR was carried out using the LightCycler system (Roche Diagnostics, Basel, Switzerland), as described previously (Iida et al. 2005). The primer sets used are listed in Supplementary Table S1.

Primer extension analysis

Total RNA was extracted from FDN1.1 cells cultured with G-CSF for 3 days, and poly(A)⁺ RNA was purified by using illustra^TM mRNA Purification Kit (GE Healthcare. Little Chalfont, UK). An anti-sense oligonucleotide (5'-CAGGTATGGCTC CTTTATGA-3') corresponding to the 3'-end sequence (+224 to +205) of exon 1 of the CCR2 gene was end-labeled with ³²P by using [-³²P]ATP and T4 polynucleotide kinase (MEGALABEL^TM, Takara Bio, Shiga, Japan). Primer extension was carried out as described previously (Seto et al. 1992) with some modifications. In brief, the labeled 20-mer oligonucleotide (0.4 pmol) and 2.5 µg of poly(A)⁺ RNA were dissolved in 10 µL of annealing buffer (6.6 mM Hepes–NaOH [pH 7.5], 6.6 mM NaCl and 0.6 mM EDTA), and denatured at 90 °C for 5 min. One µL of 1.8 M NaCl was added to the mixture and incubated at 45 °C for 2 h. The mixture was then adjusted to contain 50 mM Tris–HCl (pH 8.3), 75 mM KCl, 3 mM MgCl₂, 50 mM DTT, 0.5 mM each dNTP in a final volume of 100 µL. The cDNA was synthesized by incubation at 45 °C for 30 min followed by 50 °C 30 min with 500 units of SuperScript III (Invitrogen), and analyzed by electrophoresis through 8% polyacrylamide gel containing 7 M urea. As a size marker, a DNA fragment from the mouse CCR2 gene was sequenced by the chain termination procedure using the same ³²P-labeled primer as above and Takara Taq^TM Cycle Sequencing Kit (Takara Bio), and electrophoresed in parallel. The result of this analysis was confirmed by another primer extension experiment performed with a different primer (5'-TACCGTAAATCAGG TATGGC-3'; +234 to +215). All radioisotope experiments were carried out at the Kyoto University Radioisotope Research Center.

Electrophoretic mobility shift assay (EMSA)

Nuclear extracts were prepared as previously described (Orita et al. 1997) with some modifications. In brief, 4 x 10⁶ cells were washed with phosphate-buffered saline and made to swell by placing them in 400 µL of buffer A [10 mM Hepes–KOH (pH 7.9), 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM dithiothreitol (DTT), 10 µg/mL each aprotinin (Seikagaku Corp., Tokyo, Japan), leupeptin (Peptide Institute, Louisville, KY), and pepstatin (Sigma, St Louis, MO), 2.5 mM phenylmethylsulfonyl fluoride (PMSF; Sigma) and 10 mM 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride (pABSF; Wako)] for 15 min on ice. The cell suspension (400 µL) was layered on a two-layer sucrose cushion consisting of 400 µL of lower layer (buffer A containing 40% sucrose and 0.625% Non-idet P-40) and 200 µl of upper layer (buffer A containing 20% sucrose) in a microcentrifuge tube. After centrifugation at 20 000 g for 20 min at 4 °C, the supernatant was removed and the precipitated nuclei were washed with buffer A. Nuclei were resuspended in 50 µL of buffer B [20 mM Hepes–KOH (pH 7.9), 420 mM NaCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 10 µg/mL each aprotinin, leupeptin, and pepstatin, 2.5 mM PMSF and 10 mM pABSF] and incubated at 4 °C for 60 min with gentle shaking. After centrifugation at 20 000 g for 5 min at 4 °C, the supernatant was recovered as nuclear extract. For the preparation of DNA probes, sets of complementary oligonucleotides for the CCR2 and G-CSFR promoter region and their respective mutant sequences were annealed to yield dsDNA, and then end-labeled with biotin-N4-dCTP using the Biotin 3' End DNA Labeling Kit (Pierce, Rockford, IL). Sequences of the oligonucleotides are given in Supplementary Table S2. For the binding of DNA probes with nuclear proteins, 10 fmol of probe DNAs were incubated with nuclear extracts (2 or 10 µg protein for COS7 or FDN1.1 cells, respectively) in a 20-µL reaction mixture containing 10 mM Tris–HCl (pH 7.5), 50 mM KCl, 1 mM DTT, 5% glycerol, 2% polyvinyl alcohol and 1 µg poly(dI–dC) (GE Healthcare) for 30 min at 30 °C. For competition assay, 10, 50 and 200 fmol of unlabeled wild-type oligonucleotides or 200 fmol of mutant oligonucleotides were added before incubation. After incubation, the products were resolved by electrophoresis on a 6% polyacrylamide gel. For the supershift assay, 0.2 µg of anti-C/EBP, anti-C/EBPβ or anti-C/EBP antibody (sc-61, sc-150, sc-158; Santa Cruz Biotechnology, Santa Cruz, CA) was added after the first 15-min incubation of the binding reaction, and the mixture was further incubated for 15 min at 30 °C. After electrophoresis, the resolved DNA was electrotransferred to a nylon membrane (Hybond N+; GE Healthcare) and detected using the LightShift^R Chemiluminescent EMSA Kit (Pierce), according to the manufacturer's instructions.

Chromatin immunoprecipitation (ChIP)

ChIP was performed as previously described (Ye et al. 2001) with some modifications. Briefly, 1 x 10⁶ FDN1.1 cells in culture were fixed with 1% formaldehyde for 10 min at room temperature, and soluble chromatin was prepared by sonication. The chromatin solution (1.8 mL) was precleared with 75 µL of a mixture of salmon sperm DNA and protein A agarose beads (50% beads slurry of Salmon Sperm DNA/Protein A Agarose; Upstate Biotechnology, Lake Placid, NY), and the chromatin was immunoprecipitated with 1 µg of anti-C/EBP antibody (Santa Cruz Biotechnology), 2 µL of anti-H3K9ac antiserum (Upstate Biotechnology), 3.4 µg of anti-H3K4me3 antibody (Upstate Biotechnology), 5 µg of anti-H3K9me2 antibody (Upstate Biotechnology) or 5 µg of anti-H3K9me3 antibody (Upstate Biotechnology) overnight. As a bead control, the same operation without the antibody was performed. The immunoprecipitates were recovered by incubation with 60 µL of Salmon Sperm DNA/Protein A Agarose (50% beads slurry) for 1 h at 4 °C. Chromatin was eluted by incubation with 250 µL x 2 of elution buffer (1% SDS, 0.1 M NaHCO₃) for 15 min at room temperature. After NaCl was added to a final concentration 200 mM, the eluted chromatin was reverse-cross-linked by incubation at 65 °C for 4 h. DNA was purified by phenol extraction followed by ethanol precipitation, and subjected to quantitative, real-time PCR using the LightCycler system (Roche Diagnostics) under the conditions: 15 s at 95 °C, 5 s at 62 °C, and 20 s at 72 °C for 40 cycles. Specific amplification of each target region was confirmed by melting temperature analysis and by the presence of a single PCR product upon agarose gel electrophoresis. In all the real-time PCR analyses of ChIP experiments, cloned DNA fragments (10²–10⁵ copies per reaction) containing the respective target region were amplified in parallel and used as standards. The sample data were normalized against the standards and expressed as percent recovery of input DNA. The primer sets used are listed in Supplementary Table S3.

shRNA-mediated knockdown of C/EBP

We first constructed an shRNA expression vector, pHPU6, by modifying the pcPUR U6i plasmid (Miyagishi & Taira 2003). In brief, the two BspMI sites of pcPUR U6i were replaced with BbsI sites so that the stuffer region could be excised by BbsI digestion. Next, two BstXI (CCATTGTGCTGG) sequences were introduced into the BglII and BamHI sites that flanked the entire U6 gene unit at the 5'- and 3'-ends, respectively, to yield pHPU6. Expression plasmids for C/EBP-directed shRNAs were designed using the Whitehead siRNA Selection Web Server <http://jura.wi.mit.edu/bioc/siRNA> (Yuan et al. 2004) and a 9-base loop sequence (Brummelkamp et al. 2002). To construct the shRNA expression plasmids B and D, two double-stranded oligonucleotides, one consisting of 5'-caccGAGCtGAGATAAAGtCAAAttcaagaga TTTGGCTTTATCTCGGCTCttttt-3' and 5'-gcataaaaaGAGC CGAGATAAAGCCAAAtctcttgaaTTTGaCTTTATCTCaGCTC-3' (for construct B), and the other consisting of 5'-cacctGTGGAGAt GCAAtAGAAGttcaagagaCTTCTGTTGCGTCTCCACGttttt-3' and 5'-gcataaaaaCGTGGAGACGCAACAGAAGtctcttgaaCT TCTaTTGCaTCTCCACa-3' (for construct D), were cloned into the BbsI-digested pHPU6 to yield pHPU6-CBB and pHPU6-CBD, respectively. After these plasmids were digested with BstXI, the respective DNA fragments containing the entire U6-shRNA gene unit (0.4 kb) were tandemly multimerized and cloned into the BstXI-digested pHPU6 plasmid. The resulting plasmids were estimated to contain approximately 18 and 19 copies of tandem repeats of the respective U6 gene units, and were designated pHP(U6-CBB)₁₈ and pHP(U6-CBD)₁₉, respectively. To establish C/EBP-knockdown cells, FDN1.1 (subclone A10) cells were transfected with the ApaLI-digested pHP(U6-CBB)₁₈ and pHP(U6-CBD)₁₉ plasmids by electroporation, and the resulting puromycin (0.5 µg/mL)-resistant clones were isolated and tested for reduced C/EBP protein levels by immunoblotting using the anti-C/EBP antibody. To average out the clone-to-clone deviation in properties, six independent clones that showed reduced C/EBP expression were mixed and used for experiments.

Immunoblot analysis

Immunoblot analysis was carried out as previously described (Iida et al. 2005) with anti-C/EBP antibody (Santa Cruz), anti-lamin B antibody (Oncogene Science, Cambridge, MA), anti-tubulin antibody (Calbiochem, San Diego, CA) and anti-mouse G-CSF receptor antiserum MR-9 (Fukunaga et al. 1991).

	Acknowledgements

 
We are grateful to Dr. Shizuo Akira (Osaka University) for the pEF-C/EBP, β, and plasmids. This work was supported in part by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

	Footnotes

 
Communicated by: Tadashi Yamamoto

^* Correspondence: Email: rfukunaga{at}mfour.med.kyoto-u.ac.jp

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Received: 16 October 2007
Accepted: 21 December 2007

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