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 Previous Article

Molecular and Cellular Biology, March 2008, p. 2102-2112, Vol. 28, No. 6
0270-7306/08/$08.00+0     doi:10.1128/MCB.01943-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

C/EBPβ Induces Chromatin Opening at a Cell-Type-Specific Enhancer{triangledown}

Annette Plachetka,1 Olesya Chayka,1,{dagger} Carola Wilczek,1 Svitlana Melnik,2,{ddagger} Constanze Bonifer,2 and Karl-Heinz Klempnauer1*

Institut für Biochemie, Westfälische-Wilhelms-Universität Münster, Wilhelm-Klemm-Str. 2, D-48149 Münster, Germany,1 Division of Experimental Haematology, LIMM, University of Leeds, St. James's University Hospital, Leeds LS9 7TF, United Kingdom2

Received 29 October 2007/ Returned for modification 16 December 2007/ Accepted 1 January 2008


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ABSTRACT
 
We have used the chicken mim-1 gene as a model to study the mechanisms by which transcription factors gain initial access to their target sites in compacted chromatin. The expression of mim-1 is restricted to the myelomonocytic lineage of the hematopoietic system where it is regulated synergistically by the Myb and CCAAT/enhancer binding protein (C/EBP) factors. Myb and C/EBPβ cooperate at two distinct cis elements of mim-1, the promoter and a cell-type-specific enhancer, both of which are associated with DNase I hypersensitive sites in myelomonocytic cells but not in mim-1-nonexpressing cells. Previous work has shown that ectopic expression of Myb and C/EBPβ activates the endogenous mim-1 gene in a nonhematopoietic cell type (fibroblasts), where the gene is normally completely silent. Here, we investigated the molecular details of this finding and show that the activation of mim-1 occurs by two independent mechanisms. In the absence of Myb, C/EBPβ triggers the initial steps of chromatin opening at the mim-1 enhancer without inducing transcription of the gene. mim-1 transcription occurs only in the presence of Myb and is associated with chromatin opening at the promoter. Our work identifies a novel function for C/EBPβ in the initial steps of a localized chromatin opening at a specific, physiologically relevant target region.


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INTRODUCTION
 
The significant changes in gene expression that occur during processes such as differentiation, proliferation, and apoptosis are often controlled at the transcriptional level. In eukaryotic organisms, the transcriptional activation of genes occurs in the chromatin context. The basic unit of chromatin is the nucleosome, which consists of an octameric complex of the core histones H2A, H2B, H3, and H4 and 146 base pairs of DNA wrapped around the protein core and connected by linker DNA. Further folding of nucleosomes through the binding of histone H1 leads to compact higher-order structures in which the DNA is not easily accessible for transcription factors or the transcriptional machinery. Access by the transcription machinery to DNA is facilitated by various chromatin remodeling enzymes that cause localized changes of the chromatin structure. These remodeling complexes are themselves recruited by transcription factors to specific cis-acting sequences (for reviews see references 4, 23, 26, 41, 47, and 48). Although we have a rather detailed picture of the chromatin remodeling events that occur after transcription factors have associated with their binding sites, the mechanisms by which these proteins gain initial access to their binding sites in nucleosomally organized and tightly packed DNA are less well understood. Cirillo et al. (14) have used the term "pioneer factor" to describe a class of transcription factors that are capable of entering into silent chromatin and initiating chromatin opening. Because these pioneer factors are able to open compacted chromatin and help other proteins engage nearby sites, they are thought to play key roles in the "de novo" activation of cell-type-specific genes during differentiation and development. The best known example of a pioneer factor is FoxA (also known as HNF3), a Forkhead transcription factor which functions in liver development and occupies the albumin enhancer in undifferentiated cells prior to albumin expression (19). It has been demonstrated that FoxA is able to bind to its recognition sequences even when it is positioned on a nucleosome and to disrupt a compacted nucleosomal array in vitro (14).

The mim-1 (myb-inducible myelomonocytic 1) gene was originally identified as a target gene of the oncogenic transcription factor v-Myb, encoded by the avian leukemia virus E26, and its cellular homolog c-Myb (32, 35). Although c-Myb is expressed in all hematopoietic lineages, mim-1 is expressed only in myelomonocytic cells. The lineage-restricted activation of the mim-1 gene has been ascribed to the synergistic cooperation of Myb with members of the CCAAT/enhancer binding protein (C/EBP) family, particularly C/EBPβ, which are highly expressed in the myelomonocytic lineage (7, 28, 33). The cooperation of Myb and C/EBPβ is mediated by two distinct cis-acting elements, the mim-1 promoter (7, 33) and a cell-type-specific enhancer which is associated with a DNase I hypersensitive site (DHS) in myelomonocytic cells but has a compact chromatin structure in cells not expressing the gene (11). It was shown that the ectopic expression of Myb and C/EBPβ is sufficient to activate the endogenous, chromatin-embedded mim-1 gene in nonmyelomonocytic cells, such as fibroblasts, which normally do not express the gene, and that a dominant-negative version of C/EBPβ suppresses the activation of the gene (7, 33). These observations suggested that one of these factors is responsible for the initial opening of the chromatin structure.

Here, we have used the chicken mim-1 gene as a model system to study the initial events of chromatin opening. Our results demonstrate that C/EBPβ is able to trigger the initial steps of chromatin opening at the mim-1 enhancer in the absence of Myb, without inducing transcription of the gene. mim-1 transcription occurs only in the presence of Myb and is associated with opening of the chromatin at the promoter.


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MATERIALS AND METHODS
 
Cells. DF-1 is a chicken fibroblast line (20) and was grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. DF-1β15 is a subclone of the DF-1 line, expressing doxycycline-inducible full-length chicken C/EBPβ. To generate these cells, the coding region of C/EBPβ was cloned as a HindIII/XbaI DNA fragment into pCDNA4/TO/myc-His-A (Invitrogen). The resulting plasmid was then transfected simultaneously with pCDNA6-TR (encoding the Tet repressor) into DF-1 cells, followed by the selection of stable transfectants in the presence of 750 µg/ml zeocin and 750 µg/ml blasticidin. Doxycycline was omitted during the selection procedure to prevent C/EBPβ expression. Doubly resistant cell clones were then analyzed by Western blotting for doxycycline-inducible expression of the C/EBPβ protein, and the clone DF-1β15 was selected for further analysis. DF-1 cells constitutively expressing full-length or truncated C/EBPβ were generated by retroviral infection using the pCRNCM vector (42) (kindly provided by A. Leutz). The coding regions for full-length or truncated forms of C/EBPβ lacking the first 21 (C/EBPβ{Delta}N21), 49 (C/EBPβ{Delta}N49), and 110 (C/EBPβ{Delta}N110) amino acids were cloned between the EcoRI and XbaI sites of pCRNCM. Truncated forms were generated by PCR using the appropriate primers and were verified by sequencing. pCRNCM-based expression vectors for v-Myb and c-Myb were constructed by inserting the respective coding regions between the EcoRI and XbaI sites of pCRNCM. The v-Myb construct is derived from the pVM134 plasmid (6) and encodes a modified version of the v-Myb protein of avian myeloblastosis virus (AMV) lacking several of the AMV-specific mutations present in the DNA-binding domain. Unlike the original v-Myb encoded by AMV (32), this protein is able to activate the mim-1 gene. The resulting plasmids were then cotransfected with the RCAS helper virus vector (16) into DF-1 cells. Two days after transfection, 500 µg/ml G418 was added, and the cells were cultivated further for at least 2 weeks in the presence of G418. HD11 is a line of MC29-transformed chicken macrophages and was grown in basal Iscove's medium supplemented with 8% fetal calf serum and 2% chicken serum. BM2 is a line of AMV-transformed myeloblasts and was grown in RPMI 1640 medium supplemented with 5% fetal calf serum, 5% chicken serum, and 10% tryptose phosphate broth.

In vivo footprinting. In vivo dimethylsulfate (DMS) footprinting was performed as described by Tagoh et al. (44). DF-1β15 and HD11 cells were treated with 0.2% DMS in phosphate-buffered saline for 5 min at room temperature. The reaction was stopped by adding ice-cold phosphate-buffered saline. Cells were then lysed overnight at room temperature with proteinase K-sodium dodecyl sulfate (SDS) buffer, followed by DNA purification with phenol-chloroform, ethanol precipitation, and piperidine cleavage. DMS treatment and piperidine cleavage of naked genomic DNA were performed as described previously (12). Purified and piperidine-cleaved DMS-treated DNA (3 µg) was used as the starting material for ligation-mediated PCR (LM-PCR), as described by Kontaraki et al. (22), to visualize cleavage sites and changes in guanine(N7) DMS reactivity. PCR products were labeled by a primer extension reaction mixture using the 32P-labeled primer P3 and were analyzed on 6% denaturing polyacrylamide gels. LM-PCR-generated bands were quantified by phosphorimager analysis. The primers P1 (5'-AAACCAAAGCAGCAGCCATAATAT-3', labeled with biotin at the 5' end), P2 (5'-TCACAGGGGAAGTGGTGCAACT-3'), and P3 (5'-AAGTGGTGCAACTGCAGCTCTGTGCAA-3') were used for LM-PCR.

Electrophoretic mobility shift assays. Pairs of complementary single-stranded oligonucleotides containing wild-type (wt) and mutated (mut) C/EBP binding sites (underlined) were annealed and used for gel retardation assays, as follows: CEBP450wt (5'-TGCCTCTGTTGCCCAATGCAGGAATCCCACCAG-3' and 5'-GCTGGTGGGATTCCTGCATTGGGCAACAGAGGCA-3'); CEBP450mut (5'-TGCCTCTGTTGCCCTTTGCAGGAATCCCACCAG-3' and 5'-GCTGGTGGGATTCCTGCAAAGGGCAACAGAGGCA-3'); CEBP500wt (5'-CAGTCCTATTGCCCAACAGGCCAGGCCTG-3' and 5'-GCAGGCCTGGCCTGTTGGGCAATAGGACTG-3'); CEBP500mut (5'-CAGTCCTACAGCCCATCAGGCCAGGCCTG-3' and 5'-GCAGGCCTGGCCTGATGGGCTGTAGGACTG-3'); and CEBPcons (5'-TGTAGCTGCAGATTGCGCAATCTGCATCTA-3' and 5'-GTAGATGCAGATTGCGCAATCTGCAGCTACA-3').

After they were annealed, oligonucleotides were radiolabeled by filling in the ends using [{alpha}-32P]dCTP and Klenow polymerase. Nuclear extract containing C/EBPβ was prepared as described previously (8).

Mapping of DHS. DHS were mapped according to the method described by Sippel et al. (40), as described in detail by Braas et al. (5). The mim-1 enhancer region was analyzed by digesting the genomic DNA isolated from DNase I-treated nuclei with HindIII and probing Southern blots with a 0.4-kb SacI/HindIII DNA fragment from the mim-1 upstream region. To analyze the mim-1 promoter region, the DNA was digested with SacII, and blots were probed with a 0.5-kb SacII/SacI DNA fragment from the mim-1 upstream region.

Reporter genes and transfections. The luciferase reporter gene containing the wt mim-1 enhancer region upstream of the herpes simplex virus thymidine kinase promoter (pGL3-tk81-mimwt) has been described previously (11). Mutations of this plasmid were generated by PCR using the appropriate primers. In the tk-mimC/EBP-410luc plasmid, a C/EBP binding site (underlined) was changed from GGTTCTTTCACA to GGCCCATTCACA. In the tk-mimC/EBP-450luc plasmid, a C/EBP binding site (underlined) was changed from TGTTGCCCAATG to TGTTGCCCTTTG. In the tk-mimC/EBP-500luc plasmid, a C/EBP binding site (underlined) was changed from TATTGCCCAACA to TACAGCCCATCA. Plasmid double or triple mutations were made by sequential mutagenesis. All constructs were verified by sequencing. The β-galactosidase reporter gene pCMVβ was obtained from Clontech. DNA transfection of HD11 cells was performed by calcium-phosphate coprecipitation, as described previously (7). The preparation of cell extracts and luciferase and β-galactosidase assays were performed as described previously (7).

Chromatin immunoprecipitation. Chromatin immunoprecipitation was performed as follows. Approximately 1 x 107 to 5 x 107 cells were incubated for 10 min at room temperature with growth medium containing 1% formaldehyde and quenched by adding glycine (125 mM, final concentration) and incubating for 5 min. After cells were washed twice with ice-cold phosphate-buffered saline, they were suspended in RIPA buffer (10 mM Tris-HCl [pH 7.8], 50 mM NaCl, 1 mM phenylmethylsulfonyl fluoride [PMSF], 0.5% sodium deoxycholate, 0.5% NP-40, 0.1 to 0.5% SDS) and sonicated on ice (30-s-pulsed intervals for 12.5 min) (Diagenode sonicator). After the extract underwent centrifugation for 10 min at 14,000 rpm, it was preincubated with protein A-Sepharose for 1 h at 4°C. The supernatant was then incubated with C/EBPβ-specific rabbit antiserum raised against bacterially expressed C/EBPβ (28), or antiserum against histone H3 acetylated at lysine 9 (Abcam), or antiserum against the Flag epitope (M2; Sigma) or with no antiserum overnight at 4°C on a rotating wheel. Samples were incubated with protein A- or protein G-Sepharose for 1 h and then received three washes with ELB buffer (50 mM Tris-HCl [pH 7.5], 120 mM NaCl, 1 mM EDTA, 6 mM EGTA, 15 mM sodium pyrophosphate, 20 mM sodium fluoride, 1 mM PMSF, 0.5% NP-40), two washes with ELB buffer supplemented with 250 mM LiCl, one wash with ELB buffer supplemented with 250 mM LiCl and 0.1% SDS, and two final washes with ELB buffer. The immunoprecipitates were eluted first with elution buffer 1 (100 mM NaHCO3, 1% SDS) and then with elution buffer 2 (10 mM Tris-HCl, 1 mM EDTA, 1% SDS, 10 mM dithiothreitol). The eluates were combined and after the addition of proteinase K (500 µg/ml, final concentration), the DNA was reverse cross-linked by incubation for 12 h at 65°C. Finally, the immunoprecipitated DNA was extracted by phenol-chloroform, ethanol precipitated, resuspended in 50 to 100 µl of water, and stored at –20°C. PCR was performed by using the following primers: mim-1 enhancer (5'-CAGACTGATGTTGGAGGCAC-3' and 5'-TGTGGTGGTTGAGGCTTCTC-3'), mim-1 promoter (5'-CAGACTGATGTTGGAGGCAC-3' and 5'-TGTGGTGGTTGAGGCTTCTC-3'), and the Scl upstream region (5'-TCATGGCCTGAACCACTGTT-3' and 5'-GGTGAATTGCCTTCATCTATGC-3'). PCR products were resolved on 1.5 to 3% agarose gels and stained with ethidium bromide.

Northern blotting. Preparation of polyadenylated RNA and Northern blotting were performed as described previously (6). mim-1 mRNA was detected using a cDNA clone of the chicken mim-1 gene (32). As an internal control, a specific probe for the ribosomal protein S17 gene was used.


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RESULTS
 
The mim-1 enhancer acquires an open chromatin structure during myelomonocytic differentiation. The comparison of cell lines that represent different hematopoietic lineages has previously shown that the mim-1 enhancer colocalizes with a DNase I hypersensitive site only in cells of the myelomonocytic lineage (11). To demonstrate that this site is acquired during myelomonocytic differentiation, we used the multipotent progenitor line HD50 and a derivative of this line (HD50myl) that is committed to the myelomonocytic lineage (18). Both cell lines express the viral Myb protein of the E26 virus. As shown by Northern blotting, mim-1 was not expressed in the undifferentiated HD50 cells but was strongly expressed in HD50myl cells. C/EBPβ, which is highly expressed in myelomonocytic cells and has been implicated in myelomonocytic lineage commitment (30), is also expressed in HD50myl cells but not in HD50 cells (Fig. 1A). Figure 1B demonstrates the presence of a strong DHS site at the mim-1 enhancer in HD50myl cells, whereas this site was absent from HD50 cells. This suggests that the chromatin of mim-1 is not primed in HD50 cells and that the commitment of early hematopoietic progenitor cells to the myelomonocytic lineage is accompanied by the development of an open chromatin structure at the mim-1 enhancer. Moreover, the experiments also show that the commitment is associated with the upregulation of C/EBPβ, raising the possibility that C/EBPβ is involved in the induction of the DHS at the mim-1 enhancer.


Figure 1
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  		FIG. 1. The mim-1 enhancer acquires an open chromatin structure during myelomonocytic differentiation. (A) RNA isolated from HD50 and HD50myl cells was analyzed by Northern blotting using probes specific for mim-1, C/EBPβ, and ribosomal protein S17 mRNA. (B) The strategy for mapping DHS in the chromatin upstream of the mim-1 gene is illustrated schematically at the top. The arrow marks the transcriptional start site. Relevant restriction sites are marked as follows: H, HindIII; SI, SacI. The black bar marks the region that was used as the hybridization probe. Nuclei isolated from HD50 or HD50myl cells were treated without (lanes marked with –) or with increasing concentrations of DNase I, as indicated. DNA isolated from the nuclei was then digested with HindIII and analyzed by Southern blotting using the probe shown at the top. Bands that are not present in lanes lacking DNase I and whose intensity increases with increasing DNase I concentration represent DHS. The bands marked with white arrows represent constitutive DHS. The lengths (in kbp) of some size markers (lane M, molecular size markers) are indicated at the left.

The C/EBPβ initiates the opening of the chromatin structure at the mim-1 enhancer. To address the question of whether C/EBPβ alone or C/EBPβ in cooperation with Myb is responsible for the initial opening of the chromatin structure at the mim-1 enhancer during myelomonocytic differentiation, we established a stable fibroblast line (referred to as DF-1β15) in which the expression of C/EBPβ could be induced by doxycycline. We used chicken DF-1 cells because these cells do not express the mim-1 gene and lack a DHS at the mim-1 enhancer (11). In addition, DF-1 cells do not express c-myb or detectable amounts of C/EBPβ. The Western blot shown in Fig. 2A demonstrated that C/EBPβ expression was induced by growing the cells in the presence of doxycycline. We then examined the region upstream of the mim-1 gene for the presence of DHS (Fig. 2B). Figure 2B shows there was no DHS at the mim-1 enhancer when the cells were grown in the absence of doxycycline; however, a DHS was induced after cultivating cells in the presence of doxycycline. Several other DHSs, which were detected previously in all cell types analyzed, including the parental DF-1 line (11), were not affected by C/EBPβ induction, indicating that C/EBPβ specifically affected the chromatin structure at the mim-1 enhancer. To investigate whether it was indeed C/EBPβ that bound to the mim-1 enhancer, we performed ChIP using DF-1β15 cells grown in the presence or absence of doxycycline and confirmed that this was the case (Fig. 2C).


Figure 2
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  		FIG. 2. C/EBPβ induces chromatin opening at the mim-1 enhancer. (A) Doxycycline-inducible expression of C/EBPβ in DF-1 cells. DF-1β15 cells grown for 24 h in the presence or absence of doxycycline (Dox – and +) were analyzed by Western blotting using antiserum specific for C/EBPβ. C/EBPβ is marked by an arrowhead. (B) DHSs were mapped in the chromatin upstream of the mim-1 gene, as shown in Fig. 1. Nuclei isolated from DF-1β15 cells grown in the absence or presence of doxycycline were treated without (lanes marked –) or with increasing concentrations of DNase I, as indicated. DNA isolated from the nuclei was then digested with HindIII and analyzed by Southern blotting using the probe described for Fig. 1. In lane H, nuclei from the myelomonocytic cell line HD11 were treated similarly, except that only one concentration of DNase I was used. The region of the mim-1 enhancer is marked by the bar on the right side. The full-length genomic fragment detected by the probe is marked by a white arrowhead. DHSs not affected by doxycycline are marked with black dots on the right side. The lengths (in kbp) of some of the fragments are indicated at the right. (C) ChIP of the mim-1 enhancer region. Chromatin fragments prepared from DF-1β15 cells grown in the presence or absence of doxycycline were subjected to immunoprecipitation using C/EBPβ-specific antiserum or antiserum against the Flag epitope or without added antiserum. DNA isolated from the immunoprecipitates or from the total chromatin preparation before immunoprecipitation (input lanes) was analyzed by PCR using primers specific for the mim-1 enhancer. (D) Northern blotting analysis of endogenous mim-1 expression in DF-1β15 cells. The cells were grown in the presence (lane 1) or the absence (lane 2) of doxycycline. As a control, RNA from myeloid cells expressing Myb was loaded in lane 3. The blot was probed sequentially with probes specific for mim-1 and ribosomal protein S17 mRNA.

The cooperation of C/EBPβ and Myb has been shown to be sufficient to induce mim-1 expression. We were interested to know whether the appearance of a DHS at the mim-1 enhancer after induction with C/EBPβ alone was sufficient to induce the expression of mim-1 and found that this was not the case (Fig. 2D), indicating the crucial role of Myb in activating the mim-1 gene. To investigate whether Myb is also able to induce a DHS at the mim-1 enhancer on its own, we used retroviral infection to generate DF-1 cells stably expressing c-Myb or v-Myb. As shown in Fig. 3, v -Myb or c-Myb was readily detected in the infected cells; however, there was no DHS at the mim-1 enhancer in any of the cells. Thus, the ability to induce chromatin opening at the enhancer appears to be a specific function of C/EBPβ.


Figure 3
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  		FIG. 3. Chromatin structure at the mim-1 enhancer in DF-1 cells expressing c-Myb or v-Myb. (A and B) Nuclei were isolated from DF-1 cells constitutively expressing c-Myb or v-Myb and subjected to DHS mapping as shown in Fig. 1. (C) DF-1 cells expressing c-Myb (lane 1) or v-Myb (lane 2) were analyzed by Western blotting using Myb-specific antiserum. c-Myb and v-Myb are marked by arrowheads.

Taken together, these experiments showed that C/EBPβ does not need Myb to remodel chromatin at the mim-1 enhancer region from the silent state. However, an open chromatin structure at the enhancer itself is not sufficient for transcription of the mim-1 gene, which is Myb dependent.

Identification of functional C/EBPβ binding sites at the mim-1 enhancer. To identify the functional binding sites for C/EBPβ within the mim-1 enhancer, we performed in vivo DMS genomic footprinting experiments (Fig. 4A). The ability of DMS to induce the formation of N-7-methylguanine and N-3-methyladenine in DNA is modulated by protein-DNA interactions, bases which are close to specific binding sites or which actually participate in protein-DNA interactions and can therefore be recognized by their decreased or enhanced reactivity toward modification by DMS compared to that of purified genomic DNA subjected to the same treatment. Footprinting experiments were carried out with DF-1β15 cells grown in the presence or absence of doxycycline. As a positive control, we used HD11 cells, which express endogenous C/EBPβ and show a DHS at the mim-1 enhancer. Lanes 2 and 3 of Fig. 4A illustrate the major differences for the DMS sensitivity levels detected between DF-1β15 cells grown in the presence or the absence of doxycycline. The induction of C/EBPβ expression resulted in reproducible increases in the DMS sensitivity at positions C456 and C499 of the enhancer sequence. The corresponding guanine residues (on the opposite DNA strand) in both cases are located within the sequence TTGCCCAA, which shows a good match to the consensus C/EBP binding motif (C/EBP consensus sequence T(T/G)NNNNAA). The same footprints were also observed in HD11 cells, demonstrating that they were not the result of C/EBPβ overexpression. To further confirm that C/EBPβ is able to recognize these sites, we performed electrophoretic mobility shift assays (EMSA) (Fig. 4B). Strong mobility shifts of the radiolabeled oligonucleotides were observed when nuclear extracts from C/EBPβ-expressing cells were used, whereas there was only background binding when control extracts were used. Mutation of the binding site in both cases abolished the mobility shift. Taken together, our experiments identified two novel C/EBPβ binding sites (referred to as C/EBP450 and C/EBP500) within the mim-1 enhancer.


Figure 4
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  		FIG. 4. Identification of C/EBPβ binding sites in the mim-1 enhancer. (A) In vivo DMS footprinting. DF-1β15 cells grown in the presence (lane 2) or absence (lane 3) of doxycycline and HD11 cells (lane 4) were subjected to in vivo DMS footprinting as described in Materials and Methods. Lane 1 shows DMS treatment of isolated genomic DNA as the control. The footprinting gel encompasses the central part of the mim-1 enhancer, whose nucleotide sequence is shown at the bottom of the figure. The numbering refers to that used by Chayka et al. (11). Nucleotides showing altered DMS sensitivities in DF-1β15 cells expressing or not expressing C/EBPβ are highlighted by arrowheads, and the C/EBP consensus binding sites are marked. The mim-1 promoter and enhancer regions and the approximate position of the region covered by the footprint are shown schematically. Ovals marked MBS 1 to 4 and ovals marked C, B, and A indicate to myb-binding sites in the enhancer and promoters respectively. (B) EMSA experiments. Radiolabeled oligonucleotides corresponding to the wt or the mutated (mut) C/EBP binding sites C/EBP450 and C/EBP500 or to a consensus C/EBP binding site were incubated without nuclear extract (lane 1), with nuclear extract from untransfected cells (lane 2), or with nuclear extract from cells transfected with the C/EBPβ expression vector. Protein-DNA complexes were analyzed by native polyacrylamide gel electrophoresis. Complexes of C/EBPβ and the oligonucleotides migrate in the upper part of the gels. The intense bands at the bottom correspond to unbound oligonucleotides.

Next, we wished to know whether the C/EBP binding sites identified in vivo and in vitro were also relevant for the activity of the mim-1 enhancer. To address this issue, we mutated these sites in a luciferase reporter gene containing the mim-1 enhancer and the thymidine kinase promoter. We also included another potential C/EBP binding site (C/EBP410) in which we had detected binding of C/EBPβ in EMSA experiments (data not shown). We assessed the effects of the C/EBP site mutations on the enhancer activity by transfecting the corresponding reporter genes into HD11 cells. Figure 5 shows that each of the C/EBP site mutations abolished the activity of the enhancer, demonstrating that these sites are essential for the enhancer activity.


Figure 5
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  		FIG. 5. Activity of wild-type and mutant mim-1 enhancer constructs. (Top panel) The nucleotide sequence of the central part of the mim-1 enhancer is shown in the top row. The numbering is that used by Chayka et al. (11). C/EBP binding sites, two of which (C/EBP450 and C/EBP500) were identified by in vivo DMS footprinting, are marked by boxes. (Bottom left panel) Luciferase reporter genes (5 µg) containing the tk promoter (tk-luc), the tk promoter and the wild-type mim-1 enhancer (tk-mimwt-luc) or derivatives of this plasmid carrying mutations of one or several C/EBP sites were transfected into HD11 cells. The cells were additionally transfected with 0.5 µg of the β-galactosidase pCMVβ plasmid to control the transfection efficiency. Cells were harvested 24 h after transfection and analyzed for luciferase and β-galactosidase activity. The columns show the average luciferase activity normalized with respect to the activity of the cotransfected β-galactosidase plasmid. Thin lines show standard deviations. The activity of the reporter gene containing only the thymidine kinase promoter (tk-luc) was designated one. (Bottom right panel) ChIP of the mim-1 enhancer and promoter region. Chromatin fragments from prepared HD11 and BM2 cells were subjected to immunoprecipitation using C/EBPβ-specific antiserum or normal rabbit serum (NRS) or without added antiserum. DNA isolated from the immunoprecipitates or from the total chromatin preparation before immunoprecipitation (lane marked input) was analyzed by PCR using primers specific for the mim-1 enhancer or the promoter.

To confirm that C/EBPβ is bound to the mim-1 enhancer in untransfected myelomonocytic cells, we performed ChIP experiments using HD11 and BM2 cells, both of which express C/EBPβ endogenously. BM2 cells, in addition, express the v-Myb protein. As shown in the bottom panels of Fig. 5, C/EBPβ was bound to the mim-1 enhancer in both cell lines. Because C/EBPβ has also been shown to bind to and activate the mim-1 promoter (7, 28, 32), we also analyzed the ChIP experiment for in vivo binding of C/EBPβ to the mim-1 promoter; however, binding was apparent only in BM2 cells but not in HD11 cells. These experiments confirmed that endogenous C/EBPβ is bound to the mim-1 enhancer in myelomonocytic cells. Furthermore, the results suggested that the mim-1 enhancer and promoter behave differently with respect to C/EBPβ.

Amino terminal sequences of C/EBPβ are required for chromatin opening and histone acetylation at the mim-1 enhancer. The experiments described so far showed that the C/EBPβ sites identified above are able to induce de novo opening of the chromatin structure at the mim-1 enhancer. The transactivation domain of C/EBPβ, which occupies the N-terminal part of the protein, is known to bind several chromatin remodeling and modification activities, such as p300/CBP (29, 34) and SWI/SNF (24). This raised the question of whether C/EBPβ DNA binding per se was sufficient to induce chromatin opening or whether the recruitment of chromatin remodeling proteins was additionally required. To address this question, we generated DF-1 cells stably expressing full-length or a different N-terminally truncated form of C/EBPβ. All of these cells expressed the different forms of C/EBPβ at levels similar to those found in HD11 cells (Fig. 6B). In these cells, the C/EBPβ-specific antiserum recognizes two bands which correspond in size to the full-length protein and a slightly shorter form lacking 21 amino acids from the N terminus. Presumably, this shorter form is derived by internal translation initiation from an alternative translational start codon, as described previously (9). We then used the parental DF-1 cells and their C/EBPβ-expressing derivatives to map DHS. Figure 6C shows that a DHS appeared at the mim-1 enhancer in cells expressing full-length C/EBPβ or in the truncated form lacking 21 amino acids from the N terminus (C/EBPβ-{Delta}N21). DF-1 cells expressing the more extensively truncated forms of C/EBPβ lacking 49 or 110 N-terminal amino acids did not show a DHS at the mim-1 enhancer. These observations demonstrate that the amino terminus of the protein is essential for opening the chromatin structure at the mim-1 enhancer and that the DNA binding domain itself, which is located at the C terminus of the protein, is not sufficient.


Figure 6
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  		FIG. 6. Chromatin opening at the mim-1 enhancer by full-length or deletion mutants of C/EBPβ. (A) Schematic illustration of the C/EBPβ constructs used. The bZIP DNA-binding domain is shown by a black bar. (B) Western blotting analysis of the parental DF-1 cell line (lane 1), DF-1 cells expressing full-length C/EBPβ or deletion mutants of C/EBPβ (lanes 2 to 5), and HD11 cells (lane 6) using antiserum against C/EBPβ (top panel) or tubulin (bottom panel). (C) DHS analysis of the parental DF-1 cells and DF-1 cells expressing full-length C/EBPβ or partially deleted forms of C/EBPβ, as indicated. Details of the DHS mapping experiments are described in the legend to Fig. 1. The region of the mim-1 enhancer is marked by the bar on the right side.

Because the C/EBPβ-{Delta}N110 deletion mutant, which lacks the entire transactivation domain of the protein but retained the DNA-binding domain, failed to open the chromatin structure at the mim-1 enhancer, we asked whether this protein was able to bind to the mim-1 enhancer in vivo or whether cooperation with cofactors was necessary for stable binding. To address this issue, we performed ChIP experiments using the DF-1 cells expressing the different C/EBPβ mutants. As illustrated clearly in Fig. 7A, all forms of C/EBPβ, even those deletion constructs (C/EBPβ-{Delta}N49 and C/EBPβ-{Delta}N110) which do not induce chromatin opening, were bound to the mim-1 enhancer in transfected cells. The parental cell line did not show enrichment of the mim-1 enhancer sequences. This indicates that binding of C/EBPβ can be separated from the opening of the chromatin structure. Taken together, our data suggest that chromatin remodeling proteins which are known to associate with the amino terminus of C/EBPβ are required for chromatin opening at the mim-1 enhancer.


Figure 7
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  		FIG. 7. ChIP analysis of C/EBPβ binding to the mim-1 enhancer. (A) Chromatin fragments prepared from the parental DF-1 cell line or from DF-1 cells expressing full-length C/EBPβ or deletion mutants of C/EBPβ were subjected to immunoprecipitation using C/EBPβ-specific antiserum, antiserum against the Flag epitope, or no antiserum. DNA isolated from the immunoprecipitates or from the total chromatin preparation before immunoprecipitation (lanes marked input) was analyzed by PCR using primers specific for the mim-1 enhancer. (B) Chromatin fragments were prepared from the parental DF-1 cell line or DF-1 cells expressing full-length C/EBPβ or the {Delta}N110 deletion mutant of C/EBPβ, as marked on the left side of each panel. Chromatin fragments were then subjected to immunoprecipitation with antibodies against C/EBPβ, against acetylated H3K9, or against the Flag epitope. DNA isolated from the immunoprecipitates or from the total chromatin preparation before immunoprecipitation (input) was analyzed by PCR using primers specific for the mim-1 enhancer, the mim-1 promoter, or the upstream region of the SCL gene, as indicated on the right side of each panel.

Next, we performed ChIP experiments with antibodies specific for histone H3K9 acetylation, to test whether the changes in chromatin structure induced by C/EBPβ at the mim-1 enhancer were accompanied by the acetylation of core histones. Figure 7B shows that mim-1 enhancer sequences were precipitated with these antibodies when chromatin from DF-1 cells expressing full-length C/EBPβ was used. For a control experiment to confirm the specificity of the ChIP experiment, we performed a PCR with primers binding to an unrelated region upstream of the SCL gene; this did not result in any detectable PCR product. Finally, we also performed a ChIP experiment using DF-1 cells expressing the truncated C/EBPβ-{Delta}N110 mutation. This experiment showed only background levels of H3K9 acetylation at the mim-1 enhancer. Taken together, the experiments shown in Fig. 7B demonstrate that binding of full-length but not of truncated C/EBPβ to the mim-1 enhancer leads to histone H3K9 acetylation.

Chromatin opening by C/EBPβ does not require DNA replication. It has been hypothesized that DNA replication provides a window of opportunity for transcription factors to set up an active chromatin structure (46). To test whether DNA replication is required for C/EBPβ to gain access to the mim-1 enhancer, we investigated whether the establishment of a DHS at the mim-1 enhancer could be blocked by treating the cells with aphidicolin, an inhibitor of DNA replication. DF-1β15 cells were grown for 24 h without doxycycline or were induced with doxycycline in the absence or presence of 0.6 µg/ml aphidicolin for 24 h. Control experiments showed that the incorporation of [3H]thymidine into DNA was down to 20% of normal levels, indicating that the DNA replication activity was strongly inhibited under these conditions (data not shown). Figure 8 shows that the appearance of a DHS at the mim-1 enhancer was not inhibited by aphidicolin treatment, indicating that DNA replication is not necessary for chromatin opening by C/EBPβ.


Figure 8
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  		FIG. 8. The effect of aphidicolin on chromatin opening by C/EBPβ. DF-1β15 cells were grown in the presence (+) or absence (–) of 1 µg/ml doxycycline (dox) or 0.6 µg/ml aphidicolin (aphid.), as indicated at the bottom. The cells were then subjected to DHS mapping as described in the legend to Fig. 1. The position of the mim-1 enhancer is marked by an arrowhead.

Chromatin opening at the mim-1 promoter requires the cooperation of Myb with C/EBP. C/EBPβ has also been implicated together with Myb in the activation of the promoter of the mim-1 gene (7, 28, 33). Surprisingly, analysis of the ChIP samples of HD11 cells and DF-1 cells expressing full-length C/EBPβ with mim-1 promoter-specific PCR primers showed that C/EBPβ was unable to bind to the mim-1 promoter in these cells (see Fig. 5 bottom panels and Fig. 7, third panel from the top) and also did not lead to histone H3K9 acetylation at this element. To assess the nuclease sensitivity of the promoter region, we performed DHS-mapping experiments of the mim-1 promoter region, using HD50, HD50myl, and DF-1β15 cells grown in the absence or presence of doxycycline. HD50 and HD50myl cells both express the viral Myb protein encoded by the E26 virus. As shown in Fig. 1, HD50myl cells also express C/EBPβ. Figure 9A shows that a DHS was detected at the mim-1 promoter in HD50myl cells but not in HD50 cells, consistent with the expression pattern of the mim-1 gene. Figure 9B shows that no DHS appeared at the promoter upon the induction of C/EBPβ expression in the DF-1β15 cells, which do not express Myb. Note that under the same conditions, a DHS was clearly visible in these cells at the enhancer (Fig. 2B). This indicated that C/EBPβ might require Myb to open the chromatin at the mim-1 promoter. To test this idea, we made use of the chicken HD11 cell line which, unlike DF-1 cells, expresses C/EBPβ endogenously. We used a subclone of the HD11 line (designated HD11-E) that expressed a doxycycline-inducible Myb protein, as confirmed by Western blotting (Fig. 9C, left upper panel). Following its induction by doxycycline, the expression of the mim-1 gene was activated, as demonstrated by Northern blotting (Fig. 9C, middle and bottom panels), and this was accompanied by the appearance of a DHS at the mim-1 promoter (Fig. 9C). We therefore concluded that C/EBP-mediated chromatin opening at the mim-1 promoter, unlike the opening of the mim-1 enhancer, occurs only in the presence of Myb.


Figure 9
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  		FIG. 9. Mapping of a DHS at the mim-1 promoter. The strategy for mapping DHSs in the chromatin around the mim-1 promoter is illustrated schematically at the top. The arrow marks the transcriptional start site. Relevant restriction sites are marked as follows: H, HindIII; SII, SacII; SI, SacI. The black bar marks the region that was used as the hybridization probe. Nuclei isolated from HD50 or HD50myl cells (A), DF-1β15 cells grown in the absence or presence of doxycycline (B), or HD11-E cells grown in the absence or presence of doxycycline (C) were treated without (– lanes) or with increasing concentrations of DNase I, as indicated by the black triangles. DNA isolated from the nuclei was then digested with SacII and analyzed by Southern blotting using the probe shown at the top. The black arrowheads point to a DHS at the mim-1 promoter. DNA size markers (in kilobase pairs) are indicated on the left. Panel C shows additionally a Western blot of HD11-E cells stained with antibodies against v-Myb (top). The cells were grown in the presence or absence of doxycycline (+dox; –dox). The middle and bottom panels show Northern blots of the same cells hybridized with probes specific for mim-1 or ribosomal protein S17 mRNAs (middle and bottom).


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DISCUSSION
 
C/EBPβ is capable of activating the mim-1 enhancer from the silent state. The data reported here demonstrate that the initial contributions of C/EBPβ and Myb to the activation of the mim-1 enhancer from the silent state are fundamentally different. Our results clearly show that c-Myb and v-Myb on their own are unable to induce chromatin opening at the mim-1 enhancer. The inability of Myb to open chromatin is in concordance with the observation that the mim-1 enhancer in the early progenitor line HD50, which expresses Myb but not C/EBPβ, has a compact chromatin structure. Furthermore, we have previously shown that erythroid and lymphoid cell lines which express c-Myb lack a DHS at the mim-1 enhancer (11), again supporting the notion that c-Myb is not able to induce chromatin opening. In contrast, our data show for the first time that C/EBPβ is able to enter into silent chromatin and can induce localized opening of the chromatin structure at a physiological target site. We suspect that this novel function of C/EBPβ plays a crucial role in hematopoietic development and lineage commitment and raises the possibility that at some target genes, C/EBPβ can function as a pioneer factor. In vitro studies of the pioneer factor FoxA have shown that FoxA on its own is able to bind stably to its target sites on nucleosomes (13, 14) and to open histone H1-compacted chromatin arrays built on the enhancer region of the serum albumin gene. However, the albumin enhancer also contains a single C/EBP site, and in this context, C/EBPβ was unable to induce chromatin opening under the same conditions in vitro (14). C/EBPβ is also unable to form stable complexes with binding sites on nucleosomes (14, 43). These examples illustrate that C/EBP opens chromatin at the mim-1 enhancer by a different mechanism. Because the inhibition of DNA replication does not prevent chromatin opening, it appears unlikely that C/EBPβ acts by competing with nucleosome deposition during DNA replication. Model studies using DNA molecules with defined arrays of specific binding sites have demonstrated that transcription factors which do not bind stably to a single site positioned on a nucleosome might nevertheless cooperate to disrupt a nucleosome if multiple binding sites are present on the same nucleosome (1, 45). Our in vivo footprinting analysis and mutagenesis studies of the mim-1 enhancer shown here establish that C/EBPβ binds to three sites whose integrity is crucial for the activity of the enhancer. As shown in Fig. 3, these binding sites are of equal functional importance and occur within a DNA stretch of approximately 100 base pairs and, therefore, could be present on a single nucleosome. The presence of several closely spaced binding sites may be crucial for the ability of C/EBPβ to trigger chromatin opening at the mim-1 enhancer. Since C/EBP binding sites occur randomly and with relatively high frequency, the requirement of a cluster of several binding sites within a short distance for C/EBPβ to trigger chromatin opening could explain the specificity of this novel function of C/EBPβ.

The context-dependent function of C/EBP is also apparent at the mim-1 gene itself. Opening of the chromatin at the mim-1 promoter, in contrast to opening at the enhancer, occurs only in the presence of Myb. The failure of C/EBPβ to affect the chromatin structure at the mim-1 promoter might explain why C/EBPβ on its own does not induce transcription of mim-1. We speculate that the number and the spacing of the C/EBP binding sites are important as only two C/EBP binding sites located approximately 100 base pairs apart have been mapped in the promoter region (28).

Chromatin opening requires the presence of the C/EBPβ transactivation domain. Chromatin opening is not simply the consequence of the binding of C/EBPβ to its binding sites in the mim-1 enhancer, because C/EBPβ binding and chromatin remodeling can be separated. Our ChIP assays cannot distinguish between the occupancy of single and multiple sites; it is therefore possible that one C/EBPβ molecule can bind to an accessible site (for example in a linker region), whereas the binding of multiple proteins and the formation of a DHS require nucleosome remodeling and modification. This is illustrated by our finding that most of the transactivation domain of C/EBPβ appears to be required for its ability to induce a DHS. The observation that N-terminal sequences of C/EBPβ are required for chromatin opening makes it very likely that C/EBPβ-binding proteins such as SWI/SNF and p300/CBP, whose interaction with C/EBPβ depends on the integrity of the transactivation domain of C/EBPβ (24, 29), are involved in chromatin opening. Several isoforms of C/EBPβ (referred to as Lap*, Lap, and Lip) can be generated from C/EBPβ mRNA by alternative translation initiation (9, 15). Our data suggest that the isoforms Lap* and Lap (which correspond to the full-length and the {Delta}N21 constructs, respectively, used here) but not the shorter Lip isoform (which completely lacks the transactivation domain) are able to trigger chromatin opening. SWI/SNF interacts efficiently only with the full-length (Lap*) form of C/EBPβ (24), whereas p300/CBP binds to C/EBPβ{Delta}N21 (alias Lap) as well (29, 39), suggesting that C/EBPβ-mediated histone acetylation might be particularly relevant for the initial chromatin opening. Consistent with this, we have found that chromatin opening triggered by C/EBPβ is accompanied by histone H3 acetylation. Several studies have shown that histone acetylation precedes the recruitment of SWI/SNF and nucleosome eviction during gene activation (2, 21, 27, 37, 38, 49). Furthermore, recent work with in vitro assembled nucleosome arrays has shown that nucleosomes acetylated on lysine 9 of histone H3 are preferentially displaced by SWI/SNF (10), in agreement with the notion that histone acetylation precedes nucleosome sliding or removal by SWI/SNF.

In summary, our work identifies a novel function of C/EBPβ involved in the initial steps of localized chromatin opening at a specific, physiologically relevant target region. The expression of C/EBPβ in nonmyeloid cells is known to activate several resident myeloid cell-specific genes in addition to mim-1 (7, 24, 29, 33). Furthermore, C/EBPβ also has important functions in the lineage commitment of multipotent hematopoietic progenitors (30) and the activation of specific genes during fat and liver cell differentiation (25, 31). In addition, the ratios in which the different C/EBPβ isoforms are expressed are known to change in certain cellular processes, for example, during the lipopolysaccharide-mediated acute-phase response (3) or during neoplastic transformation of mammary epithelial cell tumorigenesis (17, 36). This suggests that the chromatin opening function of C/EBPβ can be modulated during such processes. This strongly suggests that the chromatin opening function of C/EBPβ may have a broader relevance, beyond the activation of the mim-1 gene.


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ACKNOWLEDGMENTS
 
We thank B. Michaelis for expert technical assistance.

This work was supported by grants from the DFG (Kl 461/10-2 and Kl 461/13-2) to K.-H. Klempnauer and from the Leukemia Research Fund to C. Bonifer. A. Plachetka and O. Chayka were supported by fellowships from the Graduate School of Chemistry (GSC-MS) at the University of Münster, Germany.


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FOOTNOTES
 
* Corresponding author. Mailing address: Institut für Biochemie, Westfälische-Wilhelms-Universität Münster, Wilhelm-Klemm-Str. 2, D-48149 Münster, Germany. Phone: 49-251-8333203. Fax: 49-251-8333206. E-mail: klempna{at}uni-muenster.de Back

{triangledown} Published ahead of print on 14 January 2008. Back

{dagger} Present address: Institute of Child Health, Molecular Haematology and Cancer Biology Unit, 30 Guilford Street, London WC1N 1EH, United Kingdom. Back

{ddagger} Present address: Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, United Kingdom. Back


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Molecular and Cellular Biology, March 2008, p. 2102-2112, Vol. 28, No. 6
0270-7306/08/$08.00+0     doi:10.1128/MCB.01943-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



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