Cell lines and reagents.

The RAW264.7 and J774 murine macrophage cell lines were maintained in Dulbecco's modified Eagle's medium (DMEM) (Cellgro) supplemented with 10% low-endotoxin fetal bovine serum (Omega Scientific) and penicillin/streptomycin (Omega Scientific). The B6.129/J2 macrophage line was a gift from Jill Suttles and Peter Tontonoz and was maintained in RPMI 1640 (Cellgro) supplemented with 10% low-endotoxin fetal bovine serum (Omega Scientific) and penicillin/streptomycin (Omega Scientific). This line was generated by transformation of bone marrow from wild-type mice in a mixed 129-C57BL/6 background with the murine J2 retrovirus. Superfect (QIAGEN) was used for transient transfection of RAW264.7 cells. J774 cells were stably transfected by electroporation. The parameters for electroporation were described previously (33). Stable lines were selected and maintained in DMEM supplemented with 10% fetal bovine serum, penicillin/streptomycin, and 364 μg G418 (GibcoBRL)/ml.

Bone marrow- and fetal liver-derived primary macrophages were prepared from wild-type and mutant mice as described previously (35). Macrophages were stimulated with 10 μg LPS (Sigma)/ml with or without 10 U recombinant murine gamma interferon (IFN-γ) (PharMingen)/ml. c-Rel^−/−, Oct-1^−/−, and Oct-2^−/− mice were described previously (9, 19, 46).

Reporter assays.

For CAT assays, 0.5 × 10⁶ cells/well were plated in six-well plates. The following day, the cells were washed with phosphate-buffered saline and transfected with 2 μg DNA. DNA was incubated in DMEM without serum or antibiotics in a total volume of 100 μl with Superfect at a 1:3 ratio (μg of DNA to μl of Superfect) for 5 to 10 min at room temperature. A total of 600 μl of complete DMEM supplemented with 10% fetal bovine serum (Omega Scientific) and penicillin/streptomycin (Omega Scientific) was added to the DNA/Superfect mix and then added to the cells, and the cells were incubated at 37°C for 2.5 to 3 h. Cells were washed with phosphate-buffered saline and split into two wells in 2.5 ml of complete DMEM. The cells in one of the wells were activated with 10 μg LPS (Sigma)/ml 6 h posttransfection and incubated for 24 h. Cells were then harvested and used in reporter assays as described previously (33).

Restriction enzyme accessibility and DNase I hypersensitivity assays.

DNase I hypersensitivity assays were performed as described previously (8, 52). Primers for generating probe 1 were 5′-CTCAATGCGTAAAGTGAGCAGGATTGC-3′ and 5′-TAGCTCAGGCTAGCCTCAACTCTTC-3′. Primers for generating probe 2 were 5′-GGCCAACCACCCTTGTTAATATATTG-3′ and 5′-GTCCATAGTGCTACTATATTTGGTTGAAAGG-3′. Primers for generating probe 3 were 5′-GGCTAGGTGTACATGTATGTGCATATATC-3′ and 5′-GAAAGAAATGAATGAGTTCCCACC-3′. Primers for generating probe 4 were 5′-TTGGGGCAAGTCCTTCCTTTTTCTGC-3′ and 5′-AGTGTCAAAACATTCTGGGGG-3′. The probes were labeled, and the Southern blot-based restriction enzyme accessibility assay was performed as described previously (52).

Gel shifts, Western blots, and enzyme-linked immunosorbent assays.

Nuclear extracts were prepared as described previously (33). Gel shift probes for Oct-1, Oct-2, and C/EBPβ contain sequences derived from the HSS1 site. Oct-1 (SC-232X), Oct-2 (SC-233X), and C/EBPβ (SC-150X) antibodies were from Santa Cruz Biotechnology. Gel shifts, Western blots, and enzyme-linked immunosorbent assays were performed as described previously (52).

c-Rel is not required for HSS1 and HSS2 formation.

As described above, the NF-κB family member c-Rel is critical for Il12b induction in macrophages stimulated with LPS plus IFN-γ (35, 36). However, we previously showed that nucleosome remodeling at the endogenous Il12b promoter is only slightly reduced in c-Rel^−/− macrophages, leading to the hypothesis that c-Rel acts downstream of, or parallel to, nucleosome remodeling in the Il12b activation pathway (48). One possibility was that c-Rel is required for the activation of a distant enhancer. To determine whether c-Rel is required for the formation of HSS1 and HSS2, DNase I hypersensitivity assays were performed using bone marrow-derived macrophages from c-Rel^−/− mice (19, 36, 52). Interestingly, both the inducible and constitutive hypersensitive sites were readily detected in the c-Rel-deficient macrophages (Fig. ​(Fig.1B,1B, lanes 11 to 20; Fig. ​Fig.1C,1C, lanes 11 to 20). These results demonstrate that c-Rel, although critical for Il12b transcription, is not required for the chromatin changes that are responsible for the appearance of HSS1 and HSS2.

Defining the boundaries of the inducible chromatin changes surrounding HSS1.

The DNase I hypersensitivity results suggested that the chromatin structure approximately 10 kb upstream of the Il12b start site is altered following stimulation with LPS plus IFN-γ. To analyze more precisely the locations and boundaries of these chromatin changes, a restriction enzyme accessibility assay was employed. First, to determine whether this assay can be used to monitor chromatin changes at HSS1 and to assess the reproducibility of this assay, three independent aliquots of B6.129/J2 macrophages were left unstimulated, with three additional aliquots stimulated with LPS plus IFN-γ. The B6.129/J2 line was used for this analysis because Il12b transcription is strongly and reproducibly activated in this line and HSS1 DNase I hypersensitivity was comparable to that observed in primary bone marrow-derived macrophages (data not shown). Nuclei from each of the six samples were incubated with restriction enzyme PstI, which has the potential to cleave at a recognition site located at bp −10061 (Fig. ​(Fig.2A).2A). Genomic DNA was then purified and cleaved to completion with the restriction enzyme AseI, followed by gel electrophoresis and Southern blotting.

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FIG. 2.

Defining the boundaries of LPS-induced chromatin changes in the vicinity of HSS1. (A) The positions of HSS1, relevant restriction enzyme recognition sites, and the Southern blot probes are indicated on the Il12b 5′ flanking region extending from kb −5.5 to −12 relative to the transcription start site. The thick dashed lines represent evolutionarily conserved regions located from bp −9437 to −9981 (containing HSS1), from bp −7521 to −6186, and from bp −6012 to −5327. The thin dashed line represents repetitive sequence. (B) LPS-induced chromatin changes in the vicinity of HSS1 were monitored using a restriction enzyme accessibility/Southern blot assay with nuclei from B6.129/J2 cells that were left unstimulated (−) or were stimulated with LPS plus IFN-γ (LPS/IFN-γ) for 4 h (+). Nuclei were incubated with 40 (lanes 1 and 2), 80 (lanes 3 and 4), or 160 (lanes 5 and 6) units of PstI to monitor accessibility at HSS1. Genomic DNA was then purified and cleaved with AseI. The digested DNA fragments were visualized by Southern blotting with probe 3. The PstI-AseI fragment reveals the efficiency of PstI cleavage in the isolated nuclei. Quantification of cleavage percentage was determined by phosphorimager analysis. (C) LPS-induced chromatin changes at different locations between kb −5.9 and −11.5 were monitored using the restriction enzyme accessibility/Southern blot assay. Aliquots of nuclei were incubated with different amounts of EcoRV (lanes 1 to 6), KpnI (lanes 7 to 12), AflII (lanes 13 to 18), SpeI (lanes 19 and 20), XmnI (lanes 21 and 22), BamHI (lanes 23 to 28), or BglI (lanes 29 to 34) enzyme (Enz.). Purified genomic DNA was cleaved with AseI. The digested DNA fragments were visualized by Southern blotting with probe 1. The EcoRV-AseI, KpnI-AseI, AflII-AseI, SpeI-AseI, XmnI-AseI, BamHI-AseI, and BglI-AseI fragments reveal the efficiency of nuclear cleavage with EcoRV, KpnI, AflII, SpeI, XmnI, BamHI, and BglI, respectively. (D) B6.129/J2 cells were pretreated for 15 min with cycloheximide (CHX) (+) dissolved in dimethyl sulfoxide (DMSO) (10 μg/ml) (lanes 3 and 5), with DMSO alone (lanes 2 and 4), or were left untreated (−) (lane 1). Cells were then stimulated with LPS plus IFN-γ for 1 h (lanes 4 and 5) or were left unstimulated (lanes 1 to 3). Nuclei were incubated with SpeI. Purified SpeI-digested DNA was digested with KpnI and SphI. The digested DNA fragments were visualized by Southern blotting with probe 3. The SpeI-SphI fragment reveals the efficiency of SpeI cleavage in the isolated nuclei. All data in panels B, C, and D are representative of at least three independent experiments.

When a radiolabeled probe derived from the Il12b locus was used (Fig. ​(Fig.2A,2A, probe 3), cleavage at the two AseI sites flanking HSS1 yielded a 5,600-bp product, whereas an additional cleavage at the PstI site yielded a fragment of 4,075 bp. Approximately 20% of the genomic DNA was cleaved by PstI in nuclei from unstimulated cells (Fig. ​(Fig.2B,2B, lanes 1, 3, and 5), with approximately 40% cleaved in nuclei from stimulated cells (Fig. ​(Fig.2B,2B, lanes 2, 4, and 6).

Stimulus-dependent restriction enzyme cleavage was also apparent when the nuclei were incubated with AflII (which cleaves at bp −10121), SpeI (bp −9869), and XmnI (bp −8725) (Fig. ​(Fig.2C,2C, lanes 13 to 22). In contrast, stimulus-dependent cleavages were not observed with two enzymes that cleave at positions farther from the transcription start site, EcoRV (bp −10916) and KpnI (bp −10256), or with two enzymes that cleave closer to the start site, BamHI (bp −8088) and BglI (bp −7369) (Fig. ​(Fig.2C,2C, lanes 1 to 12 and lanes 23 to 34). These results suggest that the region in which LPS-induced changes in chromatin structure take place extends from at least bp −10121 (AflII site) at the distal end to at least bp −8725 (XmnI site) at the proximal end, but not beyond bp −10256 (KpnI site) at the distal end or bp −8088 (BamHI site) at the proximal end. This region includes the 545-bp region that exhibits high evolutionary conservation (−9981 to −9437) plus 700 to 1,300 bp proximal to these conserved sequences. It is important to note, however, that no suitable restriction enzyme recognition sequence was available between the SpeI site at bp −9869 and the XmnI site at bp −8725. Thus, we cannot rule out the possibility that the XmnI site is within a control region that is independent of the control region containing the clustered AflII, PstI, and SpeI sites.

To further analyze LPS-induced nucleosome remodeling at the HSS1 site, its requirement for new protein synthesis was monitored. When the B6.129/J2 line was pretreated for 15 min with the protein synthesis inhibitor cycloheximide, the increase in restriction enzyme cleavage typically observed following stimulation with LPS plus IFN-γ was eliminated (Fig. ​(Fig.2D).2D). Similar results were recently reported in J774 macrophages (34). New protein synthesis is similarly required for Il12b transcription and for LPS-induced increases in restriction enzyme accessibility at the Il12b promoter (34, 49).

The HSS1 region functions as a transcriptional enhancer in transient-transfection experiments.

The inducible DNase I hypersensitivity and restriction enzyme accessibility at HSS1 suggest that this region might function as an inducible Il12b enhancer. To determine whether enhancer activity could be detected, seven overlapping DNA fragments of approximately 1 kb each were fused upstream of a 400-bp Il12b promoter fragment in a CAT reporter plasmid (Fig. 3A and B). Transient-transfection experiments were then performed with the RAW264.7 macrophage line, with the transfected cells left unstimulated or stimulated with LPS for 24 h. RAW264.7 cells were used for the transient-transfection experiments because the J774 and B6.129/J2 macrophage lines do not transfect with sufficiently high efficiency for detectable reporter activity (data not shown). In our hands, RAW264.7 cells express the endogenous Il12b gene poorly in response to either LPS or LPS plus IFN-γ, but they express many other LPS-induced genes and therefore support TLR4 signaling pathways (33). We do not know why the endogenous Il12b locus is not expressed in RAW264.7 cells, but we previously found that transfected Il12b promoter-reporter plasmids were efficiently induced in these cells, and we therefore suspected that the line would be equally appropriate for a basic characterization of the HSS1 enhancer. The CAT reporter gene was used for these experiments because we previously found that luciferase reporter activity is strongly induced in RAW264.7 cells, even when luciferase transcripts are driven by constitutively active promoters (33). This nonspecific induction of luciferase activity has the potential to yield misleading results. In contrast, CAT activity was only slightly induced when constitutive promoters were used to drive CAT transcription in these cells (33).

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FIG. 3.

The DNA region surrounding HSS1 exhibits enhancer activity in transient-transfection experiments. (A) Individual Il12b DNA fragments (fragments 1 through 7) that were subcloned into the CAT reporter vector are shown. (B) Critical regions of the CAT reporter plasmids are diagrammed, including the fragment being tested for enhancer activity, the 400-bp Il12b promoter fragment, and the CAT reporter gene. (C) RAW264.7 cells were transfected with plasmids containing each of the seven fragments (fragments 1 to 7 [Frag. 1 to Frag. 7]) diagrammed in panel A (lanes 1 to 14), as well as a plasmid containing only the Il12b promoter (lanes 15 and 16), and the pCAT vector lacking both a promoter and enhancer (lanes 17 and 18). Transfected cells were left unstimulated (−) or were stimulated with LPS (10 μg/ml) for 24 h (+). IFN-γ was not included during stimulation because this cytokine has no effect on promoter activity upon transient transfection of this line (33). CAT enzyme activities were measured as described previously (7). (D) The means plus standard errors of the means (error bars) from four independent experiments (after phosphorimager quantification) are shown. The activity of the Il12b promoter was set at 100%.

LPS-induced CAT activity dependent on the Il12b promoter was observed with all plasmids tested, but only fragment 2 substantially enhanced the CAT activity (Fig. 3C and D). This fragment spans the DNase I cleavage site and three of the restriction enzyme recognition sites that showed LPS-induced increases in cleavage (AflII, PstI, and SpeI) (Fig. ​(Fig.3C).3C). Fragment 3, which overlaps fragment 2 and also includes the DNase I cleavage site and the same three restriction enzyme recognition sites, supported CAT activity at an enhanced level relative to that observed with the other five 1-kb fragments. However, the activity obtained with this plasmid was comparable to that obtained with the Il12b promoter in the absence of a 1-kb insert. Figure ​Figure3D3D shows the average signals with standard deviations obtained in three independent transfection experiments.

Localization and analysis of the core HSS1 enhancer.

To localize the core enhancer within fragments 2 and 3, a series of smaller DNA fragments, as well as a larger fragment encompassing both fragments 2 and 3, were analyzed in the context of the Il12b promoter-CAT reporter plasmid (Fig. ​(Fig.4A).4A). Only one small fragment, a 370-bp fragment called fragment C, supported enhanced CAT activity (Fig. ​(Fig.4B).4B). Of further note, fragment A-E, which encompasses both fragments 2 and 3, yielded activity comparable to that of fragment 3 (Fig. ​(Fig.4B).4B). These results suggest that all sequences responsible for the HSS1 enhancer activity detectable in a transient-transfection assay reside within the 370-bp fragment C. Furthermore, fusion of these sequences to the Il12b promoter in the context of fragment 2 or fragment C is preferable to fusion in the context of fragment 3, which places the critical sequences farther from the promoter. Importantly, the 370-bp fragment C encompasses the DNase I-hypersensitive site and restriction sites that showed LPS-induced increases.

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FIG. 4.

Identification and characterization of a 105-bp core enhancer region. (A) The transient-transfection assay in RAW264.7 cells was used to analyze a variety of DNA fragments to more precisely localize the sequences responsible for enhancer activity. Fragment A-E contains sequences spanning both fragments 2 and 3 (Frag.2 and Frag.3) from Fig. ​Fig.3A.3A. Fragment C, which exhibited the strongest enhancer activity, is 370 bp long. (B) Transfected cells were stimulated with LPS (10 μg/ml) for 24 h. (No significant activity was observed with any of the plasmids in the absence of stimulation [not shown]). CAT reporter activities were then determined. The means plus standard errors of the means (error bars) from four independent experiments (after phosphorimager quantification) are shown. The activity of the Il12b promoter was set at 100%. (C) The sequences within the 370-bp fragment C that were responsible for enhancer activity were further localized by analysis of six subfragments (C1 through C6). Fragment C1 is 105 bp long. (D) CAT reporter activities of plasmids containing the fragments diagrammed in panel C were determined. The means plus standard errors of the means (error bars) from four independent experiments are shown. (No significant activity was observed with any of the plasmids in the absence of stimulation [not shown]). (E) The transient-transfection assay was used to monitor the enhancer activities of fragments C and C1 in the forward (C and C1) and reverse (Cr and C1r) orientations in the context of the Il12b promoter-CAT reporter plasmid. The Il12b promoter-CAT reporter plasmid in the absence of an inserted enhancer was analyzed as a control (promoter). Transfected cells were stimulated with LPS (10 μg/ml) for 24 h. The means plus standard errors of the means (error bars) from four independent experiments are shown. (F) The enhancer activities of fragments C and C1 in the context of a minimal heterologous core promoter containing consensus TATA and Inr elements (C/TI and C1/TI) were examined. The TI promoter-CAT reporter plasmid in the absence of an inserted enhancer fragment was tested as a control (TI). The means plus standard errors of the means (error bars) from three independent experiments are shown.

To localize more precisely the sequences within fragment C that are responsible for enhancer activity, smaller fragments were analyzed in the context of the Il12b promoter-CAT reporter plasmid (Fig. ​(Fig.4C).4C). The results revealed that enhancer activity is contained entirely within a 105-bp region (−9994 to −9890) termed fragment C1 (Fig. ​(Fig.4D).4D). Both fragments C and C1 were capable of supporting weak enhancer activity in either orientation in the context of the Il12b promoter-CAT reporter plasmid (Fig. ​(Fig.4E).4E). Furthermore, both of these fragments supported LPS-induced CAT activity when inserted into a CAT reporter plasmid containing only a minimal consensus TATA box and Inr element (Fig. ​(Fig.4F)4F) (7).

Identification of a DNA element required for HSS1 enhancer activity.

The 105-bp C1 fragment found to be required for enhancer activity (−9994 to −9890) is located at the distal edge of the 545-bp region of evolutionary conservation between DNA sequences encoding the mouse, rat, human, and dog C1 fragments (−9981 to −9437). It is interesting that a careful examination of the sequence within this 105-bp region revealed three segments that exhibit particularly strong conservation (Fig. ​(Fig.5A).5A). To determine which DNA elements are important for enhancer activity in the transient-transfection assay, 11 different substitution mutants were prepared in the context of the Il12b promoter-CAT reporter plasmid (Sub A through Sub K [Fig. ​[Fig.5A]).5A]). In these plasmids, the mutations were introduced into the 370-bp fragment C. Analysis of the mutants by transient transfection in RAW264.7 cells revealed that enhancer activity was abolished by only one mutation, Sub H, with a substantial decrease in activity observed with an adjacent mutation, Sub I (Fig. ​(Fig.5B).5B). The Sub H and Sub I mutations disrupt one of the three regions of evolutionary conservation. In a stable-transfection assay in J774 cells (see below), the Sub H mutation also abolished enhancer activity, whereas the Sub I mutation only slightly reduced enhancer activity (data not shown). These data suggest that the DNA sequence altered by the Sub H mutation represents a critical enhancer element. Importantly, a small DNA fragment encompassing only the Sub H and Sub I regions was unable to support enhancer activity (data not shown), demonstrating that additional sequences within the 105-bp fragment are essential for activity. However, these additional sequences must function in a redundant manner, at least in the transfection assay, as none of the other individual mutations significantly decreased enhancer activity.

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FIG. 5.

Localization of a DNA element required for the LPS-induced enhancer activity. (A) The DNA sequence of mouse Il12b fragment C1 (−9,994 to −9,890 bp) is shown, along with the homologous sequences from the rat, human, and dog genomes. The nucleotides altered in each of the 11 substitution mutants (Sub A through Sub K) are indicated, as are three regions that are particularly well-conserved between the species. The 11 mutations were analyzed in the context of the 370-bp fragment C in the Il12b promoter-CAT reporter plasmid. Gaps introduced to maximize alignment are indicated by dashes. (B) RAW264.7 cells were transfected with enhancer-promoter-CAT reporter plasmids containing wild-type fragment C and the 11 mutant enhancers. The Il12b promoter alone (promoter) was analyzed as a control. Transfected cells were stimulated with LPS (10 μg/ml) for 24 h. (No significant activity was observed with any of the plasmids in the absence of stimulation [not shown]). The means plus standard errors of the means (error bars) from four independent experiments are shown.

Oct proteins bind the DNA sequence altered by the Sub H mutation.

To identify transcription factors capable of binding the DNA sequence altered by the Sub H mutation, gel shift assays were performed with nuclear extracts prepared from the B6.129/J2 line and a 40-bp DNA probe (Fig. ​(Fig.6A).6A). Two major protein-DNA complexes were detected with the wild-type probe, with no differences between unstimulated and stimulated macrophages (Fig. ​(Fig.6B,6B, lanes 1 and 2). Analysis of a series of mutant probes, each of which alters 3 bp, revealed that both complexes were eliminated by three different mutations within the Sub H region (Fig. ​(Fig.6B6B).

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FIG. 6.

Oct-1 and Oct-2 bind the critical Sub H region of the HSS1 enhancer. (A) The DNA sequence of the probe used for gel shift assays is shown. Nucleotides altered in each of the eight mutant probes are underlined. The predicted Oct and C/EBP binding sites within the probe are shown. (B) ³²P-labeled wild-type (WT) (lanes 1 and 2) and mutant (lanes 3 to 18) probes (probes 1 to 8) were used for gel shift assays with nuclear extracts from unstimulated B6.129/J2 cells (−) or B6.129/J2 cells stimulated for 4 h with LPS (+). (C) The identities of the two protein-DNA complexes were determined by adding Oct-1 (lanes 7 to 12) and Oct-2 (lanes 13 to 18) antibodies (Ab) to gel shift reaction mixtures. Nuclear extracts were made from J744 (J), B6.129/J2 (B), and RAW264.7 (R) cells. (D) Oct-1 binding to the endogenous HSS1 region was monitored using a ChIP assay. (Left) Formaldehyde-cross-linked chromatin samples prepared from unstimulated peritoneal macrophages (lane 1) or peritoneal macrophages stimulated for 4 h with LPS plus IFN-γ (lane 2) were immunoprecipitated with Oct-1 antibodies (top blot) or control GST antibodies (middle blot). The presence of the HSS1 region and the Il12b promoter in the immunoprecipitates was determined by semiquantitative PCR. Input samples are shown in the bottom blot. (Right) ChIP experiments were performed with bone marrow-derived macrophages, with the Oct-1 antibody immunoprecipitates analyzed by real-time PCR, using a variety of primer pairs that amplify regions spanning the Il12b locus. Similar results were obtained in a second independent experiment (not shown). NS, not stimulated; PROM, promoter. (E) ChIP assays analyzed by both semiquantitative PCR (left) and real-time PCR (right) were performed with Oct-2 or GST antibodies. For both semiquantitative and real-time PCR analysis, chromatin samples were prepared from bone marrow-derived macrophages.

An examination of the DNA sequence within the Sub H region revealed the presence of potential binding sites for Oct and C/EBP proteins (Fig. ​(Fig.6A).6A). Antibodies against C/EBP family members had no effect on the complexes observed in the gel shift experiments (data not shown). However, the lower-mobility complex was eliminated by antibodies directed against Oct-1, and the higher-mobility complex was eliminated by antibodies against Oct-2 (Fig. ​(Fig.6C).6C). This experiment was performed with extracts derived from three different macrophage lines (J774, B6.129/J2, and RAW264.7), which revealed that Oct-1 is readily detectable in all three lines, whereas Oct-2 is readily detectable only in the J774 and B6.129/J2 lines (Fig. ​(Fig.6C).6C). Because OCA-B (also known as OBF1 and Bob-1) has been shown to interact with Oct-1 and Oct-2 in B lymphocytes (11, 22, 37, 40), the presence of OCA-B in complex 1 or 2 was examined by gel shift analysis. OCA-B antibodies failed to supershift or disrupt either complex (data not shown). Furthermore, by Western blot analysis, OCA-B was undetectable in macrophage extracts, whereas it was readily detected in extracts from B-cell lines (data not shown).

To monitor the recruitment of Oct proteins to the endogenous HSS1 enhancer in primary murine macrophages, ChIP assays were performed with peritoneal or bone marrow-derived macrophages left unstimulated or stimulated with LPS plus IFN-γ. Sheared chromatin from formaldehyde-treated cells was precipitated with antibodies directed against either Oct-1 or Oct-2 and was first analyzed by PCR with a mixture of two primer pairs. One primer pair amplified the HSS1 region, and the second pair amplified the Il12b promoter. In the DNA samples prepared from macrophages stimulated with LPS plus IFN-γ, the HSS1 PCR product was enriched relative to the promoter product when either the Oct-1 or Oct-2 antibodies were used (Fig. 6D and E, top blots). In contrast, the HSS1 product was not enriched when a glutathione S-transferase (GST) control antiserum was used (Fig. 6D and E, middle blots). The immunoprecipitated chromatin was further evaluated by real-time PCR, using PCR primer pairs that amplify seven different regions spanning the Il12b locus. The HSS1 region was selectively enriched in Oct-1 and Oct-2 antibody immunoprecipitates prepared from stimulated macrophages (Fig. 6D and E, bar graphs). These results suggest that Oct-1 and Oct-2 associate with the endogenous HSS1 enhancer upon macrophage activation. Interestingly, interactions between Oct-1 and Oct-2 and the Il12b promoter were not detected. These data contrast with evidence that some transcription factors can interact with enhancers and promoters simultaneously by DNA looping, although this possibility cannot be ruled out (12).

Il12b expression in Oct-1- and Oct-2-deficient macrophages.

Although the functions of Oct-1 and Oct-2 have been studied extensively in B cells and other cell types (26, 45, 47), little is known about the functions of these proteins in macrophages. To examine their relevance for Il12b expression, macrophages were derived from the fetal livers of one Oct-1^−/− mouse and two Oct-2^−/− mice, as well as their wild-type littermates. Il12b mRNA was then monitored by real-time RT-PCR, with mRNAs from three other LPS-induced genes (Nos2, Ccl5, and Tnf) monitored as controls. Although variable results were obtained, no significant reduction in expression of any of the four genes was observed in the mutant samples in comparison to the wild-type samples (Fig. ​(Fig.7).7). One explanation for these negative results is that Oct-1 and Oct-2 act redundantly to stimulate Il12b transcription. Unfortunately, we were unable to obtain Oct-1^−/− Oct-2^−/− double mutant fetuses to test this possibility.

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FIG. 7.

Analysis of Il12b expression in fetal liver macrophages from Oct-1^−/− and Oct-2^−/− mice. Fetal liver macrophages from one Oct-1^−/− mouse and two Oct-2^−/− mice, as well as their wild-type littermates, were left unstimulated or were stimulated with LPS plus IFN-γ (LPS/IFN-γ) for 6 h (for Oct-1^−/− mice) or for 2 and 4 h (for Oct-2^−/− mice). Real-time RT-PCR was then used to monitor Il12b mRNA levels, with Nos2, Ccl5, and Tnf mRNA levels monitored as controls. NS, not stimulated; Oct-2^−/− #1, Oct-2^−/− mouse 1.

C/EBPβ associates with the HSS1 enhancer.

As shown in Fig. ​Fig.5A,5A, the 105-bp HSS1 enhancer fragment contains three segments that exhibit particularly strong evolutionary conservation. Although only the Sub H region was important for enhancer activity in the transient-transfection assay, we suspect that the other two regions, and possibly additional regions that exhibit significant evolutionary conservation both within and adjacent to the 105-bp fragment, are important for activity of the endogenous enhancer. Interestingly, the highly conserved DNA element spanning the Sub B/Sub C region of the enhancer contains a DNA sequence that perfectly matches the consensus recognition sequence for C/EBP proteins (Fig. ​(Fig.5A5A and ​and8A).8A). In addition, gel shift experiments performed with extracts from the three macrophage lines and a probe encompassing the Sub B/Sub C region yielded complexes that comigrated with complexes formed by in vitro-translated LAP, a C/EBPβ isoform containing the bZIP DNA binding domain and N-terminal transactivation domains (Fig. ​(Fig.8B,8B, lanes 1 to 7) (6). This complex is thought to consist of LAP homodimers. A second abundant complex in the macrophage extracts comigrates with a complex consisting of a heterodimer between LAP and LIP, a smaller C/EBPβ isoform that lacks the transactivation domains (Fig. ​(Fig.8B,8B, lane 9) (6). The levels of these complexes were greatly diminished by C/EBPβ antibodies (Fig. ​(Fig.8B,8B, lanes 11 to 19).

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FIG. 8.

C/EBPβ binds the endogenous HSS1 enhancer. (A) The DNA sequence of the probe used for gel shift assays is shown, along with the C/EBP consensus recognition sequence. (B) Gel shift (lanes 1 to 10) and antibody (Ab) supershift (lanes 11 to 20) assays were performed as described in the legend to Fig. ​Fig.6.6. Nuclear extracts from J774 (J), B6.129/J2 (B), and RAW264.7 (R) cells were examined, as well as in vitro-translated LAP (C/EBPβ large isoform; lanes 7 and 17), LIP (C/EBPβ small isoform; lanes 8 and 18), and cotranslated LAP and LIP (LAP/LIP) (lanes 9 and 19). In vitro-translated luciferase (LUC) (lanes 10 and 20) was tested as a negative control. The positions of protein-DNA complexes corresponding to LAP homodimers, LAP/LIP heterodimers, and LIP homodimers are indicated. A protein-DNA complex that does not interact with C/EBPβ antibodies is also apparent (*). (C) The Il12b genomic region examined for C/EBPβ binding by semiquantitative ChIP is shown. The locations of DNA fragments amplified by PCR (fragments I through VI) are indicated. The binding of C/EBPβ to the endogenous Il12b locus was monitored by ChIP using C/EBPβ or GST antibodies and chromatin samples prepared from peritoneal macrophages. Semiquantitative PCR was used to analyze the DNA fragments present in the immunoprecipitates. (D) ChIP experiments were performed with real-time PCR as described in the legend to Fig. ​Fig.6,6, using chromatin prepared from bone marrow-derived macrophages. Similar results were obtained in a second independent experiment (not shown).

ChIP experiments provided further support for the hypothesis that C/EBPβ contributes to HSS1 enhancer activity, as DNA fragments in close proximity to HSS1 were enriched following immunoprecipitation of cross-linked chromatin from stimulated macrophages with C/EBPβ antibodies (Fig. ​(Fig.8C).8C). The immunoprecipitates were analyzed with various combinations of primer pairs from the Il12b locus to determine the approximate locations of the C/EBPβ contact sites. DNA amplified by primer pair II, which overlaps HSS1, was enriched to a much greater extent than the other five DNA fragments analyzed (Fig. ​(Fig.8C).8C). This finding was confirmed by real-time PCR analysis of C/EBPβ antibody immunoprecipitates from an independent set of unstimulated and stimulated macrophages (Fig. ​(Fig.8D).8D). Together, these results provide strong evidence that C/EBPβ associates with the endogenous Il12b locus within or in close proximity to the HSS1 enhancer.

Although mutations in the consensus C/EBP binding site at the Sub B/Sub C region had no effect on enhancer activity in transient- and stable-transfection assays (Fig. ​(Fig.5B5B and data not shown), these results suggest that C/EBP proteins may indeed contribute to enhancer activity. C/EBP proteins may not be important in the transfection experiments because of the artificial nature of these assays, or the functions of C/EBP proteins may be redundant with the functions of other enhancer binding proteins. Unfortunately, in the absence of functional data, we cannot exclude the possibility that the binding of C/EBPβ to the endogenous enhancer is fortuitous and without functional relevance.

HSS1 enhancer activity in an insulated chromatin environment.

The transient-transfection results, combined with the DNase I hypersensitivity and restriction enzyme accessibility results, support the hypothesis that DNA sequences in the vicinity of HSS1 function as an LPS-induced enhancer. However, one weakness of this hypothesis is that fragments 2, C, and C1 stimulate Il12b promoter activity by only three- to fivefold in transient-transfection experiments. To determine whether this weak activity might be due to the artificial nature of the transient-transfection assay, the activity of the HSS1 region was tested in a chromatin environment by using a stable-transfection assay. For this assay, fragments C and C1 were fused upstream of the Il12b promoter in a vector containing a GFP reporter gene (28). In this vector, the enhancer-promoter-GFP cassette is flanked on each side by two chicken β-globin insulators (Fig. ​(Fig.9A).9A). A phosphoglycerate kinase (PGK)-neomycin (Neo) drug selection cassette is located on the plasmid on the side opposite that of the insulators. The resulting plasmids (Il12b promoter alone, fragment C1 plus promoter, and fragment C plus promoter) were linearized using the restriction enzyme SalI, followed by transfection into the J774 macrophage line. (Although the J774 line, which produces large quantities of IL-12 p40, is not useful for transient-transfection experiments in our hands, it transfects with sufficient efficiency for selection of clones containing stably integrated plasmids.) Transfected cells were immediately plated on 96-well plates and were selected in growth medium containing G418, yielding monoclonal or oligoclonal colonies. After LPS stimulation for 24 h, the mean GFP fluorescence was determined for each colony (Fig. ​(Fig.9B9B).

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FIG. 9.

Enhancer activity and nucleosome remodeling in an insulated chromatin environment. (A) The insulator vector used for the generation of stable cell lines is diagrammed. Two copies of the 1.2-kb chicken β-globin insulator (Ins) are present at each end of the reporter cassette. A NotI cloning site was used for insertion of the enhancer fragments (fragment C or C1) and Il12b promoter, with a downstream GFP reporter gene. A PGK-Neo drug resistance cassette is located outside of the insulated reporter cassette. The plasmids were linearized with SalI before transfection. The location of probe 4 used for the Southern blot assay is indicated. (B) Insulator plasmids containing the Il12b promoter alone or the Il12b promoter with upstream fragment C1 or fragment C were stably transfected into J774 cells. Several monoclonal or oligoclonal drug-resistant colonies were left unstimulated (UN) or were stimulated with LPS for 24 h. The mean GFP fluorescence was then determine by flow cytometry. Eleven colonies containing the Il12b promoter alone, 10 colonies containing fragment C1, and 15 colonies containing fragment C were analyzed. (C) Three monoclonal or oligoclonal J774 lines containing stably integrated plasmids with wild-type (WT) fragment C and five lines containing integrated plasmids with the Sub H mutant version of fragment C were analyzed using the restriction enzyme accessibility assay. Cells were left unstimulated (odd-numbered lanes) or were stimulated with LPS for 4 h (even-numbered lanes) prior to preparation of nuclei. Nuclei were incubated with MluI (40 units), and genomic DNA was then purified and cleaved to completion with NotI. The digested DNA fragments were visualized by Southern blotting with probe 4. The NotI-NotI fragment corresponds to the in vitro cleavage product. The MluI-NotI fragment reveals the efficiency of MluI cleavage in the isolated nuclei.

In these experiments, the activity of the isolated Il12b promoter was very low in unstimulated cells and was stimulated only slightly by LPS (Fig. ​(Fig.9B),9B), in striking contrast to the results obtained in transient-transfection experiments and in previous stable-transfection experiments performed with promoter-GFP reporter plasmids in the absence of insulators (48). However, in the presence of fragments C1 and C, strong LPS-induced promoter activity was observed (Fig. ​(Fig.9B).9B). Although considerable clonal variability was observed, there was a consistent trend toward strong LPS-induced GFP fluorescence. We suspect that the magnitude of the enhancer effect is greater in these stable-transfection experiments than in the transient-transfection experiments because the insulator elements allow the promoter to assemble into an inactive chromatin structure that cannot be converted to an active state in the absence of the enhancer. These results greatly strengthen the hypothesis that the HSS1 region contains a biologically important enhancer that is a target of LPS signaling pathways.

We thank Gary Felsenfeld and Mark Schlissel for providing the insulator vector used for the stable-transfection experiments and Jill Suttles and Peter Tontonoz for the B6.129/J2 murine macrophage line.

S.T.S. is an Investigator with the Howard Hughes Medical Institute.