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Originally published In Press as doi:10.1074/jbc.M510700200 on January 26, 2006

J. Biol. Chem., Vol. 281, Issue 14, 9227-9237, April 7, 2006
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The Interleukin-1beta Gene Is Transcribed from a Poised Promoter Architecture in Monocytes*

Michael D. Liang{ddagger}, Yue Zhang§, Daniel McDevit§, Sylvia Marecki§, and Barbara S. Nikolajczyk§1

From the {ddagger}Department of Pathology, Boston University School of Medicine, the §Department of Medicine, Immunobiology Unit, Evans Memorial Department of Clinical Research, Boston Medical Center, and the Department of Microbiology, Boston University School of Medicine, Boston, Massachussetts 02118

Received for publication, September 30, 2005 , and in revised form, January 17, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Cytokine transcription is usually regulated by transcription factor binding and chromatin remodeling following an inducing signal. By contrast, these data showed the interleukin (IL)-1beta promoter assembles into a "poised" structure, as evidenced by nuclease accessibility and loss of core histones immediately surrounding the transcription start site. Strikingly, these properties do not change upon transcriptional activation by lipopolysaccharide. Furthermore, association of two key transcriptional activators, PU.1 and C/EBPbeta, is robust pre- and post-stimulation indicating the IL-1beta promoter is packaged into a nontranscribed but poised promoter architecture in cells capable of rapidly inducing IL-1beta. Monocyte stimulation causes recruitment of a third factor, IRF-4, to the IL-1beta enhancer. PU.1 phosphorylation at a CK2 kinase consensus element is required for this recruitment. We showed that CK2 phosphorylates PU.1, CK2 inhibitors abrogate IL-1beta induction, and CK2 inducibly associates with the IL-1beta enhancer. Taken together, these data indicate a novel two-step mechanism for IL-1beta transcription: 1) formation of a poised chromatin architecture, and 2) phosphorylation of an enhancer-bound factor that recruits other activators. We propose that this poised structure may generally characterize rapidly activated genes.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Members of the monocyte/macrophage lineage are the major IL-1beta2 source in vivo. Processes regulating the first step of IL-1beta production, transcription, have been studied in detail by transient transfection and DNA binding assays. These analyses have putatively identified transcription factors and regulatory elements playing nonredundant roles in IL-1beta transcription in response to endotoxin stimulation. IL-1beta transcription is regulated by a proximal promoter and an enhancer located at about 3 kb upstream of transcription start. Inducible transcriptional activation has been localized to both promoter and enhancer elements and appears to be regulated similarly in mouse and human (1–4).

Classical reporter and in vitro DNA binding assays have demonstrated that PU.1, C/EBPbeta, and NF-{kappa}B can activate the IL-1beta promoter (2, 5, 6). The IL-1beta enhancer is activated by PU.1, C/EBPbeta, AP-1 (Fos/Jun), and a novel STAT transcription factor dubbed LIL-STAT (7). Members of the interferon regulatory factor (IRF) family also activate IL-1beta transcription (8). However, activation by any of the enhancer-binding proteins is minimal in the absence of intact PU.1 promoter site(s) (8). Importantly, ectopic PU.1 expression in nonhematopoietic cells results in IL-1beta transcription from the endogenous (i.e. chromatinized) locus, further emphasizing the role PU.1 plays in this process (8). Work herein therefore focuses on the critical role PU.1 plays in inducible IL-1beta transcription.

Monocytic cells activate IL-1beta transcription rapidly (i.e. within minutes) following stimulation with the endotoxin lipopolysaccharide (LPS). The mechanisms driving inducible IL-1beta transcription appear to hinge on PU.1 phosphorylation at serine residue 148. Mutation of serine 148, located within a protein kinase CK2 consensus element, substantially blunts PU.1-mediated transcriptional activation of an IL-1beta-regulated reporter gene (9). Because CK2 has been shown to be activated by LPS, and CK2 can target PU.1 serine 148 in cells (10), it is likely that CK2-mediated PU.1 phosphorylation leads to IL-1beta transcription in monocyte lineage cells. Furthermore, PU.1 phosphorylation is required for transcriptional activation via recruitment of IRF-4 to an ETS/IRF composite element, found in both the {kappa}3' and IL-1beta enhancers (9, 10). This model of PU.1-activated transcription via IRF-4 recruitment has not been tested in the context of a chromatinized gene. Most analyses aimed at understanding the role PU.1 or other transcription factors play in inducible IL-1beta transcription were completed before the importance of the potential regulatory context of cellular chromatin was fully realized. The lone exception is the demonstration that PU.1 overexpression can activate IL-1beta transcription from the endogenous locus. Whether PU.1 functions through direct (i.e. IL-1beta enhancer or promoter association) or indirect mechanisms was not discerned (8).

Changes in promoter chromatin structure regulate inducible transcription of most cytokine genes tested to date. The typical scenario is that a histone octamer sequestering the transcription start site is remodeled in response to stimulation, resulting in more or less rapid gene transcription. This mechanism was described with variable rigor for the IL-2, granulocyte-macrophage colony-stimulating factor, IL-4, interferon-{gamma}, IL-12 p35, IL-12 p40, IL-13/IL-5, TNF-{alpha}, IL-10, MCP-1, and interferon-beta promoters (11–24). Whether this mechanism results in the very rapid induction of IL-1beta transcription has not been reported, despite the recent demonstration that modifications of histones packaging the locus change following monocyte stimulation (25).

We have analyzed chromatin structure and protein association at the IL-1beta locus in resting and stimulated monocyte lineage cells. Results herein demonstrate that, in resting cells capable of rapid transcriptional activation, the IL-1beta promoter is packaged into an accessible, poised chromatin architecture not yet shown at any cytokine locus. Further analyses suggest that phosphorylation of constitutively associated PU.1 by DNA-associated CK2 upon cellular stimulation results in recruitment of IRF-4 to the IL-1beta enhancer. RNA polymerase II is concomitantly recruited to the promoter to result in the robust IL-1beta transcription characteristic of stimulated monocytes. Overall, the data demonstrate that the IL-1beta gene is inducibly transcribed from a poised promoter architecture likely due to post-translational modification of DNA-associated protein.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture—Mono-Mac-6 (MM6) cells were maintained in RPMI containing 15% heat-inactivated fetal calf serum, 2 mML-glutamine, and OPI media supplement to a final concentration of 1 mM oxaloacetate, 0.45 mM pyruvate, and 0.2 units/ml insulin. 293 human embryonic kidney cells were maintained in Dulbecco's modified Eagle's medium containing 10% heat-inactivated fetal calf serum and 50 µM beta-mercaptoethanol. THP-1 and THP-1/CD14 cells were maintained in RPMI containing 10% heat-inactivated fetal calf serum and 1 mM sodium pyruvate. HL-60 cells were maintained in RPMI containing 10% heat-inactivated fetal calf serum and 2 mM L-glutamine. U937 cells were maintained in RPMI containing 10% heat-inactivated fetal calf serum. RAW 264.7 cells were maintained in Dulbecco's modified Eagle's medium containing 10% heat-inactivated fetal calf serum. All culture media contained 100 units of penicillin and 100 µg of streptomycin per ml.

Bone marrow-derived macrophages (BMDM) were derived from 6- to 8-week-old BALB/cByJ femurs according to Vicente et al. (26). Specifically, marrow from femurs and tibias was flushed out with 1–2 ml of RPMI 1640 containing 15% fetal bovine serum using a 30-gauge needle. Viable cells were purified through Lympholyte M. Cells from each mouse were plated in a T75 flask with 20 ml of BMDM media (60% irrigation media, 40% L-929 fibroblast (L cell)-conditioned medium). The nonadherent cells were further cultured in BMDM media. Macrophages were obtained as a homogeneous population of adherent cells after 7 days of culture. Homogeneity of the CD11c– Mac-1+ population was assessed by flow cytometry. Primary splenic B cells were prepared as described (27). Primary splenic T cells were prepared using the pan-T Macs depletion system according to the manufacturer's protocol. All primary cells were routinely >90% pure.

RNA Isolation and Quantitative PCR Analysis—Total RNA was isolated from 5 x 105 cells using RNeasy (Qiagen). cDNA was prepared by standard methods. PCRs amplified 10 ng of cDNA, using SYBR Green (Bio-Rad) incorporation for quantitation and the Stratagene Mx3000p Real Time PCR System (Stratagene, La Jolla, CA). Each amplification was performed in triplicate using the following conditions: hot start, 3 min at 95 °C; melting, 15 s at 95 °C and then annealing and extension at 60 °C for 1 min, for 40 cycles. The primers used for IL-1beta transcript were as follows: sense, 5'-ACGAATCTCCGACCACCACT-3', and antisense, 5'-CCATGGCCACAACAACTGAC-3'. PCRs conducted in parallel using beta2-microglobulin (beta2m) primers (sense, 5'-CTCCGTGGCCTTAGCTGTG-3', and antisense, 5'-TTTGGAGTACGCTGGATAGCCT-3') were used to normalize for differences in cDNA synthesis and RNA input. mRNA copy number was calculated according to the equation: copies = 10(Ct–40)/(–3.32).

Nuclease Probing of Chromatin Accessibility Assayed on Southern Blots—Nuclease probing of chromatin accessibility was performed as described (28). 2 x 107 cells were used for each condition. For the accessibility cut, 100 units of HindIII were added, and the nuclei were incubated at 37 °C for 1 h with agitation. Ten µg of purified genomic DNA was digested to completion with XmaI. Digestion products were separated on agarose gels. The accessibility fragment was detected on Southern blots using a radiolabeled probe specific for the IL-1beta promoter (–860 to –352 bp relative to transcription start) and standard methods.

Chromatin Accessibility by Real Time (CHART) PCR—Accessibility of DNA to digestion with MNase was analyzed using CHART-PCR as published (29) using 105–106 cells. 5–10 ng of DNA was analyzed by SYBR Green incorporation during quantitative PCR. All products were run on acrylamide gels to verify product size and displayed a single optimum on melt curve analysis. Apparent negative accessibility is a mathematical artifact likely due to error in DNA quantitation and is neither reproducible nor biologically significant.

Chromatin Immunoprecipitation (ChIP)—ChIPs were performed as published previously (27, 30), using 500,000 human or 107 murine cells per antibody. Fold enrichment was calculated by 2(Ctinput–CtChIP) after quantitative amplification of equivalent picogram amounts of DNA (31). The appropriate amount of antibody was determined empirically, and the same microgram amount of {alpha}-histidine tag antibody was added in the control samples. ChIP-competent antibodies were as follows: {alpha}-histidine tag antibody (SC-803; Santa Cruz Biotechnology) as a nonspecific isotype/species-matched control, {alpha}-acetylated histone H3 (06-599; Upstate), rabbit {alpha}-PU.1 (SC-352; Santa Cruz Biotechnology), {alpha}-C/EBPbeta (C-19; Santa Cruz Biotechnology), {alpha}-histone H3 (antibody 1791-100; Abcam), {alpha}-CK2{alpha}' (19278-100; Abcam) and {alpha}-RNA polymerase II (N-20; Santa Cruz Biotechnology). Oligonucleotides used for amplifying the precipitated IL-1beta promoter region were from promoter primer set –I (Tables 1 and 2). IL-1beta enhancer-specific primers were 5'-AGCGGTCTCCTTGGGAAGA-3' and 5'-ACCATCCCATCCATCTCAGG-3'. beta2m promoter primers for control ChlP assays were 5'-CCCAGTCTAGTGCATGCCTTC-3' and 5'-ACGCAGTGCCAGGTTAGAGAG-3'.


Figure 1
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  		FIGURE 1.
The IL-1beta promoter is packaged into a poised chromatin structure in monocytes. A, schematic of amplicons amplified by primers sets in Table 1. +1 is position of transcription start. B, CHART-PCR analysis of human MNase-resistant IL-1beta promoter DNA in 293 cells (open bars), resting Mono-Mac-6 monocytes (MM6 U, black bars), or MM6 monocytes stimulated with LPS (MM6 S, gray bars). Numbers at top represent center nucleotide of the quantitated amplicon. High levels of accessibility denote a relatively accessible chromatin structure as indicated. S.E. was < 5% over atleast three independent experiments. C, CHART-PCR of resting human monocyte lines THP-1 (black bars), U937 (gray bars), HL-60 (stippled bars), and 293 cells (open bars). Each cell line was analyzed at least twice with less than 5% deviation between experiments. * indicates value was not determined at this position in THP-1 cells. D, chromatin accessibility at the IL-1beta promoter in resting and stimulated control (293, lanes 1 and 2, respectively) and monocytes (THP-1, lanes 3 and 4, respectively) as measured by restriction endonuclease cutting. Southern blots of indirect end labeling results are shown. Arrow denotes accessibility fragment. E, chromatin accessibility at the IL-1beta enhancer in resting control (293, lane 1) and monocytes (THP-1, lane 2) and in stimulated THP-1 cells (lane 3), as in D. Data represent at least three independent analyses.

 


Figure 2
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  		FIGURE 2.
Specificity of the IL-1beta promoter poised chromatin structure. A, poised IL-1beta promoter structure is cell- and cytokine-specific. Murine amplicons designated at top of figure correspond to positions in Table 2. 3T3 cells are fibroblasts; RAW 264.7 are a macrophage cell line. B2 cells are murine splenic B cells, and T denotes murine splenic T cells. B, CHART-PCR showing accessibility of the TNF-{alpha} promoter in resting or LPS-stimulated (S) human monocytes (MM6) or control (293) cells. Amplicons centered at +27 (TNF1), –270 (TNF4), or –354 (TNF5) with respect to transcription start. Analysis is one of three similar results using 1–10 units MNase for TNF-{alpha} or IL-1beta. Error of duplicate PCRs was <10%.

 


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  		TABLE 1
Primer sequences and amplicon position along the human IL-1beta promoter

F indicates forward and R indicates reverse.

 


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  		TABLE 2
Primer sequences and amplicon position along the murine IL-1beta promoter

F indicates forward and R indicates reverse.

 
Recombinant Protein Production and Kinase Assays—Recombinant His-tagged PU.1 production has been described (32). Multiple protein preparations were analyzed in replicate experiments. Recombinant PU.1was incubated in 20 µl of incubation buffer (100 mM Tris, pH 8, 100 mM NaCl, 50 mM KCl, 20 mM MgCl2, and 100 µM sodium orthovanadate) with 1 µl of CK2 (New England Biolabs) and either 1 µl of [{gamma}-32P]ATP or 1 µl of 100 mM ATP for 10 min at 30 °C. The reaction was stopped with 4 µl of SDS sample buffer. Western blots were probed with 1:300 rabbit anti-PU.1 (sc-352; Santa Cruz Biotechnology).

Kinase Inhibitor Analyses—Human Mono Mac-6 monocytes were pretreated with pharmacological inhibitors of various intracellular signaling pathways for 3 h as detailed in Table 3. Alternatively, SP600125 and SB203580 were added 10 min prior to further treatment. LPS was then added to stimulate IL-1beta transcription. Transcripts were measured by quantitative reverse-transcriptase PCR and SYBR green incorporation as detailed above. Primers approximated >97% efficiency; hence this slope is within experimental error. Values graphed are the ratio of normalized IL-1beta copy number in stimulated cells: normalized IL-1beta copy number in resting cells for a fold induction. Error of triplicate PCRs was in no case >10%.


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  		TABLE 3
Inhibitors tested for effect on IL-1beta transcription

NA indicates not applicable; JNK is c-Jun NH2-terminal kinase.

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
The IL-1beta Promoter Is Accessible in Resting Monocyte Lineage Cells— Toward characterizing putative inducible changes in IL-1beta promoter chromatin structure in monocytes, we completed quantitative chromatin accessibility analyses. Specifically, we measured MNase sensitivity of the IL-1beta promoter chromatin in multiple cell lines by CHART-PCR. Fig. 1A shows the approximate location of amplicons used to measure MNase-resistant DNA and therefore promoter chromatin packaging. The center position of each amplicon is listed (relative to transcription start) in Fig. 1, B and C. By using this method, we showed that less than 50% of the IL-1beta promoters in the cellular population were packaged into an MNase-accessible structure in 293 cells, which cannot produce IL-1beta, at all positions tested (Fig. 1B, open bars). Similarly, monocyte IL-1beta DNA located 700 to 300 bp upstream of the transcription start site was relatively MNase-resistant (Fig. 1B, black and gray bars). In contrast, DNA positioned from –200 to +100 relative to transcription start was highly accessible to MNase digestion in human Mono-Mac-6 monocytes (up to 80%). Surprisingly, accessibility was indistinguishable in resting versus LPS-stimulated cells (black versus gray bars), despite the demonstration that only stimulated cells produced measurable levels of transcript (Fig. 3A, MM6 S). This CHART-PCR result suggests that the IL-1beta promoter is packaged into a relatively accessible chromatin structure in resting monocytes that do not transcribe this gene. Further analyses focused on confirming this "poised" chromatin structure.


Figure 3
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  		FIGURE 3.
Quantitation of human IL-1beta transcript normalized tobeta2m. A, mRNA levels in resting (open squares) or stimulated (filled squares) Mono-Mac-6 monocytes were measured by real-time PCR. B, mRNA levels were similarly measured in resting (open circle) or stimulated (filled circle) HeLa cells, stimulated THP-1 cells (oval), stimulated THP-1/CD14 cells (open squares) or stimulated U937 cells (filled squares). Results are plotted as copy number of IL-1beta transcript divided by copy number of beta2m transcript. The x axis shows time of LPS stimulation prior to RNA harvest.

 


Figure 4
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  		FIGURE 4.
The IL-1beta transcription start site is not packaged by histone H3 in Mono-Mac-6 monocytes. ChIP specific for acetylated histone H3 (A) or total histone H3 (B) is shown. Amplicons in legend box correspond to those in Table 1. Resting or LPS-stimulated cell values are shown in left and right sets of bars in each panel, respectively. Values show average and S.E. for three independent experiments.

 
To dismiss the trivial possibility that this poised chromatin structure is characteristic of a single cell line, we analyzed the structure of the IL-1beta promoter in additional resting human cell lines (Fig. 1C). THP-1 (Fig. 1C, black bars), U937 (gray bars) and HL-60 (stippled bars) monocyte lineage cells package the IL-1beta promoter into a highly MNase-accessible structure proximal to the transcription start site as compared with 293 cells (open bars). Taken together, the data strongly indicate the IL-1beta promoter is packaged into MNase-accessible chromatin in monotype lineage cells independent of transcriptional status.

To confirm further the constitutively accessible promoter structure and to determine its extent, we performed more traditional restriction endonuclease accessibility assays on THP-1 monocytes measuring the appearance of an accessibility fragment on Southern blots (33). The IL-1beta promoter was relatively accessible to restriction endonuclease in THP-1 cells (Fig. 1D, compare lanes 1 and 2 to lanes 3 and 4; arrow shows accessibility fragment), and the amount of accessibility fragment did not change upon LPS stimulation (lane 3 versus 4), consistent with the interpretation that the promoter is packaged into a poised chromatin structure. In contrast, Fig. 1E demonstrates that accessibility of the IL-1beta enhancer, as indicated by the presence of an accessibility fragment (arrow), is approximately equivalent in IL-1beta-negative 293 cells (lane 1), and resting and stimulated THP-1 cells (lanes 2 and 3, respectively). These data suggest that the chromatin packaging the IL-1beta enhancer, in contrast to the promoter, is similarly accessible in expressing and nonexpressing cell types; hence changes in promoter but not enhancer chromatin structure may regulate the IL-1beta gene in monocytes.

The Poised Promoter Chromatin Structure of IL-1beta Is Monocyte Lineage-specific—Toward understanding how specific the identified poised chromatin structure is among hematopoietic cell types, we measured IL-1beta promoter MNase accessibility in primary B and T cells by CHART-PCR. Although both types of cells have been shown to produce IL-1beta upon appropriate stimulation (34, 35), the promoter is MNase-insensitive from approximately –500 to –100 relative to the transcription start site in resting lymphocytes (Fig. 2A, dotted bars). This difference is not because of species differences, because murine RAW 264.7 macrophages package the promoter into an MNase-accessible structure (Fig. 2A, white bars). In contrast, murine 3T3 fibroblasts package the IL-1beta promoter into an inaccessible chromatin structure (Fig. 2A, black bars). Overall, the data in Figs. 1 and 2A are consistent with the interpretation that the poised IL-1beta promoter chromatin structure uniquely prepares the monocyte for rapid IL-1beta production post-stimulation.

To test further the novelty of the poised IL-1beta promoter in monocytes/macrophages, we analyzed chromatin accessibility at several positions along the TNF-{alpha} promoter (TNF1,4,5) in resting and stimulated Mono-Mac-6 monocytes. As shown previously (36), MNase accessibility of DNA proximal to the TNF-{alpha} promoter increased by ~5-fold upon LPS stimulation (Fig. 2B, TNF1). Less dramatic increases in MNase accessibility were detected upstream from transcription start (TNF4 and TNF5). Taken together, data in Fig. 2 show the poised IL-1beta promoter structure is both cytokine- and lineage-specific.

The IL-1beta Promoter Is Packaged into a Unique Poised Chromatin Structure in the Absence of Transcription—Toward ensuring that the various monocyte lines tested for IL-1beta promoter structure were capable of inducible IL-1beta transcription, we stimulated each line with LPS (100 ng/ml) and quantitated IL-1beta transcript normalized to beta2m transcript by real time PCR. Mono-Mac-6 monocytes responded rapidly to LPS (Fig. 3A, filled squares). As expected, HeLa cells do not transcribe IL-1beta (Fig. 3B, open and filled circles), consistent with results from 293 cells (data not shown). Most surprisingly, THP-1 monocytes did not transcribe IL-1beta in response to LPS (Fig. 3B, ovals), demonstrating that promoter accessibility is insufficient for inducible IL-1beta transcription. This finding is consistent with our interpretation of a poised promoter. This interpretation is bolstered by identical findings for U937 monocytes (Fig. 3B, filled squares). Stable expression of the LPS co-receptor CD14 in THP-1 enabled the cells to respond to LPS by transcribing IL-1beta (open squares), indicating THP-1 cells express many of the additional signaling molecules required for IL-1beta transcription in response to LPS. Overall, these results demonstrate that monocytes, even those incapable of transcribing IL-1beta in response to LPS, package the IL-1beta promoter into an accessible chromatin structure.


Figure 5
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  		FIGURE 5.
The IL-1beta transcription start site is accessible in murine macrophages. ChIP specific for acetylated histone H3 (A) or total histone H3 immunoprecipitated with the corresponding antibody from RAW 264.7 cells (B) is shown. Amplicons in legend box correspond to those in Table 2. C, fluorescence-activated cell sorter profiles from primary murine BMDM. Isotype control staining is shown in left panel. The cells are CD11c negative (middle panel) and Mac-1 high (right panel). D, histone H3-specific ChIP in BMDMs from C. Resting or LPS-stimulated cell values are shown in left and right sets of bars in each panel (A, B, and D), respectively. Values show average and S.E. for 2–3 independent experiments or range of points in two independent assays for D. E, IL-1beta promoter accessibility to MNase in resting primary BMDMs as analyzed by CHART-PCR. x axis shows position relative to transcription start site. Average of two independent experiments using 5 units of MNase is shown. Error bars show range of values.

 
The Poised IL-1beta Promoter Is Not Packaged by Hyperacetylated Histones—Toward further characterizing the poised chromatin structure of the IL-1beta promoter, we measured association of hyperacetylated histone H3 (AcH3) with the human promoter in Mono-Mac-6 cells. DNA relatively distal to the IL-1beta transcription start site (primer set –VII) associated with AcH3 (Fig. 4A). Both downstream regions tested (primer sets +I and +II) are also packaged by hyperacetylated histones. In contrast, DNA amplified by primer sets –Vto –I lacked hyperacetylated H3 packaging, as indicated by amplification of less than 2-fold over input. AcH3/IL-1beta association was not significantly different in stimulated versus resting monocytes. Overall, the pattern of AcH3/IL-1beta promoter association is consistent with the proposed poised chromatin structure. {alpha}-His control antibody did not precipitate the promoter in any cell tested (data not shown).

Decreased AcH3 Packaging Proximal to the IL-1beta Transcription Start Site Correlates with Decreased Histone H3 Packaging in the Poised Promoter Structure—The decreased apparent AcH3 association proximal to transcription start could be explained two ways. Either H3 acetylation is low or overall H3 packaging is absent. Loss of histone packaging upon transcriptional activation has been measured on only a few promoters at present, namely Pho5, IL-2, and granulocyte-macrophage colony-stimulating factor (37, 38). Toward differentiating between these possibilities for the IL-1beta promoter, we completed ChIPs on resting and stimulated Mono-Mac-6 cells with antibodies recognizing total histone H3. H3 is insignificantly associated with the IL-1beta promoter proximal to the transcription start site in both resting and stimulated monocytes (DNA amplified by primer sets –III, –I, and +I; Fig. 4B). DNA located more distal to the transcription start site is packaged by H3 (primer sets –VII and +II). Furthermore, H3/IL-1beta association does not change significantly upon monotype stimulation. H3/IL-1beta association patterns overall mirror those found for AcH3/IL-1beta association demonstrating that the lack of AcH3/promoter packaging is because of lack of chromatin packaging which additionally characterizes the poised promoter structure.

The IL-1beta Promoter Is Poised for Activation in Primary Murine Macrophages—To further test whether the poised IL-1beta structure is evolutionarily conserved, we measured histone packaging at the murine promoter in RAW 264.7 cells. AcH3-specific and histone H3-specific ChIP analyzing IL-1beta chromatin packaging from about 500 bp upstream to 100 bp downstream of transcription start demonstrated low association at all sites in resting and stimulated macrophages (Fig. 5, A and B, left or right panels, respectively). Like the human promoter, the lowest association was detected proximal to the murine transcription start site (+I) and was unchanged upon cellular stimulation (Fig. 5, A and B, right panels). The modest increase in AcH3 association detected at murine amplicon –V upon stimulation mirrored findings in human cells. Nonspecific antibody/DNA associations were detected at background levels, as measured by {alpha}-His-specific ChIP (data not shown). Overall, the murine association pattern recapitulated results from human monocytes, suggesting (along with Fig. 2A) the poised chromatin structure is evolutionarily conserved.

Finally, to determine whether human and murine cell line analyses accurately reflect IL-1beta chromatin packaging in primary cells, we analyzed the IL-1beta promoter structure in primary murine BMDMs. To confirm homogeneity of BMDMs harvested after 7 days in culture, we performed flow cytometry with cells stained for {alpha}-CD11c and {alpha}-Mac-1. Greater than 90% of the mature BMDM population stained appropriately for these markers (CD11c– Mac-1+, Fig. 5C, middle and right panels, respectively), ensuring that the proper primary cell population was indeed being analyzed. ChIP analyses specific for H3 showed again that the IL-1beta promoter is histone-free proximal to the transcription start site in BMDMs (Fig. 5D). Finally, IL-1beta promoter chromatin accessibility to MNase in BMDMs demonstrated a pattern highly similar to the accessibility pattern demonstrated by human monocyte lines (Fig. 5E versus Fig. 1, B and C). We therefore conclude that cell line analyses reflect a naturally poised IL-1beta promoter structure in primary monocyte lineage cells.

Transcription Factors Constitutively Associate to Define a Poised Promoter Architecture—Because PU.1 and C/EBPbeta are two important IL-1beta activators, we focused additional ChIP analyses on association of these proteins with IL-1beta regulatory elements in monocytes. Fig. 6 shows C/EBPbeta or PU.1 association with the IL-1beta promoter (A) or enhancer (C) in Mono-Mac-6 human monocytes. Both PU.1 and C/EBPbeta associated with the IL-1beta promoter approximately equivalently in resting and stimulated monocytes (Fig. 6A). PU.1 or C/EBPbeta/IL-1beta promoter association is not detected in control 293 cells (Fig. 6A, dark bars), consistent with the inaccessible, transcriptionally inactive status of the gene in these cells. Western blot analyses demonstrated C/EBPbeta is expressed at approximately equivalent levels in Mono-Mac-6 and 293 cells (data not shown), indicating specificity of this interaction in monocytes.


Figure 6
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  		FIGURE 6.
Transcription factor association with the IL-1beta gene by chromatin immunoprecipitation. Antibodies specific for the protein labeled on the x axis measured association of the factor with the IL-1beta promoter (A), beta2m promoter (B), or IL-1beta enhancer (C) are shown. y axis measures enrichment over the same quantity of input DNA amplified in parallel PCRs. Bars show average plus S.E. from 5 or more assays. The Student's t test gave a p value of >0.10 for resting versus stimulated cells for all antibodies except {alpha}-pol II in panel A.

 
In a final series of transcription factor-specific ChIP assays on the IL-1beta promoter, we assessed association of RNA polymerase II (pol II). pol II did not associate substantially with the IL-1beta promoter in resting monocytes (Fig. 6A), explaining the lack of transcript production shown in Fig. 3A. Upon monocyte stimulation pol II was recruited to the promoter, as indicated by an approximate 8-fold enrichment of promoter sequence in ChIP products (Fig. 6A, stippled bar, far right). pol II was not recruited to the IL-1beta promoter in 293 cells as expected. A series of control ChIP assays further demonstrated specificity of PU.1 and C/EBPbeta association with the IL-1beta promoter. The transcriptionally active beta2m promoter constitutively associated with AcH3 and pol II (Fig. 6B). Neither PU.1 nor C/EBPbeta associated with the monocyte beta2m promoter, indicating IL-1beta promoter specificity. Overall, the data show that association of two sequence-specific DNA binding proteins, C/EBPbeta and PU.1, does not change upon IL-1beta transcriptional activation. The data therefore show the promoter is poised by two criteria, an accessible chromatin structure and constitutive transcription factor association, and hence is termed a "poised promoter architecture."


Figure 7
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  		FIGURE 7.
PU.1 is phosphorylated by the CK2 kinase at serine residue 148 in vitro and CK2 activity is required for IL-1beta transcription. A and B, CK2 labeling using [32P]ATP as a phosphate donor. Radioactive reaction products separated by SDS-PAGE are shown. Autophosphorylated CK2, phosphorylated recombinant PU.1, and (nonmodified/nonradioactive) Ets-1 are marked to the right of A. Autophosphorylated CK2 has run off the bottom of the gel in B. C, nonradioactive CK2 kinase assay using recombinant PU.1 (lanes 1 and 2), Ets-1 (lanes 3 and 4), or PU.1 mutated serine to alanine at residue 148 (lanes 5 and 6). CK2 was omitted in mock reactions shown in lanes 1, 3, and 5. Western blots were probed with {alpha}-PU.1 (lanes 1, 2, 5, and 6)- or {alpha}-Ets-1 (lanes 3 and 4)-specific antibodies. Images represent two independent experiments. D, CK2 inhibitors block inducible IL-1beta transcription in Mono-Mac-6 monocytes. Cells were pretreated with inhibitors before stimulation. IL-1beta mRNA copy number was normalized to beta2m mRNA copy number. y axis shows normalized IL-1beta mRNA in stimulated versus unstimulated cells. Graph shows average plus range for 2–4 independent determinations.

 
It is possible that changes in protein association at the IL-1beta enhancer may play a role in recruiting pol II to the promoter. We therefore used ChIP to measure protein association with the IL-1beta enhancer in resting and stimulated Mono-Mac-6 monocytes. As for the promoter, association of C/EBPbeta and PU.1 with the IL-1beta enhancer was similar in resting and stimulated monocytes (Fig. 6C). Additional analyses detected no pol II/enhancer association in resting or stimulated monocytes (Fig. 6C, rightmost bars). This finding strongly suggests that pol II is not recruited to the promoter by the enhancer but instead by the promoter complex. Furthermore, the IL-1beta enhancer is not packaged by hyperacetylated histones (data not shown) and is not selectively accessible (Fig. 1E), suggesting large regions of the gene may not be similarly structured, in contrast to findings for Igµ and globin loci (31, 39).

Inducible PU.1 Phosphorylation at the IL-1beta Enhancer Plays an Important Role in IL-1beta Transcription from a Poised Promoter Architecture—Based on the demonstration of constitutive PU.1 association at both promoter and enhancer, we hypothesized that IL-1beta-bound PU.1 is modified to somehow trigger pol II recruitment only in stimulated monocytes. Toward testing this possibility, we focused analysis on the LPS-inducible protein kinase CK2, proposed to activate IL-1beta transcription by modifying PU.1 serine 148 (9, 40). Direct kinase assays testing the ability of CK2 to modify PU.1 in vitro demonstrate that CK2 transfers radioactive phosphate to PU.1 (Fig. 7A, lanes 2 and 3). Notably, phospho-PU.1 had decreased mobility on SDS-PAGE as compared with unmodified PU.1 (40 versus 35 kDa, respectively). Ets-1, a factor related to PU.1, was not radiolabeled in parallel reactions (Fig. 7A, lanes 4 and 5, arrow indicates position of the 54-kDa Ets-1 protein; signal is absent because Ets-1 is not phosphorylated), demonstrating specificity. CK2 autophosphorylation (Fig. 7A, lane 1) is as expected. In contrast, PU.1 mutated serine-to-alanine at position 148 was not phosphorylated by recombinant CK2 (Fig. 7B, compare lanes 2 and 3). To confirm the labeled species was PU.1, we repeated the analysis using a nonradioactive phosphate donor, detecting CK2- or mock-treated PU.1 on Western blots. Anti-PU.1 reactivity confirmed the 40-kDa band was PU.1 (Fig. 7C, lane 2). In contrast, PU.1 S148A was not extensively phosphorylated by CK2 (Fig. 7C, lane 6), as indicated by a lack of supershifting on SDS-PAGE. Because CK2 is detectable throughout the cell (41–43), these biochemical data demonstrate that CK2 can theoretically phosphorylate DNA-bound PU.1 at serine 148 in response to LPS stimulation.

Toward determining whether CK2 activity is required for IL-1beta transcription, we treated Mono-Mac-6 monocytes with a cadre of kinase inhibitors prior to LPS stimulation and then measured IL-1beta mRNA (normalized to beta2m mRNA). Treating cells with Me2SO, bisindolylmaleimide (protein kinase C inhibitor), PD98059 (MAP kinase inhibitor), or LY294002 (phosphatidylinositol 3-kinase inhibitor) did not ablate inducible IL-1beta transcript levels (Fig. 7D and data not shown). The lack of MAP kinase involvement is interesting given C/EBPbeta is phosphorylated by MAP kinases (44). Pretreating cell with the c-Jun NH2-terminal kinase inhibitor SP600125 decreased IL-1beta transcript levels modestly (~29%), indicating c-Jun NH2-terminal kinase may play a minor role in inducible IL-1beta transcription. Treating monocytes with the p38 inhibitor SB203580 resulted in a 58% decrease in IL-1beta mRNA, indicating p38, shown to phosphorylate PU.1 serine 142 (45), may act in inducible IL-1beta transcription. This result agrees with a previous demonstration that p38 plays a role in activating IL-1beta transcription in murine RAW 264.7 cells (46). The CK2 inhibitors apigenen and emodin decreased IL-1beta transcript most dramatically (by 97 or 68%, respectively). These inhibitor data suggest that CK2 activity is essential for inducible IL-1beta transcription.


Figure 8
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  		FIGURE 8.
Inducible protein association with the IL-1beta enhancer. IRF-4 (A) or CK2 (B) associates with the IL-1beta enhancer only upon monocyte stimulation. ChIP using antibodies to IRF-4/CK2 (left set of bars in A or B, respectively) or the His6 tag (right set of bars in both A and B) in Mono-Mac-6 cells is shown. Bars show average plus S.D. of three independent experiments.

 
Previous analyses demonstrated that CK2-mediated PU.1 phosphorylation at serine residue 148 is critical for recruiting a co-activator, IRF-4, to the PU.1/IRF composite enhancer element implicated in both IL-1beta and Ig{kappa} transcription (10, 47). If IL-1beta-associated PU.1 is phosphorylated in response to LPS, IRF-4 would associate with the IL-1beta enhancer in stimulated (but not resting) monocytes. ChIP using {alpha}-IRF-4 antibody demonstrated that IRF-4 fails to associate with the IL-1beta enhancer in resting Mono-Mac-6 cells (Fig. 8A, leftmost stippled bar). Upon stimulation, IRF-4 associates with the enhancer (~6.9-fold enrichment compared with input chromatin; Fig. 8A, leftmost open bar), but not with the promoter, which lacks an IRF-4-binding site (data not shown).

Because CK2 undergoes a relatively strong interaction with substrate proteins, we reasoned that if CK2 is responsible for IRF-4 recruitment via PU.1 phosphorylation, CK2 should associate with the IL-1beta enhancer in stimulated, but not resting, Mono-Mac-6 cells. To test this prediction, we performed ChIP with an antibody specific for CK2 to measure CK2/enhancer association. This association was undetectable in resting Mono-Mac-6 monocytes (Fig. 8B, leftmost stippled bar). In contrast, CK2 clearly associates with the IL-1beta enhancer in stimulated Mono-Mac-6 cells (~4.1-fold enrichment compared with input chromatin; Fig. 8B, leftmost open bar). CK2 failed to associate with the promoter in resting or stimulated monocytes (data not shown). Taken together, these data support the model that cellular stimulation triggers CK2 association and subsequent PU.1 phosphorylation for recruitment of additional activators to the IL-1beta gene.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Data herein demonstrate by multiple independent measures that the IL-1beta promoter chromatin structure is equivalently accessible and associated with transcription factors in resting and stimulated monocytes/macrophages. Additional studies support the model that CK2-mediated phosphorylation occurs on enhancer-bound PU.1 upon LPS stimulation, leading to recruitment of additional transcription factors concomitant with pol II/promoter association. This model provides a mechanistic explanation of how IL-1beta is transcriptionally activated within minutes of monocyte stimulation.

These findings markedly extend the first characterization of the monocyte IL-1beta promoter in the context of chromatin (25). These authors demonstrated that some histone tail modifications change upon LPS stimulation in THP-1 monocytes, but did not compare DNA accessibility in stimulated versus resting cells. Furthermore, single sites of chromatin were analyzed in the published work, rendering the more comprehensive structure we have presented impossible to detect. Interestingly, p50 association (which we did not test) was constitutive, in agreement with our poised promoter architecture model. One caveat to the demonstration that the poised promoter is largely devoid of histone H3 (or acetylated H3) packaging is that the resolution of the ChIP assay is limited by the length of the sheared DNA fragments, or ~500 bp. However, because shearing randomly breaks the DNA and results in amplification of randomly overlapping fragments, our results most likely underestimate differences in histone association at the transcription start site versus regions upstream or downstream. It is possible that the chromatin preferentially sheared in the accessible nucleosome-free regions to result in unexpectedly high ChIP resolution.


Figure 9
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  		FIGURE 9.
Model for IL-1beta transcription in the context of chromatin. a, the IL-1beta promoter is likely packaged into inaccessible chromatin in multipotent precursor cells. b, in resting monocytes, the promoter is accessible, based on lack of nucleosome association. Sequence-specific binding proteins such as PU.1 and C/EBPbeta may promote formation of accessible chromatin and the poised promoter architecture. c, LPS stimulation results in CK2 activation, CK2/enhancer association, and phosphorylation of PU.1 at serine 148. Phospho-PU.1 recruits IRF-4 to the IL-1beta enhancer. d, RNA polymerase is recruited to the poised promoter by unknown mechanisms resulting in IL-1beta transcription.

 
The data herein are consistent with a two-step model of IL-1beta transcriptional activation (Fig. 9). First (Fig. 9, a and b), the monocyte-specific poised chromatin architecture is established. Based on the general paradigm that chromatin of tissue-specific genes becomes selectively accessible during cell development, and the demonstration that the IL-1beta promoter is inaccessible to MNase in B and T cells, it is likely that the combination of transcription factors associated with either the promoter or enhancer established this architecture during monocyte development. Because hematopoietic stem cells, a multipotential blood cell precursor, express IL-1beta (48), we presume HSCs package the promoter into an accessible chromatin structure. If so, the poised promoter architecture must be established in a very early multipotent precursor, unless HSCs activate the locus using alternative mechanisms. Furthermore, because PU.1 can act as a chromatin accessibility factor in other contexts (28, 49), and ectopic PU.1 expression can induce IL-1beta from the endogenous locus in nonhematopoietic cells (8), PU.1 is a likely candidate for establishing the poised chromatin structure. HSCs express PU.1 (50, 51). Although lymphocytes are capable of producing IL-1beta (34, 35), our data may explain why monocytes appear uniquely sensitive in activating the IL-1beta gene in response to physiological stimuli.

The second step required for IL-1beta transcription occurs only upon monocyte stimulation (Fig. 9, c and d). Because 1) CK2 is inducibly recruited to the IL-1beta enhancer; 2) CK2 can directly phosphorylate PU.1; 3) this reaction requires serine 148 of PU.1; and 4) IRF-4 associates with the IL-1beta enhancer only upon monocyte stimulation, the data (along with published findings that CK2 is activated by LPS, and only phosphorylated PU.1 recruits IRF-4 to DNA (8–10)) strongly suggest that enhancer-associated PU.1 is phosphorylated by CK2 in situ and that this post-translational modification catalyzes transcription through recruitment of at least one additional factor. The demonstrations that PU.1 can interact directly with the basal transcription complex TFIID (53) and that PU.1 may induce assembly of a TBP-dependent complex on the IL-1beta promoter (54) raise the possibility that PU.1 may be responsible for the demonstrated pol II recruitment to the IL-1beta promoter post-stimulation.

Although we have characterized a poised promoter architecture at a cytokine promoter, partial evidence suggests similar structures at other cytokine promoters. For example, RUNX1 constitutively associates with the MIP-1{alpha} promoter in resting and stimulated Jurkat T cells (55). In a second example, analysis of the IL-8 promoter suggests that the transcription start site is nucleosome-free prior to transcriptional activation (56) and hence perhaps packaged in a poised chromatin architecture. Recent work by Smale and co-workers has demonstrated that immediate early genes are generally characterized by a poised promoter chromatin structure; whether activating proteins are constitutively bound and post-translationally modified upon macrophage stimulation remains to be determined (57). This study therefore highlights the general importance of IL-1beta analyses toward establishing paradigms for regulating rapidly inducible pro-inflammatory genes.

The most extensive, convincing example of a poised promoter architecture to our knowledge is the receptor locus c-fms. The promoter for this cytokine receptor is fully occupied by DNA binding factors prior to protein production, and as for IL-1beta, additional proteins associate at the enhancer upon transcription initiation (58). This promoter is packaged into accessible chromatin in resting macrophages (59) and monocyte precursors (58). However, in contrast to our findings for IL-1beta, recruitment of additional proteins because of post-translational modification of bound proteins has not been demonstrated for c-fms. Finally, constitutive association of both sequence-specific binding proteins and the basal transcriptional machinery to the A20 promoter suggests that pre-assembly of even more extensive complexes than that shown for the IL-1beta promoter may be required for rapid activation of this NF-{kappa}B target (60). Overall, identification of a poised promoter architecture specifically in a quick-response cytokine promoter suggests a more thorough analysis of inducible genes is important to determine the extent cells utilize this mechanism toward rapid transcriptional activation.

There are few examples in which phosphorylation of a DNA-associated sequence-specific protein, the second step in IL-1beta activation, is used to activate transcription. The yeast Sko1 transcription factor is phosphorylated while bound to DNA to activate transcription (61). In a second example, phosphorylation of promoter-associated Elk-1 occurs upon cellular stimulation, resulting in transcription of the Egr gene (62). However, cAMP-response element-binding protein, the classical example of a DNA-bound factor phosphorylated following cellular stimulation (63), has been discredited recently (64). Hence, the number of examples of transcriptional activation by phosphorylation of DNA-associated protein is extremely limited.

Overall, our data introduce a unique combination of two recently identified mechanisms enabling monocytes to inducibly achieve rapid, robust IL-1beta gene transcription. Understanding the development of the poised promoter architecture and precisely how RNA polymerase II becomes associated with the promoter are the next logical steps toward establishing additional mechanistic explanations for this model of rapid cytokine gene induction.


   FOOTNOTES
 
* This work was supported by an American Diabetes Association research grant and National Institutes of Health (NIH) Grant R01 AI54611 (to B. S. N.) and by NIH Grant T32 HL07501 and a Grunebaum Foundation fellowship (to M. D. L.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed: Dept. of Microbiology, Boston University School of Medicine, 715 Albany St. L516, Boston, MA 02118. Tel.: 617-638-7019; Fax: 617-638-4286; E-mail: bnikol{at}bumc.bu.edu.

2 The abbreviations used are: IL, interleukin; LPS, lipopolysaccharide; TNF, tumor necrosis factor; BMDM, bone marrow-derived macrophages; ChIP, chromatin immunoprecipitation; CHART, chromatin accessibility by real time PCR; MNase, micrococcal nuclease; MAP, mitogen-activated protein; IRF, interferon regulatory factor; beta2m, beta2-microglobulin. Back


   ACKNOWLEDGMENTS
 
Nichol Holodick, Sean Gurdak, and Xuemei Zhong contributed primary murine lymphocytes. Dr. Tom Rothstein kindly provided mice and Heather MacLeod provided expertise for BMDM preparation. Dr. Stu Levitz contributed the CD14-transfected THP-1 cells, and Dr. Matthew Fenton gave the XT-luc plasmid. The IRF-4 specific antibody was a gift from Dr. Michael Atchison. We thank Dr. Greg Viglianti for helpful discussions and "poised promoter architecture." Drs. Steve Smale and Gavin Schnitzler provided comments on the manuscript.



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