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Histone acetylation and chromatin conformation are regulated separately at the TNF- promoter in monocytes and macrophages. Julia Y. Lee,*,† Nahmah A. Kim ...

Histone acetylation and chromatin conformation are regulated separately at the TNF-␣ promoter in monocytes and macrophages Julia Y. Lee,*,† Nahmah A. Kim,*,† Amy Sanford,*,† and Kathleen E. Sullivan*,† * Department of Pediatrics, †Children’s Hospital of Philadelphia, University of Pennsylvania School of Medicine

Abstract: Tumor necrosis factor ␣ (TNF-␣) is a proinflammatory cytokine, which participates in a wide range of immunoregulatory activities. It is generally produced at highest levels by cells of the myeloid lineage in response to activation of pathogen recognition receptors such as Toll-like receptors. Impaired production predisposes to infection with intracellular organisms, and overproduction results in systemic or organ-specific inflammation. Control of expression is essential to maintain homeostasis, and this control is mediated via multiple strategies. We examined two separate aspects of chromatin accessibility in this study of the human TNF-␣ promoter. We examined the role of histone acetylation and chromatin remodeling in cell lines and primary cells and identified two individual steps associated with activation of TNF-␣ production. Histone H3 and H4 acetylation was found to be strongly dependent on the developmental stage of human monocytes. It did not appear to be regulated by acute stimuli, and instead, chromatin remodeling was found to occur after acute stimuli in a cell line competent to produce TNF-␣. These data suggest that there is a hierarchy of controls regulating expression of TNF-␣. Acetylation of histones is a prerequisite but is insufficient on its own for TNF-␣ production. J. Leukoc. Biol. 73: 862-871; 2003. Key Words: gene regulation 䡠 TNF-␣ 䡠 inflammation

INTRODUCTION Tumor necrosis factor ␣ (TNF-␣) is produced predominantly by monocytes/macrophages, neutrophils, activated lymphocytes, and natural killer cells. It is generally produced immediately after recognition of an invading pathogen by Toll-like receptors; however, interleukin (IL)-1, IL-3, granulocyte macrophage-colony stimulating factor, immune complexes, and interferon-␥ (IFN-␥) can also induce TNF-␣ in myeloid cells in some circumstances. The major roles of TNF-␣ include killing of tumor cells, induction of adhesion molecule expression at sites of inflammation, stimulation of bone resorption, induction of fever, and activation of B cells, neutrophils, and monocytes. As a cellular activator, it enhances the production of IL-1, 862

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IL-6, and IL-8. TNF-␣ is critical for the development of lymph nodes and modulates dendritic cell maturation [1]. TNF-␣ exhibits a complexity of regulation that is extraordinary. Transcription is regulated through the production and/or activation of transcription factors [2–7]. Another layer of regulation occurs after transcription. The TNF-␣ message 3⬘ end binds tristetraprolin, T cell intracellular antigen-1 (TIA-1), and TIA-1-related protein, which regulate message stability and translation [8 –10]. Some cells express TNF-␣ message but do not translate it [11]. TNF-␣ expression is also regulated at the level of splicing, message transport, and protein cleavage [8, 12–22]. The relative importance of each layer of regulation is largely unknown. Only one stimulus has been investigated with respect to this question. Lipopolysaccharide (LPS) induces transcription fivefold after stimulation. Message stability is increased 12-fold over unstimulated, and translation is increased two- to threefold [23]. This suggests that LPS leads to increased transcription, but increased message stability is the single most potent effect. It is not known if this finding can be generalized to other stimuli. The complex actions of TNF-␣ require sophisticated regulation to prevent inappropriate expression, which is almost always deleterious [24]. Two findings suggest that chromatin conformation might contribute to the complex regulation of TNF-␣. Some transient transfections using the TNF-␣ promoter have high basal expression, even in cells that aren’t expressing their endogenous TNF-␣ gene [25]. This could be a result of loss of a tissue-specific silencer in the construct or loss of chromatin context. In addition, fusion of an expressing cell with a nonexpressing cell leads to silencing of the active locus via DNA methylation, suggesting chromatin effects could be dominant [26]. We chose to focus on the roles of histone acetylation and nucleosome remodeling in the regulatory hierarchy controlling TNF-␣ expression. Histone H3 serine 10 phosphorylation is synergistically coupled to lysine 9 and lysine 14 acetylation [27]. This combination of H3 acetylation (ACH3) and phosphorylation is a marker for transcriptional activation, and other combinations of histone acetylation, phosphorylation, and

Correspondence: Kathleen E. Sullivan, M.D., Ph.D., Immunology, Children’s Hospital of Philadelphia, 34th St. and Civic Ctr. Blvd., Philadelphia, PA 19104. E-mail: [email protected] Received December 20, 2002; revised January 28, 2003; accepted February 13, 2003; doi: 10.1189/jlb.1202618.

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methylation are associated with other transcriptional states. This has led to the concept of combinatorial codes for histone modifications [28, 29]. Histone acetylation is a completely reversible process regulated by the dynamic interplay between histone acetyltransferases (HATs) and histone deacetylases (HDACs). In general, histone acetylation is linked to nucleosome repositioning and transcription. In the past year, it has become clear that there are at least three strategies that link histone modifications, nucleosome remodeling, and transcription. Mitotically expressed yeast genes bind an activator, which directly recruits the SWI/SNF chromatin-remodeling complex during the previous M phase. GCN5p, a HAT enzyme, binds to the promoter, acetylates the histones, and facilitates the binding of another activator [30, 31]. This nucleates the assembly of the preinitiation complex. The human gene, which has been best studied, illustrates the most common method of linking chromatin effects to transcription. Viral infection causes a complex of activators to bind the IFN-␤ promoter. This complex is responsible for the recruitment of the GCN5p HAT, which then recruits the SWI/SNF complex, which facilitates the TATA box binding protein [32]. The last known strategy has been described in the ␣1-antitrypsin gene. In this case, the activators are already present on the promoter. When the preinitiation complex forms, the HAT is recruited, and the SWI/SNF complex comes in last [33]. Thus, the linkage among histone acetylation, nucleosome remodeling, and active transcription is clear, but the order of events may vary regarding different types of promoters. This study suggests that histone acetylation at the TNF-␣ promoter is developmentally regulated and is required for TNF-␣ expression, and chromatin remodeling occurs after acute stimuli.

MATERIALS AND METHODS Cells and tissue culture THP-1.1 and U937.1 sublines were established from THP-1 and U937 obtained from American Type Culture Collection (Manassas, VA) and were maintained in RPMI supplemented with 10% fetal bovine serum (FBS), 2 mM glutamine, and 100 U/ml penicillin-6 phosphate. MonoMac 6 cells were maintained in RPMI 1640 supplemented with 10% FBS, 2 mM glutamine, 10 U/ml penicillin-6 phosphate, 10 mM minimum essential medium nonessential amino acid, 10 ␮g/ml insulin-transferrin sodium selenite, and 100 mM sodium pyruvate. Studies were performed while cells were in log-phase growth and were at a density of 0.5–1.0 ⫻ 106 cells/ml. Culture supernatants were periodically screened for endotoxin and consistently tested negative. Peripheral blood mononuclear cells (PBMCs) were isolated from healthy individuals using Ficoll-Paque Plus (Amersham Pharmacia, Uppsala, Sweden). Monocytes were purified by adherence or by the monocyte negative selection kit following the manufacturer’s instructions (Dynal A. S., Oslo, Norway). Monocytes became differentiated macrophages by adherence to plastic in tissue-culture medium after 7 days.

Stimulants and reagents Phorbol 12-myristate 13-acetate (PMA), sodium butyrate (NaBut), and trichostatin A (TSA) were purchased from Sigma Chemical Co. (St. Louis, MO). Chlamydocin (CMD) and HC-toxin (HCT) were from Calbiochem (San Diego, CA). Immune complexes were generated by incubating 5 ␮g antibovine serum albumin (anti-BSA) antibody with 0.8 ␮g fatty acid-free BSA (both from Sigma Chemical Co.) for 1 h at 37°C. Human-matched antibody pairs for TNF-␣ were purchased from Endogen (Woburn, MA) for a conventional sandwich enzyme-

linked immunosorbent assay (ELISA). Antibodies used for chromatin immunoprecipitation (ChIP) analysis and Western blotting were purchased from Upstate Biotechnology (Lake Placid, NY).

Flow cytometry Cells were stained with the following antibodies purchased from BD PharMingen (San Diego, CA): anti-human CD14, CD16, CD64, CD38, and CD11b, antimannose receptor, anti-human human leukocyte antigen (HLA)-A, -B, and -C, anti-human HLA-DR, and anti-TNF-␣. Isotype controls were run for each antibody/fluorochrome combination. Flow cytometry was performed on the FACSCaliber instrument (BD PharMingen, San Diego, CA) using Cell Quest software.

ChIP assay Five to 10 million cells in each condition were prepared for the ChIP assays following the protocol from Upstate Biotechnology with some modifications. Cells were treated with 1% formaldehyde for 10 min at room temperature. Lysed cells were sonicated and immunoprecipitated overnight at 4°C with anti-ACH3, anti-ACH4 (Upstate Biotechnology), or rabbit anti-BSA (Sigma Chemical Co.) as a control. Antibody-bound complexes were collected with a slurry of protein A (Invitrogen, Carlsbad, CA) and were washed extensively, and immune complexes were eluted. DNA was extracted by phenol-chloroform after reverse cross-linking for 6 h at 65°C and after protein removal by proteinase K (200 ␮g/ml; Roche Diagnostics, Nutley, NJ) treatment in the presence of 20 ␮g/ml glycogen. DNA was finally RNase-treated (40 mg/ml; Roche) for 30 min at 37°C and quantitated before analyses.

Micrococcal nuclease assay Twenty-five to 50 million cells were stimulated as indicated and harvested at various times after stimulation. The cells were resuspended in cold hypotonic buffer, and the nuclei were separated by douncing [34]. The nuclei were purified by washing and were resuspended in 0.1 ml micrococcal nuclease buffer. Micrococal nuclease (5U; USB Corp., Cleveland, OH) was added, and the digestion was allowed to proceed for 20 min. This time point was established after a series of titrations and resulted in 25% of DNA being found in the single nucleosome fraction for each cell line. The digestion was stopped by proteinase K treatment and phenol:chloroform extraction. After precipitation, the DNA was quantitated. DNA (100 ng) was used for each polymerase chain reaction (PCR).

Real-time PCR We used real-time PCR to quantitate the amount of DNA from specific regions of the TNF-␣ promoter and used the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter or actin as internal controls. Each DNA sample isolated from the ChIP or micrococcal nuclease assay was subjected to four PCR reactions for the four sets of primers and probes to analyze the four regions of the TNF-␣ promoter. The parentheses after the TNF-␣ and GAPDH labels represented PCR amplifications encompassing the various TNF-␣ and GAPDH promoter regions relative to the transcription start sites: TNF1 (⫹99/– 42), forward: 5⬘-CTCTCGCCCCAGGGACATAT-3⬘, reverse: 5⬘-ATGTGGCGTCTGAGGGTTGT-3⬘, probe: 5⬘-6FAM-CAGAGGACCAGCTAAGAGGGAGAGAAGCAA-TAMRA-3⬘; TNF2 (⫹32/–119), forward: 5⬘CGCTTCCTCCAGATGAGCTC-3⬘, reverse: 5⬘-TGCTGTCCTTGCTGAGGGA-3⬘, probe: 5⬘-6FAM-CCAAGGAAGTTTTCCGCTGGTTGAATG-TAMRA-3⬘; TNF3 (–100/–250), forward: 5⬘-ccccctcggaatcgga-3⬘, reverse: 5⬘-GAGCTCATCTGGAGGAAGCG-3⬘, probe: 5⬘-6FAM- TGTCCCCAACTTTCCAAATCCCCG-TAMRA3⬘; TNF4 (–195/–345), forward: 5⬘-CCCAAAAGAAATGGAGGCAAT-3⬘, reverse: 5⬘-AAGCATCAAGGATACCCCTCAC-3⬘, probe: 5⬘-6FAM-CTACACACAAATCAGTCAGTGGCCCAGAAG-TAMRA-3⬘; GAPDH (–152/–211), forward: 5⬘-CGGTGCGTGCCCAGTT-3⬘, reverse: 5⬘-CCCTACTTTCTCCCCGCTTT-3⬘, probe: 5⬘-JOE-ACCAGGCGGCTGCGGAAAAAA-TAMRA-3⬘. All primers and labeled probes were synthesized by IDT (Coralville, IA) except the actin primers/probe, which were purchased from Applied Biosystems (Foster City, CA). Real-time PCRs were run on the ABI 7700 Taqman thermocycler. All Taqman reagents were purchased from Applied Biosystems. Optimal primer and probe concentrations were predetermined in separate PCR reactions. Known concentrations of genomic DNA were used as standards and positive controls with each Taqman run. Percent input was determined as

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follows: % Input ⫽ 2 (CT 1% input fraction–CT sample), and CT is the threshold cycle detected by the thermocycler.

Western analyses Approximately 1 million stimulated cells were lysed and dounced to isolate nuclei. Nuclear proteins were acid-extracted from the nuclei. Equal amounts of protein, determined by the Bradford assay (BioRad, Hercules, CA), were separated on 12% Bis-Tris gels, transferred onto nitrocellulose membrane, and blotted with antibodies against ACH3 or ACH4. Membranes were stripped and reblotted with antibody against total histone H3 or total histone H4. Densitometric quantitation was determined on the BioRad Gel Doc system and the Quantity One program (BioRad).

Statistical analyses Paired Student’s t-tests were performed to compare the quantitative differences of TNF-␣ production in the ELISAs and the densitometric analyses of the Western blots. A P value of less than 0.05 was used to indicate statistical significance. In place of multiple Student’s t-tests, the ANOVA (one-way or two-way ANOVA with replication) was used to compare the multivariable results. P values less than 0.05 determined statistical significance. In all graphs, error bars represent SD.

RESULTS Characterizing monocytic cell lines and primary cells THP-1.1, U937.1 sublines, and MonoMac 6 cells were characterized by flow cytometry to assess their maturation status relative to each other and to primary monocytes and macrophages (Table 1). ELISAs were used to define differences in TNF-␣ protein production (Table 2). MonoMac 6 cells were the most mature, based on the expression of CD14 and HLADR, and the THP-1.1 subline was the least mature with the U937.1 subline intermediate. All three cell lines were less mature than primary monocytes and macrophages. Differences in the levels of CD14, mannose receptor, and MHC class II expression also confirmed that monocytes have differentiated into macrophages after adherence to plastic for 7 days. There is a rough correlation between competence to produce TNF-␣ after stimulation and level of maturity.

HDAC inhibitors up-regulated PMA-induced TNF-␣ production To determine whether histone acetylation regulates TNF-␣ expression, cell lines were treated with HDAC inhibitors, which artificially induce histone acetylation. To artificially induce hyperacetylation, cells were simultaneously treated

TABLE 1.

with PMA and HDAC inhibitors: NaBut, CMD, HCT, and TSA. Dose-response studies were performed, and the results reflect the optimal PMA and HDAC inhibitor concentration. Cells were cultured at 2 ⫻ 105 cells per well for 18 –24 h with 10 ng/ml PMA and the individual HDAC inhibitors (Table 3). All cell lines exhibited PMA responsiveness, and responses to LPS and immune complexes were cell-specific (Table 2). ELISA (n⫽4) measured soluble TNF-␣ protein in triplicate cultures. In THP-1.1 cells, all of the HDAC inhibitors tested up-regulated PMA-induced TNF-␣ production. The inhibitors never induced expression of TNF-␣ in the absence of PMA. NaBut was the most potent, as it induced a 22-fold increase in TNF-␣ production comparing PMA alone with PMA with NaBut (P⬍0.02). HCT increased TNF-␣ production by 1.4-fold (P⬍0.02). CMD induced an approximately twofold increase of the PMA-induced TNF-␣ production (P⬍0.02). TSA also induced an approximate twofold increase, but the difference was not statistically significant. However, TSA demonstrated a significant dose-response effect (P⬍0.001 by one-way ANOVA). NaBut was the only HDAC inhibitor of the four tested that significantly up-regulated PMA-induced TNF-␣ protein expression in U937.1 and MonoMac 6 cells. In U937.1 cells, NaBut induced, on average, a ninefold increase of PMAinduced TNF-␣ production (P⬍0.05). MonoMac 6 cells did not produce TNF-␣ in response to a low dose of PMA but readily secreted TNF-␣ in the presence of PMA and NaBut (P⬍0.01). These studies strongly suggested that histone acetylation plays a critical role in the regulation of TNF-␣ expression.

HDAC inhibitors increase TNF-␣ expression by increasing the frequency of TNF-␣-expressing cells Cell lines are generally homogenous populations; however, gene expression has a stochastic component [35]. To determine whether HDAC inhibitors were causing all cells to increase their TNF-␣ expression or whether a subpopulation became competent to produce TNF-␣, intracellular cytokine staining was performed. There is a strong correspondence between the effect of the HDAC inhibitors on secreted TNF-␣ protein detected by ELISA and the effect seen with intracellular cytokine staining (Fig. 1). U937.1 and MonoMac 6 cells have increased frequencies of TNF-␣-producing cells (over that seen with PMA alone) with NaBut treatment only. In contrast, THP-1.1 cells exhibited lower frequencies of responding cells, as expected, but responded to TSA and NaBut. The effect of the

Maturity of Cell Types

Cell type

CD14

CD11b

CD38

CD16

CD64

MHC I

MHC II

Mannose R

THP-1.1 U937.1 MonoMac 6 Primary monocytes Primary macrophages THP-1 parent line U937 parent line

⫺ ⫺ ⫹ ⫹⫹⫹ ⫹⫹ ⫺ ⫹⫹⫹

⫹/⫺ ⫹ ⫹ ⫹⫹ ⫹⫹ ⫹ ⫹

⫺ ⫹⫹ ⫹ ⫹⫹ ⫹⫹ ⫹⫹ ⫹⫹

⫺ ⫺ ⫺ ⫹⫹ ⫹ ⫺ ⫺

⫺ ⫹⫹ ⫹⫹ ⫹ ⫹⫹ ⫺ ⫹⫹⫹

⫹⫹ ⫹⫹⫹ ⫹⫹⫹ ⫹⫹⫹⫹ ⫹⫹⫹⫹ ⫹ ⫹⫹⫹

⫺ ⫺ ⫹ ⫹⫹ ⫹⫹⫹⫹ ⫹ ⫺

⫺ ⫺ ⫺ ⫹/⫺ ⫹⫹ ⫺ ⫺

MHC, Major histocompatibility complex.

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TABLE 2. Cell type

TNF-␣ Protein Production after Stimulationa

Unstimulated

THP-1.1 U937.1 MonoMac 6 Primary monocytes Primary macrophages THP-1 parent line U937 parent line

Immune complex

PMA

LPS

Pg/ml

Pg/ml

Pg/ml

Pg/ml

37 ⫾ 2.5 24 ⫾ 2.6 21 ⫾ 1.1 0.3 ⫾ 0.5 2⫾4 0 0

38 ⫾ 0.6 30 ⫾ 3.5 23 ⫾ 0 91 ⫾ 11* 100 ⫾ 100* 6.8 ⫾ 0.02* 2.4 ⫾ 1.8

47 ⫾ 5* 37 ⫾ 0.6* 44 ⫾ 3.2* 6 ⫾ 3* 24 ⫾ 24* 83 ⫾ 7* 4.6 ⫾ 0.04*

39 ⫾ 3.5 39 ⫾ 2* 98 ⫾ 16* 211 ⫾ 22* 1063 ⫾ 443* 722 ⫾ 90* 22.2 ⫾ 1*

a Cells were plated at 1 ⫻ 105/well in triplicates and treated with 1000 ng/ml immune complexes, 100 ng/ml PMA, or 1000 ng/ml LPS. Supernatants were harvested at 8 h. TNF-␣ protein determined by ELISA is expressed as pg/ml. * A significant difference (P⬍0.05) compared with unstimulated.

HDAC inhibitors was to increase the number of cells competent to produce TNF-␣. The effect was not necessarily uniform throughout the population.

Histone acetylation levels were dependent on cell maturation and region of the TNF-␣ promoter HDAC inhibitors were not uniformly effective in the three cell lines. As these cells represented different stages in monocyte maturation, histone acetylation regulating TNF-␣ production might be dependent on the maturation state of the cells. To investigate the acetylation status at the TNF-␣ promoter, the ChIP assay was used. Formaldehyde-cross-linked DNA–protein complexes from unstimulated THP-1.1, U937.1, and MonoMac 6 cells were immunoprecipitated with antibodies against ACH3, ACH4, or BSA. DNA isolated from the immunoprecipitates was analyzed by real-time PCR. Each sample was subjected to four PCR reactions for the four regions of the TNF-␣ promoter designated TNF1–TNF4 (Fig. 2). Two-way ANOVA indicated that the levels of ACH3 were dependent on the cell type and region of the TNF-␣ promoter (P⬍0.0001; Fig. 3A). The extent of ACH3 was highest in MonoMac 6 cells, the most mature of the three cell lines, and acetylation was lowest in the least mature THP-1.1 cells. One-way ANOVA revealed that differences of ACH3 levels in TNF1 and TNF2 were significantly different among the three cell lines (P⬍0.001 and P⬍0.05, respectively). Furthermore, in all three cells, ACH3 levels were greater in regions of the promoter that were proximal to the transcription start site (TNF1 and TNF2) compared with the more distal regions (TNF3 and TNF4). Conversely, levels of ACH4 were dependent on cell type only (Fig. 3B). ACH4 in the TNF-␣ promoter region was greater in MonoMac 6 cells compared with U937.1 cells by two-way TABLE 3. Cell line THP-1.1 U937.1 MonoMac 6

ANOVA. ACH3 and ACH4 levels in a region of the GAPDH promoter, the internal-positive control, were similar in all three cell lines; no statistically significant differences were found. DNA isolated from anti-BSA immunoprecipitates was also amplified as the negative control for the ChIP assay; as expected, DNA was barely detectable or not detectable (data not shown).

NaBut hyperacetylated histone H3 and H4 THP-1.1 cells were relatively hypoacetylated in histone H3, and all HDAC inhibitors were effective in THP-1.1 cells. To directly demonstrate that increased histone acetylation occurred at the TNF-␣ promoter with HDAC treatment, ChIP assays were performed for NaBut- and TSA-treated THP-1.1 cells in comparison with untreated THP-1.1, MonoMac 6, and U937.1 cells. NaBut was able to significantly up-regulate the levels of ACH3 and ACH4 in THP-1.1 cells (P⬍0.05 for both; Fig. 4). TSA, conversely, did not augment either level in THP-1.1 cells, although it was able to up-regulate PMAinduced TNF-␣ protein production. This could reflect different kinetics, specificity, or potency of HDAC inhibition. To determine whether the differences in histone acetylation seen in the three cell lines reflected global differences or were specific for the TNF-␣ promoter, total nuclear extracts were analyzed by Western blot (Table 4). There were no significant differences in the overall levels of ACH3 and ACH4 among the three cell lines (ANOVA, P⫽0.99, comparing ACH3 levels in untreated THP-1.1, U937.1, and MonoMac 6 cells; P⫽0.83, comparing ACH4 levels among the cells). NaBut enhanced acetylation of total histone H3 (P⬍0.05, comparing untreated and NaButtreated THP-1.1; P⬍0.005, comparing treated and untreated U937.1; P⬍0.05, comparing treated and untreated MonoMac 6 cells). Conversely, NaBut had no significant effects on global

HDAC Inhibitors Enhance TNF-␣ Production by Monocyte Cell Lines

a

PMA

PMA ⫹ NaBut

PMA ⫹ TSA

PMA ⫹ CMD

PMA ⫹ HCT

1 1 1

22 ⫾ 22* 8.7 ⫾ 4.9* 17.3 ⫾ 14.5*

1.9 ⫾ 0.8 2.1 ⫾ 1.6 0.8 ⫾ 0.1

2.1 ⫾ 0.6* 1.1 ⫾ 0.2 2.3 ⫾ 1.0

1.4 ⫾ 0.3* 1.5 ⫾ 1.5 0.4 ⫾ 0.4

a Data are expressed as fold-increase over PMA alone. * Significant with P ⬍ 0.05. Dose-response analyses were performed, and the optimal combination of PMA and inhibitor is shown in each case: PMA at 1 ng/ml for U937.1 and MonoMac 6 cells and for THP-1.1 in combination with NaBut. PMA at 10 ng/ml for other inhibitors in THP-1.1. NaBut was used at 100 mM, HCT was used at 1 nM, TSA was used at 100 nM, and CMD was used at 1 nM.

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Fig. 1. Intracellular cytokine staining demonstrates that not all cells are induced equally by HDAC inhibitors. Cells were incubated with 100 ng/ml PMA with or without HDAC inhibitors for 6 h in the presence of monensin. Untreated cells were used as a negative control, and an isotype control was run using untreated cells. This figure is representative of two independent experiments. The percent of cells positive for TNF-␣ is indicated in each histogram.

levels of histone H4 in the three cell lines (P values were all greater than 0.05). This suggests that the different levels of histone acetylation seen in different cell lines are specific for the TNF-␣ promoter.

Histone acetylation levels were increased in differentiated, primary macrophages The ChIP assays on the three cell lines suggested that ACH3 increases with increasing maturation. To extend these studies, we compared histone acetylation levels in primary human monocytes and differentiated macrophages. Monocytes were isolated from PBMCs and became differentiated macrophages after adherence to plastic for 7 days (Table 1). ChIP assays showed that differentiated macrophages had significantly greater levels of ACH3 and ACH4 (two-way ANOVA, P⬍0.01 for both; Fig. 5, A and B). It is interesting that ACH4 in the distal regions of the TNF-␣ promoter (TNF3 and TNF4) was significantly increased in the primary monocytes (one-way ANOVA, P⬍0.01; Fig. 5B). In general, our results in primary 866

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monocytes and macrophages reflected the effects we observed in the monocytic cell lines. As monocytes mature, histones in the TNF-␣ promoter region become increasingly acetylated.

Acetylation changes were not a result of acute stimulation To investigate whether increased histone acetylation levels were also induced by acute stimulation, U937.1 cells were treated PMA for various times. Examining the TNF-␣ promoter region by the ChIP assay showed that ACH3 and ACH4 levels were unaltered in PMA-stimulated cells at all time points (0, 20, 60, 120, 240 min). At each of the time points, the levels of ACH3 and ACH4 were comparable (data not shown). Additionally, THP-1.1, U937.1, and MonoMac 6 cells stimulated with PMA for 18 h showed no significant alterations in levels of ACH3 and ACH4 (data not shown). MonoMac 6 cells were also acutely stimulated for 4 h with PMA, LPS, or immune complexes. Although TNF-␣ protein was produced, ACH3 and ACH4 levels remained unchanged (data not shown). http://www.jleukbio.org

Fig. 2. Schematic diagram of the TNF-␣ promoter with selected binding motifs for specific transcription factors [2– 4, 6, 36, 37]. The indicated motifs have demonstrated importance by transfection studies or footprinting in T cells or monocytes. Black bars labeled TNF1–TNF4 indicate the regions of the promoter analyzed for ChIP and real-time PCR. CBP, Cyclic AMP response element binding protein-binding protein; Egr-1, early growth response 1; AP-1, activated protein-1; CRE, cyclic adenosine monophosphate-responsive element; C/EBP, CCAAT/enhancer-binding protein.

Chromatin remodeling occurs after acute stimulation Histone acetylation is clearly increased with maturation, but we were unable to show any acetylation changes after acute stimulation. To determine whether another type of chromatin modification was occurring with stimulation, we examined nucleosome remodeling after various stimuli. Initially, nuclei were digested with micrococcal nuclease for different times, and Southern blots were used to demonstrate that a nucleosome within the TNF-␣ proximal promoter region at approximately –70 bp was being remodeled. In Figure 6, a modified, micrococcal nuclease access assay was performed using the same four real-time PCR probes as the ChIP analyses. In this assay, micrococcal nuclease digesting between nucleosomes within an amplimer results in decreased amplification [38]. The three

cell lines corresponding to different levels of maturity were examined after PMA, LPS, or immune complex stimuli (Fig. 6). Increased accessibility to micrococcal nuclease was seen just 30 min after stimulation in MonoMac 6 cells. This was true for all four probes and all three stimuli. In the figure, a standard amount of micrococcal nuclease was used, which resulted in 25% of the DNA being digested into single nucleosomes. Each bar represents a time point after stimulation (0, 30, 60, 90 min). U937.1 does not exhibit any increase in accessibility after stimulation, with the exception of the probe 4 region. THP-1.1 may exhibit some mild increase in accessibility. Some differences in nucleosome remodeling could relate to surface receptors or signaling molecules. U937.1 and MonoMac 6 cells express Fc receptors for immunoglobulin G (Fc␥Rs), but only MonoMac 6 cells express CD14. TNF-␣ protein production

Fig. 3. Levels of ACH3 were dependent on cell type and region of the TNF-␣ promoter. Comparable numbers of THP-1.1 (THP-1), U937.1 (U937), and MonoMac 6 (MM6) were prepared for the ChIP analysis as described in Materials and Methods. DNA–protein complexes were immunoprecipitated with anti-ACH3 (A) or anti-ACH4 (B). Four sets of primers and probes (TNF 1– 4) were used for real-time PCR (A and B). Each reaction also included a set of GAPDH primers and probe as an internal control. Real-time PCR output data were expressed as percent of the input fraction, and statistical analyses were performed. *, Significant differences. Error bars represent standard deviations from nine independent experiments.

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Fig. 4. NaBut hyperacetylated histone H3 and H4 in THP-1.1 cells. THP-1.1 (THP-1) cells were treated with 100 mM NaBut or with 100 nM TSA for 4 h. Unstimulated THP-1.1, U937.1 (U937), and MonoMac 6 (MM6) cells were prepared simultaneously for comparison. DNA–protein complexes were immunoprecipitated with anti-ACH3 (A) or anti-ACH4 (B). Four sets of primers and probes (TNF 1– 4) were used for real-time PCR (A and B). Real-time PCR output data were expressed as percent of the input fraction, and statistical analyses were performed. *, Significant differences between treated and untreated THP-1.1 cells; †, significant differences between TNF-␣ promoter regions. Error bars represent standard deviation from three separate experiments.

after each stimulus is shown in Table 2. MonoMac 6 cells, which increase their chromatin accessibility the most, are also the cells with the greatest production of TNF-␣.

DISCUSSION This is the first study to examine the role of chromatin conformation in the regulation of TNF-␣ expression. Given the known complexity of the regulation of TNF-␣ expression, it should not be surprising that gene expression is regulated at two additional levels. The least mature cell line, THP-1.1, could be induced to express greater levels of TNF-␣ when treated with all HDAC inhibitors tested. This suggested very strongly that histone acetylation facilitates expression of TNF-␣. Only NaBut increased TNF-␣ expression in the more mature cell lines, which presumably reflects their higher basal level of acetylation. It is interesting that not all cells within a population responded equally, suggesting a stochastic component. The fraction of induced cells did not correlate exactly with the amount of protein detected by ELISA, which could reflect variation in TNF-␣ production within the responding population or differences in the final export stages. ChIP assays were used to show that the level of histone acetylation correlated with the developmental state, which in turn correlated with competence to produce TNF-␣ after PMA stimulation. Furthermore, treatment with NaBut increased TNF-␣ production and TABLE 4.

simultaneously increased acetylation at the TNF-␣ promoter. These data suggest very strongly that histone acetylation at the TNF-␣ promoter facilitates expression of TNF-␣. Importantly, HDAC inhibitors had no effect on their own. Increased histone acetylation may act by regulating access of transcription factors to promoter motifs directly or by signaling competence for nucleosome remodeling. Histone acetylation was clearly developmentally and positionally regulated. Acute stimuli had no effect on histone acetylation. Even prolonged stimulation with PMA, which can induce phenotypic changes in monocyte cell lines, did not alter the histone acetylation levels at the TNF-␣ promoter. Thus, the precise signals governing the developmental progression require additional study. Transcription factors or coactivators present on the very proximal promoter could explain the high level of histone acetylation between –119 and ⫹99. CBP, a coactivator with known histone acetylation activity, has been reported to bind between –117 and 80 [3]. Histone acetylation was developmentally regulated in monocytes in our study but was refractory to several common types of stimuli. To examine other chromatin effects at the TNF-␣ promoter, we used a modified micrococcal nuclease assay to show that chromatin remodeling was seen with all stimuli in MonoMac 6 cells, which is a cell line capable of responding to LPS and PMA. U937.1, which produces modest levels of TNF-␣ after stimulation with PMA or LPS, showed no clear chromatin remodeling in the region examined. Thus, there is imperfect concordance between TNF-␣ protein production and

ACH3 and ACH4 Are Not Markedly Different among the Three Cell Linesa

Histone

Cell line

No treatment (normalized densitometric units)

NaBut (normalized densitometric units)

TSA (normalized densitometric units)

CMD (normalized densitometric units)

H3 H3 H3 H4 H4 H4

THP-1.1 U937.1 MonoMac 6 THP-1.1 U937.1 MonoMac 6

114 200 156 134 285 190

225 491 208 259 796 290

125 207 244 119 300 222

93 198 152 ND 254 175

a

Normalized densitometric units; n ⫽ 3.

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Fig. 5. Differentiated macrophages had higher levels of ACH3. Fresh monocytes and differentiated macrophages were isolated as described. DNA–protein complexes were immunoprecipitated with antiACH3 (A) or anti-ACH4 (B). Four sets of primers and probes (TNF 1– 4) were used for real-time PCR (A and B). Real-time PCR output data were expressed as percent of the input fraction, and statistical analyses were performed. ACH3 is markedly higher in macrophages than in the monocytes or cell lines. *, Statistical significance comparing monocytes and macrophages by two-way ANOVA; †, significant differences among TNF-␣ promoter regions by one-way ANOVA. Unmatched monocytes (n⫽6) and macrophages (n⫽6) were analyzed for this figure. Error bars represent standard deviations.

nucleosome remodeling. It may be that only a subset of U937.1 cells is producing TNF-␣, and the micrococccal nuclease assay was not able to detect changes occurring in that small subset. This explanation is supported by the flow cytometry showing that only a subset of cells responded to the HDAC inhibitors. If nucleosome remodeling is dependent on previous histone acetylation, then only a subset would have been competent to remodel. The explanation for the MonoMac 6 cells remodeling in response to immune complexes when they fail to produce TNF-␣ protein may be that they could potentially transduce a chromatin remodeling signal via Fc␥Rs without inducing TNF-␣ protein production. Alternatively, histone acetylation and nucleosome remodeling may be prerequisites for transcription but insufficient on their own, or transcription may be occurring but not translation. One possible model to explain these results is that a certain level of histone acetylation is a prerequisite for nucleosome remodeling. Histone acetylation is driven by maturation, and

nucleosome remodeling is dependent on stimulation. Additional signals induce required transcription factors. A similar hypothesis has been proposed to explain the effect of histone acetylation at the IFN-␤ locus [32]. We have not shown directly that histone acetylation is required for nucleosome remodeling nor that nucleosome remodeling is important for transcription-factor access to the TNF-␣ promoter; however, such a relationship has been described for a few other genes [33, 39 – 44]. Our study suggests very strongly that there is a hierarchy of control at the TNF-␣ promoter. Similar to the IFN-␤ promoter, histone acetylation appears to precede nucleosome remodeling at the TNF-␣ promoter. Regulation of DNA methylation, histone methylation, histone phosphorylation, or histone ubiquitination could also participate in the complex control of this locus. The advantages of such a system could relate to the extremely potent effects of TNF-␣. In the systemic inflammatory response in sepsis, dramatic overproduction of TNF-␣ is del-

Fig. 6. Stimulation of MonoMac 6 cells caused chromatin remodeling. Micrococcal nuclease analyses of chromatin accessibility are shown with THP-1.1 (THP-1), U937.1 (U937), and MonoMac 6 cells. Each cell line was treated with 100 ng/ml PMA, 1000 ng/ml LPS, or 1000 ng/ml immune complexes. The four bars for each cell line represent DNA purified and digested 0, 30, 60, and 90 min after stimulation (left to right; dark bars to light bars). All nuclei were digested to achieve 25% in the single nucleosome fraction. Micrococcal nuclease digests between nucleosomes, and digestion diminishes PCR amplification. DNA amplified with each of the TNF-␣ real-time probes from Figure 2 is expressed as a ratio with amplified actin DNA. Means of triplicates from two independent experiments are shown. Remodeling of chromatin occurs soon after stimulation and across all four regions in MonoMac 6 cells. (Amplification shown in the gray bars is diminished compared with unstimulated in the left-hand black bars.) U937.1 and THP-1.1 exhibit little change after stimulation.

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eterious to the host [45, 46]. By restricting TNF-␣ production to mature macrophages, histone acetylation may serve as an important control against systemic bloodstream overproduction, such as is seen in the pathologic systemic inflammatory response.

14.

15.

ACKNOWLEDGMENTS 16.

This work was supported by NIH RO1 AI/AR44127, the Wallace Chair of Pediatrics (K. E. S.), and NIH 5T32EYO7131 (J. Y. L.). The authors thank Joie Cutilli for advice regarding large-scale monocyte purification and Craig North and Donald Campbell for flow cytometry advice.

17.

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