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Department of Obstetrics & Gynecology (M.S.S., D.L.C., Y.-J.J., G.D.A.), Sealy Center for Molecular Science (M.S.S.), University of Texas Medical Branch, Galveston, Texas 77555-1062
Address all correspondence and requests for reprints to: Melvyn S. Soloff, Department of Obstetrics and Gynecology, University of Texas Medical Branch, 301 University Boulevard, Galveston, Texas 77555-1062. E-mail: msoloff{at}utmb.edu.
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Abstract |
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B (NF-B) or CCAAT/enhancer binding protein (C/EBPß), or both acting cooperatively. To determine the mechanisms of COX-2 (cyclooxygenase or prostaglandin endoperoxide synthase) induction in cultured human myometrial cells in situ, we examined the cross-linking of the RelA subunit of NF-B and C/EBPß to the cox-2 promoter and flanking sequences. As a control, we inspected the interaction of these transcription factors with the IL-8 gene, which has been shown in other cell types to be activated by the cooperative interaction of NF-B and C/EBPß. Indeed, both transcription factors were cross-linked to the il-8 promoter after IL-1 treatment, but only RelA was cross-linked to cox-2 DNA. The il-8 promoter was also found to physically interact with proteins cross-linked to sites further upstream. IL-1 treatment also increased polymerase II cross-linking to both promoters and increased histone H4 acetylation at specific sites. These results indicate that modification of chromatin structure is part of the response to IL-1 stimulation. Chromatin immunoprecipitation thus provides critical insight into the mechanisms of COX-2 and IL-8 expression in human myometrial cells. |
Introduction |
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B (NF-B) family members (5, 6), which induce more than 100 known stress-related target genes and other genes such as vimentin, laminin, and gelatinase (7). Because most NF-B target genes are involved in the host immune response, only a fraction of potential target genes are probably induced in myometrial cells, and these may be activated in ways that are distinct from those existing in immune cell types. To elucidate the mechanisms of IL-1-induced gene expression in the myometrium, we used the in situ method of chromatin immunoprecipitation (ChIP) to examine two known NF-B targets genes, cox-2 and il-8, in cultured human myometrial cells. Prostaglandin endoperoxide synthase 2 (cyclooxygenase 2, or COX-2) catalyzes the rate-limiting step in the inducible production of prostaglandins in a number of cell types, including cultured human myometrial cells (8, 9, 10). Because prostaglandins can stimulate uterine contractions, it has been suggested that induction of COX-2 expression in the myometrium at the end of pregnancy is involved in labor initiation (11, 12, 13, 14). Understanding the mechanisms of COX-2 induction may therefore be important in recognizing events leading to both spontaneous and preterm labor. COX-2 has also been shown to play a role in diverse pathophysiological processes, such as inflammation, tissue damage, and tumorigenesis. However, despite the broad interest in COX-2 regulation, the complex mechanisms by which COX-2 transcription is activated require further clarification.
The human COX-2 gene in different cell types has three cis-acting elements that allow a variety of agents to induce COX-2 expression (see Ref. 15 for references). Two NF-B binding sites are located in the cox-2 5'-flanking sequence between positions -448 to -439 and -223 to -214 relative to the transcriptional start site. CCAAT/enhancer binding protein ß (C/EBPß, also referred to as NF-IL6) binds to a site at -132 to -124; a third element at -59 to -53, lacks a consensus binding site but binds C/EBPß via an overlapping cAMP response element (CRE)/E-box.
Depending on the cell type, IL-1 induction of cox-2 transcription can involve either B, C/EBP-CRE elements, or both. Apart from differential tissue expression of transcription factors, methodological considerations may account for different findings within the same cell type. Some investigations suggest that IL-1 induces cox-2 expression via NF-B in amnion-derived cells (16, 17), whereas others indicate that the C/EBP-CRE element alone mediate IL-1 signaling in this cell type (18).
IL-8, a potent chemokine for lymphocytes, is expressed by myometrial tissue and cultured myometrial cells (19). Whereas the primary role of IL-8 is thought to be chemotaxis, chemokines are also involved in cellular proliferation, differentiation, apoptosis, and a variety of other functions, including angiogenesis (20). Treatment of the cells with IL-1 results in about a 15-fold increase in IL-8 mRNA levels after 12 h (19). The il-8 promoter contains a single transcriptional initiation site and a consensus TATA box (21). Functional studies indicate that only about 100 bp of 5'-flanking sequence are required for a rapid transcriptional response to proinflammatory mediators. This region contains adjacent binding sites for NF-B and C/EBPß, and the two transcription factors act synergistically by direct physical interaction (see Ref. 22 for references).
Virtually all studies characterizing cox-2 and il-8 promoters and adjacent cis-acting elements have involved transient transfection of promoter-driven reporter constructs and EMSAs. Transient transfection studies, using mutated or deleted cis-acting sequences in promoter-driven reporter constructs and cotransfection with transcription factor expression vectors, might not take into account transgene overexpression, which may cause squelching or promiscuous interactions in some instances; and, along with mobility shift assays, do not allow an examination of the contributions of distant elements and chromatin structure on transcriptional activity. Although these assays have provided useful preliminary information, the ability to perform experiments within the natural cell environment would provide a more accurate picture of gene regulation. Thus, ChIP has been useful in either confirming or rejecting models based on transfection and in vitro studies (23), and has furthered our knowledge of the importance of chromatin structure in the interaction of trans-factors with DNA sequences. We have therefore examined cross-linking of the RelA subunit of NF-B and C/EBPß to cognate sites in human myometrial cells in primary culture. Because acetylation of histones H3 and H4 are usually associated with gene expression, we also examined the cross-linking of these acetylated histones to cox-2 and il-8.
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Materials and Methods |
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B site), 5'-tgcgaagaagaaaagacat-3' and 5'-agactgaaaaccaagccca-3' (-300 to -32, proximal NF-B site), and 5'-tgggcttggttttcagtct-3' and 5'-gcgggggtaggctttgctg-3' (-31 to +111, transcriptional start site). The specificities of the PCR-amplified products were verified by cloning into pCRII (Invitrogen, Carlsbad, CA), and determining the DNA sequence of the inserts in isolated plasmids. Rabbit polyclonal antibodies to p65 (sc-109), C/EBPß (sc-150), C/EBP
(sc-7658), and polymerase II (sc-899) were obtained from Santa Cruz Biotechnology, Inc., (Santa Cruz, CA), as was protein A/G PLUS-agarose. Polyclonal antibodies to acetylated histone H3 (06-599) and histone H4 (06-598) were obtained from Upstate (Waltham, MA). With the exception of the C/EBP antibody, the specificities of the other antibodies have been verified in previous studies using either ChIP or EMSAs (Ref. 24 and references provided in the Upstate catalog). Recombinant human IL-1
was purchased from R&D Systems (Minneapolis, MN). Plasmid DNA containing full-length human COX-2 cDNA was obtained from Dr. Timothy Hla (Vanderbilt University, Nashville, TN).
Myometrial cell culture
The University of Texas Medical Branch Committee on Research Involving Human Subjects approved the use of human tissue. Myometrial samples were taken from nonlaboring women by cesarean section near the end of gestation. Cells were prepared as described previously and maintained in MEM containing 10% (vol/vol) FBS, 1 mM sodium pyruvate, 2 mM L-glutamine, penicillin G (100 IU/ml), streptomycin sulfate (100 µg/ml), and amphotericin B (15 µg/ml) at 37 C (95% humidity) in the presence of 5% CO2 (25). The cells, which were used at confluency between passages 2 and 10, were serum-starved overnight (about 16 h) before treatment. Cells were treated with IL-1 (500 ng/ml) for the times indicated in the figures.
Ribonuclease protection assay (RPA)
RNA was isolated using TRIzol reagent (Life Technologies, Inc.-BRL Life Technologies, Rockville, MD), and COX-2 and IL-8 mRNA concentrations were measured by RPA after treatment with IL-1. The DNA fragment amplified using the IL-8 primer set (-102 to +81) was cloned into pCRII (Invitrogen), the plasmid linearized, and labeled cDNA was transcribed using T7 polymerase. Full-length COX-2 inserted into pCRII, was linearized, and transcribed with T7 polymerase to give a 325 nucleotide probe. A reference cyclophilin probe was purchased from Ambion, Inc. (Austin, TX). After electrophoresis, polyacrylamide gels were dried and exposed to a Cyclone phosphor screen (Packard Instrument Co., Meriden, CT) for image analysis. Cells from three different patients were used, and the data are expressed relative to cyclophilin mRNA concentrations. Differences between the means of each treatment group was analyzed statistically by Student’s t test. P < 0.05 was considered significant.
Nuclear run-on
Nuclei were isolated from confluent monolayers of cells, and run-on transcription was carried out as previously described (26). Nuclei (1 x 107) were mixed with an equal volume of 2x reaction buffer [10 mM Tris-HCl (pH 8.0), 0.3 M KCl, 5.0 mM dithiothreitol, 5.0 mM MgCl2, 1.0 mM GTP, 1.0 mM uridine triphosphate, 1.0 mM ATP, and 100 µCi [á-32P]CTP (800 Ci/mmol)] and incubated for 30 min at 30 C. After the labeling reaction, 50 µg carrier tRNA were added and samples were subjected to sequential deoxyribonuclease I and proteinase K digestion. Labeled transcripts were then purified by trichloroacetic acid precipitation. Plasmid pCRII, and plasmids containing the complete coding sequence of human cox-2, the first 81 bp of transcribed il-8, and the complete coding sequence of human ß-actin cDNA (American Type Culture Collection, Manassas, VA) were immobilized on nitrocellulose and used as probes. Hybridizations were carried out as previously described (26), and transcription levels were determined by using PhosphorImager (Molecular Dynamics, Sunnyvale, CA) analysis of the hybridized membranes. Based on the degree of variability of our initial results, the experiments were repeated with cells from four different patients. Values are expressed as the mean amount of nascent transcript ± SE relative to ß-actin transcript. Statistical analyses were carried out as described for RPA.
Western blotting
Changes in the concentration of IB in cell lysates were determined by immunoblotting. Cells were rinsed in cold PBS and lysed in immunoprecipitation assay solution [Tris-HCl, 50 mM (pH 7.4); Nonidet P-40, 1%; Na-deoxycholate, 0.25%; NaCl, 150 mM; EDTA, 1 mM; phenylmethylsulfonyl fluoride, 1 mM; aprotinin, leupeptin, pepstatin,1 µg/ml each; Na3VO4, 1 mM; and NaF, 1 mM. The lysates, containing 10–20 µg of protein, were mixed with an equal volume of 2x Laemmli sample preparation buffer and were subjected to electrophoresis on 10% sodium dodecyl sulfate-polyacrylamide gels. Proteins were transferred to polyvinylidene difluoride membranes. The membranes were incubated in PBS containing 3% BSA and 0.1% Tween 20 for 1 h, followed by incubation with primary antibody for 2 h and secondary IgG antibody for 1 h. Immunocomplexes were visualized by enhanced chemiluminescence (Amersham). The membranes were stripped of antibody, reprobed with antibody directed against ERK2, and bands detected in the same manner as used for IB. All operations were carried out at room temperature.
ChIP
In ChIP analysis, transcription factors are cross-linked in situ to cognate DNA-binding sites and associated nuclear proteins using formaldehyde. The cells are then lysed and chromatin is sheared into 200- to 1000-bp fragments by sonication. The presence of a particular cross-linked protein is determined by addition of its specific antibody, and the chromatin fragments containing the cross-linked protein are purified by immunoadsorption and elution from protein A/G beads. After reversal of the cross-links and purification of the DNA, the DNA region cross-linked to the protein is determined by PCR analysis. Different regions of the same gene can be amplified, using the same or different antibodies. ChIP analysis was carried out as described previously (27). Pooled lysates were prepared from cells from 10 10-cm plates in immunoprecipitation assay solution, and samples were aliquoted and stored at -70 C until used for immunoprecipitation. In this way, the same lysate pool was used with each antibody. After immunoprecipitation and reversal of the cross-links, the DNA was purified using a QIAquick Nucleotide Removal kit (QIAGEN Inc., Valencia, CA). PCR was performed with doubling concentrations of DNA to ensure that amplification was linear, and amplicons were detected by ethidium bromide staining after agarose gel (2.5%) electrophoresis. The experiments were repeated with each antibody several times to ensure reproducibility, and separate lysate pools from cells from two different patients were analyzed. The results depicted in the figures are representative of multiple analyses.
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Results |
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(500 ng/ml) stimulated increases in steady-state COX-2 and IL-8 mRNA levels in human myometrial cells (Fig. 1
). The increases in both mRNA concentrations were significant at the earliest time point taken, 30 min (Fig. 1). To determine whether the effects of IL-1 were at the transcriptional level, nuclear run-on experiments were performed. There was increased labeling of both COX-2 and IL-8 run-on transcripts after 30 min of treatment (Fig. 2).
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B levels in myometrial cellsB concentration after 15 and 30 min, indicating release of NF-B from the inhibitor IB (Fig. 3). IB levels were restored by 60 min. These findings indicate that IL-1 increased NF-B activation during the time when inhibitor levels were reduced.
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. Specific antibodies to polymerase II, the RelA subunit of NF-B, C/EBPß, C/EBP, acetylated histone H3, or acetylated histone H4 were added separately to the same chromatin samples. As controls, we examined the +153/+423 region of the HSP70 gene, and the U6 snRNA promoter, which is transcribed by polymerase III.
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). Polymerase was not cross-linked to either the -571/-311 or -309/-32 regions of cox-2, illustrating the resolution of the method (Fig. 5A). However, polymerase II was cross-linked to the upstream (-1026/-811) region of the IL-8 gene, suggesting that proteins bound to this region interact with the preinitiation complex (Fig. 5A). The selectivity of the antibody for polymerase II was demonstrated by the observation that polymerase was constitutively associated with the hsp promoter, as is known from studies in Drosophila and in mammalian cells (24); and polymerase II was not cross-linked to the transcriptional start site region of the U6 snRNA gene, which is transcribed by polymerase III (Fig. 5A).
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B binding sites in cox-2 (-571/-311 and -309/-32), and the promoter region (-31/+111), which lacks a B element (Fig. 5B
). RelA was also cross-linked to both regions of the IL-8 gene examined (Fig. 5B). The interactions between NF-B and cognate sites were specific, as evidenced by the absence of RelA cross-linking to the U6 snRNA gene. These findings are consistent with data indicating that the binding of NF-B is associated with transcriptional activation of both cox-2 and il-8 promoters. IL-1 treatment also increased RelA cross-linking to the hsp70 promoter—a unique finding that was not explored further in the present studies (Fig. 5B).
We did not detect an effect of IL-1 treatment on C/EBPß cross-linking to any of the three cox-2 DNA regions examined (Fig. 5C). However, C/EBPß cross-linking to the il-8 promoter and upstream sequences was increased after IL-1 treatment (Fig. 5C). As a negative control, we examined the cross-linking of C/EBP with both genes, using a specific antibody to the -isoform. Because with C/EBPß there was no evidence of C/EBP cross-linking to any of the three cox-2 regions (Fig. 5D). Unexpectedly, C/EBP was cross-linked to il-8 in unstimulated cells, and this interaction was lost with IL-1 treatment (Fig. 5D). The significance of these findings was not examined further.
Activation of transcription can involve modulation of the chromatin environment in combination with the binding of transcriptional activators. A number of studies have shown that acetylation of core histones modifies histone-DNA contacts within nucleosomes to facilitate transcriptional activation. The elevated expression of COX-2 after IL-1 treatment for 30 min was associated with a minimal increase in histone H3 acetylation at the cox-2 promoter region (-31/+111) (Fig. 5E). There was neglible histone H3 acetylation in the two il-8 regions probed, with or without IL-1 treatment (Fig. 5E). In contrast, IL-1 selectively induced histone H4 acetylation of both COX-2 and IL-8 genes. Histone H4 acetylation of the COX-2 gene was specifically associated with the region containing the proximal B element (-300/-32) (Fig. 5F). Histone H4 acetylation of the distal region of il-8 was also strongly induced by IL-1 (Fig. 5F). However, there was no acetylation in the promoter region (Fig 5F).
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Discussion |
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IL-1 treatment results in the activation of NF-B, by signaling the dissociation and degradation of the inhibitory protein IB (28) (Fig. 3). The liberated cytosolic NF-B, composed of p50 and p65 (RelA) subunits, is then translocated to the nucleus where it binds to target DNA elements (B) in more than 100 different genes (7), including cox-2 (15) and il-8 (29, 30). Inhibition of NF-B activation in some cell types impairs both COX-2 (31, 32) and IL-8 (29) expression. IL-1 treatment of human myometrial cells resulted in the cross-linking of RelA to regions containing both NF-B binding sites on cox-2 and did not stimulate any significant C/EBPß cross-linking to any of the regions examined. In contrast, IL-1 treatment resulted in the cross-linking of both RelA and C/EBPß to the IL-8 gene. Consistent with the nuclear run-on data, polymerase II was cross-linked to the promoter regions of both cox-2 and il-8 after IL-1 treatment.
The resolution of the ChIP method is limited by the size of chromatin fragments that are produced by sonication. The larger the fragment, the more difficult it would be to discriminate between the two NF-B binding sites in cox-2, which lie about 220 bp apart. Because IL-1-induced histone H4 acetylation was apparent only with PCR amplification of the cox-2 proximal NF-B binding site region, it is likely that the DNA fragments were of sufficiently small size to allow resolution between the two NF-B elements. Likewise, cross-linking of polymerase II occurred only in the region of the transcriptional initiation site of cox-2 with IL-1 treatment. Thus, both NF-B binding sites appear to bind RelA, suggesting that both play a role in the regulation of transcriptional activity of the COX-2 gene. The association of RelA with the cox-2 TATA box region (-31/+111), which lacks a B site, is suggestive that NF-B bound to either one or both cis elements, or recruited to the promoter, can interact with the preinitiation complex. Indeed, the carboxyl-terminal transactivation domain of RelA has been shown to interact with TATA-binding protein, transcription factor IIB, and coactivators (33). The absence of cross-linking of polymerase II to the upstream regions of the COX-2 gene indicates that RelA bound to its cognate sites does not interact with proteins that bind polymerase II (Fig. 6). These findings suggest that NF-B is recruited to the preinitiation complex itself.
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B and C/EBPß binding sites, and maximal transcriptional activation requires both (29). As expected, RelA, C/EBPß, and polymerase II were cross-linked to the il-8 promoter. It was surprising that the same results were obtained when the il-8 -1026/-811 fragment was probed, because this region lacks cognate sites for RelA and polymerase II. Because there is no evidence for the presence of more than one transcriptional initiation site, the findings suggest that the transcription factors and polymerase II bound to the il-8 promoter interact with proteins, possibly C/EBPß, located further upstream. Several studies have shown that C/EBPß recruits the coactivators p300 and cAMP response element binding protein (CREB)-binding protein (CBP) (34, 35), which promote transcription by acetylating histones and recruiting basal transcription factors. Perhaps CBP/p300 serve to bridge proteins associated with the promoter and the upstream C/EBPß cognate sites (Fig. 6). EMSAs indicate that NF-B and C/EBPß form a ternary complex with the il-8 promoter (29, 30). Ternary complexes between NF-B, C/EBPß, and DNA have also been seen in the promoters of the major acute phase reactant serum amyloid A2 (36), HIV-I (37), and granulocyte colony/stimulatory factor (38) genes. Transient cotransfection analyses also indicate that the cooperative association of RelA and C/EBPß with the il-8 promoter results in synergistic transcriptional activation (29). The same situation may exist in myometrial cells, but because both transcription factors appear to also interact with sites further upstream from their cognate sites the situation appears to be more complex.
Histone H4 acetylation occurred with IL-1 stimulation of COX-2 and IL-8 expression. Both RelA and C/EBPß can recruit histone acetyltransferases to target genes (34, 39, 40, 41, 42), possibly accounting for the increases in histone H4 acetylation seen. The acetylated sites were specific, being restricted to the region of the proximal NF-B binding site of cox-2, and the region upstream from the il-8 promoter. The significance of this specificity remains to be determined. The effects of IL-1 on histone H3 acetylation were minimal.
The results of these studies reinforce the obvious conclusion that not all NF-B target genes are regulated in the same fashion. In cultured human myometrial cells, COX-2 expression appears to be regulated by NF-B alone, whereas IL-8 requires both NF-B and C/EBP, presumably acting synergistically (Fig. 6). The cross-linking results also indicate that cox-2 and il-8 differ with respect to the interaction of the preinitiation complex with proteins bound to upstream regions (Fig. 6). Immunoprecipitation analysis has therefore provided new insights into the mechanisms of IL-1 induction of COX-2 and IL-8 expression, pointing out the importance of chromatin in assessing transcriptional regulation. The present studies were carried out with cultured cells, which may not accurately represent myometrial cells in vivo. However, an asset of the ChIP method is that excised tissue samples that can also be used to determine occupancy of NF-B and C/EBPß binding sites on cox-2, il-8, and other selected genes in future studies.
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Acknowledgments |
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Footnotes |
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Abbreviations: CBP, CREB (cAMP response element binding protein)-binding protein; C/EBP, CCAAT/enhancer binding protein; ChIP, chromatin immunoprecipitation; COX-2, cyclooxygenase or prostaglandin endoperoxide synthase; CRE, cAMP response element; HAT, histone acetyltransferases; NF-B, nuclear factor-B; RPA, ribonuclease protection assay.
Received September 30, 2003.
Accepted for publication November 17, 2003.
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References |
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