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The Journal of Immunology, 2003, 170: 4139-4147.
Copyright © 2003 by The American Association of Immunologists

Communication Between NF-{kappa}B and Sp1 Controls Histone Acetylation Within the Proximal Promoter of the Monocyte Chemoattractant Protein 1 Gene1

Gunther H. Boekhoudt, Zhu Guo, Guy W. Beresford and Jeremy M. Boss2

Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, GA 30322


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The induction of the monocyte chemoattractant protein 1 gene (MCP-1) by TNF occurs through an NF-{kappa}B-dependent distal regulatory region and an Sp1-dependent proximal regulatory region that are separated by 2.2 kb of sequence. To investigate how these regions coordinate activation of MCP-1 in response to TNF, experiments were performed to examine the role of coactivators, changes in local chromatin structure, and the acetylation of histones at the MCP-1 regulatory regions. An E1a-sensitive coactivator was found to be required for expression. In vivo nuclease sensitivity assays identified changes in response to TNF at both the proximal and distal regions that were dependent on the p65 subunit of NF-{kappa}B and Sp1. Chromatin immunoprecipitations used to analyze factor assembly and histone acetylation at the distal and proximal regions showed that Sp1 binding to and histone acetylation of the proximal region was dependent on NF-{kappa}B p65. Conversely, Sp1 assembly at the proximal region was required for p65 binding to and acetylation of the distal region, suggesting communication between the two regions during gene activation. These data and the NF-{kappa}B p65-dependent histone acetylation of a middle region sequence suggest a potential order for the assembly, acetylation and accessibility of the MCP-1 regulatory regions in response to TNF.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Monocyte chemoattractant protein 1 (MCP-1),3 a 76-aa peptide, is a member of the C-C chemokine family that induces chemotaxis of monocytes and macrophages to areas of infection and inflammation (1, 2, 3, 4). Expression of MCP-1 has been associated with a variety of human diseases, such as atherosclerosis, rheumatoid arthritis, HIV replication, and prostate adenocarcinoma (5, 6, 7, 8). MCP-1 expression is induced in numerous cell types by inflammatory mediators, such as TNF (9), platelet-derived growth factor (PDGF) BB (10), IL-1 (11), and IFN-{gamma} (12). Agents that suppress inflammation, including retinoic acid (10), dexamethasone (13), and estrogen (14) can suppress the induction of MCP-1.

Of the above inducers of MCP-1 expression, TNF is perhaps the most potent (10). Regulation of MCP-1 by TNF occurs primarily at the level of transcription initiation (15). Two upstream regulatory regions (Fig. 1), distal and proximal, separated by 2.2 kb of DNA are required for TNF-induced expression (9). The proximal regulatory region, which is required for all aspects of MCP-1 gene expression and is sufficient for PDGF-induced expression (10), is comprised of three elements: {kappa}B-3, site B, and a GC box. The {kappa}B-3 site, which was named after its homology to an NF-{kappa}B binding site, does not bind NF-{kappa}B family members in vitro (9). The factor that binds to the {kappa}B-3 site under TNF- and PDGF-induced conditions remains unknown. Interestingly, the {kappa}B-3 site of the human MCP-1 gene functions as a IFN-{gamma} activation sequence element, which binds STAT1 and is required for IFN-{gamma} induction of MCP-1 (12). The factor(s) that interacts with site B has also not been defined. Along with {kappa}B-3, site B becomes occupied during TNF and PDGF induction of MCP-1; however, these sites were not required for expression from transfected reporter plasmids (16), and thus their specific role in MCP-1-mediated induction by these stimuli is not known. In contrast, the GC box is critical for MCP-1 regulation. The proximal GC box, which binds Sp1 and Sp3 in vitro, requires Sp1 for full induction by TNF in vivo (17).



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  		FIGURE 1. Schematic of MCP-1 promoter. 5' and 3' boundaries of the regulatory regions are indicated.

 
The distal regulatory region contains four elements: site A, a pair of {kappa}B sites termed {kappa}B-1 and {kappa}B-2, and a dimethylsulfate-hypersensitive sequence termed HS. Site A, which is required for maximal TNF induction, is constitutively occupied in vivo by the transcription factor NF-1 (18). The region encompassing the two {kappa}B sites and the HS region is critical for MCP-1 induction by TNF. In vitro, the {kappa}B sites are able to bind NF-{kappa}B (16). In vivo genomic footprinting on wild-type BALB/3T3 cells and fibroblasts isolated from animals containing targeted disruption of their NF-{kappa}B p65 genes showed that p65 was required for assembly of the distal regulatory region in vivo (16), suggesting that p65 is a bona fide regulator of MCP-1 induction by TNF. Additionally, these studies showed that the absence of p65 reduced the efficiency of the proximal regulatory region to assemble and bind Sp1 upon stimulation by TNF, implying that the two regions communicate despite their 2.2-kb physical separation (16). The mechanism of this communication is not known.

The packaging of eukaryotic chromatin into higher order structures allows for the possibility that these regulatory regions are in close proximity for either direct physical interactions or interactions through bridging molecules. Recently, the role of chromatin remodeling and histone modification has been implicated in the activation and repression of many eukaryotic genes (19). Several lines of evidence suggest that before activation, some form of chromatin remodeling of the MCP-1 gene takes place. First, the proximal region DNA-binding proteins, including Sp1 are all present in the nucleus of cells and can bind their sequences in vitro without the need for activation by TNF (9, 10). Yet, in vivo footprinting shows that this region is unoccupied before TNF or PDGF treatment. Second, trichostatin A (TSA), a histone deacetylase inhibitor, is able to trigger MCP-1 expression (10). This trigger is associated with the in vivo occupancy of the Sp1 GC box in the proximal regulatory region.

To determine the role of chromatin in controlling the accessibility of factors to the MCP-1 gene, we examined changes in nuclease-hypersensitive sites, the requirement for coactivators that have the potential to modify chromatin, and examined changes in histone acetylation at both the proximal and distal regulatory regions of the MCP-1 gene in response to TNF. The results showed that TNF induced changes in the local chromatin structure and that both p65 and Sp1 were important for these changes. CREB-binding protein (CBP) and p300 but not p300/CBP-associated factor (P/CAF) enhanced TNF-induced expression, whereas E1a, an adenovirus inhibitor of CBP/p300 activity, blocked the induction. In a p65-dependent manner, TNF was found to induce rapid changes in histone acetylation at both the distal and proximal regulatory regions. Interestingly, sequences located between these regions were also found to be hyperacetylated in response to TNF. Sp1 was found to be required for histone acetylation at the distal region. The combined data suggest that MCP-1 activation by TNF is a complex process requiring chromatin remodeling and histone acetylation that ultimately leads to the assembly of the transcription factor complexes at both the distal and proximal regulatory regions, events necessary for gene activation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell lines and culture

NIH3T3 fibroblasts and BALB/3T3 clone A31 were purchased from the American Type Culture Collection (Manassas, VA). NF-{kappa}B p65-/- cells were provided by Dr. D. Baltimore (California Institute of Technology, Pasadena, CA). We previously showed that both the NIH3T3 and BALB/3T3 lines, which like the p65-/- cells are embryonic fibroblasts, produced similar TNF induction profiles and in vivo genomic footprints to a wild-type embryonic fibroblast line that was derived in parallel with the p65-/- cells (16). Because the NIH3T3 and BALB/3T3 lines had been used in all of our other studies on MCP-1 regulation, they were used here as controls (9, 16, 17). Cells were grown in DMEM supplemented with 10% calf serum (HyClone, Logan, UT), penicillin (50 U/ml), streptomycin (50 µg/ml), and L-glutamine (1 mM; Life Technologies, Grand Island, NY). An Sp1-/- cell line derived from an embryonic stem cell line containing a homozygous disruption of the Sp1 gene was also used (17). Although no wild-type equivalent of this line was available, TNF-induced expression of the IFN-{gamma}-inducible protein 10 gene, which does not require Sp1, was identical to the BALB/3T3 line as was an in vivo genomic footprint of the occupied sites in the IFN-{gamma}-inducible protein 10 promoter (17). Thus, the BALB/3T3 line was used as a control for the Sp1-derived cell line. The Sp1-/- cell line was grown in {alpha} minimum Eagle’s medium supplemented with 5% (v/v) FBS (HyClone), antibiotics, 1 mM L-glutamine (Life Technologies), 1 ng/ml basic fibroblast growth factor (Roche Molecular Biochemicals, Indianapolis, IN), and 4 µg/ml insulin (Life Technologies). Human rTNF was purchased from Genzyme (Cambridge, MA) and used at 500 U/ml for the indicated times.

Transient transfection

Transient transfections of NIH3T3 cells were conducted by using electroporation as described previously (9, 16) or with FUGENE 6 (Roche Molecular Biochemicals) according to the manufacturer’s instructions. Plasmids expressing E1a, CBP, p300, and, P/CAF were provided by Drs. L. Gooding (Emory University, Atlanta, GA), R. Goodman (Vollum Institute, Oregon Health Science Center, Portland, OR), and Y. Nakatani (Dana Farber Cancer Institute, Boston, MA), respectively.

Adenovirus infection

NIH3T3 cells grown to ~70% confluency were infected with the wild-type adenovirus strain dl520 at multiplicities of infection ranging from 30 to 200 PFU/cell. After 24 h, TNF (500 U/ml) was added and the cells were incubated for 4 h. RNA was then prepared from control and infected cells and analyzed as described below.

Western blot analysis

Whole cell lysate (50 µg/lane) was separated by SDS-7.5% PAGE and transferred to a polyvinylidene difluoride membrane as described previously (20). The membrane was probed with anti-E1a (a gift from Dr. L. Gooding, Emory University), followed by HRP-labeled secondary Ab, and visualized with ECL substrate (Amersham Life Sciences, Arlington Heights, IL).

Micrococcal nuclease (MNase) sensitivity assays

BALB/3T3 cells were permeabilized as described elsewhere (21). BALB/3T3 cells were used because NIH3T3 cells have a polymorphism in the MCP-1 promoter region that prevents a discernible footprint pattern (9, 16). Cells were grown to ~70% confluency and washed once in a solution containing 150 mM sucrose, 80 mM KCl, 35 mM HEPES (pH 7.4), 5 mM K2HPO4, 5 mM MgCl2, and 0.5 mM CaCl2, followed by permeabilization in the above solution containing 0.5 mg/ml lysolecithin (Sigma-Aldrich, St. Louis, MO). Cells were carefully washed with nuclease digestion buffer (150 mM sucrose, 50 mM NaCl, 50 mM Tris-HCl (pH 7.5), and 2 mM CaCl2) and then incubated with MNase (10 U/ml; Roche Molecular Biochemicals) for 10 min at room temperature in the digestion buffer described above. The MNase solution was removed, Stop solution (20 mM Tris-HCl (pH 8.0), 20 mM NaCl, 20 mM EDTA, 1% SDS, and 600 µg/ml proteinase K) was added, and genomic DNA was purified. Free 5' ends of genomic DNA samples were phosphorylated with T4 polynucleotide kinase and ligation-mediated PCR was conducted using the primer sets previously described for in vivo genomic footprinting (9).

Chromatin immunoprecipitation (ChIP) assays

ChIP assays were performed essentially as described previously (22). One-tenth of each preparation from 4 x 107 cells was used for each ChIP. Immunoprecipitations were performed at 4°C overnight with 5 µg of primary Ab. Antiacetylated H3 and antiacetylated H4 were purchased from Upstate Biotechnology (Lake Placid, NY) and anti-p65, anti-Sp1, and anti-Sp3 were purchased from Santa Cruz Biotechnologies (Santa Cruz, CA). Immune complexes were harvested with protein A-Sepharose beads (60 µl/precipitation) as described. Following immunoprecipitation, washing, and purification of DNA, the samples were dissolved in water and used as templates in real-time PCR as described below.

RNA analysis

Cells were split and grown to ~70% confluency. Total RNA was isolated from cells using the RNAeasy kit (Qiagen, Valencia, CA). Reverse transcription was conducted using the GeneAmp RNA PCR kit (PerkinElmer, Boston, MA) according to the manufacturer’s instructions. Two micrograms of RNA was used per sample and each reaction contained a parallel control with no reverse transcriptase added. One-fortieth of the reverse transcriptase reactions were analyzed by real-time PCR.

Real-time PCR

Quantitative PCR analysis was performed using an iCycler with the optical assembly unit (Bio-Rad, Hercules, CA). For analysis of ChIP assay products, the following previously described primer sets were used to amplify the distal regulatory region, proximal regulatory region, and the middle site of the MCP-1 gene or the GAPDH gene, respectively: JENF-{kappa}B3-1 and JENF-{kappa}B5-2, JE5-9 (9) and JE3-9: 5'-GAGTTGGCTGGTGCTGGTGCTGG; JECHR3: 5'-CTGTTCTCTTCCAAGCATCCCC and JECHR2 5'-CATTTCAGCAGACTCTTGATAA; and MGAP5-3: 5'-TGCACCACCAACTGCTTAG and MGAP3-3: 5'-GATGCAGGGATGATGTTC. SYBR green incorporation into dsDNA was measured as described elsewhere (23). Each primer set was tested on genomic DNA before use and found to produce a single PCR product. All real-time PCR assays were performed in duplicate and averaged. The amount of MCP-1 genomic DNA that coimmunoprecipitated in the ChIP assay was quantitated by comparing the threshold cycle values from each assay to a standard curve generated using genomic DNA and the indicated primer sets. To correct for potential differences in chromatin preparations between samples, the above PCR values were normalized to the amount of chromatin added to each Ab immunoprecipitation reaction. All real-time ChIP assays were performed from three or more independent experiments. The results were averaged and plotted with the SEM. Student t tests were performed to determine the significance between samples.

For RT-PCR, parallel assays with primers directed to MCP-1 and {beta}-actin genes were performed. The results from MCP-1 RT-PCR assays were normalized to results obtained for the corresponding {beta}-actin RT-PCR assays, providing a relative quantitation value. The sequences of the MCP-1 primers are as follows: MCP5-2, 5'-GAGTAGCAGCAGGTGAGTGGGGCGTTA and MCP3–2, 5'-CAGCACCAGCACCAGCCAACTCTCA. Primers for the {beta}-actin RT-PCR, {beta}-actin5-1 and {beta}-actin3-1, were as described by Schmittgen and Zakrajsek (24). All RT-PCR assays were performed three or more times from independently prepared RNA samples. The data were averaged and plotted with the SEM.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
E1a represses MCP-1 induction by TNF

Previous analyses of MCP-1 regulation suggested that the distal and proximal regulatory regions interact either directly or through some intermediate factor(s) (9, 10, 16). Coactivators, such as p300 and CBP, have been shown to coactivate gene transcription by acting as bridges between transcription factors and the basal transcription machinery (reviewed in Ref. 25). The adenovirus E1a protein is known to repress the expression of some cellular genes by inhibiting the function of p300 and CBP (26, 27). Therefore, to determine whether an E1a-sensitive coactivator plays a role in MCP-1 induction by TNF, NIH3T3 cells were either infected or not with adenovirus-expressing E1a. After 24 h of infection, TNF was added. RNA was isolated 4 h later and subjected to quantitative real-time RT-PCR using primers specific to MCP-1 and {beta}-actin cDNA. The results showed that adenovirus infection repressed TNF induction of MCP-1 by >90% (Fig. 2A). The levels of {beta}-actin mRNA, as well as GAPDH mRNA, which was measured by Northern blot, were not altered by adenovirus infection (data not shown). To determine whether this observation was attributable directly to E1a and to rule out any effects of adenovirus infection, the consequence of transiently expressing E1a in NIH3T3 cells was determined. The results showed that E1a alone was able to repress TNF-induced expression of MCP-1. E1a-mediated repression was dose dependent, displaying a 76% reduction in MCP-1 expression at its highest dose (Fig. 2B). The observed dose-dependent repression correlates with the amount of E1a being expressed (Fig. 2C). These data therefore suggest that MCP-1 expression requires the function of a coactivator such as p300 or CBP. Differences in the amount of overall repression between the experiments presented above are most likely because of the differences between adenovirus infection and transfection efficiency of E1a expression plasmids.



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  		FIGURE 2. E1a represses MCP-1 induction by TNF. MCP-1 and {beta}-actin mRNAs were quantitated by real-time RT-PCR from mRNA isolated from NIH3T3 cells that were either infected or not infected with a wild-type strain of adenovirus (A) or transiently transfected with an E1a expression vector (B). Cells were treated with TNF for 4 h or left untreated (Control). The relative quantitation value represents the fold stimulation of MCP-1 mRNA determined from the change in crossover threshold between RT-PCR samples following normalization to the crossover threshold determined for {beta}-actin cDNA. The mock infection sample treatment of TNF was arbitrarily set to 100%. The average of three experiments is shown with the SEM. C, A Western blot for the presence of E1a was performed on equal amounts of whole cell lysates generated from NIH3T3 cells that were transiently transfected with an E1a expression vector.

 
CBP and p300 enhance TNF induction of MCP-1

To investigate further the coactivator requirement for MCP-1 expression, the effect of coactivator overexpression was determined. CBP, p300, or P/CAF expression vectors were transiently cotransfected with a NF-{kappa}B p65 expression vector into NIH3T3 cells. Cotransfection of a NF-{kappa}B p65 expression vector mimics activation by TNF, but at a lower level so that the effects of other gene products can be assessed. RNA was isolated and quantitated for the MCP-1 transcripts by real-time RT-PCR (Fig. 3). CBP and p300 overexpression led to statistically similar and reproducible increases in MCP-1 expression. However, P/CAF overexpression in this system had no observable effect on MCP-1 expression. Expression of the {beta}-actin control gene was not altered during these assays (data not shown). These results indicate that CBP and p300 can function as a coactivator during the induction of MCP-1 by TNF.



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  		FIGURE 3. CBP and p300 enhance TNF induction of MCP-1. RNA was isolated from NIH3T3 cells that were cotransfected with either a NF-{kappa}B p65 expression vector and/or in combination with vectors expressing CBP, p300, or P/CAF. A transfection with empty vector was also performed to provide a background level of MCP-1 expression. Real-time RT-PCR assays were performed, as in Fig. 2, to determine the amount of MCP-1 mRNA produced. The values of the samples transfected with NF-{kappa}B p65 expression vector was arbitrarily set to 1. Values obtained between p300 and CBP were found to be statistically similar.

 
TNF alters chromatin accessibility at the proximal and distal regulatory regions

Studies performed on the expression of the IL-12, IL-4, and IFN-{gamma} genes indicated that chromatin structure and remodeling plays an important role in their expression (12, 28, 29, 30). To determine whether chromatin structure plays a role in the TNF-induced expression of MCP-1, MNase sensitivity assays were performed (Fig. 4). MNase tends to cleave unprotected chromatin DNA at regions that are between nucleosomes (31). Limited MNase digestion was performed on permeabilized BALB/3T3 cells that were pretreated or not treated with TNF. The purified MNase-digested genomic DNA was analyzed by ligation-mediated PCR using primers that amplified the proximal and distal regulatory regions of the MCP-1 gene. The lack of a defined nucleosomal banding pattern (~150–200 bp) in the proximal regulatory region in untreated cells suggests that the nucleosomes may be randomly deposited (Fig. 4A). In the distal region, a strong hypersensitive banding pattern was found 3' to the {kappa}B-2 site. This pattern can be seen on both the template and nontemplate strands, suggesting that there is some order to the nucleosomes (Fig. 4B). Within 30 min of TNF treatment, the MNase cleavage patterns at both the proximal and distal regions became altered. In the proximal regulatory region, the hypersensitive bands between the Sp1 binding site and the start of transcription became less pronounced, while the hypersensitive bands upstream of the Sp1 site maintained their intensity. In the distal regulatory region, the bands between the {kappa}B sites faded, while the bands surrounding these sites maintained their intensity. The less dramatic changes in the distal region may reflect the stabilization of nucleosomes at specific sequences or alternatively the assembly of transcription factor complexes into compact, nuclease-resistant structures.



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  		FIGURE 4. TNF induces changes in overall chromatin structure. Permeabilized BALB/3T3, NF-{kappa}B p65-deficient (p65-/-), and Sp1-deficient (Sp1-/-) cells, which were untreated (0 min) or treated with TNF (5, 30, 120 min), were digested with MNase (10 U/ml). Following the isolation of genomic DNA, ligation-mediated PCR was performed using primers for the proximal or distal regulatory regions of the MCP-1 gene. The start site of transcription is indicated by a single arrow and transcription factor binding sites are indicated by boxes. Vertical arrows indicate the regions where the sensitivity patterns change following TNF treatment. The numbers indicate the region protected with respect to the transcription initiation site. Asterisks indicate bands that were used for loading control. wt, wild type.

 
TNF-induced chromatin structural changes are dependent on NF-{kappa}B p65 and Sp1

To determine whether the TNF-induced changes in chromatin structure detected in the above experiments were dependent on Sp1 or NF-{kappa}B p65, the key regulatory factors in each of their respective regions, MNase hypersensitivity assays were performed on Sp1- and NF-{kappa}B p65-deficient fibroblast cell lines. The MNase digestion patterns observed in the control untreated samples (Fig. 4) from both mutant cell lines were identical to that of the wild-type BALB/3T3 cell line. In contrast, the mutant cell lines displayed limited changes in their MNase sensitivity pattern following TNF treatment (Fig. 4, note vertical lines). These data imply that both NF-{kappa}B p65 and Sp1 were required for the changes in chromatin structure observed during TNF induction and that the observed alterations are involved in the induction process of MCP-1 by TNF.

Factor assembly occurs rapidly in response to TNF

TNF has been shown to rapidly induce a variety of inflammatory-related genes, including MCP-1 (32). Nuclear run-on studies showed that MCP-1 message is induced 56-fold within 30 min of TNF treatment (15). As shown previously (16, 17) and as suggested in the above experiments, MCP-1 induction by TNF requires NF-{kappa}B p65 and Sp1. Although it is presumed that the distal {kappa}B and proximal Sp1 sites previously shown to bind their respective factors in vitro are involved specifically in the regulation of MCP-1 (9, 16, 17), rigorous proof that these factors actually bind these regions in vivo has not been provided. To obtain such data, ChIP assays coupled with real-time PCR quantitation of the precipitated DNA were performed. Abs against p65 and Sp1 were used in ChIP assays (Fig. 5). The results showed that TNF induces the binding of Sp1 to the proximal regulatory region and p65 to the distal regulatory region. Additionally, binding of these factors occurs within 30 min. Both p65 and Sp1 can be detected as early as 10 min after TNF induction (data not shown). The ChIP assays are specific as the proximal region does not bind p65 and the distal region does not bind Sp1. These data suggest that during TNF induction of MCP-1, NF-{kappa}B p65 binds specifically to the distal region while Sp1 binds specifically to the proximal region.



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  		FIGURE 5. TNF induces rapid factor assembly at the MCP-1 promoter. ChIP assays coupled with real-time PCR were performed on NIH3T3, p65-/-, and Sp1-/- cells using Abs specific to p65 and Sp1. The proximal and distal regulatory regions of the MCP-1 gene were analyzed. A no-Ab control was included as well (data not shown). Threshold cycles from real-time PCR were plotted on a standard curve of genomic DNA amplified using either the distal or proximal region primers and normalized to the amount of MCP-1 chromatin added to the initial immunoprecipitation. The results are expressed as fold stimulation of binding over untreated cells. The average of three to four experiments derived from separate chromatin preparations is shown. A Student’s t test demonstrated that the values obtained between p65 or Sp1 and control binding were significant.

 
It was suggested from previous observations that the binding of the transcription factors that bind the proximal and the distal regulatory region occurs in a coordinate manner. To determine whether this is the case in vivo, ChIP assays were performed on Sp1-/- and NF-{kappa}B p65-/- cell lines (Fig. 5). Following TNF treatment, the NF-{kappa}B p65-/- cell line displayed no binding of p65 to the distal regulatory region as expected. Interestingly, Sp1 was also not detected at the proximal region. This suggests that in order for Sp1 to bind, NF-{kappa}B p65 is required. To further investigate the relationship between NF-{kappa}B p65 and Sp1, a similar analysis was performed in the Sp1-/- cell line. As expected, Sp1 was not detected at the proximal regulatory region in this knockout cell line. Surprisingly NF-{kappa}B p65 was detected at the distal region and the level of precipitated DNA was TNF dependent. These results suggest a sequence of events that occurs during TNF induction of MCP-1, where NF-{kappa}B p65 binds to its site in the distal regulatory region before Sp1 binding in the proximal region.

Inhibition of histone deacetylation is sufficient to activate MCP-1 gene expression

The coactivators CBP and p300 contain intrinsic histone acetyltransferase (HAT) activity, which has been suggested as one of the ways in which these coactivators enhance transcription (33). It has been previously shown that TSA, a histone deacetylase inhibitor, induces MCP-1, suggesting that histone acetylation alone can induce MCP-1 expression (10). Coupled with this activation was the in vivo assembly of the proximal but not the distal regulatory region. To determine whether TSA treatment results in the acetylation of histones at the regulatory regions of MCP-1, ChIP assays using specific antiacetylated histone H3 or antiacetylated histone H4 Abs were performed on NIH3T3 cells that were treated or not treated with TSA (Fig. 6). The results showed that both histone H3 and H4 became acetylated compared with the untreated control. This acetylation is seen in both the distal and the proximal regulatory region at 30 min and is maximal at 60 min. Changes in histone acetylation of the control GAPDH gene were minimal over this time course. Together with previous expression data (10), these results link histone acetylation of the MCP-1 regulatory regions with its expression.



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  		FIGURE 6. TSA treatment of NIH3T3 cells leads to histone acetylation. Real-time ChIP assays were performed on the proximal and distal regulatory regions of the MCP-1 gene on chromatin isolated from NIH3T3 cells before and after TSA treatment. Abs specific to acetylated histones H3 and H4 were used for both the MCP-1 and GAPDH genes. The samples are expressed as fold induction over the control untreated cells as described for Fig. 5.

 
TNF induces histone H3 and H4 acetylation at both the proximal and distal regulatory region

To determine whether TNF induction also results in histone acetylation at the MCP-1 locus, ChIP assays were performed on NIH3T3 cells that were treated or not treated with TNF (Fig. 7). As above, Abs specific for acetylated histone H3 or H4 were used. The results showed a maximum of 2- to 3-fold increase in acetylation of both histone H3 and H4 at the distal regulatory region. At the proximal region, histone H3 acetylation increases by 2.4-fold at 30 min and declines to near baseline by 60 min following TNF treatment. In contrast, histone H4 acetylation continues to increase to a maximum of ~6-fold at 1 h following TNF treatment. To determine whether histone acetylation changes are specific to the proximal and the distal region or are continuous throughout the MCP-1 promoter, a randomly chosen region located between the two regulatory modules (-1277 to -1061) was examined. Interestingly, acetylation of both histone H3 and H4 increased 7- to 10-fold in this "middle" region. Histone H3 and H4 acetylation of the GAPDH gene were unchanged during the TNF treatment, indicating that the modest changes observed are specific to the MCP-1 gene (Fig. 7). These results demonstrate that induction of MCP-1 by TNF results in increased histone acetylation not only at the proximal and the distal regulatory regions but also at the sequences bridging these regions. Moreover, changes in histone H4 acetylation at the proximal region appears to be preferential over that of H3.



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  		FIGURE 7. Histone acetylation of the proximal and distal regulatory regions of the MCP-1 gene requires NF-{kappa}B p65. A, Using anti-acetyl-histone H3 and H4 Abs, real-time ChIP assays were performed on NIH3T3, NF-{kappa}B p65-deficient, and Sp1-deficient cells before and after TNF treatment. Primers specific to the MCP-1 proximal and distal regulatory regions, the middle sequence, and the GAPDH gene were used and analyzed as in Fig. 5. The average nanograms of precipitated chromatin for acetylated histone H3 in control untreated cells for the proximal, middle, and distal regulatory regions were 17, 7, and 7, respectively. The average nanograms of precipitated chromatin for acetylated histone H4 in control untreated cells for the proximal, middle, and distal regulatory regions were 1.6, 6, and 1.2, respectively. The difference in absolute quantity of precipitated MCP-1 DNA from control cell experiments among NIH3T3, p65-/-, and Sp1-/- cell lines was statistically insignificant. B, A schematic of the MCP-1 promoter indicating relative positions of primers used in A.

 
Histone acetylation at the proximal regulatory region is dependent on NF-{kappa}B p65

To determine whether the histone acetylation of the MCP-1 gene by TNF is dependent on Sp1 or NF-{kappa}B p65, ChIP assays were performed on Sp1-/- and NF-{kappa}B p65-/- cell lines (Fig. 7). In response to TNF, the NF-{kappa}B p65-/- cell line displayed no increase in histone acetylation at the proximal or distal regulatory region. However a small increase was observed in the middle region. This suggests that the changes in histone acetylation at the proximal, distal, and middle region following TNF treatment are greatly influenced by the transcription factor NF-{kappa}B p65. In contrast, a unique pattern of acetylation following TNF induction was observed in the proximal regulatory region in the Sp1-/- cell line. In this study, TNF treatment induced a 7-fold increase in H4 acetylation at 30 min, which was reduced to 3-fold at 60 min. Histone H3 acetylation remained unchanged. The middle region sequence showed both an increase in histone H3 and H4 acetylation (6.7- and 9.9-fold, respectively) at 30 min, which decreased (3.8- and 5.2-fold, respectively) at 60 min. This result suggests that histone acetylation at the proximal regulatory region and the middle region is not due to Sp1 but rather due to the other factors that assemble at the proximal regulatory region or to factors that assemble at the distal region. The binding of NF-{kappa}B p65 at the distal regulatory region in the Sp1-/- cells correlates with the increase of H4 acetylation in this cell line. Similar to the NF-{kappa}B p65-deficient cells, the distal regulatory region in Sp1-/- cells failed to increase its level of histone acetylation in response to TNF. These results and the result from Fig. 5 are consistent with the hypothesis that the distal and proximal regulatory regions communicate to increase transcription factor binding either directly or through the alteration and configuration of the local chromatin structure.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we have examined the changes in chromatin structure that occur during the induction of the MCP-1 gene by TNF. The results suggest potential roles for the key factors as well as a possible order or kinetics of their action. The model of activation begins in cells that have not been stimulated to express their MCP-1 gene. Before activation of the MCP-1 gene, the distal regulatory region is accessible and binding of NF-{kappa}B to that region is permitted. Following TNF treatment, NF-{kappa}B is translocated to the nucleus and can bind the distal regulatory region {kappa}B sites. Once bound, NF-{kappa}B recruits a coactivator or coactivator complex with HAT activity, such as CBP/p300. Interactions between NF-{kappa}B and CBP have been documented in other systems (34, 35). The recruited coactivator-HAT complex acetylates histone H4 at the proximal region. Because TSA alone can induce expression and assembly of the proximal regulatory region (10), the model predicts that the initial acetylation events result in the opening of chromatin structure, allowing assembly of the proximal regulatory factors, including Sp1. The binding of Sp1 initiates and stabilizes the assembly of the factors at this region as well as the factors at the distal regulatory region. This part of the model is supported by in vivo footprinting data that revealed reduced occupancy of the distal regulatory region in Sp1-deficient cells (17), and here from ChIP using Abs against Sp1 and NF-{kappa}B p65 in Sp1-/- and NF-{kappa}B p65-/- cell lines. Concurrent with or following the binding of Sp1 to the proximal region, the proximal region H3 histones and both H3 and H4 histones at the distal region become acetylated. Because Sp1-deficient cells display H4 acetylation at the proximal region, it is likely that histone H3 acetylation in the proximal region is mediated by another coactivator-HAT complex than the one recruited by NF-{kappa}B. Lastly, the combined activities of the DNA-binding factors, coactivators, and HATs result in the recruitment of the basal transcription machinery and the activation of transcription.

The current data are consistent with a role for coactivators in bridging the activities between the distal and proximal regulatory regions, which are separated by a significant distance of 2.2 kb. The sensitivity of MCP-1 expression to E1a suggests that CBP or p300 or complexes that contain these factors are involved in regulation of MCP-1. Because the coactivators CBP and p300 contain multiple binding sites for a vast number of transcription factors, which includes NF-{kappa}B (34, 35), and can interact with components of the general transcription factors, they can interact with the components in the MCP-1 regulatory system. This interpretation poses the question of how the coactivators physically bridge the regions? One way to view this problem is to consider the distances in the terms of chromatin. Two kilobases separate the proximal and distal regions. If packaged into nucleosomes, this region would represent ~10 nucleosomes and the linear distance between the two regions could be substantially reduced such that coactivators might be able to directly bridge the distal and proximal regions. Alternatively, the finding of histone acetylation in the middle region suggests an intriguing mechanism in which TNF-dependent accessibility of the proximal region could result from the progressive histone modification of nucleosomes from the distal to the proximal region, leading to increased DNA accessibility across the 5' flanking DNA of MCP-1.

Methylation, phosphorylation, and acetylation are among the posttranslational modifications known to occur on the N-terminal tails of histones. These modifications have been correlated with a number of biological events (36). In the case of acetylation, hyperacetylation of lysine residues correlates with gene expression, while hypoacetylation correlates with gene silencing and heterochromatic chromosomal regions. The balance between HAT and histone deacetylase activities is important in maintaining the steady-state levels of histone acetylation in vivo (37). Previously, we showed that TSA can induce MCP-1 expression (10). In this previous work, TSA induced occupancy at the proximal regulatory region, suggesting that the initially closed region was now accessible to factors like Sp1. Although not unexpected, here it was found that TSA did in fact lead to acetylation of histones H3 and H4 at both the proximal and distal regulatory regions, supporting the original interpretation. TNF treatment also resulted in acetylation of histones H3 and H4 at the distal and proximal regions. However, whereas TSA treatment resulted in similar fold increases in both histones H3 and H4 acetylation, TNF-mediated acetylation was less in magnitude and differed in specificity. At the distal regulatory region, there was only a moderate increase in both H3 and H4 acetylation. In contrast, the proximal regulatory region showed a substantial increase in histone H4 acetylation while H3 acetylation was moderate and transient. The differences in acetylation levels between TSA and TNF are likely to reflect the exact specificity of lysines that are acetylated. The Abs used in this study were generated to recognize Lys9 and 14 of histone H3 and Lys5, 8, 12, and 16 of histone H4. Thus, the ability to distinguish between whether one or multiple lysines were acetylated was not possible. By inhibiting histone deacetylases, TSA should allow a general increase in acetylation of all available lysines than would occur following TNF treatment, which would be expected to have a discrete acetylation pattern that was dependent on which HATs were recruited. For example, CBP/p300 have been shown to modify Lys14,18 at histone H3 and Lys5,8 at histone H4 (38). Thus, in the current system these modifications would be expected to occur and be indicative of an active promoter complex. Although the "histone code" is not yet solved, it has been proposed that the different modifications are associated with different levels of activity of the chromatin and potentially the rate at which a region is open or transcribed (39).

The remodeling and the opening of chromatin are important aspects in the control of inducible gene transcription (40, 41). Studies done on a number of genes showed a constant and orderly positioning of nucleosomes, which upon activation are repositioned, allowing an open chromatin structure (29, 42). This was not the case with MCP-1 since MNase sensitivity studies suggested that the nucleosome positioning was not strictly ordered. However, like other inducible genes (12, 28, 29, 30), specific changes occurred in response to the stimulus. In this study, TNF treatment induced a specific change in the nuclease sensitivity pattern at the proximal regulatory region in the vicinity of the transcriptional start site. The results could imply that during TNF treatment a nucleosome is positioned near these sequences. Thus, TNF seems to switch the nucleosome positioning from a random to a more orderly position. This change required both NF-{kappa}B p65 and Sp1. Another interpretation is that MNase is detecting the assembly of a preinitiation complex at the promoter of the MCP-1 gene. This interpretation could also describe the dynamics of assembly of the distal regulatory region factors into a stable complex as well. Regardless of the interpretation, TNF treatment and the assembly of the factors alters the sensitivity of the both the proximal and distal regions to MNase. This rearrangement is accomplished by factor assembly and is concurrent with histone acetylation and expression of the gene.


   Acknowledgments
 
We thank Dr. D. Reines and members of the laboratory for helpful suggestions and discussions of this work.


   Footnotes
 
1 This work was supported by National Institutes of Health Grant CA74271. Back

2 Address correspondence and reprint requests to Dr. Jeremy M. Boss, Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, GA 30322. E-mail address: boss{at}microbio.emory.edu Back

3 Abbreviations used in this paper: MCP-1, monocyte chemoattractant protein 1; CBP, CREB-binding protein; ChIP, chromatin immunoprecipitation; HAT, histone acetyltransferase; MNase, micrococcal nuclease; P/CAF, p300/CBP-associated factor; TSA, trichostatin A; PDGF, platelet-derived growth factor. Back

Received for publication October 23, 2002. Accepted for publication February 6, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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