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Mol Cell Biol. 2010 June; 30(12): 2874–2886.
Published online 2010 April 19. doi:  10.1128/MCB.01527-09
PMCID: PMC2876687

Repression of IP-10 by Interactions between Histone Deacetylation and Hypermethylation in Idiopathic Pulmonary Fibrosis[down-pointing small open triangle]

Abstract

Targeted repression of a subset of key genes involved in tissue remodeling is a cardinal feature of idiopathic pulmonary fibrosis (IPF). The mechanism is unclear but is potentially important in disease pathogenesis and therapeutic targeting. We have previously reported that defective histone acetylation is responsible for the repression of the antifibrotic cyclooxygenase-2 gene. Here we extended our study to the repression of another antifibrotic gene, the potent angiostatic chemokine gamma interferon (IFN-γ)-inducible protein of 10 kDa (IP-10), in lung fibroblasts from patients with IPF. We revealed that this involved not only histone deacetylation, as with cyclooxygenase-2 repression, but also histone H3 hypermethylation, as a result of decreased recruitment of histone acetyltransferases and increased presence of histone deacetylase (HDAC)-containing repressor complexes, histone methyltransferases G9a and SUV39H1, and heterochromatin protein 1 at the IP-10 promoter, leading to reduced transcription factor binding. More importantly, treatment of diseased cells with HDAC or G9a inhibitors similarly reversed the repressive histone deacetylation and hypermethylation and restored IP-10 expression. These findings strongly suggest that epigenetic dysregulation involving interactions between histone deacetylation and hypermethylation is responsible for targeted repression of IP-10 and potentially other antifibrotic genes in fibrotic lung disease and that this is amenable to therapeutic targeting.

Idiopathic pulmonary fibrosis (IPF) is a debilitating lung disease characterized by exaggerated extracellular matrix deposition and aggressive structural remodeling. The prognosis of IPF is poor, with less than a 50% 5-year survival rate, the etiology of the disease remains unknown, and the main therapies of antiinflammatory corticosteroids and immunosuppressive agents are ineffective (6, 21). It is therefore essential that the mechanisms that orchestrate this disease are defined to allow the development of novel, targeted, and effective therapeutic options. Angiogenesis is critical in physiological and pathological processes such as wound healing, tumor growth, and inflammation (32). There is evidence that aberrant angiogenesis may be an important factor in supporting tissue remodeling and contribute to IPF in a manner similar to that seen in tumorigenesis (39). Angiogenesis is a highly coordinated process that is regulated by an opposing balance between locally produced angiogenic and angiostatic molecules. However, an imbalance in the levels of angiogenic and angiostatic chemokines that favors net angiogenesis has been shown in animal models and tissue specimens from patients with IPF (11-14). The CXC chemokine gamma interferon (IFN-γ)-inducible protein of 10 kDa (IP-10; CXCL10) is a potent chemoattractant for mononuclear leukocytes and a strong inhibitor of angiogenesis (40). Recent studies have demonstrated that lung tissues from IPF patients and isolated fibroblasts from IPF patients (F-IPF) constitutively express less IP-10 and more interleukin-8 (IL-8), thereby inducing greater angiogenic activity than tissues from control subjects and fibroblasts from nonfibrotic lungs (F-NL) (12). Furthermore, lung tissue from a murine model of bleomycin-induced pulmonary fibrosis demonstrates a significant decrease in IP-10, which is inversely correlated to total lung collagen and a greater angiogenic response than that from controls (13). Collectively, these findings suggest that IP-10 is an important regulatory molecule in angiogenesis as well as fibroblast migration and proliferation. Therefore, repression of IP-10 may play a key role in the aberrant tissue remodeling and the development of IPF and other fibrotic diseases. However, the molecular mechanisms for repressed IP-10 expression in IPF have not been explored.

The 5′-flanking promoter region of human IP-10 contains multiple regulatory elements, including two interferon-stimulated responsive element (ISRE), two signal transducer and activator of transcription (STAT), two CCAAT/enhancer-binding protein β (C/EBP-β), and two nuclear factor κB (NF-κB)-binding sites (38). Induction of IP-10 in response to cytokines is dependent primarily on transcription (28, 29), which is critically governed by various transcription factors in a cell-type-specific and stimulus-specific manner. IFN-induced IP-10 transcription is characteristically mediated by the transcription factors IFN regulatory factor 1 (IRF-1), p48, and STAT-1α via ISRE, whereas IL-β- and tumor necrosis factor alpha (TNF-α)-induced IP-10 transcription is typically mediated by NF-κB (5, 23, 46).

Posttranslational modifications of histone proteins in chromatin structure play a central role in the epigenetic regulation of gene transcription. Histone acetylation and methylation are the two most common and best-characterized modifications that function as specific transcription regulators. Histone acetylation on lysine residues by histone acetyltransferases (HATs) and histone deacetylation by histone deacetylases (HDACs) are associated with transcriptional activation and repression, respectively. Several transcription coactivators, including p300, CREB binding protein (CBP), p300/CBP-associated factor (PCAF), and general control nonderepressible 5 (GCN5), have been found to possess intrinsic HAT activity (8), and at least 13 HDACs have been identified. HDAC complexes are generally recruited to transcription factors by “bridging” factors, such as nuclear receptor corepressor (NCoR), co-RE1-silencing transcription factor (CoREST), and mammalian SIN3 homolog A (mSin3a). NCoR exists in core repression complexes with HDAC3; the CoREST complex and the mSin3a complex contain, in addition to CoREST and mSin3a, respectively, HDAC1 and HDAC2 (43, 47), providing a mechanism to mediate the repression of transcription. Histone methylation of lysine residues can be either mono-, di-, or trimethylated, leading to different functional consequences depending on the site of methylation and number of methyl groups added (34). Heterochromatin is specifically enriched with trimethylated lysine 9 on histone H3 (H3K9me3) and H3K27me3, whereas H3K9me1, H3K4me3, etc., are found in euchromatin (36). Several histone methyltransferases (HMTs) have been characterized, including SUV39H1 and G9a (33, 41). G9a is able to methylate lysine 27 as well as mono- and dimethylate lysine 9 in H3, compared with SUV39H1, which is only able to trimethylate lysine 9 (34, 41). H3K9me3 provides a specific binding site for heterochromatin protein 1 (HP1), including HP1α, -β, and -γ (3, 18), whose association with other HP1 molecules and the recruitment of additional HMTs, DNA methyltransferases (Dnmts), and HDACs spread heterochromatin and repress gene transcription (15). Altered gene expression plays a causal role in certain human diseases, and aberrant epigenetic modifications of the chromatin and direct interactions between them may result in the altered expression (16, 25). We have previously reported that defective histone acetylation is responsible for the repression of the antifibrotic cyclooxygenase-2 (COX-2) gene (7). So far, epigenetic regulation of IP-10 gene transcription has not been explored in detail, and whether alterations in histone acetylation/deacetylation and methylation result in repression of IP-10 transcription in IPF is unknown. In this study, we extended our study to IP-10 and aimed to compare lung fibroblasts obtained from patients with IPF (F-IPF) with fibroblasts from nonfibrotic lungs (F-NL) to explore the role of histone deacetylation and methylation in IP-10 repression in F-IPF in response to IFN-γ and IL-β. We report here that IP-10 gene expression was defective in F-IPF compared with F-NL due to histone deacetylation and hypermethylation as a result of decreased recruitment of HATs and increased recruitment of the HDAC-containing transcriptional repressor complexes NCoR, CoREST, and mSin3a, the H3 lysine 9-specific HMTs G9a and SUV39H1 and the H3K9me3 binding protein HP1 to the IP-10 promoter. These findings indicate that interactions between histone deacetylation and hypermethylation at the IP-10 promoter are responsible for the reduced IP-10 expression in IPF and that HDAC and G9a inhibitors could restore IP-10 expression by reorganizing the heterochromatin-associated proteins at the IP-10 promoter.

MATERIALS AND METHODS

Fibroblast cell culture.

F-IPF and F-NL from the explanted lungs of patients with IPF who underwent lung transplantation at the University of Pittsburgh Medical Center and from normal lung tissues obtained from organ donors under a protocol approved by the University of Pittsburgh Institutional Review Board (31) were cultured in Dulbecco's modified Eagle's medium (DMEM; Sigma, Dorset, United Kingdom) supplemented with 10% fetal bovine serum (Seralab, West Sussex, United Kingdom), penicillin, streptomycin, and an antimycotic agent (Sigma) as described previously (31). F-IPF and F-NL cells (six cell lines each) were used at passages 5 and 6, respectively, to ensure purity and maintain the differences present in vivo. Comparisons of the responses of F-IPF and F-NL to IL-1β and IFN-γ (R & D Systems, Abingdon, United Kingdom) were used throughout the study. The cells were growth arrested in serum-free medium for 24 h prior to stimulation with either recombinant human IL-1β (1 ng/ml) or IFN-γ (10 ng/ml) in serum free medium. At the indicated time points, the cells were harvested for subsequent analysis.

Quantitative real-time RT-PCR.

Cells were cultured and treated in 6-well plates, and cell lysates were collected at the times indicated in the text. Total RNA was isolated using the RNeasy minikit (Qiagen, West Sussex, United Kingdom) following the manufacturer's protocol. A total of 2 μg of total RNA was then reverse transcribed. The cDNA was then subjected to real-time reverse transcription-PCR (RT-PCR) analysis with the MXPro 3000 detection system (Stratagene, Amsterdam, Holland) using Excite master mix and Sybr green (Biogene, Cambridge, United Kingdom) and the following primer sequences: IP-10 sense, 5′-GAAATTATTCCTGCAAGCCAATTT-3′; and antisense, 5′-TCACCCTTCTTTTTCATGTAGCA-3′. β2-Microglobulin (β-2M) was used as the housekeeping gene, and its expression was determined using the following primer sequences: β-2M sense, 5′-AATCCAAATGCGGCATCT-3′; and antisense, 5′-GAGTATGCCTGCCGTGTG-3′. IP-10 mRNA expression was normalized to housekeeping gene expression by dividing the mean IP-10 value by the mean β-2M value as we described previously (7, 27).

IP-10 protein measurement.

An enzyme-linked immunosorbent assay (ELISA; R&D Systems, Oxon, United Kingdom) was used to measure IP-10 protein release from cell culture supernatants. The ELISA was performed according to the manufacturer's instructions.

Transient transfection and reporter gene assays.

The 5×NF-κB sequence reporter construct was kindly donated by Robert Newton (University of Calgary, Alberta, Canada) and was described previously (1); the 5×ISER sequence reporter construct was kindly provided by David Proud (University of Calgary, Alberta, Canada) and was described previously (48). The internal control Renilla luciferase reporter construct pRL-SV40 was obtained from Promega (Southampton, United Kingdom). Transfections were performed in cells at 75% confluence as performed previously (42). Briefly, 0.25 μg of the testing plasmid DNA and 0.25 ng of the internal control Renilla luciferase DNA were added to each well (24-well plate) using a 1:1 ratio of DNA/Transfast in serum-free medium. Cells were incubated with the transfection mix for 1 h before DMEM containing 10% fetal calf serum (FCS) was added to a volume of 1 ml, and cultures were left for a further 4 h. The cells were then serum starved overnight prior to stimulation with either IL-1β (1 ng/ml) or IFN-γ (10 ng/ml) for 2 h. The cells were then washed in phosphate-buffered saline (PBS) and harvested in 100 μl of reporter lysis buffer (Promega). Firefly luciferase activity from the test DNA constructs and Renilla luciferase activity from the internal control reporter were measured using the dual-luciferase reporter assay system (Promega) with a MicrolumatPlus LB96V automatic microplate reader (Berthold Technologies, Herts, United Kingdom). Relative luciferase activity was obtained by normalizing the firefly luciferase against the Renilla luciferase activity. Fold change was then calculated by comparing treated groups to the untreated controls. Data from Renilla luciferase activity showed that there was no difference in transfection efficiencies between F-NL and F-IPF.

ChIP and re-ChIP assays.

The chromatin immunoprecipitation (ChIP) assay was performed using reagents and protocols from the ChIP-IP express kit (Active Motif, Rixensart, Belgium) as described previously (7). Antibodies against NF-κB p65 (New England Biolabs, Herts, United Kingdom), IRF-1, p48, CBP, PCAF, GCN5, CoREST, NCoR, mSin3a, and HP1 (which recognizes HP1α, -β, and -γ; Santa Cruz Biotechnology), acetylated histones H3 and H4, total histones H3 and H4, and trimethylated histone H3 lysine 9 (H3K9me3) (Millipore Corporation, Temecula, CA) and the respective control antibodies were used for immunoprecipitation (IP). Purified DNA from the immunoprecipitated antibody-protein-chromatin complexes was subject to real-time PCR amplification with the following primers designed specifically for the IP-10 promoter region (positions −224 to −90), which spanned both the NF-κB and ISRE binding sites: forward, 5′-TTTGGAAAGTGAAACCTAATTCA-3′; and reverse, 5′-AAAACCTGCTGGCTGTTCCTG-3′. The amounts of IP-10 promoter DNA that were present in the bound (immunoprecipitated) fractions were calculated relative to the input control by using the 2−ΔΔCT method, where ΔΔCT is the difference between the threshold cycle (CT) for the bound fraction and the CT for the input fraction. The associations of acetylated histones H3 and H4 and H3K9me3 with the IP-10 promoter DNA were further normalized to the association of total histones H3 and H4 with the IP-10 DNA. To determine the specificity of the ChIP assay, distal controls upstream and downstream of the IP-10 promoter were applied to analyze changes in histone acetylation and histone methylation with primers specifically designed for different regions of the IP-10 gene: forward, 5′-ATGCTTCCCATTTGTTAGCG-3′, and reverse 5′-ATGCAAAGACAGCGTCCTCT-3′, for the region −12061 to −11808; forward, 5′-GAAGGATCCCTCCATTGTCA-3′, and reverse, 5′-GTTGCTTGGGGTAAATGGAA-3′, for the region −1716 to −1471; forward, 5′-CTCCTGAAAGGCCCATCATA-3′, and reverse, 5′-CAGGTTTCTGGTTTGGGAAA-3′, for the region +2527 to +2961; and forward, 5′-GGGCCACACTTGTGAAAACT-3′, and reverse, 5′-ATTCTCTCAGGAAGCAGGCA-3′, for the region +11337 to +11644. The amount of IP-10 promoter DNA that was present in the bound (immunoprecipitated) fractions was then calculated and normalized to the association of total histones H3 and H4 with the IP-10 DNA. Basal histone H3 and H4 acetylation was observed in almost all regions in untreated F-NL cells. After IL-1β treatment, marked increase of histone H4 acetylation was only observed in the minimum IP-10 promoter region, whereas increase of histone H3 acetylation was observed in the minimum promoter and the surrounding region (−1716 to −1471 and +2527 to +2961) but not the distal region of the gene. Constitutive and IL-1β-induced H3K9Me3 was also only observed in the IP-10 promoter region (data not shown). The results indicate that cytokine-induced histone modifications occur mainly at the IP-10 promoter region.

To analyze p65 binding to the MCP-1 promoter by ChIP assay, the following specific primers spanning the regulatory region of the human MCP-1 promoter containing the NF-κB and AP-1 binding sites were used: forward, 5′-CCCATTTGCTCATTTGGTCTCAGC-3′; and reverse, 5′-GCTGCTGTCTCTGCCTCTTATTGA-3′.

The re-ChIP assay was conducted by using a re-ChIP-IT express kit (Active Motif) according to the manufacturer's instructions. After the first IP using antibody against HP1, the immunoprecipitated chromatin was removed from the magnetic beads in a buffer which prevents the majority of the first antibody from participating into the second IP reaction. The chromatin was desalted, and a second ChIP step was performed using specific antibodies against NCoR, CoREST, and mSin3a (Santa Cruz). Purified DNA from the sequentially immunoprecipitated antibody-protein-chromatin complexes was analyzed by real-time PCR as described above.

HDAC and HAT activity assay.

Nuclear protein extraction from F-NL and F-IPF cells was performed using the CelLytic NuCLEAR extraction kit according to the manufacturer's instructions (Sigma). Global HDAC activity from 30 μg of the cell nuclear extract was assayed in a microtiter plate according to the manufacturer's instructions (Millipore, Temecula, CA) as described previously (7). HDAC activity associated with specific proteins was conducted with 100 μg of nuclear extracts using the Nuclear Complex co-IP kit (Active Motif) according to the manufacturer's instructions. Briefly antibodies against HP1, CoREST, NCoR, and mSin3a and their respective control antibodies (Santa Cruz) were used for IP. The antibody-protein complexes were retrieved with protein G magnetic beads and washed five times with complete co-IP wash buffer and resuspended in 30 μl of HDAC assay buffer. HDAC activity associated with the immunoprecipitates (IPs) was assayed as described above. Global HAT activity from 50 μg of the cell nuclear extract was assayed in a microtiter plate precoated with reconstituted histone H3 or histone H4 in the presence of acetyl coenzyme A (acetyl-CoA) for 1 h according to the manufacturer's instructions (Millipore). Reference standards were prepared for unmodified and acetylated histone H3 and histone H4 to generate standard curves. Substrate reactions were measured with a plate reader at a wavelength of 450 nm and 570 nm. The 570-nm values were subtracted from the 450-nm values to remove any well-to-well plate variation.

Western blotting.

G9a and SUV39H1 protein expression in resting and IL-1β-stimulated cells was examined with 30 μg of nuclear protein by Western blotting as previously reported (27), using polyclonal antibody for G9a (78 kDa) (Santa Cruz) and monoclonal antibody for SUV39H1 (45 kDa) (Santa Cruz). Lamin A/C (69/62 kDa) was used as a loading control.

HDAC and G9a inhibitor study.

To assess the role of HDACs and G9a HMT in IP-10 repression in F-IPF, one general HDAC inhibitor and one G9a inhibitor were used. The HDAC inhibitor LBH589 (panobinostat) was generously provided by Peter Atadja (Novartis Pharmaceuticals). The G9a HMT-specific inhibitor BIX-01294 {(2-(hexahydro-4-methyl-1H-1,4-diazepin-1-yl)-6,7-dimethoxy-N-[1-(phenylmethyl)-4-piperidinyl]-4-quinazolinamine trihydrochloride hydrate} (17) was purchased from Sigma. F-NL and F-IPF cells were treated without or with BIX-01294 (100 nM), LBH589 (10 nM), or both in medium with serum for 2 days before they reached confluence and then treated without or with the inhibitors in serum-free medium for 1 day before being incubated without or with IL-1β (1 ng/ml) in the presence or absence of the inhibitors for 4 and 24 h. Samples were then collected for analyses of histone H3 and H4 acetylation, H3K9me3, and HP1 association with the IP-10 promoter by ChIP assay, IP-10 mRNA expression by real-time RT-PCR, and IP-10 protein expression by ELISA.

Statistical analysis.

Data were expressed as means ± standard errors of the means (SEM) from three separate experiments (3 different cell lines) performed in duplicate or triplicate. Student's t test was conducted on IP-10 mRNA expression and protein production data to determine significant differences using GraphPad Prism 4; P values of <0.05 were regarded as statistically significant.

RESULTS

Repressed IP-10 protein and mRNA expression in F-IPF.

To confirm that IP-10 protein and mRNA expression was reduced in F-IPF, we first treated both F-NL and F-IPF with IFN-γ (10 ng/ml) and IL-1β (1 ng/ml) for 24 h and measured IP-10 protein in culture medium by ELISA. We found that IP-10 protein was undetectable in unstimulated F-NL and F-IPF. Both IFN-γ and IL-1β induced marked increases in levels of IP-10 protein compared with unstimulated cells in F-NL but failed to induce meaningful IP-10 protein expression in F-IPF; the difference between F-NL and F-IPF was significant (Fig. (Fig.11 A). To assess whether IP-10 mRNA expression was also impaired in F-IPF, we treated both F-NL and F-IPF with IFN-γ and IL-1β for 4 h and measured IP-10 mRNA by real-time RT-PCR. As shown in Fig. Fig.1B,1B, IL-β and IFN-γ induced a small increase in IP-10 mRNA in F-IPF compared with untreated cells. In contrast, both cytokines induced marked increases in IP-10 mRNA in F-NL; the difference between F-NL and F-IPF was significant. The results thus confirmed that these F-IPF cells exerted significantly repressed IP-10 expression in response to both IFN-γ and IL-1β and were suitable for the proposed study to explore the underlying molecular mechanisms.

FIG. 1.
Cytokine-induced IP-10 protein and mRNA expression is repressed in F-IPF. Confluent and serum-starved F-NL and F-IPF cells in 24-well plates were incubated without or with IFN-γ (10 ng/ml) or IL-β (1 ng/ml) for 24 h (A) and 4 h (B) prior ...

Unaltered transcription factor activation in F-IPF.

As IL-1β-induced IP-10 transcription is typically mediated by NF-κB, whereas IFN-γ-induced IP-10 transcription is usually mediated by transcription factors IRF-1, p48, and STAT-1α via ISRE, we went on to study whether there was any difference between F-NL and F-IPF cells in the activation by IL-1β and IFN-γ of transcription factors that bind to NF-κB and ISRE. We transiently transfected F-NL and F-IPF cells with a luciferase reporter construct containing 5 copies of the consensus NF-κB or ISRE sequence. Following stimulation with IFN-γ, there was no induction of NF-κB luciferase activity, but a marked increase in ISRE luciferase activity was observed in both F-NL and F-IPF cells (Fig. 2A and B). In contrast, when IL-1β was used as the stimulus, marked increases in both NF-κB and ISRE luciferase activity were observed in both F-NL and F-IPF cells (Fig. 2A and B). However, no significant difference was observed between F-NL and F-IPF cells. The results suggest that the effect of IL-1β, but not IFN-γ, is mediated through the NF-κB binding site and that ISRE is implicated in both IFN-γ- and IL-1β-induced IP-10 expression. We also analyzed NF-κB p65 nuclear translocation by immunocytochemistry after treatment with IFN-γ and IL-1β in both F-NL and F-IPF and observed that IL-1β, but not IFN-γ, caused p65 nuclear translocation in both F-NL and F-IPF cells and that there was no difference in the speed and amount of p65 translocated to the nucleus between F-NL and F-IPF cells (data not shown). Altogether, the results strongly suggest that there is no difference in the activation of the transcription factors required for IP-10 gene transcription between F-IPF and F-NL.

FIG. 2.
Cytokine-induced transcription factor activation is not altered in F-IPF. Seventy-five percent confluent F-NL and F-IPF in 24-well plates were cotransfected with Renilla luciferase internal control reporter construct (2.5 ng/well), and constructs containing ...

Impaired native binding of transcription factors to the IP-10 promoter in F-IPF.

As no difference was observed between F-NL and F-IPF in the activation of transcription factors that bind to NF-κB and ISRE by reporter gene assays, transcription factor activation therefore could not contribute to the repressed IP-10 expression in IPF. We then went on to investigate whether the binding of transcription factors to the IP-10 promoter in the native chromatin environment was different in F-IPF from that in F-NL by performing the ChIP assay. We looked at p65 binding to the NF-κB regulatory element and IRF-1 and p48 binding to the ISRE regulatory element on the IP-10 promoter. After treatment of the cells with IL-1β (1 ng/ml) and IFN-γ (10 ng/ml) for up to 4 h, PCR amplifications were conducted on a fixed amount of antibody-immunoprecipitated DNA with the specific primer pairs encompassing the region from position −224 to −90 of the human IP-10 promoter. Immunoprecipitates derived from F-NL cells with an antibody to p65, IRF-1, or p48 resulted in different patterns of enrichment of the IP-10 promoter DNA in a time- and stimulus-specific manner after IL-1β and IFN-γ treatment (Fig. 3A and C to E). IL-1β induced a maximum of between a 2- and 4-fold increase of p65, IRF-1, and p48 binding to the IP-10 promoter at 2, 0.5, and 2 h after stimulation, respectively, in F-NL, and the effect decreased thereafter (Fig. (Fig.3A,3A, C, and E). IFN-γ induced an increase of between 2- and 13-fold of p48 and IRF-1 binding to the IP-10 promoter at 2 and 0.5 h after stimulation, respectively, in F-NL, and the effect remained relatively stable for p48 but decreased for IRF-1 thereafter (Fig. 3F and D). In contrast, IL-1β-induced p65 (Fig. (Fig.3A)3A) and IL-1β- and IFN-γ-induced IRF-1 (Fig. 3C and D) and p48 (Fig. 3E and F) binding to the IP-10 promoter was markedly lower in F-IPF compared to F-NL. We also analyzed p65 binding to the promoter of monocyte chemoattractant protein 1 (MCP-1), an NF-κB-driven gene whose expression was increased in IPF (2). We found that, in contrast to the decreased p65 binding to the IP-10 promoter in F-IPF (Fig. (Fig.3A),3A), constitutive and IL-1β-induced p65 binding to the MCP-1 promoter was markedly higher in F-IPF compared to F-NL (Fig. (Fig.3B).3B). The results clearly demonstrate that transcription factor binding to the specific IP-10 promoter in the native chromatin environment, rather than transcription factor activation, was significantly impaired in F-IPF, thus providing an explanation for IP-10 repression in IPF.

FIG. 3.
Cytokine-induced native transcription factor binding to the IP-10 promoter is impaired in F-IPF. Confluent and serum-starved F-NL and F-IPF cells in 150-cm2 flasks were incubated with IL-1β (1 ng/ml) (A, B, C, and E) or IFN-γ (10 ng/ml) ...

Insufficient histone H3 and H4 acetylation at the IP-10 promoter in F-IPF.

Histone deacetylation is closely associated with repressive chromatin states and decreased transcription factor binding to specific gene promoters. To explore whether the impaired transcription factor binding to the IP-10 promoter in F-IPF was due to restricted access to the promoter as a result of reduced histone acetylation at the IP-10 promoter, we analyzed histone H3 and H4 acetylation at the IP-10 promoter site by ChIP assay. Both IL-1β and IFN-γ caused marked increases of histone H3 and H4 acetylation at the IP-10 promoter in F-NL with different time patterns after 0.5 to 4 h of treatment (Fig. 4A to D). In contrast, histone H4 acetylation, and to a lesser extent histone H3 acetylation, at the IP-10 promoter in F-IPF was consistently lower than that in F-NL, although modest increases were observed at some time points after cytokine treatment (Fig. 4A to D). The results suggest that histones H3 and H4 are insufficiently acetylated at the IP-10 promoter in F-IPF compared to F- NL.

FIG. 4.
Cytokine-induced histone H3 and H4 acetylation at the IP-10 promoter is reduced in F-IPF. Confluent and serum-starved F-NL and F-IPF cells in 150-cm2 flasks were incubated with IL-1β (1 ng/ml) (A and C) or IFN-γ (10 ng/ml) (B and D) for ...

Imbalanced HAT/HDAC recruitment to the IP-10 promoter in F-IPF.

Since histone acetylation and deacetylation are regulated by HATs and HDACs, respectively, we anticipated that the insufficient histone H3 and H4 acetylation at the IP-10 promoter in F-IPF could be due to reduced recruitment of HATs and/or increased recruitment of HDACs. We went on to analyze the association of the HATs PCAF and CBP and the HDAC-containing transcription inhibitory complexes CoREST, NCoR, and mSin3a with the IP-10 promoter using ChIP assay. IL-1β treatment caused a marked increase of PCAF, CBP (2 h), and GCN5 (0.5 h) association with the IP-10 promoter in F-NL compared to the control (0 h) (Fig. 5A and C and E), whereas IFN-γ treatment caused a marked increase of PCAF (2 h), CBP, and GCN5 (0.5 h) association with the IP-10 promoter (Fig. 5B and D and F). In contrast, the level of association of PCAF, CBP, and GCN5 with the IP-10 promoter before or after cytokine treatment was consistently lower in F-IPF than in F-NL, although modest increases were observed at some time points (Fig. 5A to F). Since HDACs are usually associated with transcriptional repressor complexes, we therefore analyzed the association of the major proteins of the three complexes CoREST, NCoR, and mSin3a with the IP-10 promoter. As shown in Fig. 6A to F, under unstimulated conditions, the association of CoREST, NCoR, and mSin3a with the IP-10 promoter was markedly higher in F-IPF than in F-NL. Treatment with IL-1β increased the association in both F-IPF and F-NL with different time patterns, while treatment with IFN-γ increased NCoR and mSin3a association in F-NL and CoREST association in F-IPF. However, the association of all three proteins was consistently higher in F-IPF than in F-NL (Fig. 6A to F). We have previously shown that global HDAC activity was markedly higher in the nuclear extracts of F-NL than that of F-IPF under both unstimulated and IL-1β-stimulated conditions (7). We performed this assay again with IFN-γ treatment and found that global HDAC activity was 4-fold higher in the nuclear extracts of F-NL than that of F-IPF and that IFN-γ treatment had no effect (data not shown). We also performed the HAT activity assay and found that there was no difference between F-NL and F-IPF in global HAT activity under both unstimulated and IL-1β- and IFN-γ-stimulated conditions (data not shown). The results suggest indirectly that despite the lower global HDAC activity in F-IPF than in F-NL, there is an imbalanced HAT/HDAC recruitment to the IP-10 promoter in F-IPF that correlates with IP-10 repression in F-IPF.

FIG. 5.
Cytokine-induced HAT recruitment to the IP-10 promoter is reduced in F-IPF. Confluent and serum-starved F-NL and F-IPF cells in 150-cm2 flasks were incubated with IL-1β (1 ng/ml) (A, C, and E) or IFN-γ (10 ng/ml) (B, D, and F) for the ...
FIG. 6.
Transcriptional repressor complex association with the IP-10 promoter is increased in F-IPF. Confluent and serum-starved F-NL and F-IPF cells in 150-cm2 flasks were incubated with IL-1β (1 ng/ml) (A, C, and E) or IFN-γ (10 ng/ml) (B, D, ...

Increased H3K9me3, HMT, and HP1 recruitment to the IP-10 promoter in F-IPF.

H3K9me3 is a typical epigenetic mark for repressive chromatin. We analyzed H3K9me3 at the IP-10 promoter by ChIP assay and found that under both unstimulated and IL-1β-stimulated conditions, H3K9me3 at the IP-10 promoter was markedly higher in F-IPF than in F-NL (Fig. (Fig.7A).7A). In addition, the association of the two major HMTs responsible for H3K9 methylation, SUV39H1 and G9a, with the IP-10 promoter was also consistently higher in F-IPF than in F-NL with or without IL-1β treatment (Fig. 7B and C). Western blot analysis revealed no difference in global SUV39H1 and G9a protein expression between F-NL and F-IPF, either with or without IL-1β treatment (data not shown), suggesting the increased recruitment of SUV39H1 and G9a to the IP-10 promoter is gene specific. Since hypermethylated histone H3K9 serves as a binding platform for HP1, we anticipated that HP1 association with IP-10 promoter could be increased in F-IPF cells. Using the ChIP assay, we found that the association was also consistently higher in F-IPF than in F-NL with or without IL-1β treatment (Fig. (Fig.7D).7D). HP1 also associates with a variety of other factors, including transcriptional repressors and HDACs. To analyze the association of HP1 with transcriptional repressors NCoR, CoREST, and mSin3a, we performed the re-ChIP assay and found that HP1 was associated with NCoR, CoREST, and mSin3a at the IP-10 promoter in F-IPF (Fig. (Fig.7E).7E). As NCoR, CoREST, and mSin3a are HDAC-containing transcriptional repressors, we then went on to measure HDAC activity of immunoprecipitates (IPs) of HP1, NCoR, CoREST, and mSin3a in F-NL and F-IPF. As stated before, global HDAC activity was lower in unimmunoprecipitated samples from F-IPF than those from F-NL; however, although HDAC activity was observed in IPs of HP1, NCoR, CoREST, and mSin3a, there was no difference in HDAC activities in these IPs between F-NL and F-IPF (Fig. (Fig.7F).7F). The results suggest that HP1, NCoR, CoREST, and mSin3a are associated with HDACs and are likely to be responsible for the insufficient acetylation of histone H3 and H4 at the IP-10 promoter in F-IPF.

FIG. 7.
H3K9me3, HMT recruitment, and HP1 binding at the IP-10 promoter are increased in F-IPF. Confluent and serum-starved F-NL and F-IPF cells in 150-cm2 flasks were incubated with IL-1β (1 ng/ml) (A to D). The protein-DNA complexes were cross-linked ...

Reversal of repressive chromatin and restoration of IP-10 expression in F-IPF by G9a and HDAC inhibitors.

To determine whether there was a direct link between increased G9a and HDAC recruitment to the IP-10 promoter and IP-10 repression in F-IPF, we first examined the effect of the G9a inhibitor BIX-01294 and the HDAC inhibitor LBH589 on histone acetylation at the IP-10 promoter. When F-IPF cells were treated with the inhibitors alone, a slight increase in histone H3 and H4 acetylation at the IP-10 promoter was observed compared with that in untreated cells (Fig. 8A and B). However, when the cells were treated with the inhibitors and IL-1β, a marked increase in histone H3 and H4 acetylation was observed compared to the level in cells treated with IL-1β alone (Fig. 8A and B). The two inhibitors also markedly reduced H3K9me2, H3K9me3, and HP1 association with the IP-10 promoter compared with untreated cells in both control and IL-β-stimulated cells (Fig. 8C to E). Treatment of F-NL cells with BIX-01294, LBH589, and both caused a modest increase in IP-10 mRNA and protein expression, whereas IL-1β alone induced a marked increase in IP-10 mRNA and protein expression with no further increase observed by the inhibitors, either alone or in combination (Fig. 8F and G). In F-IPF cells, a slight increase in IP-10 protein expression was observed when the cells were treated with BIX-01294 and LBH589 in combination or treated with IL-1β alone (Fig. (Fig.8G);8G); however, when the cells were treated with the inhibitors and IL-1β together, a marked increase in IP-10 mRNA and protein expression was observed, but no further increase was observed when the inhibitors were used in combination (Fig. 8F and G). The results show that IP-10 transcription in response to cytokine stimulation in F-IPF can be restored by inhibition of either HDAC activity or HMT G9a activity and therefore strongly suggest that histone deacetylation and hypermethylation and their interactions at the IP-10 promoter are responsible for repressed IP-10 expression in IPF.

FIG. 8.
HDAC and G8a inhibitors increase histone H3 and H4 acetylation, decrease H3K9me2 and H3K9me3 and HP1 binding at the IP-10 promoter, and restore IP-10 expression in F-IPF. F-IPF cells in 150-cm2 flasks were incubated without or with BIX-01294 (100 nM) ...

DISCUSSION

The major findings of our present study are that IP-10 gene expression was defective in F-IPF compared with F-NL due to histone deacetylation and hypermethylation as a result of decreased recruitment of HATs and increased recruitment of the HDAC-containing transcriptional repressor complexes and the H3K9 HMTs to the IP-10 promoter and that IP-10 expression was restored by HDAC and G9a inhibitors by reorganizing the heterochromatin-associated proteins and modifying repressive epigenetic modifications at the IP-10 promoter. These findings suggest that interactions between histone deacetylation and hypermethylation are responsible for the reduced expression of IP-10, and potentially other antifibrotic genes, in IPF and thus further our understanding of the molecular mechanisms of pulmonary fibrosis.

Our study confirmed previous findings that lung tissues from IPF patients and bleomycin-induced pulmonary fibrosis murine models, as well as lung fibroblasts isolated from IPF patients, express significantly less IP-10 than tissues from control subjects and fibroblasts from nonfibrotic lungs (12, 13). The repressed IP-10 expression of F-IPF in response to different inducers such as IFN-γ and IL-1β strongly suggests that the defect resides not at the level of cytokine receptors or the immediate postreceptor signaling pathways, but at more distal efferent steps of IP-10 expression. We then investigated the activation of transcription factors involved in IP-10 transcription in both F-IPF and F-NL. By applying reporter gene assays, we demonstrated that there was no difference in the activation of transcription factors that bind to the NF-κB and ISRE regulatory elements of the IP-10 promoter. This is consistent with our previous findings that both F-IPF and F-NL utilize the same set of regulatory elements and transcription factors for cytokine-induced COX-2 promoter activity and that there is no difference between the cells in transcription factor protein expression and activation (7). The results indicate clearly that postreceptor signaling up to the stage of transcription factor activation is not impaired in IP-10 transcription in F-IPF and therefore could not contribute to the repressed IP-10 expression in IPF. The results are also consistent with the findings that other NF-κB-dependent inducible genes, such as MCP-1 (2), are overexpressed in IPF.

Since the binding of transcription factors to the NF-κB and ISRE regulatory elements in the reporter gene assay is not controlled by histone modifications and chromatin states, we therefore went on to investigate whether the binding of transcription factors to the IP-10 promoter in native chromatin environment was impaired in F-IPF by ChIP assay. We found that the binding of transcription factors NF-κB, IRF-1, and p48 to the IP-10 promoter in native chromatin environment was significantly impaired in F-IPF. As there is no difference between F-NL and F-IPF in transcription factor expression and activation, the impaired transcription factor binding to the IP-10 promoter in F-IPF is likely to be gene specific. We then analyzed p65 binding to the promoter of another NF-κB-driven gene MCP-1, whose expression was increased in IPF (2). The results show that, in contrast to the decreased p65 binding to the IP-10 promoter in F-IPF, constitutive and IL-1β-induced p65 binding to the MCP-1 promoter was markedly higher in F-IPF than in F-NL, demonstrating that specific transcription factor binding to the IP-10 promoter in the native chromatin environment, rather than transcription factor activation and expression, is significantly impaired in F-IPF, thus providing an explanation for the repressed IP-10 transcription in IPF.

It is well established that binding of activated transcription factors to specific gene promoter sites is tightly controlled by the chromatin state as a result of histone modifications, particularly the balances between histone acetylation/deacetylation and histone methylation/demethylation. Histone deacetylation and hypermethylation are usually associated with repressive chromatin and lead to restricted access of the transcription factors to specific gene promoters. Acetylation of histones at the IP-10 promoter site may be attributed to the recruitment of coactivators with intrinsic HAT activity such as CBP, p300, GCN5, and PCAF, all of which have been demonstrated to be able to acetylate both histones H3 and H4 (8). We therefore anticipated that the impaired transcription factor binding to the IP-10 promoter in F-IPF could be due to decreased HAT recruitment and/or increased HDAC recruitment to the IP-10 promoter and consequently reduced histone acetylation at the IP-10 promoter site. We indeed demonstrated in this study that histone H3 and H4 acetylation was reduced in F-IPF compared with F-NL. However, the difference between F-NL and F-IPF in histone H3 acetylation was not as big as that in histone H4 acetylation and the increase in histone H3 acetylation in F-NL did not occur until 2 h after cytokine treatment, suggesting that histone H3 acetylation is not as important histone H4 acetylation in mediating transcription factor binding to the IP-10 promoter in these cells. The deacetylation was likely caused by reduced recruitment of HATs to the IP-10 promoter. There were clear differences between IFN-γ and IL-1β in the time points and the magnitude of specific HAT recruitment to the IP-10 promoter in F-NL, which may reflect different signaling pathways and HATs utilized by the two different cytokines in inducing IP-10 transcription and the fact that IFN-γ is a weaker IP-10 inducer than IL-1β in these cells. We also showed in this study that the association of the transcriptional repressor complexes CoREST and mSin3a (containing HDAC1 and -2) and NCoR (containing HDAC3) with the IP-10 promoter under both unstimulated and stimulated conditions was markedly greater in F-IPF than in F-NL. We have previously revealed that global HDAC activity is markedly lower in F-IPF than in F-NL under both unstimulated and stimulated conditions (7), and we found in this study that there was no difference between F-NL and F-IPF in global HAT activity with or without cytokine stimulation. These findings strongly suggest that decreased recruitment of HATs and increased recruitment of HDACs to the IP-10 promoter, rather than decreased global HAT activity and/or increased global HDAC activity, are responsible for histone deacetylation and repression of IP-10 gene transcription in F-IPF.

Histone K9 methylation has been shown to correlate with transcriptional repression and can be induced by HMTs that possess activity toward K9 such as G9a, SUV39H1, SUV39H2, and ESET/SETDB1 (35, 41, 45). It has been demonstrated that G9a is mainly responsible for mono- and dimethylation of H3K9, whereas SUV39H1 and SUV39H2 direct trimethylation of H3K9 (30). Both H3K9me2 and H3K9me3 serve as a binding platform for HP1 (3). In turn, HP1 associates with a variety of other factors, including HMTs, HDACs, Dnmts, transcriptional repressors, and chromatin-remodeling enzymes (22, 37). Thus, by multiple different interactions, HP1 is believed to create and compact chromatin structure that does not permit transcription. This structure is effectively heterochromatin and is accompanied by H3K9 methylation and histone deacetylation. To explore whether H3K9 methylation correlates with the transcriptional repression of IP-10 in IPF, we analyzed H3K9me3, the H3K9 HMTs G9a and SUV39H1, and HP1 association with the IP-10 promoter. We found that H3K9me3 at the IP-10 promoter and HP1 association with the IP-10 promoter were markedly increased in F-IPF compared with F-NL under both unstimulated and IL-1β-stimulated conditions and that the recruitment of both G9a and SUV39H1 to the IP-10 promoter was also increased in F-IPF compared with F-NL. As we also revealed there was no difference in levels of global SUV39H1 and G9a protein expression between F-NL and F-IPF, with or without IL-1β treatment, the increased recruitment of SUV39H1 and G9a to the IP-10 promoter is likely to be gene specific. In addition, we also demonstrated that HP1 was associated with NCoR, CoREST, and mSin3a at the IP-10 promoter in F-IPF and that HP1, NCoR, CoREST, and mSin3a were all associated with HDACs and were likely to be responsible for the hypoacetylation of histones H3 and H4 at the IP-10 promoter in F-IPF.

To further demonstrate the direct link between histone deacetylation/hypermethylation and IP-10 repression, we applied the specific G9a inhibitor BIX-01294 (17) and the well-characterized HDAC inhibitor LBH589 (24). As shown by ChIP assay, treatment with both HDAC and G9a inhibitors created an active chromatin structure at the IP-10 promoter manifested as accumulation of acetylated histones H3 and H4, a decrease of H3K9me2 and H3K9me3, and impaired binding of the heterochromatin mark HP1 at the IP-10 promoter, eventually resulting in the restoration of IP-10 mRNA and protein expression in F-IPF in response to cytokine stimulation. The results strongly indicate that histone deacetylation as a result of increased local HDAC activity and decreased HAT activity and histone hypermethylation as a result of increased local G9a activity at the IP-10 promoter are responsible for IP-10 repression in IPF. The finding that both HDAC and G9a inhibitors had no or only a modest effect on IP-10 mRNA and protein expression in both F-NL and F-IPF on their own suggests that IP-10 expression in F-NL and restoration in F-IPF require not only HDAC and G9a inhibition to induce histone acetylation and reduce histone methylation at the promoter but also cytokine stimulation to induce transcription factor activation and promoter binding to initiate gene transcription. The findings that both HDAC and G9a inhibitors did not further enhance the effect of IL-1β in F-NL but were only effective in the presence of IL-1β in F-IPF suggest that IL-1β treatment is enough to induce histone acetylation/demethylation and transcription factor activation and promoter binding and to initiate IP-10 transcription in F-NL but requires HDAC and G9a inhibition to achieve the same in F-IPF, further indicating that intrinsically aberrant local HDAC and G9a activities are responsible for IP-10 gene repression in F-IPF.

Our results show that HDAC inhibitors also inhibit H3K9me2 and H3K9me3 at the IP-10 promoter and HP1 recruitment to the IP-10 promoter. This is consistent with previous findings that the HDAC inhibitor LBH589 releases SUV39H1 from silenced estrogen receptor alpha (ER) gene promoter, which is associated with a decrease in H3K9 methylation and impaired binding of HP1 at the promoter, resulting in the restoration of ER gene expression (49) and that the HDAC inhibitor depsipeptide activates silenced genes by suppressing the expression of G9a and SUV39H1 and reducing H3K9me2 and H3K9me3 at these gene promoters and HP1 loading to H3K9me3 (44). However, whether HDAC inhibitors restore IP-10 gene expression by suppressing the expression of G9a and SUV39H1 in F-IPF cells requires further investigation. BIX-01294 is a specific G9a inhibitor that does not affect SUV39H1 (17). G9a and SUV39H1 mainly cause H3K9me2 and H3K9me3, respectively. However, our present study showed that BIX-01294 inhibited both H3K9me2 and H3K9me3 at the IP-10 promoter. Although the mechanism for H3K9me3 is not clear, it is possible that G9a provides H3K9me2 for subsequent trimethylation by SUV39H1 as demonstrated by a recent study (20). If this is the case, reduced H3K9me2 by G9a inhibition could lead to reduced H3K9me3 by SUV39H1. As HP1 can bind to both H3K9me2 and H3K9me3 (3), G9a inhibition can therefore result in reduced HP1 binding to both H3K9me2 and H3K9me3 and consequently reduced association of HDAC-containing transcriptional repressor complexes such as NCoR, CoREST, and mSin3a at the IP-10 promoter in F-IPF, leading to cytokine-induced histone acetylation and IP-10 expression. The fact that the combination of HDAC and G9a inhibitors did not enhance the effect of each inhibitor alone suggests that strong interdependence of histone deacetylation and hypermethylation upon each other plays a key role in IP-10 repression in IPF. Further detailed studies are required to clarify this.

Accumulating evidence has indicated that in addition to histone deacetylation and methylation, DNA methylation is also involved in dysregulated gene expression. Methylation of CpG dinucleotides is catalyzed by three essential DNA methyltransferases, Dnmt1, Dnmt3A, and Dnmt3B. In vivo studies have demonstrated that Dnmt1 physically binds to HDACs and H3K9 HMT SUV39H1 at silenced promoters (9, 10, 26). HDAC-mediated histone deacetylation is necessary for Dnmt1 to carry out its methylation function (10), and the presence of deacetylated histones allows SUV39H1-mediated H3K9 trimethylation to take place (4, 19). Indeed, HDAC inhibitors have been shown to reduce Dnmt1 binding to silenced gene promoters and reduce DNA methylation, resulting in the restoration of silenced gene expression (44, 49). Thus, cross talk between histone deacetylation, methylation, and DNA methylation is believed to be necessary for the generation of a stable and long-term epigenetically silenced state in cancer. However, there is no study showing the existence of CpG islands at the IP-10 promoter. This is supported by our finding using MethPrimer software (www.urogene.org) that there is no CpG island at the IP-10 promoter. In addition, our study using the DNA methylation inhibitor 5-aza-2′-deoxycytidine did not show any effect on IP-10 mRNA and protein expression in F-IPF (data not shown). It is therefore likely that DNA methylation is not involved in repressed IP-10 expression in IPF; however, the possibility of its involvement cannot be ruled out completely at the moment.

In conclusion, our data suggest that HDACs interact with histone H3K9 HMTs to maintain a stable repressive chromatin state at the IP-10 promoter, resulting in targeted repression of this gene in IPF. Inhibition of either of these interacting epigenetic programs can reactivate the silenced IP-10 gene. These findings provide direct evidence for dysregulated epigenetic control of IP-10 gene repression in IPF and could improve our understanding of the molecular mechanisms of the targeted repression of antifibrotic genes in fibrotic lung diseases.

Acknowledgments

This work was supported by the Medical Research Council (grant no. G0600890) and the Wellcome Trust (grant no. 088751).

Footnotes

[down-pointing small open triangle]Published ahead of print on 19 April 2010.

The authors have paid a fee to allow immediate free access to this article.

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