Epigenetic Regulation of Early- and Late-Response Genes in Acute Pancreatitis Juan Sandoval, Javier Pereda, Salvador Pérez, Isabela Finamor, Azahara Vallet-Sánchez, José Luis Rodríguez, This information is current as Luis Franco, Juan Sastre and Gerardo López-Rodas of September 28, 2021. J Immunol published online 19 October 2016 http://www.jimmunol.org/content/early/2016/10/19/jimmun ol.1502378 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2016 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published October 19, 2016, doi:10.4049/jimmunol.1502378 The Journal of Immunology

Epigenetic Regulation of Early- and Late-Response Genes in Acute Pancreatitis

Juan Sandoval,*,1 Javier Pereda,† Salvador Pe´rez,† Isabela Finamor,† Azahara Vallet-Sa´nchez,*,‡ Jose´ Luis Rodrı´guez,* Luis Franco,*,‡ Juan Sastre,† and Gerardo Lo´pez-Rodas*,‡

Chromatin remodeling seems to regulate the patterns of proinflammatory genes. Our aim was to provide new insights into the epigenetic mechanisms that control transcriptional activation of early- and late-response genes in initiation and development of severe acute pancreatitis as a model of acute inflammation. changes were studied by chromatin immunoprecipitation analysis, positioning, and determination of modifications in promoters of proinflammatory genes in vivo in the course of taurocholate-induced necrotizing pancreatitis in rats and in vitro in rat pancreatic AR42J acinar cells stimulated with taurocholate or TNF-a. Here we show that the upregulation of early and late inflammatory genes rely on histone Downloaded from associated with recruitment of histone acetyltransferase CBP. Chromatin remodeling of early genes during the inflammatory response in vivo is characterized by a rapid and transient increase in H3K14ac, H3K27ac, and H4K5ac as well as by recruitment of chromatin-remodeling complex containing BRG-1. Chromatin remodeling in late genes is characterized by a late and marked increase in histone , particularly in H3K4. JNK and p38 MAPK drive the recruitment of factors and the subsequent upregulation of early and late inflammatory genes, which is associated with nuclear translocation of the early gene Egr-1. In conclusion, specific and strictly ordered epigenetic markers such as histone acetylation and methylation, as well as http://www.jimmunol.org/ recruitment of BRG-1–containing remodeling complex are associated with the upregulation of both early and late proinflamma- tory genes in acute pancreatitis. Our findings highlight the importance of epigenetic regulatory mechanisms in the control of the inflammatory cascade. The Journal of Immunology, 2016, 197: 000–000.

hromatin dynamic changes toward open and more ac- site-specific using acetyl-CoA as donor, and the erasers, cessible euchromatin involve the disruption of interac- histone deacetylases (HDACs), which hydrolyze the acetamide tions with histone-DNA, which in turn include DNA bond. One of the primary functions of HDACs is to keep chromatin

C by guest on September 28, 2021 demethylation and histone modification, mainly acetylation, methyl- transcriptionally silent due to the removal of acetyl groups from the ation, and (1–8). These modifications create dock- N terminal tails of H3 and H4 (8). Although other mod- ing sites for the recruitment of proteins or complexes leading to ifications of core histones have similar effects, the acetylation active gene transcription (1–3, 5–8). The epigenetic balance of of histones H4 and H3, and the trimethylation of 4 of H3 histone acetylation/deacetylation is regulated by the writers, histone (H3K4me3), generally promote gene activation, whereas di- or tri- acetyltransferases (HATs), which incorporate the to methylation of lysine 27 of histone H3 (H3K27me2 and H3K27me3) lead to gene silencing (9, 10). *Department of Biochemistry and Molecular Biology, Faculty of Biology, University HATs and HDACs play a central role in gene regulation in in- of Valencia, 46100 Valencia, Spain; †Department of Physiology, Faculty of Phar- flammation (11). Indeed, histone acetylation via CBP/p300 HATs ‡ macy, University of Valencia, 46100 Valencia, Spain; and Institute of Health Re- [also known as KAT3A (CBP) and KAT3B (p300)] coordinates the search INCLIVA (Foundation for Research of the Clinic Hospital of the “Comunidad Valenciana”), 46010 Valencia, Spain expression of proinflammatory cytokines, particularly through NF-kB 1Current address: Epigenomics Lab, Biomarkers and Precision Medicine Unit, Hos- and STAT pathways (12–14). It is noteworthy that NF-kBisableto pital La Fe, Valencia, Spain. access regions of condensed chromatin and trigger transcriptional ORCIDs: 0000-0002-2041-4038 (J. Sandoval); 0000-0001-9610-6448 (L.F.); 0000- activation because TNF-a rapidly and substantially reduced HDAC1 0001-8367-653X (G.L.-R.) protein levels through protein degradation (15). Received for publication November 13, 2015. Accepted for publication September In addition, a shift in epigenetic marks seems to occur in H3 to 16, 2016. change the repressed state of transcription to competent transactivation This work was supported by Grants SAF2012-39694 and SAF2015-71208-R with of proinflammatory genes. The basal, transcriptionally inactive state of Fondo Europeo de Desarrollo Regional funds from the Spanish Ministry of Economy and Competitiveness to J. Sastre and Consolider-Ingenio CSD2006-49 (Network chromatin in the promoters of these genes is characterized by the Group Valencia) from the Spanish Ministry of Science and Technology to G.L.-R. presence of methylated H3 lysine 9 coupled with unphosphorylated I.F. received a fellowship from Programa de Po´s-Doutorado no Exterior, which belongs to the Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico. H3 serine 10, which shifts to the transcriptionally active state of demethylated H3 lysine 9 and phosphorylated H3 serine 10 (16, 17). Address correspondence and reprint requests to Prof. Juan Sastre, Department of Physiology, Faculty of Pharmacy, University of Valencia, Avenida Vicente Andre´s During the inflammatory cascade, many genes seem to be ac- Estelle´s s/n, 46100 Burjassot, Valencia, Spain. E-mail address: [email protected] tivated following two different patterns. The primary response The online version of this article contains supplemental material. (early) genes can be induced without de novo protein synthesis, Abbreviations used in this article: ChIP, chromatin immunoprecipitation; Ct, thresh- although some exhibit delayed kinetics (18, 19). The secondary old cycle; HAT, histone acetyltransferase; HDAC, ; RT-PCR, real- response (late) genes require de novo protein synthesis and are time PCR; TSS, transcriptional start site. induced more slowly because that synthesis needs signaling mol- Copyright Ó 2016 by The American Association of Immunologists, Inc. 0022-1767/16/$30.00 ecules or cytokines (20). Chromatin remodeling regulates the

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1502378 2 EPIGENETIC REGULATION IN ACUTE PANCREATITIS

FIGURE 1. Gene expression pattern during taurocholate- induced acute necrotizing pancreatitis in rats. (A) Early gene expression (Egr-1, Atf-3, IL-1b, and Myd118), (B) late gene expression (Nos-2, IL-6, Pap, and Tnf-a), and (C) control Downloaded from gene expression (b-actin). Steady-state mRNA levels were measured by quantitative RT-PCR in the pancreas of mice treated with saline (control) or taurocholate acid. Quanti- tative RT-PCR values were normalized against those ob- tained for rRNA 18S analysis, giving an arbitrary value of 1 to the control sample. The error bars correspond with the SD of three to four independent RT-PCR measurements. The http://www.jimmunol.org/ statistical significance is indicated as ***p , 0.001, **p , 0.01, *p , 0.05. by guest on September 28, 2021

expression of primary response genes with delayed kinetics and of under basal conditions, which results in an open chromatin secondary response genes at least through the recruitment of SWI/ structure (19–21). Acetylation of and H3K4me3 are SNF remodeling complexes that contain either BRG1 or BRM as found at the promoters of both primary and secondary response the catalytic subunit (19). Promoters of rapid primary response genes in macrophages stimulated with LPS (22). Nevertheless, the genes exhibit a high level of histone acetylation and H3K4me3 large number of and the complexity of the regulatory

Table I. Primers used for the analysis of gene expression by RT-PCR

Gene Primers Atf-3 59-GGGTCACTGGTGTTTGAGGATTGCTTGTTCTGGATGGCGAAT-39 Egr-1 59-CAGTGGCCTTGTGAGCATGAGACGATGAAGCAGCTGGAGAA-39 c-Fos 59-TGGAGCCGGTCAAGAACATTAGCCGGAAACAAGAAGTCATCA-39 Gadd45b 59-CCTGATCCAATCGATAGACGAATTGTCGCAGCAGAACGATT-39 Il-1b 59-CACAGCAGCATCTCGACAAGAGCATAGGTAGCTGCCACAGCTTCT-39 Il6 59-ATATGTTCTCAGGGAGATCTTGGATGCATCATCGCTGTTCATACAA-39 Nos-2 59-AGCGGCTCCATGACTCTCATGCACCCAAACACCAAGGT-39 Pap1 59-GCTTGGCCTTCCCAGTCATGATGTCTTACCTTGCACCTGTGAT-39 Tnf-a 59-CAGCCGATTTGCCATTTCATTCCTTAGGGCAAGGGCTCTT-39 b-Actin 59-TTCAACACCCCAGCCATGTGTGGTACGACCAGAGGCATACA-39 rRNA 18S 59-AGTCCCTGCCCTTTGTACACAGATCCGAGGGCCTCACTAAAC-39 The Journal of Immunology 3

Table II. Abs used for ChIP and RNApol-ChIP analysis

Ab Company Reference Ab Company Reference

a-ATF-2 Santa Cruz (sc-187) a-H3 K4 me2 Abcam (ab 7766) a-ATF-3 Santa Cruz (sc-188) a-H3 K4 me3 Abcam (ab 8580) a-BRG1 Santa Cruz (sc-10768) a-H3 K9 me2 Upstate (05-685) a-CBP Santa Cruz (sc-369) a-H3 K9 ac Abcam (ab 4441) a-C/EBP b Santa Cruz (sc-150) a-H3 K14 ac Upstate (07-353) a-CREB Santa Cruz (sc-186) a-H3 K18 ac Abcam (ab 1191) a-EGR-1 Santa Cruz (sc-110) a-H3 K23 ac Abcam (ab 1768) a-ELK-1 Santa Cruz (sc-355) a-H3 K27 ac Upstate (07-360) a-mSin3A Santa Cruz (sc-994) a-H3 R17 me2 Upstate (07-214) a-NF-kB Santa Cruz (sc-109) a-H3S10Ph-K14 ac Upstate (07-081) a-RNApol II Santa Cruz (sc-899) a-H4 K5 ac Abcam (ab 1758) a-SP1 Santa Cruz (sc-59) a-H4 K8 ac Abcam (ab 1760) a-SRF Santa Cruz (sc-335) a-H4 K16 ac Upstate (07-329) effects hinder the assignment of direct biological roles to a single grown in DMEM (Invitrogen, Carlsbad, CA) containing 25 mM glucose, acetylation event (23). 100 mg/ml penicillin, 100 mg/ml streptomycin, and 25 mg/ml fungizone, a The present work focuses on severe acute pancreatitis as an supplemented with 10% FBS. To activate the Egr-1 and Tnf- genes, Downloaded from AR42J cells were treated with 0.3% of sodium taurocholate (Sigma) experimental model of acute inflammation that is initially localized and50ng/mlofTNF-a (Sigma) for the indicated times. To inhibit the in the pancreatic gland but evolves into a systemic inflammatory cell signaling pathways, AR42J cells were pretreated with cyclohexi- response, and eventual multiple organ failure (24–26). Previous mide (25 mg/ml) for 1 h, and incubated with the following compounds: evidence from our group suggests that epigenetic mechanisms PD98059 (100 mM), SB203580 (100 mM), Wortmanin (1 mM), H89 (10 mM), JNK (100 mM), ERKs (20 mM), NF-kB(25mM), STAT3 control TNF-a expression and may have a key role in this disease (100 mM), and JAK-STAT1 (S14-95 5 mg/ml) inhibitors (Calbiochem). (14, 27). Indeed, activation of Tnf-a gene was accompanied by a Subsequently, the cells were incubated with sodium taurocholate or http://www.jimmunol.org/ strictly ordered recruitment or release of transcriptional factors TNF-a. (ELK-1, SP1, NF-kB and EGR-1), and chromatin modification Acute necrotizing pancreatitis complexes [HDAC1, HDAC2, GCN5 (KAT2A), PCAF (KAT2B) and CBP (KAT3A)], as well as by an ordered, increased level of Animals were anesthetized with an i.p. administration of ketamine histone H3K9ac, H3K14ac, H3K18ac, H4K5ac, and H3K4me. In (80 mg/kg body weight) and acepromazine (2.5 mg/kg body weight)). Acute necrotizing pancreatitis was induced by retrograde injection into the the current study, our aim has been to gain new insights into the biliopancreatic duct of sodium taurocholate (3.5%) (Sigma) in a volume common epigenetic mechanisms that control transcriptional acti- of 0.1 ml/100 g body weight using an infusion pump (Harvard Instru- vation of early- and late-response genes in the initiation and de- ments) (27). In control rats, 0.9% NaCl (0.1 ml/100 g body weight) was velopment of severe acute pancreatitis. infused into the biliopancreatic duct. For each group 6–12 animals were by guest on September 28, 2021 used for the in vivo experiments. Rats were anesthetized as previously described then sacrificed at the indicated times after the infusion of Materials and Methods taurocholate or saline. Serum lipase activity was measured, and histo- logical studies were performed to confirm the appropriate induction of Animals necrotizing pancreatitis. Male Wistar-Furth rats (250–300 g body weight) were used. They were fed a standard laboratory diet and tap water ad libitum, and were subjected to a RNA isolation and analysis of mRNA level by real-time PCR 12 h light–dark cycle. All animals were cared for and handled according to and quantitative PCR the criteria outlined in the Guide for the Care and Use of Laboratory Total RNA from pancreatic tissue or AR42J cells was isolated by the Animals prepared by the National Academy of Sciences and published by guanidinium thiocyanate method (28). In the case of the pancreas, the the National Institutes of Health (NIH Publication 86-23 revised 1985). tissue was previously immersed in 1 ml of RNA-later solution (Ambion) to The Research Committee of the School of Medicine (University of Valencia, stabilize the RNA. The isolated RNA (2 mg/lane) was size-fractionated by Spain) approved the study protocol. electrophoresis in a 1% agarose/formalin gel, and stained with ethidium AR42J acinar cells bromide to assess the quality of the RNA. The cDNA used as template for amplification in the PCR assay was obtained by the reverse transcription The acinar cell line AR42J, derived from an exocrine rat pancreatic tu- reaction using SuperScript II (Invitrogen), with random hexamers as pri- mor (CRL 1492; American Type Culture Collection, Manassas, VA) was mers, starting with equal amounts of RNA. As a PCR internal control, 18S

Table III. Primers used for the ChIP and RNApol-ChIP analysis at the promoter and coding region of the genes

Gene Primers Atf-3 (promoter) 59-CGTGCGAGGCGCGTAGGTGACTGGGAGCGCGTTGC-39 Atf-3 (coding region) 59-GGGTCACTGGTGTTTGAGGATTGCTTGTTCTGGATGGCGAAT-39 Egr-1 (promoter) 59-GTAGAACCCCGGCCTGACTCAGGCTCCTGGAGTTCCCAGC-39 Egr-1 (coding region) 59-CTTGCCCTGTTGAGTCCTGCCACAGGCAAAAGGCTTCTCG-39 Il-6 (promoter) 59-ATCAGCCCCACCCACTCTGGCGCCTGATGCTGGCTGCTGG-39 Il-6 (coding region) 59-AGAGGCACCTCAGTGGCTGCTGGGCTGACCTGAGACCTGC-39 Nos-2 (promoter) 59-GGTGCAGCTAAGAAAAGCCTCCTTTATACCCATCCACGCTCTGC-39 Nos-2 (coding region) 59-ACTGGACCACCGCTGTCAGGCCTGCTTTGCCACTTGCCAG-39 a-actin (promoter) 59-AGGGACTCTAGTGCCCAACACCCCCACCTCCACCCTACCTGC-39 a-actin (coding region) 59-AGGATTCCTACGTGGGCGACTAGAGAGACAGCACCGCCTG-39 b-actin (coding region) 59-TTCAACACCCCAGCCATGTGTGGTACGACCAGAGGCATACA-39 4 EPIGENETIC REGULATION IN ACUTE PANCREATITIS

Table IV. Oligonucleotides for the analysis by RT-PCR of overlapping amplicons of Egr-1 promoter

Amplicon Primer Sequence (Forward/Reverse) Size (bp) 2859-TTGAAATAGAGGCCGATTCGGGGAAGCTGGCGGCGGCGGCGAA-39 71 252 59-GGCCATGTACGTCACGGCGGATGCGCGCTGGGATCTCTCGC-39 73 2107 59-CGTCACTCCGGGTCCTCCCGGGGTCTGAAACAGCACGGGCC-39 85 2148 59-GTCGCAGGGTGGAAGCGCCCAGGGAAGCAGGAAGCCCTAATATGGAAG-39 93 2216 59-GCCGCTGTTCCAATACTAGGCTTTCAAGACCTCCCATCCAAGAGCGGTG-39 82 2253 59-GGGGGCAAGCTGGGAACTCCAACCGGCTCCGGCACCCTGA-39 84 2298 59-CCGCTTCCGGCTCTGGGAGGATGGAACAGCGGCAGTGGCCTC-39 77 2412 59-GGGAGGGTGGGTGCGCCGACCACTCCATATAAGGCGCTGCCC-39 75 2470 59-GGCGTCCCCGACTCCCGCGCCCTGCTCCTTATATGGTGTTTCCGGG-39 64 The name of the amplicons corresponds with the position of their center in relation to transcriptional start site (TSS). RT-PCR, real-time PCR. rRNA was simultaneously amplified. To obtain similar PCR band in- Chromatin immunoprecipitation assay and RNApol-chromatin tensities, competitor oligonucleotides were added to the assay in a 3:7 immunoprecipitation proportion of normal 18S rRNA oligonucleotides/competitor 18S rRNA oligonucleotides. The competitors correspond to the same 18S rRNA oli- Cross-linking of chromatin, chromatin immunoprecipitation (ChIP), and

gonucleotide sequence, except that their 39 termini were blocked with an RNApol-ChIP procedures were performed using the method of Sandoval Downloaded from amino group. et al. (27). Briefly, isolated nuclei from formaldehyde–cross-linked pan- Real-time quantitative PCR was performed using dsDNA binding dye creas or AR42J cells were lysed and cross-linked chromatin was soni- Syber Green PCR Master mix in an ABI GeneAmp 7000 Sequence De- cated to yield fragments of ∼500 bp. Diluted soluble chromatin fragments tection System (Applied Biosystems). Each reaction was run in triplicate, were precleared with blocked Protein A/G–Sepharose, to discard non- and the melting curves were constructed using Dissociation Curves Soft- specifically bound chromatin fragments. Immunofractionation of com- ware (Applied Biosystems) to ensure that only a single product was am- plexes was carried out by adding 2 mg of the corresponding Abs (see plified. As real-time quantitative PCR control 18S rRNA or ACTB was also Table II) to aliquots containing 50 mg of DNA each. The immunocom- analyzed. The specific primers used for real-time PCR (RT-PCR) analysis plexes were recovered by centrifugation at 13,500 3 g for 1 min after http://www.jimmunol.org/ are shown in Table I. The threshold cycle (Ct) was determined, and the adding blocked Protein A/G–Sepharose and washing extensively. Immu- relative gene expression was expressed as follows: fold change = 22 noselected chromatin was eluted, and the formaldehyde cross-linking was D(DCt), where DCt = Cttarget2Cthousekeeping and D(DCt) = DCttreated2 reverted at 65˚C overnight. The DNA from all samples was purified with a DCtcontrol. PCR purification kit (Qiagen), and used for PCR analysis. The analysis was by guest on September 28, 2021

FIGURE 2. Actual transcription rate for selected early, late, and control genes in taurocholate-induced acute necrotizing pancreatitis in rats. Upper panels show steady-state mRNA levels were measured by semiquantitative RT-PCR for (A)early(Egr-1 and Atf-3), (B)late(Nos-2, IL-6), and (C) control genes (a-actin as negative and b-actin as positive). The rRNA 18S was used as an internal control, and a-andb-actin genes were used as negative and positive control, respectively, of the RT-PCR analysis. The middle and lower panels depict RNApol ChIP analysis for all selected genes in promoter and tran- scriptional regions, respectively. The Journal of Immunology 5 carried out using the primers shown in Table III. PCR fragments were size- by Western blotting and chemiluminescence detection using the Phototope- fractionated by 2% agarose gel electrophoresis, stained with ethidium bro- HRP detection kit (Cell Signaling Technology). As loading control, b-tubuline mide, and analyzed with an FLA3000 electronic autoradiography system (Abcam) was used. (Fujifilm), using ImageJ software (http://rsbweb.nih.gov/ij/). The ChIP assay of histone H3 was used as an internal control for in vivo and in vitro ex- Nucleosomal occupancy periments to confirm the lack of significant fluctuation (Supplemental Fig. 1). To analyze nucleosomal occupancy, briefly, nuclei from AR42J cells were suspended in RBS buffer [15 mM TrisHCl pH 7.5, 60 mM KCl, 15 mM Western blotting for total and nuclear protein NaCl, 3 mM MgCl2, 20% (v/v ) glycerol, 5 mM b-mercaptoethanol and AR42J cell samples were resuspended in a PBS buffer supplemented with 1 mM PMSF]. Eighty micrograms of chromatin DNA were digested with protease inhibitor mixture (Sigma), added immediately before its use, at a micrococcal nuclease for 5 min and blocked with EDTA 50 mM and SDS concentration of 5 ml/ml. For total protein isolation, samples were incu- 1% (v/v). DNA from digested nuclei were enriched in fragments of mono- bated in total extraction buffer [10 mM Tris–HCl (pH 7.5), 0.25 M sucrose, nucleosomal size (150–200 bp). These fragments are used as a template for 5 mM EDTA, 50 mM NaCl, 30 mM sodium pyrophosphate, 50 mM so- quantitative PCR using tiled amplicons encompassing the promoter region of dium fluoride, 100 mM sodium orthovanadate, and 1% Igepal]. Debris was Egr-1 gene (see Table IV). To normalize for the different efficiency of pri- removed by centrifugation at 900 3 g at 4˚C for 10 min, and the super- mers, sonicated genomic DNA was also amplified by PCR. To evaluate natant obtained was used for Western blot analysis. For nuclear protein the affinity score for located in the Egr-1 promoter region, purification, AR42J cell samples were incubated with cell lysis buffer the nucleosome positioning prediction software (NuPoP) was used (http:// (5 mM HEPES pH 8, 85 mM KCl, 0.5% NP40 supplemented with protease nucleosome.stats.northwestern.edu/). inhibitor mixture) for 10 min, centrifuged at 1500 3 g at 4˚C for 10 min, Immunolocalization and the nuclear pellet was subsequently incubated in nuclear lysis buffer (50 mM TrisHCl pH 8.1, 10 mM EDTA, 1% SDS) for 10 min. For immunolocalization analysis AR42J cells were cultured in chamber SDS-PAGE was used to separate 30 mg of protein, which was transferred slides and fixed with formalin 10% (v/v), permeabilized with 10% Triton Downloaded from to nitrocellulose membranes (Schleicher & Schuell). EGR-1 was determined X-100 (v/v) in PBS and blocked with goat normal serum at 10% (v/v) in PBS. http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 3. Epigenetic promoter characterization by ChIP of selected early, late, and control genes during taurocholate-induced acute necrotizing pancreatitis in rats. (A) Histone modification occupancy analyzed with indi- cated Abs were identified using specific primers in pro- moters of early (upper panel), late (middle panel), and control genes (lower panel). (B) Recruitment of histone modification complexes were analyzed by ChIP using Abs recognizing histone acetyltransferase complex CBP, and histone deacetylase complex Sin3A in the aforementioned promoter genes. 6 EPIGENETIC REGULATION IN ACUTE PANCREATITIS

EGR-1 was determined by immunofluorescence. AR42J cells were incu- creasing at 6 h, whereas this modification was completely absent bated with DAPI 3 mM (Invitrogen). Visualization images were acquired in the promoter of Nos-2. The H3K9ac mark increased at 6 h in with a Laser Scanning Spectral Confocal Microscope (Leica TCS-SP2) Nos-2; however, it decreased markedly at 6 h in Il-6. H4K5ac connected with a Leica DM1RB phase contrast microscope. increased transiently at 1 h in Il-6 but it did not change in Nos-2 Statistical analysis (Fig. 3A, middle panel). Neither of these changes was found in the Results are expressed as mean 6 SD with the number of experiments given gene of a-actin, which was used as a negative control (Fig. 3A, in parentheses. Statistical analysis was performed in two steps. First, a one- lower panel). way ANOVAwas carried out to find significance in the overall comparison The recruitment of HATs or HDACs was also studied in early of groups. Then, differences between individual groups were investigated and late genes. The HAT CBP (KAT3A) was the histone modifier ´ by the T (Scheffe) test. Differences were considered to be significant at complex mainly associated with upregulation of early and late p , 0.05. genes, except in the case of Egr-1. It should be highlighted that Results Gene expression profiles for early and late genes in the pancreas during acute pancreatitis The different profiles of gene expression for early (Egr-1, Atf-3, Il- 1b, MyD118) or late genes (Nos-2, Il-6, Pap, Tnf-a) in the head of pancreas during acute pancreatitis are depicted in Fig. 1. Similar findings were also obtained in the pancreas tail (data not shown). Downloaded from Early genes exhibited a rapid upregulation, reaching the maximum mRNA stationary level 1 h after taurocholate infusion (Fig. 1A), and progressively decreasing thereafter. However, late genes in- creased their mRNA stationary levels progressively, reaching a maximum at 3 h or after (Fig. 1B). Gene expression was studied using 18S rRNA as a reference. No significant differences in the http://www.jimmunol.org/ expression of 18S rRNA occurred in the course of pancreatitis using ACTB as reference in this case (Fig. 1C, Tables I–IV). We further analyzed the real-time transcriptional activity of early- or late-response genes by measuring the actual transcription rate using RNApol ChIP, a technique that detects elongating RNA polymerase II at the desired genes, avoiding interference with polymerase paused at the promoter. Fig. 2 shows the recruitment of RNA polymerase to the coding regions (elongating), and to the promoter of early (Fig. 2A) and late genes (Fig. 2B). The maxi- by guest on September 28, 2021 mum binding of RNA polymerase II to the coding region was found at 0.5–1 h for early genes, whereas the maximum recruit- ment for late genes was at 3–6 h. Hence, the upregulation of early and late genes was due to active transcription following the profile of gene expression shown in Fig. 1. ACTA1 and ACTB were used, respectively, as negative and positive controls of gene transcrip- tion (Fig. 2C). Early- and late-response genes were also upregulated in other tissues, such as liver and lung, although with a delayed profile (Supplemental Fig. 2). These findings show the different time-course between the local and the systemic inflammatory response charac- teristics of severe acute pancreatitis. Histone modifications in early and late genes in the pancreas during pancreatitis The levels of relevant histone modifications in the promoter region of early and late genes were estimated by ChIP assay. These histone modifications were selected according to their prominent role in chromatin remodeling (9, 10, 16, 17, 19–22), as mentioned in the Introduction. Fig. 3A shows acetylation or methylation of histones H3 and H4 in two representative genes for each group, i.e., Egr-1 and Atf3 for early genes, and Nos-2 and Il-6 for late genes. The modification characteristics of early genes were the transient acetylations H3K14ac, H3K27ac, and H4K5ac with a maximum at 1 h. In addition, H3K4me3, and H3K9ac were markedly reduced at 6 h in early genes (Fig. 3A, upper panel). The major features of late genes were the increased levels of H3K14ac, and H3K4me3, FIGURE 4. Transcriptional factor recruitment by ChIP in selected early, the latter particularly at 6 h. Nevertheless, Nos-2 and Il-6 exhibited late, and control genes during taurocholate-induced acute necrotizing different profiles concerning H3K4me2, H3K4ac, and H4K5ac. pancreatitis in rats. ChIP analysis of transcriptional factors occupancy in Il-6 exhibited a high H3K4me2 level under basal conditions de- (A) early, (B) late, and (C) control genes. The Journal of Immunology 7 binding of HAT CBP occurred at 1 h in the early gene Atf3 but at gene. Fig. 6A shows that recruitment of transcription factors EGR-1, 3–6 h in the late genes Nos-2 or Il-6. None of the HAT complexes SP1, and CREB as well as remodeling complex containing BRG1 was found at the a-actin gene (Fig. 3B). was associated with induction of Egr-1 expression in vitro. Impor- tantly, the negative feedback regulation mediated by EGR-1 in vivo Recruitment of transcription factors to promoters of early and was not observed in AR42J cells, which additionally exhibited a late genes in the pancreas during pancreatitis different profile in the recruitment of transcription factors and modi- It is well known that histone modifier complexes are recruited to fier complexes. Fig. 6B shows nucleosomal remodeling measured specific regions of genes through transcriptional factors, or even by micrococcal nuclease protection assay, which characterized Egr-1 through other histone modifier complexes, allowing gene activation upregulation in AR42J cells in vitro. It is worth noting that nucle- in response to cellular signals. After in silico analysis of the pu- osomal movement or eviction at 30 min and 1 h favored the ac- tative binding sites for transcription factors (TRANSFAC analysis), cessibility of transcription factors and therefore transcriptional the candidate transcription factors were analyzed by ChIP assay. activation of Egr-1. Fig. 4A shows that the rapid upregulation of Egr-1 was associated with recruitment of ELK1 and SP1, in particular. It is noteworthy ERK1/2 and JNK mediate Egr-1 upregulation leading to that EGR-1 was recruited to its promoter under basal conditions EGR-1 nuclear translocation but was rapidly released when Egr-1 was upregulated, returning To identify the signaling network that drives the inflammatory stimulus to the promoter when Egr-1 expression progressively decreased. to the Egr-1 promoter, we analyzed the major signaling pathways Hence, EGR-1 seems to exert a negative feedback mechanism in related to inflammation by blocking them with specific inhibitors. the regulation of its own expression. The recruitment of tran- Fig. 7A shows that taurocholate-induced Egr-1 upregulation in Downloaded from scription factors was different for the early gene Atf3 (Fig. 4), AR42J cells was blocked by inhibition of MEK or JNK, but not by which was characterized by binding of EGR-1 and ATF3 at early inhibition of ERK, p38, PI3K, protein kinase A, NF-kBorSTAT3 time points. Therefore, in opposition to EGR-1, ATF3 seems to pathways. In addition, the induction of Egr-1 expression was asso- exert a positive feedback mechanism in the control of its expression. ciated with nuclear translocation of EGR-1 in AR42J cells as shown The upregulation of late genes was associated with recruitment by Western blot (Fig. 7B–C) and immunohistochemistry (Fig. 7D).

of C/EBPb and NF-kB, in particular, concomitant with the highest http://www.jimmunol.org/ Epigenetic changes of Nos-2 promoter induced in vitro by expression of these genes (Fig. 4B). None of the transcription TNF-a in pancreatic acinar cells factors studied were recruited to the promoter of a-actin as control gene (Fig. 4C). Taurocholate only upregulated Egr-1 in vitro but not the other inflammatory mediators typically induced in vivo. Indeed, Atf-3, Chromatin remodeling of Egr-1 promoter upon stimulation of Nos-2, Pap,andTnf-a were not upregulated by taurocholate in vitro pancreatic acinar cells in vitro (data not shown). Hence, we decided to investigate the classical The bile salt taurocholate also triggered a rapid upregulation of inflammatory cascade triggered by TNF-a in vitro. Fig. 8A–D Egr-1 in vitro in AR42J pancreatic acinar cells (Fig. 5), similar to shows that the TNF-a–induced upregulation of Nos-2 in AR42J

the induction found in vivo in the pancreas upon taurocholate- cells was associated with recruitment of NF-kB, and C/EBPb to its by guest on September 28, 2021 induced acute pancreatitis. The upregulation found in vitro was promoter, as well as with K14H3ac, and K18H3me3 epigenetic marks. also due to active transcription, as RNA polymerase II was in- These histone modifications were also associated with recruitment tensely recruited to the promoter and to the coding region of the of HAT CBP together with release of HDAC1 and HDAC3. This

FIGURE 5. The early Egr-1 gene expression in taurocholate-activated AR42J acinar cells. (A) and (B) RNApol ChIP analysis in transcriptional and promoter regions, respectively. (C)and(D) Semiquantitative RT-PCR and quantitative RT-PCR analysis of the Egr-1 mRNA level. rRNA 18S was used as an internal control and b-actin as negative control of the RT-PCR analysis, and a-actin as negative control of ChIP as- say. The statistical significance is ***p , 0.001. 8 EPIGENETIC REGULATION IN ACUTE PANCREATITIS Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 6. Transcriptional factor occupancy and nucleosomal remodeling in EGR-1 during taurocholate-activated AR42J acinar cells. (A) ChIP assay of transcription factors and chromatin remodeling complex binding in Egr-1 promoter in taurocholate-activated AR42J cells. The a-actin gene has been included as negative control of the ChIP experiment. (B) Changes in nucleosome occupancy in the promoter of the Egr-1 gene at different times after taurocholate treatment. (B) (upper panel) Sequence-based prediction of nucleosome affinity carried out using the NuPoP software tool. (B) (middle panel) The micrococcal nuclease protection was determined and plotted against the distance to TSS. The plotted experimental points correspond to the means 6 SE of three determinations. (B) (lower panel) Map of the Egr-1 region under study, showing the location of cis elements, and the experimental positions of the nucleosomes, in non-induced (solid line) and induced (dashed line) Egr-1 gene. At the bottom, the position of their centers in relation to TSS are shown. in-depth and detailed study of epigenetic changes in vitro under very to the coding region (Fig. 9A–C). TNF-a–induced upregulation of precise conditions provided similar findings to those observed in vivo Nos-2 was completely blocked by the inhibition of p38 MAPK, and with the exception of Egr-1. partially restrained by inhibition of MEK/ERK, JNK, NF-kB, or JAK-STAT1. However, inhibition of PI3K, STAT3, or PKA did not TNF-a triggers Nos-2 up-regulation through MAPK and significantly affect Nos-2 upregulation (Fig. 9D). NF-kB in pancreatic acinar cells in vitro Fig. 9 shows that the slow and progressive Nos-2 upregulation in- Chromatin remodeling profile for early and late genes in vitro ducedbyTNF-a in AR42J pancreatic acinar cells in vitro was also Fig. 10 summarizes the pattern of chromatin remodeling for early mediated by active transcription as RNA polymerase II was recruited genes in vitro taking Egr-1 as reference. The rapid upregulation of The Journal of Immunology 9 Downloaded from http://www.jimmunol.org/

FIGURE 7. Upstream Egr-1–activated pathways and nuclear translocation in taurocholate-activated AR42J acinar cells. (A) Egr-1 fold induction after taurocholate treatment compared with DMSO treated AR42J cells, and the abrogated activation or not using the indicated pathways inhibitors. (B) Coomassie gel with nuclear and total protein purification staining and different BSA protein at different concentrations. (C) Western blot analyzing EGR-1 protein levels at nuclear and total fractions. TUBULIN protein levels were used as reference. Lower panel depicts ImageJ analysis of Western blot levels. by guest on September 28, 2021 (D) Confocal laser scanning microscope images using Ab–recognizing EGR-1 (green) during taurocholate-activated AR42J acinar cells. Nuclei were immunostained using DAPI (red). early genes would be mediated by recruitment of transcription through NF-kB (11). Indeed, histone acetyltransferases CBP factors EGR-1 and SP1, as well as histone modifiers CBP and (KAT3A), and p300 (KAT3B) are coactivators of p65 (RelA) (30, BRG1, together with phosphorylation of transcription factors 31). Thus, recruitment of CBP and/or p300 complexes and their mediated by MEK1/2 and JNK and changes in nucleosomal po- corresponding histone acetyltransferase activity are required for sitioning. On the other hand, Fig. 11 shows that the slow upreg- the transcriptional activation of numerous NF-kB driven pro- ulation of late genes would be first mediated by release of HDAC1 moters, such as those of Il-6, Il-8,andNos-2 (30, 32, 33). At and HDAC3, and subsequently by recruitment of NF-kB, C/EBPb, the transcriptional level, selectivity is conferred by interactions CBP, as well as phosphorylation events mediated by all MAPK, between NF-kB and other transcription factors, and coactivators especially p38. that form specific enhanceosome complexes in association with specific promoters (34). It was previously reported that NF- Discussion kB cooperates with STAT1 for Nos-2 upregulation induced by Common inflammatory cascades seem to be involved in the eti- proinflammatory cytokines (35, 36). Here we show that NF-kB ology of acute pancreatitis, and cytokines play a major role in it contributes to Nos-2 upregulation in pancreatic acinar cells to- (14). IL-1b and TNF-a are considered initiators of the inflam- gether with JAK-STAT1, ERK, and JNK, but p38 seems to be the matory cascade because they activate NF-kB, trigger the secretion major driver of this induction. of other proinflammatory mediators, and induce their own secre- In epithelial cells NF-kB induces acetylation, preferentially of tion by a positive feedback mechanism, leading to amplification of lysines 8 and 12 of histone H4, targeting primarily at NF-kB the inflammatory cascade. However, they follow different patterns responsive regulatory elements on proinflammatory genes (37). of secretion through independent pathways. IL-1b is mainly in- Sepsis induced by cecal ligation and puncture in mice increased volved in the early response associated with the inflammasome, both histone H3 and H4 acetylation levels in lungs, in parallel with whereas TNF-a is a major driver of the late response (14, 29). the inflammatory response (38). In our in vivo model of acute inflammation the induction of early Histone acetylation coordinates the induction of early and late genes, such as Egr-1 and Atf-3, was associated with increased inflammatory genes histone acetylation, particularly H3K14, H3K27 and H4K5. In HATs and HDACs coordinate the dynamic transcriptional reg- addition, c-Fos, MyD118, and Il-1b were also identified as early ulation of genes involved in the inflammatory response mainly genes. The upregulation of late genes, such as Nos-2 and Il-6, was 10 EPIGENETIC REGULATION IN ACUTE PANCREATITIS Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 8. Promoter characterization by ChIP of Nos-2 TNFa-activated AR42J acinar cells. The immunoprecipitated samples were analyzed by PCR using primers of the Nos-2 promoter region with indicated Abs that recognize (A) transcription factors, (B) histone modification and (C) histone modification complexes. A-actin was used as negative control of ChIP assay. also associated with increased histone acetylation, particularly binds to the Tnf-a promoter during gene activation (27). As we in H3K14. Pap and Tnf-a were also identified as late genes. In have previously shown, TNF-a in turn activates inflammatory late addition, the upregulation of both early and late genes, with the genes, such as Nos-2, Icam or Il-6, in the pancreas during acute exception of Egr-1, was associated with recruitment of HAT CBP, pancreatitis, because their upregulation was abrogated in TNF-a which was rapid for early genes and slow for late genes. knockout mice (27). The upregulation of early and late genes was due to active In contrast, ATF-3 is a member of the ATF-3/CREB subfamily of transcription following the profile of gene expression. mRNAs of the basic-region leucine zipper family that may interact directly most inflammatory mediators exhibit short half-lives often caused with HDAC1 and recruit HDAC1 to the ATF/NF-kB sites in the by the presence of AU-rich elements in the 39untranslated region Il-6 gene promoter, leading to a blockade of NF-kB binding, and (39UTR), which promotes mRNA decay (39, 40). In agreement, inhibition of inflammatory gene transcription (43). our findings show the profile of active transcription using RNA- Tnf-a is considered by some authors an early-response gene, pol ChIP was parallel with that of steady state levels obtained by because it is rapidly transcribed following exposure to pathogens RT-PCR. or signals of inflammation and stress (44, 45). It has been reported Among the early response genes, Egr-1 and Atf-3 were more that Tnf-a exhibits rapid transcription after stimulation, indepen- deeply studied due to their relevant role in the control of the initial dent of new protein synthesis (44). However, our results clearly phase of the inflammatory cascade. EGR-1 plays a major role in show that Tnf-a expression follows the profile of late-response TNF-a expression in monocytes (41), and it is also capable of genes in vivo. Consequently, the role of TNF-a as a strict early- enhancing p300 expression by itself (42). We have shown that response mediator during the inflammatory response in vivo should EGR-1 upregulation is associated with its nuclear translocation, be revised. which may induce not only early-expressed genes, such as Atf-3 as Our in vitro experiments show that histone modifications change it binds to its promoter (Fig. 4), but also late genes, as EGR-1 in the promoter of early-response genes, particularly for Egr-1,in The Journal of Immunology 11

FIGURE 9. The late Nos-2 gene expression and up- stream pathways activated in TNF-a activated AR42J acinar cells. (A) RNApol ChIP analysis for Nos-2 and a-actin was used as negative control of ChIP assay. (B) and (C) Semiquantitative RT-PCR and quantitative RT- PCR analysis of the Nos-2 mRNA level. rRNA 18S was used as an internal control and b-actin as negative control of the RT-PCR analysis. (D) Nos-2 fold induction after TNF-a treatment compared with DMSO treated AR42J cells and the abrogated activation or not using the indicated pathways inhibitors. The statistical significance is indicated as follows: ***p , 0.001, **p , 0.01, *p , 0.05. Downloaded from

comparison with the in vivo conditions, whereas they were shift from a higher nucleosomal affinity position to a lower one, maintained for late-response genes, with a general increase in revealing the CREB binding site during gene activation. http://www.jimmunol.org/ H3K14ac. Importantly, the in vitro upregulation of Egr-1 was The in vitro histone modifications of late genes were associated associated with recruitment of the HAT CBP– and the BRG-1– not only with the recruitment of HAT CBP but also with the release containing modifier complex, which drives a marked nucleosomal of HDAC1 and HDAC3. Interestingly, secondary genes appear to by guest on September 28, 2021

FIGURE 10. Schematic model for early Egr-1 gene activation during acute necrotizing pancreatitis. Pathways activation, transcription factor occupancy, RNApol II (red oval) and nucleosomes (gray ovals) are depicted at different times. Traffic lights on the right indicated gene expression status (red: silencing, and green: activated). 12 EPIGENETIC REGULATION IN ACUTE PANCREATITIS Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 11. Schematic model for late Nos-2 gene activation during acute necrotizing pancreatitis. Pathway activation, transcription factor occupancy, RNApol II (red oval) and histone modifications are depicted at different times after TNF-a treatment. Traffic lights on the right indicate gene expression status (red: silencing, yellow: potentially activated, and green: activated). be more sensitive to changes in histone acetylation as the synthetic active transcription and dimethylation signaling a state of compound I-BET, which mimics acetylated histones, interferes readiness or competence (48, 49). The upstream trimethylation with the recognition of acetylated histones by the BET family of mark promotes phosphorylation of the C terminal domain of proteins disrupting chromatin complexes, reducing the levels of H3 RNA polymerase II, which is required for transcription ini- and H4 acetylation on LPS-induced gene promoters, and sup- tiation (50). Nevertheless, exceptions to this pattern have been pressing many secondary response genes (46). described (51, 52). Thus, Ash1l (KMT2H), an H3K4 methyl- transferase, suppressed IL-6 and TNF-a production in TLR- Histone methylation during the inflammatory cascade triggered macrophages, and, in addition, protected mice from Specific histone methylation marks also arise during the inflam- sepsis (53). Furthermore, trimethylation in other positions may matory cascade. Thus in a monocytic cell line stimulated with TNF-a, be repressive, and thus Jumonji domain-containing protein 3 methylation of H3R17 together with the acetylation of H3K9 and (JMJD3), also called lysine-specific demethylase 6B (KDM6B), H3K14 was induced at the promoters of NF-kB target genes in a enhances gene expression by demethylating repressive H3K27me3 CBP/p300 dependent manner (47). Furthermore, stimulation of epigenetic marks (54). In vitro experiments in macrophages monocytes led to increased histone acetylation as well as to showed that the pattern of chromatin remodeling varies be- methylation of H3K4 (44). tween primary response genes and secondary response genes Importantly, H3K4me2 and H3K4me3 occur at the tran- (19). Particularly, a high level of histone acetylation and H3K4me3 scription initiation site, with trimethylation correlating with were found under basal conditions in primary response genes The Journal of Immunology 13

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