IL-1 Transcriptional Responses to Lipopolysaccharides Are Regulated by a Complex of RNA Binding

This information is current as Lihua Shi, Li Song, Kelly Maurer, Ying Dou, Vishesh R. of October 1, 2021. Patel, Chun Su, Michelle E. Leonard, Sumei Lu, Kenyaita M. Hodge, Annabel Torres, Alessandra Chesi, Struan F. A. Grant, Andrew D. Wells, Zhe Zhang, Michelle A. Petri and Kathleen E. Sullivan J Immunol published online 17 January 2020 http://www.jimmunol.org/content/early/2020/01/16/jimmun Downloaded from ol.1900650

Supplementary http://www.jimmunol.org/content/suppl/2020/01/16/jimmunol.190065 http://www.jimmunol.org/ Material 0.DCSupplemental

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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 © 2020 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published January 17, 2020, doi:10.4049/jimmunol.1900650 The Journal of Immunology

IL-1 Transcriptional Responses to Lipopolysaccharides Are Regulated by a Complex of RNA Binding Proteins

Lihua Shi,* Li Song,* Kelly Maurer,* Ying Dou,*,1 Vishesh R. Patel,* Chun Su,† Michelle E. Leonard,† Sumei Lu,† Kenyaita M. Hodge,† Annabel Torres,‡,x,{,‖ Alessandra Chesi,† Struan F. A. Grant,†,‡,x,{ Andrew D. Wells,†,‖ Zhe Zhang,# Michelle A. Petri,** and Kathleen E. Sullivan*

The IL1A and IL1B lie in close proximity on 2 near the for their natural inhibitor, IL1RN. Despite diverse functions, they are all three inducible through TLR4 signaling but with distinct kinetics. This study analyzed transcriptional induction kinetics, chromosome looping, and enhancer RNA production to understand the distinct regulation of these three genes in human cells. IL1A, IL1B, and IL1RN were rapidly induced after stimulation with LPS; however, IL1B mRNA production was

less inhibitable by iBET151, suggesting it does not use pause-release regulation. Surprisingly, chromatin looping contacts between Downloaded from IL1A and IL1B were highly intermingled, although those of IL1RN were distinct, and we focused on comparing IL1A and IL1B transcriptional pathways. Our studies demonstrated that enhancer RNAs were produced from a subset of the regulatory regions, that they were critical for production of the mRNAs, and that they bound a diverse array of RNA binding proteins, including p300 but not CBP. We, furthermore, demonstrated that recruitment of p300 was dependent on MAPKs. Integrator is another RNA binding recruited to the promoters and enhancers, and its recruitment was more dependent on NF-kB than MAPKs. We

found that integrator and NELF, an RNA polymerase II pausing protein, were associated with RNA in a manner that facilitated http://www.jimmunol.org/ interaction. We conclude that IL1A and IL1B share many regulatory contacts, signaling pathways, and interactions with enhancer RNAs. A complex of protein interactions with enhancer RNAs emphasize the role of enhancer RNAs and the overall structural aspects of transcriptional regulation. The Journal of Immunology, 2020, 204: 000–000.

he IL-1 family of cytokines is comprised of 11 members the genes for which are located on chromosome 2q14 in close that all function in the regulation of inflammation (1). apposition. IL-1a is a dual function cytokine regulating tran- T Among the IL-1 family members are IL-1a and IL-1b, scription directly through a DNA binding function as well as regulating the inflammatory response through the binding of a by guest on October 1, 2021 cell membrane receptor (2). IL-1a in the nucleus binds chromatin *Division of Allergy Immunology, Children’s Hospital of Philadelphia, Philadelphia, PA 19104; †Center for Spatial and Functional Genomics, Children’s Hospital of Phil- and is generally induced through proapoptotic signals. Necrotic adelphia, Philadelphia, PA 19104; ‡Division of Human Genetics, Children’s Hospital of signals, in contrast, cause IL-1a to distribute primarily in the Philadelphia, Philadelphia, PA 19104; xDivision of Endocrinology and Diabetes, Child- { a ren’s Hospital of Philadelphia, Philadelphia, PA, 19104; Department of Pediatrics, cytoplasm (3). When released from the cell, IL-1 stimulates a Children’s Hospital of Philadelphia and University of Pennsylvania Perelman School of robust inflammatory response, driving chemokine expression ‖ Medicine, Philadelphia, PA, 19104; Department of Pathology and Laboratory Medi- that regulates the influx of neutrophils and monocytes. cine, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA # b 19104; Department of Biomedical and Health Informatics, Children’s Hospital of IL-1 has similar but nonoverlapping functions, and it functions Philadelphia, Philadelphia, PA 19104; and **Division of Rheumatology, Johns Hopkins as a cytokine. It was discovered as a key inflammatory agent University School of Medicine, Baltimore, MD 21205 driving fever in rabbits (4). IL-1b is transcriptionally and 1 Current address: Division of Hematology and Oncology, Children’s Hospital of posttranslationally regulated and is primarily induced after Chongqing Medical University, Chongqing, China. recognition of microbial patterns (5, 6). Monocytes appear to be ORCIDs: 0000-0001-6388-8666 (C.S.); 0000-0002-5490-3884 (S.L.); 0000-0002- b 0954-7446 (A.C.); 0000-0003-2025-5302 (S.F.A.G.); 0000-0003-1441-5373 (M.A.P.); the main cell that produces IL-1 following stimulation with LPS. 0000-0003-4018-1646 (K.E.S.). The transcriptional induction of IL-1b leads to the accumulation Received for publication June 11, 2019. Accepted for publication December 5, 2019. of an inactive protein precursor in the cytoplasm (pro–IL-1b that This work was supported by National Institutes of Health (NIH) Grant R01 ES017627 is processed by the activation of nucleotide binding domain and and the Wallace Chair of Pediatrics (K.E.S.). S.F.A.G. is funded by the Daniel B. leucine-rich repeat pyrin containing protein-3) to active IL-1b (7). Burke Endowed Chair for Diabetes Research, Children’s Hospital of Philadelphia, Near the IL1B gene, encoding IL-1b, lies the gene IL1RN that and NIH Grant R01 HG010067. A.D.W. is supported by NIH Grants R01 AI130115 and R01 AI123539. encodes the IL-1R antagonist (IL-1ra). IL-1ra is a potent inhibitor a b Address correspondence and reprint requests to Dr. Kathleen E. Sullivan, Division of of both IL-1 and IL-1 (8) by blocking the IL-1R1. This agent is Allergy Immunology, Children’s Hospital of Philadelphia, 34th Street and Civic Center used to treat inflammatory conditions such as arthritis and dis- Boulevard, Philadelphia, PA 19104. E-mail address: [email protected] orders of the inflammasome (9). Despite the fact that IL-1ra The online version of this article contains supplemental material. functions as an antagonist of IL-1a and IL-1b, it too is induced Abbreviations used in this article: ASO, antisense oligonucleotide; ATAC-seq, assay for primarily by microbial byproducts. The close approximation of transposase-accessible chromatin using sequencing; ChIP, chromatin immunoprecipi- tation; eRNA, enhancer-derived RNA; FWD, forward; IL-1ra, IL-1R antagonist; these three genes with similar regulatory patterns offers an oppor- INTS11, integrator 11 complex; OCR, open coding region; PRB, probe; qRT-PCR, tunity to dissect commonalities and differences in the transcriptional quantitative RT-PCR; REV, reverse; RNA IP, RNA immunoprecipitation; SLE, sys- regulatory pattern of these three genes. Understanding the regu- temic lupus erythematosus. lation of these three genes is of critical importance in chronic Copyright Ó 2020 by The American Association of Immunologists, Inc. 0022-1767/20/$37.50 inflammatory conditions in which increased expression of the

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1900650 2 IL-1 TRANSCRIPTIONAL RESPONSES TO LPS antagonist might provide clinical benefit. We noted increased ex- 59-TAGAGCTAGCGAATTATACAAAATTAGCTGGGCATGG-39; CD831A299- pression of all three mRNAs in cells from patients with systemic lupus FWD, 59-TAGAGCTAGCGAATTCCCAAAGCTGGACGTGCTG-39; 9 erythematosus (SLE), and the use of the therapeutic IL-1ra antagonist CD831A299-reverse (REV), 5 -ACGCGGCCGCGGATCAATTGCAGTG- TAAAGTGTAAAACAC-39; and CD831A327-REV, 59-ACGCGGCCGCG- has shown promise in SLE (10). Thus, there is a clinical rationale for GATCGATGAACTTTCCAATAAGACAAACC-39. the examination of transcriptional pathways regulating this locus. Inhibitors and stimuli There is also a strong scientific rationale for the study of enhancer– promoter interactions at this locus. Much of what is understood The inhibitors included 20 mM C646 (Santa Cruz Biotechnology, Dallas, regarding the mechanism by which enhancers regulate transcription TX). It is an inhibitor of p300-induced histone acetylation (29, 30). Twenty has come from genome-wide studies of developmentally regulated micromolars of iBET151 (Life Science Research, Billerica, MA) was used at a specific inhibitor of BRD3/4 that is important for transcriptional ac- systems (11, 12). The IL-1 locus, in contrast, exhibits a poised tivation and elongation at paused genes (31). Forty micromolars of DRB status, and the mechanisms of enhancer function are not well un- (Sigma-Aldrich, Allentown, PA) was used as an inhibitor of elongation derstood in this setting (13). In developmental systems, enhancers regulating the eviction of NELF via P-TEFb (32). Twenty micromolars of are active in a cell-specific and development-specific fashion. Upon JSH-23 (Santa Cruz Biotechnology) was used as a specific inhibitor of NF-kB translocation (33), and 10 mM IT901 (Bio-Techne, Minneapolis, activation driven by developmental signals, most enhancers are MN) was used as a specific antagonist of p65 NF-kB (34). Ten micromolars marked by the histone modification H3K27ac, and they produce SP600125 (Calbiochem, Darmstadt, Germany) was used as an inhibitor of regulatory RNAs (14, 15). The enhancer-derived RNA (eRNA) is the JNK MAPK. Toxicity analyses confirmed minimal cell death at these important for bridging the enhancer to the proximity of the pro- concentrations. HPLC-purified LPS was purchased from Sigma-Aldrich. moter and plays a role in elongation by RNA polymerase II (16, 17). Transcriptional analysis The contacts are often stabilized by cohesin and CTCF; loops are Total RNAwas extracted by TRI Reagent (Sigma-Aldrich), and the Direct-zol Downloaded from not only critical for the activation of transcription, but CTCF can RNA MiniPrep Kit (Zymo Research, Irvine, CA) was used for cleanup. The also act to insulate promoters from enhancer effects, contributing to Clontech Advantage RT-PCR Kit (Takara Bio, Mountain View, CA) was the specificity of the enhancer effect. Three-dimensional chro- used to make cDNA. Transcript levels were detected by real-time PCR using matin loops actively regulate the transcriptional program (18–20), the TaqMan 7900 normalizing to the 18S signal. Noncoding RNAs were and the final transcriptional state of any given promoter integrates detected with custom SYBR green primers and normalized to actin. Primer sequences are listed as follows: IL1-327 FWD, 59-GCATG- signals from multiple enhancers (whose effects are additive) (21), GAACTGTGCTCTACAGGAA-39; IL1-327 REV, 59-TTCTATCC- promoter–proximal transcription factors, and insulators and other CATGCAAAGTTGCTCA-39; IL1-288 FWD, 59-GCA AGC CGA CTA http://www.jimmunol.org/ negative regulators. Very few studies have examined enhancer ATG TGT TAT TT-39;IL1-288REV,59-GGT CCT TCT AAT CCC GTT 9 9 9 effects in settings in which the induction of expression is rapid, GTG-3 ; IL1-299 FWD, 5 -CTTGTGTCCTGTTGAGAGAGAG-3 ;IL1- 299 REV, 59-CTTGAGTCCCTGCTTCCATATT-39; IL1-338 FWD, 59- and promoters are already poised (22). These genes are of critical AGGCTCAGGAATTGGGTATTT-39; IL1-338REV, 59-GGGTGGAA- importance in inflammatory settings, yet the rules governing en- AGGTAGAGAAAGAG-39; 896_FWD, 59-ATCCCACCTCTTCCGACT- hancer activation in such a setting are largely unexplored. Another 39;896_REV,59-CAGTGACGCTGAATGAATGAATAC-39;477FWD, compelling reason to better understand the dynamics and mech- 59-TGCTCTTCACTATGGCTCTTTAC-39; and 477 REV, 59-GTCA- 9 anisms of this process is the recent recognition that the most GCGTTGTTGTCAGTTTAC-3 . common variants implicated in disease susceptibility lie in regu- Capture-C by guest on October 1, 2021 latory regions and impact the level of transcription, functioning as Promoter connectome analysis was performed essentially as in Chesi et al. expression quantitative trait loci (23–25). (35). Briefly, 1 3 107 primary monocytes were fixed with 1% formalde- Our data demonstrate that these three genes in the IL-1 locus (IL1A, hyde. After 10 min, 1 M glycine was added to stop the process. Cells were IL1B,andIL1RN) lie within a chromosome region with pre-established multiply washed before ultimately being snap frozen in lysis buffer chromatin loops and a poised transcriptional status. LPS treatment (10 mM Tris [pH 8.0], 10 mM NaCl, and 0.2% NP40 with protease in- hibitor) and stored in 280˚C. The Chromatin Conformation Capture appears to drive eRNA production and nucleation of RNA binding process was followed as described (35). The pellet was centrifuged, and proteins that collectively regulate expression. Despite similar kinetics lysis buffer was removed. Protease inhibitor mixture was added, and the and signaling pathways, transcriptional mechanisms are distinct at the resuspended samples were divided into six tubes with a predigestion three genes. control reserved (control 1). Digestion of DNA used DpnII (New England BioLabs) overnight at 37˚C. Before starting the ligation reaction, 100 mlof each digestion was removed and combined to create the digest control Materials and Methods (control 2). DpnII was inactivated at 65˚C. T4 DNA Ligase (Thermo Fisher Cells Scientific) was added. Proteinase K (Invitrogen) was used to de–cross-link at 65˚C, and RNase A (MilliporeSigma) was added. The efficiency of the Primary monocytes were obtained from a campus core facility under an digestion process was tested through quantitative PCR and a 0.9% agarose Institutional Review Board–approved protocol. Subjects gave informed gel. A 10-mg aliquot of each Chromatin Conformation Capture library was consent. The SLE samples were previously reported, and all subjects then sonicated using a QSonica Q800R. Once the DNA was sheared, gave informed consent (26–28), although these analyses are new. The AMPure XP beads (Agencourt) were used to clean the samples, and size MonoMac6 cell line is a human monocyte line maintained in RPMI 1640 was assessed on a Qubit fluorometer (Life Technologies) and a Bio- with 10% cosmic calf serum with OPI supplement (Sigma-Aldrich, St. analyzer 2100. Capture-C libraries were made using the Agilent SureSelect Louis, MO). MonoMac6 cells were obtained from the German source and m XT Library Prep Kit (Agilent Technologies). After the precapture PCR, thawed from primary vials every 2–3 mo. LPS treatment used 1 g. the concentration and size of the sample were measured. One micro- Transfection of cells gram of each library was hybridized and captured following the SureSelect XT method. The concentration was tested on the Qubit, and Transfection of cells was performed by electroporation with the Amaxa the size was measured by Bioanalyzer using a DNA High Sensitivity Cell Line Lonza Nucleofector Kit (Amaxa Biosystems, Gaithersburg, Chip. Five replicate libraries were barcoded using Nextera Index and MD) or Lipofectamine 2000 (Thermo Fisher Scientific, Waltham, MA). free adaptor blocking kits and sequenced to 1.2 billion 50-bp reads per Two million cells were transfected with 500 nM antisense oligonucleotide library on the Illumina NovaSeq 6000 platform. Paired-end reads from (ASO) using nucleofection. ASO used were as follows: IL1-299ASO1, five biological replicates for both LPS-treated and untreated monocytes 59-G*G*A* A*G*T* T*C*T* T*G*C* T*G*T* G*C*A-39;IL1- were preprocessed using the HICUP pipeline (v0.5.9) (36), with bowtie2 as 299ASO2, 59-A*G*A*A*A*T*A*T*C*C*A*C*A*T*T*T*T*T-39; aligner and hg19 as the reference genome. Significant promoter interac- IL1ALNC327_ASO2, 59-C*C*C* T*T*T* C*T*G* T*A*C* T*C*T* tions at 1-DpnII fragment resolution were called using CHiCAGO (v1.1.8) T*C*C* C*T*C-39; and IL1ALNC327_ASO7, 59-G*T*T* C*T*C* (37) with default parameters except for binsize set to 2500. Significant T*C*C* T*T*C* C*T*C* C*T*T* T-39. The following overexpres- interactions at 4-DpnII fragment resolution were also called at artificial 4 sion primers for vectors were used: CD831A327-forward (FWD), fragment resolution in which DpnII fragments were concatenated in silico The Journal of Immunology 3 into four consecutive fragments using default parameters except for with glycine. The fixed cells were lysed in RIPA buffer (1% NP40, 0.5% remove Adjacent set to False. The significant interactions (CHiCAGO sodium deoxycholate, 0.05% SDS, 1 mM EDTA, and 150 mM NaCl) and score .5) from both 1-fragment and 4-fragment resolutions were sonicated. The precleared lysates were incubated with Abs and protein exported in ibed format and merged into a single file using a custom A agarose at 4˚C. Immune complexes were washed and reverse cross- PERL script to remove redundant interactions and to keep the maximal linked. Direct-zol RNA MiniPrep Kit was used to extract the immuno- CHiCAGO score for each interaction. precipitated RNA. To test the role of RNA in protein–protein interactions, we treated lysates Assay for transposase-accessible chromatin using sequencing with RNases before coimmunoprecipitation. MonoMac6 cells were stim- and data analysis ulated with HPLC-purified LPS (1 mg/ml; Sigma) for 0, 10, and 30 min. Cells were lysed and precleared with protein A agarose beads (Thermo Chromatin profiling was performed by assay for transposase-accessible Fisher Scientific). One thousand and five hundred micrograms of protein chromatin using sequencing (ATAC-seq) as described previously (38). was used in each immunoprecipitation. Two micrograms of Ab (NELF-A; Briefly, 12,000–50,000 cells were washed in cold PBS and lysed in cold Santa Cruz) was added to the protein and incubated overnight at 4˚C. lysis buffer (10 mM Tris-HCl [pH 7.4], 10 mM NaCl, 3 mM MgCl , and 2 Thirty microliters of protein A agarose beads were added for 60 min the 0.1% IGEPAL). Transposition was performed at 42˚C for 45 min with next day, and the beads were washed. Beads were resuspended in 30 mlof Nextera DNA Library Prep kit (Illumnia, San Diego, CA). DNA was pu- lysis buffer with 50 mg/ml of RNase A (Roche) and incubated at 37˚C for rified with the MinElute PCR Purification Kit (QIAGEN, Hilden, Germany) 15 min. After the incubation, they were spun down, and both the super- and was amplified for 12 cycles with the primers from Nextera Index Kit natant and beads were collected. The beads were washed twice in PBS and (Illumina). Additional PCR cycles were evaluated by real-time PCR. Final resuspended in lysis buffer, and both supernatant and beads were run on a product was cleaned by Ampure Beads. Libraries were sequenced on a 4–12% Bis-Tris Novex gel (Thermo Fisher Scientific). The membrane was HiSeq 2500 1T in a 50 bp/50 bp paired-end run, using the TruSeq SBS Kit probed with Ab to the integrator 11 component (INTS11) (Bethyl Labo- v3 (Illumina). LPS-treated and control monocyte ATAC-seq peaks were ratories). To test the ability of RNA to increase INTS11–NELF-A inter- called independently using the Encyclopedia of DNA Elements ATAC-seq actions, both proteins were produced as GST-tagged proteins and purified pipeline. Briefly, paired-end reads from four biological replicates for each Downloaded from using glutathione beads. In vitro production of eRNAs used SP6 or T7. condition were aligned to hg19 genome using bowtie2, and duplicate reads 2 Proteins were mixed 1:1 at 1 3 10 8 M with a 33 molar excess of eRNAs were removed from the alignment. Narrow peaks were called independently and incubated at 37˚C for 15 min. Anti–NELF-A was used for the recovery for each replicate using macs2 (-p 0.01–nomodel–shift-75–extsize 150-B– of the complexes on protein A beads overnight. Blotting with Ab to INTS11 SPMR–keep-dup all–call-summits), and Encyclopedia of DNA Elements was performed as above. blacklist regions (ENCSR636HFF) were removed from peaks in individual replicates. Peaks from all replicates were merged by bedtools (v2.25.0) Statistical analyses within each cell type, and the merged peaks present in less than two bio- logical replicates were removed from further analysis. Finally, ATAC-seq The results represent a minimum of three separate biological experi- http://www.jimmunol.org/ peaks from both conditions were merged to obtain reference open chromatin ments. Each figure legend describes the number of individual exper- regions. To determine whether an open coding region (OCR) is present in LPS iments. Comparisons across treatments were performed using Student or control cells, we first intersected peaks identified from individual replicates t test. in each condition with reference OCRs. If any peaks from at least one replicate overlapped with a given reference OCR, we considered the region as open. Results Chromatin immunoprecipitation assays Increased nucleotide binding domain and leucine-rich repeat pyrin containing protein-3 activation leading to IL-1b production has 3 6 The 5–10 10 cells in each condition were treated with 1% formalde- been observed in SLE patients and is thought to be critically hyde for 10 min at room temperature to cross-link. Glycine was used to important in lupus nephritis (42–45). In our previously published quench the cross-linking, as previously described (39, 40). Lysed cells by guest on October 1, 2021 were sonicated and immunoprecipitated overnight at 4˚C. Immune com- RNA sequencing analysis (27), we noted that the IL-1 family plexes were collected with protein A (Invitrogen, Carlsbad, CA), washed genes were upregulated in monocytes from patients with SLE extensively, and eluted. DNA was extracted by phenol–chloroform and (Supplemental Table I) (27). IL1RN, encoding IL-1ra, was in- m m treated with proteinase K (200 g/ml; Roche) and RNase (40 g/ml; creased ∼2-fold over control monocytes, whereas the IL1A RNA Roche). Abs used included those to CDK9 (a component of P-TEFb), NELF-A, c-JUN, NF-kB p65, CBP, and p300 Abs (all from Santa Cruz abundance was also significantly increased in monocytes from Biotechnology, Santa Cruz, CA); H3K4me3 Ab (Active Motif, Carlsbad, patients with SLE. IL1B was increased, but the difference did not CA) and H3K27ac Ab (Active Motif) were used in chromatin immuno- reach statistical significance, and the nearby IL37 gene was not precipitation (ChIP) assays and RNA immunoprecipitation (RNA IP). The expressed in monocytes. Based on these data, we hypothesized GST Ab (Invitrogen, Camarillo, CA) was used as a negative control for all ChIP assays. Cells were harvested at 30 min after LPS stimulation. Signals that there may be abnormal transcriptional induction of the IL-1 are reported as units normalized to 10% input according to the equation gene family in SLE and designed our studies to understand 2^(10% input cycle threshold–Sample cycle threshold). The primers for commonalities and discordant mechanisms of gene regulation the ChIP assays are as follows: IL1-299 enhancer FWD, 59-GCATGT- in the IL-1 family at this locus. 9 9 TACAGCTGTGGATAAC-3 ; IL1-299 enhancer REV, 5 -GACATGGGT- We first established the three-dimensional architecture of the GAAGGGAAAGA-39; IL1-299 enhancer probe (PRB), 59-/56-FAM/ AGCATCCTATTGTGTTTAGATGAGCCTGG/3IABkFQ/-39;IL1-338 IL-1 locus in primary monocytes. The arrangement of the genes enhancer FWD, 59-TTTAGATGAGCCTGGTCTTTCC-39; IL1-338 en- in the context of the one-dimensional sequence of the human IL-1 hancer PRB, 59-/56-FAM/ATGCACATTAAAGGCCTCCCTGGA (sense)/ family locus is depicted in Fig. 1A. To better understand the 9 9 3IABkFQ-3 ; IL1-338 enhancer REV, 5 -TGCTATTGTCTGCCTCACTTC- transcriptional regulation at this locus, we defined the relationship 39; IL1-327 enhancer FWD, 59-TTTCCTGAAATCTAGAGAGCATACA-39; IL1-327 enhancer PRB, 59-56-FAM/TAGGCAGTGCTCACCAAATACCTGC/ of the enhancers to the target promoter using a genome-wide 3IABkFQ-39; IL1-327 enhancer REV, 59-GCAAGGGAGACCAGGAAATTA- promoter-focused chromatin conformation capture approach we 39; IL1A promoter FWD, 59-GACTCAAACGCCAATGAAATGA-39; recently developed (35). The resulting Capture-C data were used IL1A promoter PRB, 59-/56-FAM/AAAGGCGAAGAAGACTGACTCAGGC/ to specifically understand the local chromatin folding at this locus. 9 9 9 3IABkFQ-3 ; IL1A promoter REV, 5 -GTAGCCACGCCTACTTAAGAC-3 ; Interactions between regulatory elements and the IL1B, IL1A, and IL1B promoter FWD, 59-GCCATGCACTGGATGCTG-39;IL1Bpromoter REV, 59-TGGCTGCTTCAGACACCT-39;IL1BpromoterPRB,59-/56-FAM/ IL1RN promoters were identified in cells stimulated with LPS and ACACATGAACGTAGCCGTCATGGG/36-/3IABkFQ-39; GAPDH FWD, unstimulated cells. Two distinct three-dimensional regulatory 59-CGGTGCGTGCCCAGTT-39; GAPDH PRB, 59-/56-FAM/ACCAG- domains were apparent (Fig. 1A). The first notable finding is that 9 9 GCGGCTGCGGAAAAAA/36-3IABkFQ-3 ; and GAPDH REV, 5 -CC- IL1A and IL1B share numerous contacts with putative regulatory CTACTTTCTCCCCGCTTT-39. elements, whereas IL1RN, despite similar transcriptional kinet- RNA–protein analyses ics, uses its own unique set of regulatory contacts. IL37,whichis The RNA IP assay was modified as described (41). Briefly, 20–40 million not expressed in these cells, has no loops. The second notable cells were cross-linked using 1% formaldehyde for 10 min and quenched finding is that the looping pattern is more focused after LPS. 4 IL-1 TRANSCRIPTIONAL RESPONSES TO LPS Downloaded from http://www.jimmunol.org/

FIGURE 1. Long-range chromatin interactions at the IL-1 region. Primary monocytes were mock treated (blue) or treated with 1 mg/ml LPS (red) for 30 min. (A) Promoter contacts from IL1A, IL1B, IL37, and IL1RN were analyzed by Capture-C in five separate biological replicates. Interactions were designated based on the geometric mean of the replicates. (B) ATAC-seq signal profile across IL1A, IL1B, and IL37. This was performed on the same cells. (C) H3K27ac, H3K4me1, and H3K4me3 peaks from primary monocytes treated with or without LPS for 1 h. The ChIP sequencing data were extracted from a published study (80). (D) The long-range chromatin contacts defined by Capture-C and eRNA locations are displayed as an expanded view for IL1A, IL1B, and IL37. by guest on October 1, 2021

IL1RN loses all of its downstream contacts after LPS, and IL1A transcriptional regulation (46, 47). Both primary monocytes and loses a major set of loops from the promoter to a cluster of MonoMac6 cells exhibited induction of IL1A and IL1B after 90 min regulatory sites that lie between IL1B and IL37. Loss of loops of LPS. The eRNAs were similarly induced after LPS with the could imply release from a negative regulatory effect or simply exception of 477, which we use as a negative off-target control. The the refocusing of loops on the strongest positive regulatory ef- 477 is an enhancer RNA located on but not induced fect. Also noted were loops from each promoter to the 39 end of by LPS and was used as a negative control. TNF was included as a the gene. These were stable after stimulation. For better visuali- positive control for LPS induction (40, 48). There were differences zation of IL1A and IL1B, expanded views are shown in Fig. 1B–D. between cell types and between inhibitors. The NF-kB inhibitor, ATAC-seq tracks from the same cells as the Capture-C studies are IT901, and the BRD4 inhibitor, iBET, had the most consistent in- shown in Fig. 1B, and ChIP sequencing tracks from published data hibitory effects across mRNAs and eRNAs and both cell types. The are shown in Fig. 1C. Fig. 1D is an expanded view of the data on magnitude of the LPS effect and the inhibitors was greater in Fig. 1A. We selected regulatory elements with diverse behaviors, MonoMac6 cells, and the need for high cell numbers for the ChIP stable contacts (299 and 338 for IL1B) or contacts lost with LPS studies led us to use this model cell line for subsequent studies. In (327 for IL1B, and 299 and 338 for IL1A), for further study based MonoMac6 cells, the 327 RNA species was more inhibitable by on these findings. All three regulatory elements are embedded in iBET and C646 than the other eRNAs. The 299 and 338 eRNAs H3K27ac peaks that increase after LPS (Fig. 1C). All three have behaved similarly with respect to inhibitor effects, and they lie in a detectable ATAC-seq peaks at baseline that increase after LPS single complex regulatory region upstream of IL1B. (Fig. 1B). The 338 and 299 lie in a complex web of contacts Although eRNA production is not a universal requirement for upstream of IL1B and have baseline contacts with IL1A and IL1B enhancer activation, the majority of active enhancers are actively that simplify to IL1B alone after LPS. The 327 eRNA, in contrast, transcribed in either a unidirectional or bidirectional pattern (49, has contacts with IL1B at baseline, which are lost after LPS. 50). We defined the location of the RNA transcripts associated In monocytes, both IL1A and IL1B mRNAs were induced by with these putative regulatory contact regions using MonoMac6 LPS. We compared responses in a cell line model of peripheral cells. The RNAs were found in chromatin, and they increased after blood monocytes and primary monocytes from healthy donors LPS stimulation (Supplemental Fig. 2). Chromatin localization (Fig. 2). Kinetics for the effect of the inhibitors in MonoMac6 supports a regulatory role for these RNA species. Actin (ACTB) cells are shown in Supplemental Fig. 1. One key finding is that in is shown as a typical mRNA localized to cytoplasm, and XIST the kinetic analysis, IL-1B is less inhibitable by iBET151 com- is shown as a noncoding regulatory RNA localized to chromatin as pared with IL1A and IL1RN, an effect previously described for controls for cell fraction purity. These results indicate that these IL1B, which does not use pause release in the standard fashion for RNA species associated with the loop contacts are chromatin The Journal of Immunology 5

FIGURE 2. Analysis of LPS induction of RNAs. MonoMac6 cells and primary mono- cytes were treated with 1 mg/ml LPS for 90 min. Total RNA was extracted, and qRT-PCR was performed. IL1A, IL1B,andTNF were ex- amined as mRNAs. The inhibitors JSH-23, iBET151, C646, SP600125, 6-dichloro-1-a-D- ribofuranosylbenzimidazole, and IT901 were tested for effects on RNA abundance. These graphs represent the mean of n = 3 bio- logical replicates (error bars represent SD). The asterisks (*) indicate a consistent statistical significance across all measured RNA species (except TNF for mRNAs and 477 eRNAs, which represent controls). Supplemental Fig. 1 provides additional detailed views of the data in a time course. Downloaded from associated and embedded in chromatin with regulatory charac- region. These data suggest that a complex of interactions occurs in a teristics. Thus, we will refer to these RNAs as eRNAs. stimulus-inducible manner, centered on RNA, uniquely for each re- A recognized function of eRNAs is to activate enzymatic activity gion, a concept with growing support (62–64). of the RNA binding histone acetyltransferases CBP and p300 (51). To test whether the eRNAs recruit additional protein complexes, We hypothesized that one or both of these proteins play a role in we analyzed the binding pattern of other known RNA binding http://www.jimmunol.org/ enhancer activation at LPS stimulation. To determine if CBP and proteins to the eRNAs using RNA IP (Supplemental Fig. 3). J2 is p300 are localized to the enhancer or promoter of these genes, we an Ab that recognizes dsRNA. S9.6 recognizes RNA:DNA hy- performed a ChIP analysis for CBP and p300 using anti-GST as a brids. RBFOX bridges PRC2 to chromatin and is required for control (Fig. 3A). We observed increased binding of p300, but not transcriptional repression, and binding is RNA dependent, similar CBP, to the enhancers and promoters of IL1A and IL1B following to CBP and p300. SUZ12 is a Zn finger protein component of LPS treatment in MonoMac6 cells. SP600125 (JNK inhibitor) PRC2, and it binds nascent transcripts. EZH2 is a methyl- inhibited p300 binding to chromatin, indicating that LPS-driven transferase component of PRC2, and binding RNA inhibits en- recruitment of p300 to chromatin is JNK dependent. p300 is di- zymatic activity. TIP60 recognizes R loops (RNA:DNA at by guest on October 1, 2021 rectly phosphorylated by MAPKs (52–54), and it is known to transcription bubbles). TIP60 binding was not induced by LPS. interact with a variety of transcription factors (55–57). Our data EZH2 binding went down with LPS, and SUZ12 showed in- place p300 recruitment and activation at an early point in tran- creased binding to the eRNAs after LPS. Therefore, there are scriptional activation. CBP surprisingly does not appear to be considerable protein–RNA interactions after LPS even if there involved. are no major changes in chromatin loops. We concluded that the To further test whether CBP and/or p300 regulate IL-1 family eRNAs participate in multiple protein complexes at the enhancer– members via eRNA binding, we performed RNA IP by pulling promoter interface. down CBP and p300 and analyzing the bound RNA by quantitative We hypothesized that eRNAs, produced before mRNA tran- RT-PCR (qRT-PCR) (Fig. 3A). GFP is a negative control Ab. scription, bind p300, driving increased histone acetylation. This is a Consistent with the chromatin binding results, eRNA binding to recognized model for eRNA function, but this setting is concep- p300 was significantly increased after LPS treatment, whereas tually different from that seen in development in which the acti- eRNA binding to CBP was not detected. These results together vation of enhancers and eRNA production underlies chromatin loop suggest that LPS-driven enhancer activation of IL-1 family genes formation to bring the enhancer into proximity of the regulated is mediated by JNK-dependent p300 binding to chromatin and promoter(s) (16, 65, 66). Our Capture-C data suggested that the rapid eRNA interaction with p300. loops are largely pre-established at these loci. To test whether To additionally understand whether other RNA binding proteins the eRNAs participate in mRNA regulation, we used an ASO nucleate at the promoter-enhancer interface, we examined binding knockdown approach to examine the role of the eRNAs on mRNA of the CDK9 component of P-TEFb and the E component of NELF, expression. We first confirmed that each ASO led to diminished two proteins involved in pause–release (Fig. 3B). We had previ- levels of the cognate eRNA (data not shown). The knockdown of ously shown that the SERPINB2 promoter was regulated in a eRNA 327, which contacts the IL1B promoter in unstimulated pause–release manner (58). The IL-1 family is similarly induced monocytes and gains a connection to IL1A after LPS, resulted in rapidly after stimulation, and we tested whether NELF and the diminished IL1A mRNA after LPS treatment, with less of an effect CDK9 component of P-TEFb bind to these eRNAs in a manner on IL1B transcript levels (Fig. 4A). In contrast, the knockdown of similar to that observed at the SERPINB2 locus. Recent data eRNA 299, which is part of a large enhancer complex that loops support a role for enhancers at pause–release promoters (59–61). to both IL1B and IL1A promoters in unstimulated monocytes, NELF rapidly bound to all the eRNAs, whereas CDK9 bound only significantly impaired expression of both IL1B and IL1A after LPS 299 and 338 after LPS. The kinetics of binding were very rapid induction (Fig. 4B). In addition, IL1RN mRNA levels remained and were selective for eRNAs induced by LPS. The 327 did not unchanged when knocking down these two eRNAs, as anticipated, bind CDK9 and had a distinct pattern of inhibition in Fig. 2 because IL1RN does not loop to these two regions. This study compared with 338 and 299, which share a single complex regulatory demonstrates that the eRNAs are required for full transcriptional 6 IL-1 TRANSCRIPTIONAL RESPONSES TO LPS Downloaded from http://www.jimmunol.org/ by guest on October 1, 2021

FIGURE 3. LPS induces p300 but not CBP binding to eRNAs and chromatin. (A) ChIP analysis of p300 and CBP binding. MonoMac6 cells were pretreated with JNK inhibitor SP600125 (SP) for 30 min and then stimulated with 1 mg/ml LPS for 15 min. ChIP assays were performed using p300, CBP, or GST (negative control) Abs. Bound promoter (IL1B and IL1A) and enhancers (327, 338, and 299) were detected by quantitative PCR. Results are the means plus SD (n =4,*p , 0.05 comparing LPS treatment to unstimulated [large asterisks] and inhibitor plus LPS to LPS alone [small asterisks]). RNA IP was used to identify RNA binding to proteins. MonoMac6 cells were treated with 1 mg/ml LPS for 0, 10, or 30 min. RNA IPs were performed using p300, CBP, or GFP (negative control) Abs. Bound RNAs were detected by qRT-PCR. The 477 is an enhancer RNA located on chromosome 1 but not induced by LPS and was used as a negative control. Results displayed represent the means plus SD (n = 4, asterisks (*) indicate the same as for ChIP assays). (B) MonoMac6 cells were treated with 1 mg/ml LPS for 0, 10, or 30 min. RNA IP was performed using NELF-A or CDK9. Bound RNAs were detected by qRT-PCR. The eRNAs were observed to significantly bind to CDK9 and NELF upon LPS treatment, peaking at 10 min. The 477 is a control eRNA that is not LPS inducible. Results displayed represent the means with SD (n =4,*p , 0.05). induction after LPS even without large changes in chromatin loops LPS was less susceptible to inhibition. This is again consistent and suggests a hierarchy of effects of the eRNAs on mRNA with the low-level baseline transcription of IL1B and less depen- regulation. dence on pause–release. We hypothesized that such a large com- Finding that the eRNAs are structurally important for mRNA plex, including enhancer DNA, eRNA, promoter DNA and at least transcription at the IL-1 locus and that they bound to pause–release five proteins besides the RNA polymerase complex, might require proteins suggested to us that integrator may be involved in IL1A a scaffold to maintain structural integrity. To test whether the and IL1B transcriptional regulation. Integrator is known to reg- protein complexes might be held by a structural aspect of the ulate NELF eviction from the paused RNA polymerase complex eRNAs, we digested cell lysates with RNase A and then immu- (67). We first performed a ChIP assay for INTS11 and found that noprecipitated with NELF and blotted with an Ab to INTS11 integrator was bound to both promoters and enhancers after LPS (Fig. 5B). Although INTS11 was easily coimmunoprecipitated in stimulation. Furthermore, blockade of NF-kB with JSH-23 led the absence of RNase treatment, RNase treatment led to the re- to markedly compromised recruitment of integrator at all sites, lease of integrator from the complex. These data suggest that the whereas JNK inhibition had a more modest effect, the opposite of eRNAs may act as scaffolding for some of the proteins complexed what was observed for p300 recruitment to chromatin (Fig. 5A). at the enhancer–promoter interface. To further test this, we pro- We observed greater integrator occupancy at the IL1B promoter duced NELF and integrator as GST-linked proteins and incubated at baseline compared with the other targets, and recruitment after them with various RNA species (Fig. 5C). All of the RNA species The Journal of Immunology 7

FIGURE 4. IL1 eRNAs regulate transcription. MonoMac6 cells were transfected with the indi- cated 20-bp phosphorothioate ASO designed to knock down eRNAs (two different ASOs each) or an ASO to GFP for 3 h and stimulated with or without 1 mg/ml LPS for 60 min. Total RNA was isolated, and qRT-PCR was performed to measure levels of eRNAs and mRNAs. (A) Knockdown of 327 eRNA significantly decreased the LPS induc- tion of IL1A but had less effect on IL1B and no B effect on IL1RN.() Knockdown of 299 eRNA Downloaded from significantly decreased the LPS induction of IL1A and IL1B mRNAs but did not affect the IL1RN transcript levels. Fold change indicates levels compared with the GFP ASO in the unstimulated cells (n =3, *p , 0.05). PCRs were normalized to actin. http://www.jimmunol.org/ by guest on October 1, 2021 increased the protein–protein interaction, although the eRNAs promoter at baseline and declines after LPS, whereas H3K4me3 is appeared to have a greater effect than actin. more moderate at baseline at the IL1A promoter and increases after LPS. The IL1B gene has been described as having Spi-1/PU.1 Discussion bound to the promoter at baseline and then stimulus induction of These studies of acute activation of transcription define a distinct NF-kB to the same region of the promoter (46). Based on our data, model for the role of chromatin in transcriptional regulation. In recruitment of integrator is an additional step downstream with developmental systems, eRNAs are critical for loop formation, and PU.1→NF-kB→integrator representing a central regulatory differentiation depends on the correct approximation of enhancers pathway that is in turn part of a larger complex of RNA binding and promoters related to fate determination and differentiation (68, proteins. IL1A may require recruitment of a histone methyl- 69). Many inflammatory genes use a pause–release transcriptional transferase, usually SET1 at proinflammatory genes (72). Re- mechanism, presumed to facilitate rapid activation of transcription quirement for this extra step, compared with IL1B, may explain in response to threat (70, 71). Our data defined a complex set of the delayed kinetics of IL1A mRNA accumulation. We also loops at baseline that focused after LPS, a conceptually different found that the BRD4 inhibitor, iBET151, had markedly different view of looping than that seen in developmental systems. This effects on IL1B compared with IL1A. IL1B mRNA expression may be a systematic effort to promote the rapid transcriptional was delayed, but expression of IL1A was nearly completely response of these critical cytokines. With enhancers already ap- abrogated with iBET151 treatment. The role of BRD4 is to proximated to their cognate promoters, transcription of mRNAs activate P-TEFb and release RNA polymerase II from pausing. These can be very rapid. With this information, it was possible to ask data suggest that IL1A is more dependent on BRD4 for pause–release about the role of the eRNAs. If the loops are established at than IL1B, and coupled with the requirement to recruit a histone baseline, what might the role be for eRNAs in promoting tran- methyltransferase, certainly contributes to the slower kinetics of ex- scription? To address this question, we first established the critical pression. IL1B has previously been found to have less dependence on pathways regulating the eRNAs and mRNAs. We hoped to exploit pause–release (46). Commonalities noted for IL1A and IL1B are their commonalities and differences between IL1A and IL1B to dissect reliance on NF-kB and MAPK pathways for mRNA production and pathways regulating enhancer–promoter interactions. their overall rapid induction. Collectively, our data identify central The transcriptional patterns of IL1A and IL1B are subtly dif- differences in mechanisms of regulation that may help to explain ferent. IL1A mRNA accumulates more slowly than that of IL1B differences in their kinetics of expression and highlight the critical and its final abundance is lower. IL1B mRNA level at baseline is role of chromatin in regulating transcription. substantially higher than that of IL1A. In accordance with the With this detailed information on transcriptional induction of baseline transcription of IL1B, H3K4me3 is very high at the IL1B the mRNAs, we went on to similarly dissect eRNA production. 8 IL-1 TRANSCRIPTIONAL RESPONSES TO LPS

FIGURE 5. Integrator interac- tions. (A) An INTS11 (INT11) ChIP assay was performed to analyze INTS11 binding to the promoters and enhancers. Inhibition of the NF-kB pathway by JSH-23 (JSH) and the JNK pathway with SP600125 (SP) led to diminished binding with greater effects observed with JSH-23 (n = 4). (B) Coimmunoprecipitation was used to show the interaction of INTS11 and NELF-A. MonoMac6 cells were treated with LPS (1 mg/ml) for 0, 10, or 30 min. Lysates were

collected and treated with RNase A Downloaded from as indicated, and anti-NELFA was used for immunoprecipitation. After RNase treatment, INTS11 interac- tions with NELF were decreased. The NELF blot of the same gel is shown below for quantitative com- parison. (C) Purified proteins were http://www.jimmunol.org/ incubated with various types of RNAs in an effort to test the ability of RNAs to facilitate interactions between integrator and NELF. Vari- ous RNAs increased the interaction between the two proteins. There were at least five biological repli- cates of these analyses. “+” repre- sents the lysate, and “2” represents the negative control of mouse IgG. by guest on October 1, 2021

In general, the eRNAs were less inhibitable than the mRNAs but for p300. The MAPK pathway was critical for p300 recruitment to were again dependent on MAPKs and NF-kB. The three eRNAs chromatin. Several studies identified a role for MAPK in acti- we focused on in this study arose from H3K27ac peaks that in- vating p300 binding in very specific settings, but it is not yet clear creased after LPS. In terms of expression patterns, two eRNAs if MAPKs represent the major pathway for p300 activation (55, behaved similarly with subtly different responses to the inhibitors 74, 75). Our study contributes to the emerging understanding of compared with 327, which is located in a different region. We early assembly of transcriptional complexes at enhancers. These went on to demonstrate a key role for the eRNAs in IL1A and IL1B data allow the sequence of events to be established at least at the mRNA production by knocking them down with an antisense IL-1 locus. NF-kB may be responsible for the initial eRNA technology. This confirmed, as has been seen in many systems, a production and integrator recruitment to the promoter, but mechanistic effect of eRNAs; however, the precise role and se- MAPKs appear to be important for recruitment of p300 to the quence of events involving eRNAs is not yet defined for pause– enhancers. Upon recruitment of p300 and binding eRNAs, p300 release genes. We therefore identified key binding partners and acetylates H3K27ac. This is thought to act as a platform for the dependencies. CBP and p300 are highly homologous histone assembly of the transcriptional activation complex. acetyltransferases (73). The enzymatic activity is activated by Integrator has been characterized as a multimolecular complex RNA, but the functional niches in which each is dominant remain that serves as a scaffold and has recently been implicated in the to be defined (51). The p300 protein was inducibly recruited to pause–release transcriptional regulation (67, 76). Both enhancers both enhancers and promoters after LPS and bound eRNAs. LPS- and promoters bound INTS11 in an LPS-inducible manner, al- inducible CBP binding to chromatin was not identified. These data though the IL1B promoter appeared to have some INTS11 bound suggest that p300 may be more critical for inflammatory gene at baseline. This is consistent with the overall impression that expression than CBP, and in our previous study of a different IL1B transcription is active at baseline. INTS11 assembly into a locus, we also found that the eRNAs bound to p300 after LPS and higher order complex with NELF, one of the main pausing pro- not CBP (64), supporting the inflammatory gene set as a key niche teins, was facilitated by RNA, although not wholly dependent on The Journal of Immunology 9

RNA. INTS11 has been previously shown to interact with NELF blueprint, we exploited differences to better understand key mech- using coimmunoprecipitation from nuclear extracts, and it may be anisms regulating expression. that unrecognized RNA was enhancing the interaction (67). These key findings lead to a model in which eRNAs act in many and Disclosures varied structural roles. Through binding to p300, histone acetyl- The authors have no financial conflicts of interest. transferase activity is regulated by eRNAs, and the very landscape of H3K27ac is therefore dependent on eRNA production. In ad- dition, the higher order complex of enhancer–promoter–integrator References appears to have an RNA dependency. How the eRNAs correctly 1. Dinarello, C. A. 2018. Overview of the IL-1 family in innate inflammation and acquired immunity. Immunol. Rev. 281: 8–27. partner with the proteins in each promoter–enhancer pair is not 2.Werman,A.,R.Werman-Venkert,R.White,J.K.Lee,B.Werman, known, and the Capture-C data suggest that multiple enhancers Y. Krelin, E. Voronov, C. A. Dinarello, and R. N. Apte. 2004. The precursor can participate in the transcriptional regulation of a single pro- form of IL-1alpha is an intracrine proinflammatory activator of transcrip- tion. Proc. Natl. Acad. Sci. USA 101: 2434–2439. moter. This leads to a dynamic model in which the chromatin is 3. Cohen, I., P. Rider, Y. Carmi, A. Braiman, S. Dotan, M. R. White, E. Voronov, variably looped, and interactions may be transient with the sta- M. U. Martin, C. A. Dinarello, and R. N. Apte. 2010. Differential release of chromatin-bound IL-1alpha discriminates between necrotic and apoptotic cell bilization of favorable interactions after LPS. death by the ability to induce sterile inflammation. Proc. Natl. Acad. Sci. USA We selected this locus specifically to compare IL1A and IL1B.In 107: 2574–2579. a previous study, we identified that these pivotal cytokines cluster 4. Dinarello, C. A., N. P. Goldin, and S. M. Wolff. 1974. Demonstration and characterization of two distinct human leukocytic pyrogens. J. Exp. Med. 139: together with IL-6 in terms of their kinetics of expression and their 1369–1381. behavior with various inhibitors (77). IL-1a and IL-1b are func- 5. Schindler, R., J. Mancilla, S. Endres, R. Ghorbani, S. C. Clark, and tionally similar, although structurally very different. Surprisingly, C. A. Dinarello. 1990. Correlations and interactions in the production of Downloaded from interleukin-6 (IL-6), IL-1, and tumor necrosis factor (TNF) in human blood their promoter contacts to regulatory regions were highly inter- mononuclear cells: IL-6 suppresses IL-1 and TNF. Blood 75: 40–47. mingled but completely distinct from those of IL1RN. Although 6. Schindler, R., P. Ghezzi, and C. A. Dinarello. 1990. IL-1 induces IL-1. IV. IFN-gamma suppresses IL-1 but not lipopolysaccharide-induced tran- we identified their coincreased expression in SLE, common fea- scription of IL-1. J. Immunol. 144: 2216–2222. tures by clustering analysis, and rapid induction after LPS, there 7. Perregaux, D. G., P. McNiff, R. Laliberte, M. Conklyn, and C. A. Gabel. 2000. were differences in the mechanisms of transcriptional regulation. ATP acts as an agonist to promote stimulus-induced secretion of IL-1 beta and J. Immunol. IL-18 in human blood. 165: 4615–4623. http://www.jimmunol.org/ IL1B appeared to have completed more steps in the path to expression 8. Hannum, C. H., C. J. Wilcox, W. P. Arend, F. G. Joslin, D. J. Dripps, than IL1A, and low levels of mRNA were seen at baseline. This is P. L. Heimdal, L. G. Armes, A. Sommer, S. P. Eisenberg, and R. C. Thompson. consistent with emerging models in which pause–release is not a 1990. Interleukin-1 receptor antagonist activity of a human interleukin-1 in- hibitor. Nature 343: 336–340. binary quality but is part of a spectrum of pausing (78, 79). According 9. Aksentijevich, I., M. Nowak, M. Mallah, J. J. Chae, W. T. Watford, to this model, IL1B is not highly paused and is therefore less sus- S. R. Hofmann, L. Stein, R. Russo, D. Goldsmith, P. Dent, et al. 2002. De novo CIAS1 mutations, cytokine activation, and evidence for genetic heterogeneity in ceptible to inhibition by iBET151, as was observed. Although the patients with neonatal-onset multisystem inflammatory disease (NOMID): a new enhancers we focused on also had many similarities and either at member of the expanding family of pyrin-associated autoinflammatory diseases. baseline or after LPS were found to interact with both promoters, Arthritis Rheum. 46: 3340–3348. 10. Ostendorf, B., C. Iking-Konert, K. Kurz, G. Jung, O. Sander, and M. Schneider. there were also differences between the enhancers. The 327

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B

A/B) MonoMac6 cells were treated with 1μg/ml LPS for 30, 60,180, and 240 minutes and total RNA was extracted. The eRNA and mRNA expression levels were measured using qRT-PCR at various time-points and normalized to 18S in MonoMac6 cells. A) The data in this panel maximizes the aspect of kinetics. B) The same data as panel A displayed with error bars. N=3, error bars indicate SD. Supplemental Figure 2

MonoMac6 cells were mock or LPS treated. RNA was extracted from fractions: cytoplasm (Cyto)), nucleoplasm (NP), and chromatin (Ch) fractions. qRT-PCR was performed after reverse transcription. 477 is a non-LPS inducible eRNA. ACTIN and XIST are shown as positive controls for cytoplasmic and chromatin fractions, respectively (n=3, error bars in panels denote SD). Supplemental Figure 3

MonoMac6 cells were treated with 1μg/ml LPS for 60 minutes. RNA immunoprecipitations were performed using TIP60, RB-FOX, SUZ12, EZH1, J2 or S9.6 antibodies. Bound RNAs were detected by qRT-PCR. 477 is a control eRNA not induced by LPS. Results are the means plus standard deviation (n=4, error bars in panels denote SD).