- Research article
- Open Access
Dynamic reorganization of the AC16 cardiomyocyte transcriptome in response to TNFα signaling revealed by integrated genomic analyses
© Luo et al.; licensee BioMed Central Ltd. 2014
- Received: 30 September 2013
- Accepted: 5 February 2014
- Published: 24 February 2014
Defining cell type-specific transcriptomes in mammals can be challenging, especially for unannotated regions of the genome. We have developed an analytical pipeline called groHMM for annotating primary transcripts using global nuclear run-on sequencing (GRO-seq) data. Herein, we use this pipeline to characterize the transcriptome of an immortalized adult human ventricular cardiomyocyte cell line (AC16) in response to signaling by tumor necrosis factor alpha (TNFα), which is controlled in part by NF-κB, a key transcriptional regulator of inflammation. A unique aspect of this work is the use of the RNA polymerase II (Pol II) inhibitor α-amanitin, which we used to define a set of RNA polymerase I and III (Pol I and Pol III) transcripts.
Using groHMM, we identified ~30,000 coding and non-coding transcribed regions in AC16 cells, which includes a set of unique Pol I and Pol III primary transcripts. Many of these transcripts have not been annotated previously, including enhancer RNAs originating from NF-κB binding sites. In addition, we observed that AC16 cells rapidly and dynamically reorganize their transcriptomes in response to TNFα stimulation in an NF-κB-dependent manner, switching from a basal state to a proinflammatory state affecting a spectrum of cardiac-associated protein-coding and non-coding genes. Moreover, we observed distinct Pol II dynamics for up- and downregulated genes, with a rapid release of Pol II into productive elongation for TNFα-stimulated genes. As expected, the TNFα-induced changes in the AC16 transcriptome resulted in corresponding changes in cognate mRNA and protein levels in a similar manner, but with delayed kinetics.
Our studies illustrate how computational genomics can be used to characterize the signal-regulated transcriptome in biologically relevant cell types, providing new information about how the human genome is organized, transcribed and regulated. In addition, they show how α-amanitin can be used to reveal the Pol I and Pol III transcriptome. Furthermore, they shed new light on the regulation of the cardiomyocyte transcriptome in response to a proinflammatory signal and help to clarify the link between inflammation and cardiomyocyte function at the transcriptional level.
- AC16 Cell
- lncRNA Gene
- Unannotated Transcript
- Hg19 Human Reference Genome
- Crude Nuclear Pellet
The repertoire of coding and non-coding transcripts expressed in a given cell type - the “transcriptome” - reflects the specific biology of that cell type, including responses to external stimuli. Thus, information about the transcriptome can provide deep biological insights with relevance to physiology and disease. Determining and analyzing the complete transcriptome, however, can be challenging, especially with respect to unannotated cell type-specific transcripts. This endeavor, however, has been facilitated by computational genomics approaches that leverage deep sequencing technologies. Herein, we apply these approaches to the study of the cardiomyocyte transcriptome, which has revealed interesting new information related to cardiovascular disease (CVD).
CVD is the leading cause of death worldwide . Many of the underlying pathologies of CVD are directly or indirectly associated with inflammation. Many studies have focused on the effects of inflammation on endothelial function and atherosclerosis [2–4]. However, the detrimental effects of inflammation are not limited to the vascular system, but also occur in cardiomyocytes. The progression from heart injury to heart failure is closely linked to necrosis, apoptosis, or autophagy in cardiomyocytes [5, 6]. During heart failure, cardiomyocytes serve as the major source of cytokine secretion, and the secreted cytokines not only interfere with the function of the cardiomyocytes, but also recruit cardiac fibroblast cells, causing fibrosis and eventually heart damage and infarction [7, 8].
Although the effects of inflammation in cardiomyocytes have been examined previously [9, 10], the detailed mechanisms underlying these effects are poorly understood. NF-κB, a key transcriptional regulator of inflammation, has been shown to play a dual role in CVD through its actions in various cell types of the cardiovascular system. It promotes an anti-apoptotic cardioprotective effect during hypoxia and reperfusion injury by repressing genes involved in cell death pathways, but also supports the secretion of detrimental cytokines during acute or chronic inflammatory injury, leading to cell death and fibrosis [11, 12]. The specific regulatory effects of NF-κB on gene expression programs in cardiomyocytes are not well understood.
Cellular functions and processes are largely determined by carefully orchestrated cell type-specific gene-expression programs. For example, a recent study has characterized an extensive estrogen-regulated gene expression program in breast cancer cells that alters a large fraction of the transcriptome and promotes a mitogenic growth program [13, 14]. A greater understanding of the NF-κB-dependent proinflammatory gene expression program in cardiomyocytes will provide a greater understanding of the links between inflammation and impaired cardiomyocyte function. Non-coding RNAs (ncRNAs) should be a key component of this analysis since previous studies have demonstrated key roles for ncRNAs, including microRNAs (miRNAs) and long non-coding RNAs (lncRNAs), in cardiovascular function [15, 16]. Further mapping and characterization of all functional transcripts, including those generated by RNA polymerases I and III, are necessary for a complete picture of the cardiomyocyte transcriptome.
In the studies described herein, we have used a combination of genomic approaches, including GRO-seq and ChIP-seq, to characterize the transcriptome of AC16 immortalized adult human ventricular cardiomyocyte cells in response to tumor necrosis factor (TNFα). Our studies shed new light on the regulation of the cardiomyocyte transcriptome in response to a proinflammatory signal and help to clarify the link between inflammation and cardiomyocyte function at the transcriptional level.
AC16 cells respond to TNFα stimulation by activating an NF-κB-dependent signaling pathway
The proinflammatory AC16 transcriptome includes a diverse array of coding and non-coding transcripts
To identify all transcripts in the proinflammatory AC16 transcriptome, including previously unannotated transcripts, we combined GRO-seq with a bioinformatics approach called groHMM, which uses a two-state hidden Markov model to identify active transcription units genome-wide . Using this approach, we identified 29,695 transcripts that are expressed in AC16 cells during at least one time point during the course of TNFα treatment (see Methods for details). To ascertain the potential functional role of each transcript, we compared the genomic locations of the identified transcription units with existing genomic annotations. We found that approximately half of the transcription units discovered in our GRO-seq data can be mapped to annotated regions, including genes encoding proteins, long non-coding RNAs (lncRNAs), microRNAs (miRNAs), tRNAs, snRNAs, and repeat elements (Figure 2C), many of which are relevant to cardiac biology (e.g., the mRNA CFLAR, the lncRNA MALAT1, the microRNA 21 precursor MIR21; Additional file 1). The remaining transcription units map to genomic loci that were previously unannotated, but may harbor important genetic information and support important functions within the TNFα response in cardiomyocytes (Figure 2C). We categorized these unannotated transcription units based on their orientation and location relative to annotated genes, including divergent, antisense, and intergenic (Figure 2D). The intergenic transcripts include a category of short, bidirectionally transcribed eRNAs, as we have described previously .
AC16 cells rapidly and dynamically reorganize their transcriptomes in response to TNFα
When analyzing the response of individual classes of transcripts, we found that small non-coding RNAs, lncRNAs, divergent RNAs, and antisense RNAs are up- and down-regulated with similar ratios and kinetics as protein-coding transcripts (Figure 3C). Conversely, intergenic and enhancer transcripts are enriched for upregulation (p < 4 × 10-25; Fisher’s exact test) at every time point of TNFα treatment, which is consistent with their putative gene activation function (Figure 3C). Overall, these analyses reveal a dynamic regulation of the AC16 transcriptome by TNFα that fits with the logic of a proinflammatory stress response: broad repression of transcription, with rapid and robust activation of a selected set of target genes. This pattern of regulation is distinct from the mitogenic transcriptional response that we have characterized previously [13, 14].
GRO-seq reveals different dynamics for the TNFα-dependent activation and repression of transcription
α-Amanitin identifies Pol I, Pol II, and Pol III activity across the AC16 transcriptome
Three different RNA polymerases produce the mammalian cell transcriptome: Pol I transcribes a large transcript from each of the ribosomal DNA (rDNA) loci, which is later cleaved into 18 s, 5.8 s, and 28 s rRNAs, accounting for 50% of the total synthesized RNAs in the cell ; Pol II synthesizes the precursor RNAs for mRNAs and most lncRNAs, microRNAs and snRNAs; and Pol III transcribes the 5 s rRNAs, tRNAs, and other small RNAs closely associated with housekeeping functions . Although the regulation and function of Pol II has been well studied, and recent mapping of the localization of the Pol III transcription machinery genome-wide has shed some light on its transcription profile [25–27], many questions remain regarding the coordination of Pol I, II, and III activities. For example, which polymerase controls synthesis of novel unannotated transcripts? How are Pol II- and non-Pol II-transcribed regions distributed across the genome?
To obtain a greater understanding of the AC16 transcriptome and to investigate coordination among the different RNA polymerases in TNFα-induced inflammatory responses in cardiomyocytes, we used α-amanitin to distinguish between the activities of Pol II (sensitive to the concentration of α-amanitin used) and Pol I/III (not sensitive). Nuclei isolated from AC16 cells were incubated on ice with α-amanitin for 15 min. prior to the run-on reaction that generates the short bromouridine-labeled transcripts for detection by GRO-seq. Since the final read density of each gene is normalized to the total reads obtained in each condition, non-Pol II transcripts are relatively enriched due to the loss or reduction of Pol II transcripts in the α-amanitin-treated condition (Figure 5D). As expected, we observed a relative enrichment of GRO-seq signals from rDNA repeats (Pol I) and tRNA genes (Pol III), as well as a reduction of the GRO-seq signal from annotated RefSeq genes (mostly Pol II) (Figure 5E, F, and G). This pattern serves as a validation of the reliability of our approach in mapping Pol II and non-Pol II transcripts.
Next, we compared the GRO-seq reads at uniquely mapped transcripts between α-amanitin- and vehicle-treated nuclei to determine which types of transcripts were produced by Pol II or Pol I/III (“non-Pol II transcripts”). These results indicate that most of the recently defined types of long non-coding transcripts, such as lncRNAs, eRNAs, divergent RNAs, and antisense RNAs are transcribed by Pol II (Figure 5H; Additional file 2), whereas annotated short non-coding transcripts are distributed between the Pol II and non-Pol II categories (Figure 5I). For example, the majority of small nucleolar RNAs (snoRNAs) and small cytoplasmic RNAs (scRNAs) are transcribed by Pol II, whereas small nuclear RNAs (snRNAs) are transcribed by both Pol II and Pol III, as expected (Figure 5I).
Characterization of the Pol I/III transcriptome in AC16 cells
To further characterize the non-Pol II transcriptome in AC16 cells, we mapped 739 non-Pol II transcripts from GRO-seq data generated ± α-amanitin (Figure 5J). We assume that most are transcribed by Pol III, since Pol I mainly controls transcription from the rDNA repeats, although we did not confirm this experimentally. This set of non-Pol II transcripts includes mainly tRNAs, rRNAs, some snRNAs, and transcripts generated from SINE repeat elements, as well as 172 novel, previously unannotated transcripts (Additional file 3). The lengths of the majority of the 739 primary non-Pol II transcripts are <400 nucleotides, which indicates that they are short, non-coding RNAs (Figure 5K). These transcripts originate mostly from intergenic regions and, to a lesser extent, intronic regions. Only a few transcripts were mapped to the exons of genic regions, concentrated in the 5′ or 3′ UTRs (Figure 5L; Additional file 3).
As expected, a large fraction of the non-Pol II transcripts that we identified overlap with the Pol III machinery (49% and 39% respectively), as indicated by ChIP-seq of the Pol III subunit RPC155 or the Pol III transcription factor TFIIIC (ENCODE data from K562 cells) (Figure 5M), further verifying our ability to identify Pol III transcripts. Many of the transcripts also overlap with CTCF binding sites (33%), which suggests an insulator-like function related to the genes encoding these transcripts. Interestingly, with exception of the aforementioned upregulated tRNA genes (Figure 5B), the expression pattern of the rest of the non-Pol II transcripts remained fairly constant across the time course of TNFα treatment [data not shown; edgeR failed to identify statistically significant regulated genes at any time point of TNFα treatment with a false discovery rate (FDR)-corrected q value threshold (q < 0.001)]. Thus, during the TNFα-induced inflammatory response, transcriptional regulation occurs mostly for Pol II transcripts, but not to a great extent for Pol I and Pol III transcripts.
GRO-seq identifies enhancers in TNFα-stimulated cardiomyocytes
We have recently developed a computational approach for identifying functional enhancers based on these patterns of transcription in GRO-seq data . Using this approach, we identified 1,146 sites of paired intergenic eRNA production in AC16 cells (Figure 6B). Metagene analyses of ChIP-seq data from adult human heart, fetal human heart, and human skeletal muscle myotubes (HSMM) for the 1,146 putative enhancers showed expected patterns of enrichment for well characterized enhancer features, such as p300, H3K4me1, and H3K27ac (Figure 6C). Remarkably, the putative enhancers identified in AC16 cells by GRO-seq match well with enhancer features in the ChIP-seq data from related, but distinct, cell types.
MEME-based motif analyses [32, 33] of the putative NF-κB and non-NF-κB enhancers defined by GRO-seq revealed enrichment of different DNA sequences, which were assigned to specific transcription factors using STAMP . The NF-κB enhancers were highly enriched for the RELA/NF-κB motifs (Figure 6D, left panel) and NF-κB p65 binding (Additional file 4), as expected, as well as AP-1 and FOS motifs (Figure 6D, left panel). The latter is consistent with previous demonstrations that AP-1 augments the NF-κB regulatory program . Interestingly, both NF-κB and AP-1 are activated during heart failure . The non-NF-κB enhancers were enriched in motifs for the transcription factors Sp1, Krüppel-like factor 4 (KLF4), SMAD3, and ZNF143 (Figure 6D, right panel). Other motifs are consistent with previous literature as well. For example, Sp1 has consistently been found in searches for cardiac transcription factors and is associated with the regulation of many cardiac genes [37–39], KLF4 is a critical transcriptional regulator of stress responses in cardiomyocytes [25–27], Smad3 is a key mediator of cardiac inflammation and fibrosis , and ZNF143 is critical for heart development in zebrafish .
Signal-regulated expression of eRNAs is a common theme [13, 20, 29, 30], an effect that we observed with the AC16 enhancers (Figure 6E). Specifically, our analyses revealed that 114 out of 208 (~55%) NF-κB binding site eRNAs are regulated by TNFα, with almost all upregulated, whereas only 169 out of 938 (~18%) non-NF-κB binding site eRNAs are regulated by TNFα, with two-thirds upregulated (Figure 6B, E, and F). Thus, the non-NF-κB binding site enhancers may represent a class of constitutive enhancers that control housekeeping functions in AC16 cells.
To further investigate the potential gene regulatory functions of the predicted NF-κB and non-NF-κB enhancers in AC16 cells, we assayed transcription levels by GRO-seq at the enhancers and their nearest annotated neighboring putative target genes with and without TNFα treatment (Figure 6F). We analyzed separately (1) upregulated NF-κB enhancers (left), (2) upregulated non-NF-κB enhancers (middle), and (3) downregulated non-NF-κB enhancers (right). Interestingly, transcription of the enhancers and target genes were well correlated (i.e., upregulation of enhancer transcription was correlated with an upregulation of target gene transcription, whereas downregulation of enhancer transcription was correlated with a downregulation of target gene transcription) (Figure 6F). These results provide further support for the functionality of the NF-κB and non-NF-κB enhancers predicted by GRO-seq.
The TNFα-induced transcriptional response in AC16 cells reveals a functional link between inflammation and the biology of cardiomyocytes
We augmented this analysis using a database from the Cardiovascular Gene Ontology Annotation Initiative project, which contains more than 4,278 genes critical for cardiac physiology and pathology. A large fraction of both up- and downregulated genes are in the cardiac-associated gene list (Figure 7B) and ~20% are regulated by TNFα treatment (Figure 7C). Interestingly, 166 of the 1,146 enhancers predicted by GRO-seq are located near genes critical for cardiac physiology (data not shown). Collectively, our analyses of the TNFα-altered transcriptome indicate that the AC16 cellular state switches from maintenance of basal housekeeping functions to defense against inflammatory stress.
TNFα-induced transcriptome changes result in corresponding alterations in the steady-state levels of mRNAs and proteins
Role of non-coding RNAs and the TNFα-induced proinflammatory transcriptome
Protein-coding genes represent only part of the AC16 transcriptome; the functions carried out by the non-coding transcripts that we identified may also play critical roles in the inflammatory response in cardiomyocytes. Discerning the potential functions of ncRNAs can be difficult due to limited annotations and direct information available. To overcome these limitations, we performed gene ontology analyses using the Genomic Regions Enrichment of Annotations Tool (GREAT), which aids in predicting the molecular functions, associated biological processes, and disease associations based on the genomic region of interest and nearby genomic regions . As such, GREAT has proven to be a powerful tool for studying cis-regulatory elements. Using GREAT, we found that TNFα-induced lncRNAs, eRNAs, and antisense transcripts are enriched in the same biological processes as the TNFα-induced protein-coding genes (Additional file 6). For example, both CASP8 and FADD-like apoptosis regulator (CFLAR) and its antisense transcript are upregulated upon TNFα stimulation (Additional file 1A). CFLAR is a crucial component of the signaling pathway involved in cardiac remodeling and heart failure . In addition, the lncRNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), which is predicated to be downregulated in many types of heart disease by the NextBio-Disease Atlas (http://www.nextbio.com/b/search/da.nb), is downregulated upon TNFα stimulation (Additional file 1B). In addition to these antisense and lncRNAs, several primary microRNA transcripts that are associated with cardiac function and deregulated in pathological conditions of the cardiovascular system are regulated by TNFα. For example, microRNA-21 (miR-21), an abundant microRNA whose primary transcript is upregulated by TNFα (Additional file 1C), is upregulated in many types of heart disease and may be a useful therapeutic target [49, 50]. Moreover, the non-coding RNA MIRLET7BHG, which is a precursor for five microRNAs including let-7a3 and let-7b, is downregulated upon TNFα treatment (Additional file 1D). Let-7 family members are highly expressed in the heart and are essential for cardiac function and development . Collectively, these data suggest that non-coding RNAs are a key component of the TNFα-mediated proinflammatory transcriptome in cardiomyocytes.
Understanding the proinflammatory cardiomyocyte transcriptome using AC16 cells as a model
Heart disease remains the primary cause of mortality worldwide. Understanding the biology and function of cardiomyocytes is critical to discovering and reducing the causes of cardiac diseases and preventing the progression from heart injury to eventual heart failure. A global view of the proinflammatory cardiomyocyte transcriptome is a key component of our overall understanding. Our integrated genomic analyses using AC16 human cardiomyocytes as a model have helped to identify ~30,000 expressed transcripts under basal and proinflammatory stress conditions, including protein-coding, as well as a wide variety of non-coding, transcripts. Our analyses serve as a guide for studying functionally annotated as well as unannotated Pol I, II, and III transcripts, and our data are an excellent resource for understanding the cardiomyocyte transcriptomes and the transcriptomes of related cell types.
Dynamic regulation of the AC16 transcriptome
A large fraction (18%) of the AC16 Pol II transcriptome is regulated by TNFα, which occurs extremely rapidly, with a large number of transcripts affected within 10 minutes of TNFα treatment (Figure 3). This regulation is highly dynamic, with the maximum regulation for most up- and downregulated transcripts occurring after 30 minutes of TNFα treatment, with a return to basal state at 120 minutes (Figure 4). Such a dynamic regulatory pattern is consistent with the oscillatory pattern of NF-κB nuclear translocation and gene activation  and is likely to play a key role in the signaling outcomes that drive cardiomyocyte biology in response to TNFα. Unlike a mitogenic growth response, which upregulates the expression of Pol I and III transcripts to meet the increased protein synthesis demands of proliferating cells [13, 14], the proinflammatory response has little effect on the expression of Pol I and III transcripts (Figure 5). These differences reflect the different needs of cells responding to mitogenic and stress signals.
Strikingly, we observed that a large fraction (~80%) of TNFα-responsive transcription is downregulated following TNFα treatment (Figure 3C). In this regard, the loss of GRO-seq signal (i.e., active Pol II-mediated transcription) was accompanied by a loss of Pol II protein on the downregulated genes (Figure 4), as expected. Little is known about the mechanisms of gene repression during proinflammatory responses, especially at a global level. Potential mechanisms of repression include: (1) active repression associated with the recruitment of transcriptional corepressors to target genes, (2) release of transcriptional activators, or (3) passive redistribution of the Pol II transcription machinery to other highly induced genes. Interestingly, in general, NF-κB is recruited to TNFα-activated genes, but not TNFα-repressed genes in response to TNFα treatment (Figure 4C), suggesting that the gene repression we observed is not due to modulation of NF-κB binding. Other transcription factors may play a role in downregulation; for example, motif analyses of the downregulated gene promoters revealed a significant enrichment of transcription factor Sp1 binding sites (data not shown).
A functional link between inflammation and cardiomyocyte function at the transcriptional level
The temporal regulation of gene expression in AC16 cells in response to TNFα, as reflected in our GRO-seq analyses, serves as a transcriptional readout of the sequential shift in cellular responses during the time course of treatment (Figure 7A). The upregulated and downregulated biological responses are closely related to cardiac function and indicate a shift from a basal cellular state to a proinflammatory stress-defense state. The transcripts driving these biological responses include those encoding proinflammatory mediators (e.g., NFKB1, IL8) and cell death-related factors (e.g., TNF, CFLAR, APLF), which are induced during the acute proinflammatory response. In addition, the expression of transcripts critical for maintaining normal cardiomyocyte function (e.g., TCF21, CALM1) is disrupted by TNFα treatment. Co-regulated transcripts may share a similar regulatory mechanism or be functionally related. For instance, we identified TNFα-regulated microRNA precursors, which are further processed into several critical cardiac-associated microRNAs (e.g., mir-21 and let-7 family members) that target mRNAs required for cardiac function [49–51].
Cell culture and treatments
AC16 human adult ventricular cardiomyocyte cells  were purchased from the American Type Cell Culture (ATCC). The cells were maintained in DMEM F-12 supplemented with 12.5% fetal bovine serum. TNFα was purchased from PeproTech (cat. no. 300-01A) and the IKKα/β inhibitor BAY-11-7082 was purchased from Calbiochem (cat. no. 196870). For TNFα treatments, the cells were grown to 75% confluence, switched to serum-free medium for 24 hours, and then treated with TNFα (25 ng/ml) for the indicated time. For experiments with BAY-11-7082, the cells were pretreated with the inhibitor (5 μM) or DMSO vehicle for 1 hour prior to treatment with TNFα for 30 minutes.
The antibodies used were as follows: NF-κB p65 (Abcam; cat. no. ab7970), Pol II (Santa Cruz; cat. no. SC-899 and SC-900, mixed in a 1:4 ratio), β-tubulin (Abcam; cat. no.ab6046), SNRP70 (Abcam; cat. no ab51266), c-Fos (H-125, Santa Cruz; cat. no. sc-7202), c-Jun (H-79, Santa Cruz; cat. no. sc-1694), and NF-κB p50 (Abcam; cat. no. ab7971).
Cell fractionation, extraction, and western blotting
For the cytoplasmic and nuclear extraction experiments shown in Figure 1A, AC16 cells were seeded at ~3 × 106 cells per 15 cm diameter plate and treated as described above. After collecting the cells, extracts of the cytoplasmic and nuclear fractions were made according to the protocol provided with the Sigma CelLytic™ NuCLEAR™ Extraction Kit. Specifically, the cells were swollen in isotonic buffer [10 mM Tris–HCl, pH 7.5, 2 mM MgCl2, 3 mM CaCl2, 0.3 M sucrose, 1 mM DTT, and 1x Roche Complete Protease Inhibitor Cocktail (RCPIC)] on ice for 15 minutes and lysed by the addition of 0.6% IGEPAL CA-630 detergent with vortexing. The lysates were centrifuged and the supernatants were collected as the cytoplasmic fraction. The crude nuclear pellet was washed once with isotonic buffer, resuspended in extraction buffer (20 mM HEPES, pH 7.9, 1.5 mM MgCl2, 0.42 M NaCl, 0.2 mM EDTA, 25% v/v glycerol, 1 mM DTT, and 1× RCPIC), and vortexed vigorously for 20 minutes at 4°C. The resuspended nuclear material was then centrifuged and the supernatant taken as the nuclear extract. For each fraction under the indicated conditions, 40 μg protein was analyzed on an 8% polyacrylamide-SDS gel and transferred to a nitrocellulose membrane. Western blotting was performed with the appropriate primary and secondary antibodies.
For the whole cell extraction and Western blotting experiments shown in Figure 8, AC16 cells were seeded at ~1 × 106 cells per 10 cm diameter plate and treated as described above. After collecting the cells, whole cell extracts were made in lysis buffer [50 mM Tris–HCl, pH 7.9, 150 mM NaCl, 1% NP-40, 0.5% Na deoxycholate (DOC), 1% SDS, 1 mM DTT, and 1x Roche Complete Protease Inhibitor Cocktail (RCPIC)] on ice for 30 minutes. The cell lysates were sonicated by using a Bioruptor UC200 at the high setting for a 5-minute cycles of 30 seconds on and 60 seconds off to release the chromatin bound proteins. The lysates were centrifuged and the supernatants were collected as the whole cell extracts. 20 μg protein was analyzed on an 12% polyacrylamide-SDS gel and transferred to a nitrocellulose membrane. Western blotting was performed with the appropriate primary and secondary antibodies.
Bio-plex cytokine assay
AC16 cells were seeded at ~1 × 105 cells per well in 6-well plates and treated as described above. The cell culture supernatants were collected and BSA was added as a carrier protein to a final concentration of 0.6%. The supernatants were centrifuged at 1,000 × g for 15 minutes at 4°C and 10,000 × g for 10 minutes at 4°C to remove cells and cell debris, respectively. The supernatants were assayed on a custom designed Bio-Plex Pro™ cytokine assay chip from Bio-rad to quantify the secretion of IL-6, IL-8 and MCP-1 according to the manufacturer’s instructions and run on a Bio-Plex 200 reader.
RNA isolation and RT-qPCR
AC16 cells were seeded at ~1 × 105 cells per well in 6-well plates and treated as described above. After collecting the cells, total RNA was isolated using TRIzol Reagent (Life Technologies) according to the manufacturer’s protocol. Total RNA was reverse transcribed using oligo (dT) primers and M-MLV reverse transcriptase, and was then subjected to real-time quantitative PCR (qPCR) using gene-specific primers:
All target gene expression was normalized to TBP expression. Each experiment was conducted with a minimum of three biological replicates.
Chromatin immunoprecipitation-sequencing (ChIP-seq)
ChIP was performed as described previously [53, 54] with a few modifications . AC16 cells were seeded at ~3 × 106 cells per 15 cm diameter plate and treated as described above. The cells were cross-linked with 1% paraformaldehyde in PBS for 10 minutes at 37°C and quenched in 125 mM glycine in PBS for 5 minutes at 4°C. The cells were then collected and lysed in Farnham lysis buffer (5 mM PIPES pH 8.0, 85 mM KCl, 0.5% NP-40, 1 mM DTT, and 1x RCPIC). A crude nuclear pellet was collected by centrifugation, resuspended in lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris–HCl, pH 7.9, 1 mM DTT, and 1x RCPIC), and incubated on ice for 10 minutes. The chromatin was sheared at 4°C by sonication using a Bioruptor UC200 at the high setting for four 5-minute cycles of 30 seconds on and 60 seconds off to generate chromatin fragments of ~300 bp in length. The soluble chromatin was diluted 1:10 with dilution buffer (20 mM Tris–HCl, pH 7.9, 0.5% Triton X-100, 2 mM EDTA, 150 mM NaCl, 1 mM DTT and 1x RCPIC) and pre-cleared with protein A agarose beads. The pre-cleared supernatant was used in immunoprecipitation reactions with antibodies against the factor of interest or with rabbit IgG as a control. The immunoprecipitated material was washed once with low salt wash buffer (20 mM Tris–HCl, pH 7.9, 2 mM EDTA, 125 mM NaCl, 0.05% SDS, 1% Triton X-100, 1 μM aprotinin, and 1 μM leupeptin), once with high-salt wash buffer (20 mM Tris–HCl, pH 7.9, 2 mM EDTA, 500 mM NaCl, 0.05% SDS, 1% Triton X-100, 1 μM aprotinin, and 1 μM leupeptin), once with LiCl wash buffer (10 mM Tris–HCl, pH 7.9, 1 mM EDTA, 250 mM LiCl, 1% NP-40, 1% sodium deoxycholate, 1 μM aprotinin, and 1 μM leupeptin), and once with 1x Tris-EDTA (TE). The immunoprecipitated material was eluted in elution buffer (100 mM NaHCO3, 1% SDS) and was then digested with proteinase K and RNase H to remove protein and RNA, respectively. The immunoprecipitated genomic DNA was then extracted with phenol:chloroform:isoamyl alcohol and precipitated with ethanol.
ChIP-seq library preparation
The immunoprecipitated DNA was purified further using the MinElute PCR Purification Kit from Qiagen. After purification, 50 ng of ChIPed DNA for each condition was used to generate libraries for sequencing, as previously described , with some modifications. Briefly, the DNA was end-repaired and a single “A”-base overhang was added using the Klenow fragment of E. coli DNA polymerase. The A-modified DNA was ligated with Illumina sequencing adaptors using the Illumina TruSeq DNA Sample Prep Kit. The ligated DNA (250 ± 25 bp) was size-selected by agarose gel electrophoresis and extraction, amplified by PCR, and purified using AmPure beads (Beckman Coulter). The final libraries were subjected to QC (size, purity, adapter contamination) and sequenced using an Illumina Hiseq 2000 per the manufacturer’s instructions.
ChIP-seq data analyses
NF-κB p65 and Pol II ChIP-Seq data in control and TNFα-treated AC16 cells were generated in the experiments described herein. In addition, existing datasets were downloaded from the NCBI’s GEO (Gene Expression Omnibus) database as listed below, and analyzed:
GSM807734 HumanAdultHeart_acCBP-p300_ChIP-seq 
GSM706848 Fetal_Heart.H3K4me1 
GSM733755 Bernstein_HSMM_H3K27ac 
GSM1022657 UW_ChipSeq_HCM_CTCFRep1 
GSM1022677 UW_ChipSeq_HCM_CTCFRep2 
GSM935372 Harvard_ChipSeq_K562_RPC155_std 
GSM935343 Harvard_ChipSeq_K562_TFIIIC-110_std 
The ChIP-seq reads were aligned to the hg19 human reference genome using the Bowtie software package . Mapped reads were further converted to (1) “bed” files for later Metagene and read-density analyses and (2) “wiggle” files counting reads in non-overlapping 200-bp windows across the genome for presentation as genome browser tracks by using the BEDTools software package .
Global run-on-sequencing (GRO-seq)
Isolation of nuclei
AC16 cells were seeded at ~3 × 106 cells per 15 cm diameter plate and treated as described above. The cells were washed three times with ice-cold PBS, swollen osmotically, and collected in ice-cold lysis buffer [10 mM Tris–HCl, pH 7.4, 0.5% NP-40, 3 mM CaCl2, 2 mM MgCl2, 1 mM DTT, 1x RCPIC, and SUPERase•In™ (Ambion)] and centrifuged at 500 × g for 5 min at 4°C. The cells were then resuspended in 1.5 ml of lysis buffer and pipetted up and down through a narrow tip opening 20 times to release the nuclei. The nuclei were washed twice with a large volume of lysis buffer, and the washed nuclear pellets were resuspended in freezing buffer (50 mM Tris–HCl, pH 8.3, 40% glycerol, 5 mM MgCl2, 0.1 mM EDTA), counted, and stored in 100 μl aliquots containing 5 × 106 nuclei.
GRO-seq library preparation
GRO-seq libraries were generated from two biological replicates of AC16 cells under the indicated treatment conditions, as previously described , but with limited modifications described previously . The TNFα time course GRO-seq libraries were sequenced using an Illumina Genome Analyzer (GAIIx). For the α-amanitin experiments, the isolated nuclei were treated with 1 μg/ml α-amanitin (Sigma, cat. no. A2263) for 15 minutes on ice prior to the run-on reaction. The libraries generated from α-amanitin-treated nuclei were amplified with indexed primers containing barcodes according to the Illumina TrueSeq small-RNA library prep kit, then sequenced using an Illumina Hiseq 2000.
GRO-seq data analyses
GRO-seq data were processed and mapped using a computational pipeline described previously , with limited modifications. Briefly, all reads longer than 32 bp were aligned to the hg19 human reference genome (including autosomes, X chromosome, and a complete copy of rDNA repeats) using the SOAP2.21 software package .
Unbiased transcript calling was performed using an algorithm based on a two-state hidden Markov model as described previously . A shape parameter value of 5 was used for the non-transcribed-state emission probability and a value of 200 was used as the negative log of the transition probability from the transcribed state to the non-transcribed state. To map the relatively smaller non-Pol II transcripts more accurately in the α-amanitin GRO-seq datasets, values of 5 and 10, respectively, were used. In order to capture non-Pol II transcripts more effectively, the control signal was subtracted from the α-amanitin signal using a running maximum of window-size three (25 bp) and adding the baseline of the mean positive signal of the control. Transcripts were then called using an algorithm based on a two-state hidden Markov model as described above.
Functional definitions of called transcripts
Protein-coding transcript. A transcript with more than 20% of its sequence overlapping any well annotated protein-coding gene.
Non-coding transcript. A transcript overlapping an annotated non-coding RNA gene, such as those encoding a miRNA, tRNA, snRNA, or lncRNA, without any restrictions on the size of the transcript or the quality of the overlap. By the standard definition, lncRNAs are non-protein-coding transcripts equal to or longer than 200 nucleotides in the mature (processed) form, whereas short ncRNAs are non-protein-coding transcripts shorter than 200 nucleotides in the mature (processed) form.
Intergenic transcript. A transcript that does not overlap with an annotated gene. Examples are likely to include: (i) novel unannotated protein-coding and non-coding genes, (ii) enhancer transcripts, or (iii) post poly (A) transcription for some well-annotated Pol II genes with low expression levels.
Enhancer transcripts (eRNAs) . A pair of short (< 9 kb) bidirectionally transcribed intergenic transcripts that do not significantly overlap annotated transcripts . We call those that overlap an NF-κB binding site (i.e., ±1000 kb from the center of an NF-κB p65 peak as defined by ChIP-seq) “NF-κB binding site eRNAs” and those that do not overlap an NF-κB binding site “non-NF-κB binding site eRNAs”. Putative target genes for the identified enhancers marked by the eRNAs were defined by searching for the nearest protein-coding or lncRNA gene in either direction.
Divergent transcript. A transcript that overlaps the 5′ promoter driving expression of a detected primary transcript, such as an mRNA or a lncRNA. A divergent transcript was only included if (1) >10% of the transcript overlapped the proximal region of a promoter (± 500 bp relative to the TSS) driving expression of a primary transcript >1 kb in size on the opposite strand and (2) the transcript was <50% the size of the primary transcript, which effectively excluded divergent enhancer-transcript pairs.
Antisense transcript. A transcript that runs antisense to a protein-coding gene or lncRNA gene and has >20% of its sequence overlapping >20% of an annotated protein-coding gene or lncRNA gene on the opposite strand.
Repeat transcript. A transcript with more than 50% of its sequence overlapping genomic regions identified in the RepeatMasker track in the UCSC Genome Browser.
Other genic transcript. A transcript that has a poor match to existing annotations, but cannot be unambiguously classified as “unannotated” or “intergenic”. Transcripts in this category overlap any segment of a gene annotation on either strand, but shows <20% matching to the annotation. Examples in this category may include: (1) genes with promoter proximal RNA Pol II pausing, but very low levels of transcription in the gene body, (2) divergent transcripts from internal start sites (antisense), (3) intronic enhancer transcripts, or (4) short cryptic transcripts of unknown function.
Determining regulation by TNFα
Other genomic data analyses
Metagene analyses were performed to illustrate the distribution of average GRO-seq and ChIP-seq read densities ±5 kb surrounding fixed genomic landmarks (e.g., TSSs, the midpoint of paired eRNAs, center of ChIP-seq peaks) using the metagene functions in our GRO-seq package, as previously described [13, 20, 66].
De novo motif analyses for a 1 kb region around the center of the overlap of paired eRNAs were performed using MEME  with a “–zoops” setting (zero or one occurrence per sequence) and a motif size between 8 and 15. The outputs of MEME were matched to known motifs using STAMP  with default settings.
Gene ontology analyses
Gene ontology (GO) analyses were performed using the Genomic Regions Enrichment of Annotations Tool (GREAT), version 2.0.2 , with the following association rule: Basal + extension: 5000 bp upstream, 1000 bp downstream, 1,000,000 bp max extension, curated regulatory domains included.
Gene set enrichment analyses
Gene Set Enrichment Analysis (GSEA), version 2.0.12 , was used to identify all enriched GO terms at each TNFα treatment time point using the 0 minute condition as a control with a set of GO terms from humans (http://download.baderlab.org/EM_Genesets/September_02_2011/Human/symbol/GO/Human_GO_bp_no_GO_iea_symbol.gmt). For ranked inputs to GSEA, we used pre-ranked gene lists based on edgeR differential analysis after filtering out gene sets whose size was greater than 500 or less than 25. Specifically, significantly regulated genes (FDR < 0.1%) were placed at the top or bottom of the list and ordered by descending or ascending fold changes, respectively. Less significantly regulated genes (FDR ≥ 0.1%) were placed in the middle of the list and ordered by ascending or descending p-values.
Hierarchical clustering and heatmaps
Hierarchical clustering was performed using the results of the GSEA. GO terms with the top ten Normalized Enrichment Scores (NESs) were selected and combined from both the upregulated and downregulated GO terms in each time point, compared to the 0 min treatment condition. Heatmaps were generated using the heatmap.2 function in the gplots package in R with default parameters. Inputs for the heatmaps included (1) normalized GRO-seq signals (median-centered and scaled relative to the 0 min time point for expression analyses; e.g. Figure 3B) and (2) NESs (e.g., Figure 7A). For the latter, heatmaps comparing later to earlier time points (e.g., 10 min vs. 30 min, or 30 min vs. 120 min) were generated in a similar manner, but using different edgeR outputs for the comparisons.
The GRO-seq and ChIP-seq data sets described herein are available from the NCBI Gene Expression Omnibus (GEO) (http://www.ncbi.nlm.nih.gov/geo/) using accession number GSE51225.
We thank Shrikanth Gadad, Ziying Liu, and Bryan Gibson for critical comments on this manuscript, and Anusha Nagari for submitting the data files to NCBI/GEO. This work was supported by predoctoral fellowships from the DOD Breast Cancer Research Program (BC093731) to X.L. and the American Heart Association to R.K.; an NIH training award (T32HD052471) and a postdoctoral fellowship from the PhRMA Foundation to C.G.D.; and a grant from the NIH/NIDDK (DK058110) to W.L.K.
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