Expression and regulation of long noncoding RNAs in TLR4 signaling in mouse macrophages
© Zuo et al.; licensee BioMed Central. 2015
Received: 26 July 2014
Accepted: 22 January 2015
Published: 5 February 2015
Though long non-coding RNAs (lncRNAs) are emerging as critical regulators of immune responses, whether they are involved in LPS-activated TLR4 signaling pathway and how is their expression regulated in mouse macrophages are still unexplored.
By repurposing expression microarray probes, we identified 994 lncRNAs in bone marrow-derived macrophages (BMDMs) and classified them to enhancer-like lncRNAs (elncRNAs) and promoter-associated lncRNAs (plncRNAs) according to chromatin signatures defined by relative levels of H3K4me1 and H3K4me3. Fifteen elncRNAs and 12 plncRNAs are differentially expressed upon LPS stimulation. The expression change of lncRNAs and their neighboring protein-coding genes are significantly correlated. Also, the regulation of both elncRNAs and plncRNAs expression is associated with H3K4me3 and H3K27Ac. Crucially, many identified LPS-regulated lncRNAs, such as lncRNA-Nfkb2 and lncRNA-Rel, locate near to immune response protein-coding genes. The majority of LPS-regulated lncRNAs had at least one binding site among the transcription factors p65, IRF3, JunB and cJun.
We established an integrative microarray analysis pipeline for profiling lncRNAs. Also, our results suggest that lncRNAs can be important regulators of LPS-induced innate immune response in BMDMs.
KeywordsTLR4 LPS elncRNA plncRNA Histone modification
TLR4, a founding member of the TLR family, is a pattern recognition receptor for lipopolysaccharide (LPS) that can induce inflammatory response and cause septic shock . Stimulation of TLR4 by LPS results in the rapid activation of transcription factors, the best characterized of which are interferon regulatory factors (IRFs), the nuclear factor-kappa B (NF-κB) and activator protein 1 (AP-1) families.
In recent years, tens of thousands of long non-coding RNAs (lncRNAs) have been identified in the mammalian genomes, many of which have been implicated in a range of developmental processes and diseases [2-5]. Though most of lncRNAs have been primarily studied in the context of genomic imprinting, developmental process and cancer, lncRNAs are now emerging as important regulators of both innate and adaptive immune responses . Mammalian CD11c + dendritic cells produce many thousands of lncRNAs when stimulated with LPS . The lncRNA Ptprj-as1 is highly expressed in macrophage-enriched tissue and transiently induced by TLR ligands with similar pattern to Ptprj . TLR signaling also induces lncRNA-Cox2, which serve as both repressor and activator of genes through interactions with various regulatory complexes . Li et al. identified a lncRNA THRIL regulating TNFα expression through its interaction with hnRNPL during innate activation of THP1 macrophages . Using a global clustering algorithm based on ChIP-seq signals of RNA polymerase II and H3K4me3, Garmire et al. identified a list of putative lincRNAs in mouse macrophages . Most recently, Ilott et al. discovered that both canonical lncRNAs and enhancer lncRNAs regulated the LPS-induced inflammatory response in human monocytes . However, systemic characterization of LPS-regulated lncRNAs in mouse BMDMs is lacking so far.
More and more studies have suggested that although lncRNAs are not specifically targeted in the original array design, a large portion of probes can be reannotated for interrogating lncRNA expression [13-19]. Compared to RNA-seq of low sequencing coverage, microarray data have lower technical variations and higher sensitivity for transcripts with low abundance [20,21], which is a markedly feature of lncRNAs . Additionally, microarray datasets contain strand information, thus allow for interrogating the expression of antisense lncRNAs.
In this study, we aim to explore the activities and potential functions of lncRNAs in LPS-induced innate immune response in mouse BMDMS. To this end, we firstly repurposed different expression microarray platforms to identify lncRNAs from reannotated probes. We then performed an integrative expression analysis of these identified lncRNAs on publicly available expression datasets on LPS-stimulated BMDMS. By using qRT-PCR, we validated the expression changes of some lncRNAs. We classified the lncRNAs to elncRNAs and plncRNAs according to chromatin status defined by relative levels of H3K4me1 and H3K4me3 surrounding transcription start sites. We further examined the correlation of the expression change between lncRNAs and nearest neighboring protein-coding genes. Crucially, several lncRNAs are near to immune response genes, and these pairs are significantly co-expressed, such as lncRNA-Nfkb2/Nfkb2, lncRNA-Rel/Rel. The majority of LPS-regulated lncRNAs have at least one binding site among the transcription factors p65, IRF3, JunB and cJun, further indicating their potential roles in immune response.
Reannotating microarray probes for lncRNAs in BMDMs
We classified lncRNAs based on their proximity and relative orientation to protein-coding genes (Figure 1C). The 994 lncRNAs with TSS evidence were classified as follows: exonic sense (overlapping a protein-coding gene exons on the same strand), intronic sense (only overlapping a protein-coding gene introns on the same strand), antisense (overlapping a protein-coding gene locus on the opposite strand), biodirectional (on the opposite strand to a protein-coding gene locus and the distance of TSSs is within 1 kb), and intergenic (no-overlapping with a protein-coding gene locus and besides biodirectional) (Figure 1C). The number and distribution of lncRNAs among the different classes were: exonic sense (49, 4.9%), intronic sense (28, 2.8%), antisense (402, 40.4%), bidirectional (224, 22.5%), intergenic (291, 29.3%) (Figure 1D; Additional file 7). Since majority of exonic sense lncRNAs may simply represent fragments of 5′ and 3′ UTRs or nonsense-mediated mRNA decay (NMD) isoforms of protein-coding genes , we excluded exonic sense lncRNAs from further analysis.
LPS-regulated lncRNAs in BMDMs
Chromatin signatures separate elncRNAs and plncRNAs
It has been well established that histone modification changes are associated with changes of lncRNA expression, which is confirmed in our finding. We observed that differences in H3K4me3 and H3K27Ac were positively correlated with changes in both elncRNAs and plncRNAs expression (Figure 4C). H3K4me1 was not associated with expression change upon LPS stimulation for both elncRNAs and plncRNAs (Figure 4C). PolII signal for LPS regulated elncRNAs and plncRNAs was significantly changed upon LPS stimulation (Figure 4C).
Correlation between lncRNA and neighboring protein-coding gene expression
LPS-regulated lncRNAs closely related to inflammatory response
LPS-induced transcriptional regulation of lncRNAs in BMDMs
Previous studies mainly focused on the study of LPS-regulated protein-coding genes but ignored the function of lncRNAs involved. To explore the potential role of lncRNAs in the activation of TLR4 signaling, we constructed a comprehensive bioinformatics pipeline to reannoate probes to lncRNA from literature expression microarray datasets in BMDMs. Giving that large number of such datasets are available in public repositories, the pipelines we generated will be useful for reannotating array probes to address different biological questions.
Our integrated lncRNA and protein-coding gene expression profiles are valuable resources for understanding the LPS-stimulated program, as well as their co-regulation. Having established that LPS induced widespread changes in the expression of lncRNAs in mouse macrophages situated close to differentially expressed immune response-related genes, it was important to determine whether these were functionally relevant. Of great interest was the identification of differentially expressed lncRNAs that are located close to two members of Ref/Nfkb family, Nfkb2 and Ref, which are classical proinflammatory transcription factors known to play critical roles in both innate and adaptive immune response. Nfkb2 was reported to be upregulated upon LPS stimulation in human monocytes . Our qRT-PCR experiments confirmed the co-expression of lncRNA-Nfkb2 and Nkfb2. It is unlikely that the co-regulation of lncRNAs and Ref/Nfkb family was a random phenomenon since two members of this family were found to be co-regulated with lncRNAs. Given the importance of Ref/Nfkb family in immune response, a further examination of the function and mechanism for their co-located and co-expressed lncRNAs is worth doing. However, our study has limitations to detect known LPS-regulated lncRNAs due to the lack of probes. Previous study indicated that Cox2 and lncRNA-Cox2 were markedly induced after TLR4 stimulation in BMDMs . Due to the lack of probes for lncRNA-Cox2, we did not detect lncRNA-Cox2 in this study. We performed qRT-PCR to confirm the co-expression of Cox2 and lncRNA-Cox2 (Additional file 15). It should be noted that we applied a stringent strategy to derive a confident list of LPS-regulated lncRNAs. Some interesting lncRNAs are also filtered out, such as lncRNA-Lyn-intron1. Previous study has demonstrated that lncRNA-Lyn spans the first exon and first intron region of Lyn and the expression is increased along with Lyn upon LPS stimulation in BMDMs . We identify a new Lyn associated lncRNA, lncRNA-Lyn-Intron1 (lncR.2430; Additional file 3), located at the first intron of Lyn and 25 Kb away from lncRNA-Lyn, is also upregulated (Additional file 15). This lncRNA was filtered out because of no clear TSS evidence.
Recent investigation in erythroid cells has suggested that the lncRNAs transcripts are almost evenly divided between elncRNAs and plncRNAs differentiated by chromatin signatures of H3K4me3 and H3K4me1 surrounding transcription start sites . Consistent with this observation, we found that BMDMs expressed elncRNAs and plncRNAs were also evenly distributed. A number of elncRNAs and plncRNAs can be regulated by LPS stimulation. Nevertheless, plncRNAs are more inclined to downregulation upon LPS stimulation compared to elncRNAs. Several previous studies suggested that lncRNA expression changes are regulated by epigenetic mechanism including histone modifications such that H3K27ac, H3K4me3 and H3K36me3 are related to enhancer activity . Similarly, we demonstrated that histone modifications also play important roles in the regulation of lncRNAs upon LPS stimulation in BMDMs. We found that H3K4me3 and H3K27Ac are associated with directionally consistent changes in not only elncRNAs, but also plncRNAs expression. Our studies demonstrate that although distance to nearest neighboring is much nearer in elncRNAs compared to plncRNAs, these both of two kinds of lncRNAs significantly co-expressed with neighboring protein-coding genes. Bidirectional transcription has been shown to be a defining feature of a subset of active enhancers in mouse cortical neurons and human fetal lung fibroblasts [35,36]. We have shown that the transcription of bidirectional plncRNAs, as well as elncRNAs, were LPS-stimulation dependent in mouse BMDMs.
The gene program stimulated in TLR4 signaling pathway requires the coordinative activation of transcription factors, of which the most well characterized are p65, IRF3, and AP-1 family members JunB and cJun. Here we demonstrate for the first time that these transcription factors also bind to lncRNAs and regulate their expression upon LPS stimulation. Also, the regulation does not differ between elncRNAs and plncRNAs. The majority of LPS-regulated lncRNAs are bound with at least one of these transcription factors. Markedly, we also identify the up-regulated and down-regulated lncRNAs that are bound by all the four transcription factors, suggesting the widely cooperation of these transcription factors. Recent study suggests that Bcl6 antagonizes p65 bindings under rest condition to prevent the hyper-activation of inflammatory genes . Interestingly, we found that Bcl6 also binds to a portion of lncRNAs and the binding sites can overlap with not only p65, but also IRF3, JunB and cJun. We speculate these transcription factors may regulate lncRNAs in a similar manner to protein-coding genes upon LPS stimulation in BMDMs.
Taken together, we have provided a valuable resource of LPS-regulated lncRNA expression profile, together with many potential co-regulated candidate protein-coding genes. Among them, we have identified lncRNAs such as lncRNA-Nfkb2 and lncRNA-Rel that are upregulated along with their corresponding protein-coding genes, which are crucial genes in immune response. Although the mechanisms are currently unknown, we speculate that many of the identified elncRNAs and plncRNAs are important participants of LPS-stimulated innate immune response. We also established an integrative microarray analysis pipeline, which opens new avenues for repurposing published genomic data to study the functions and mechanisms of lncRNAs in interested biology fields.
Re-annotation of array probes
The mouse gene annotations were collected from four sources: NCBI RefSeq , UCSC knownGene , FANTOM3  and Ensembl . For NCBI RefSeq, the mm10 version of mouse refGene was downloaded, and transcripts beginning with “NR” were treated as non-coding RNAs, while transcripts beginning with “NM” were treated as coding RNAs. For UCSC knownGene, the mm10 version was downloaded and transcripts annotated with “noncoding” were considered as non-coding RNAs, while transcripts annotated with “coding” were considered as coding RNAs. A stringent set of FANTOM3 non-coding RNAs was selected based on the conservation and noncoding votes. The fasta sequences of the stringent FANTOM3 non-coding RNAs were aligned against mm10 genome using blat  to obtain mm10 annotation of FANTOM3 non-coding RNAs. For Ensembl, the release 77 for mouse was downloaded, and the transcripts annotated with “protein_coding” were treated as coding RNAs, otherwise as non-coding RNAs. We excluded non-coding RNAs with length < 200 nt from the four sources, and defined others as long non-coding RNAs (lncRNAs). We reannotated probes of six different platforms from Affymetrix, Agilent and Illumina arrays (Additional file 1: Table S1) for lncRNAs using the following procedure. Firstly, the bed format annotations of all array probes were generated. The mm10 bed files for Affymetrix arrays were directly downloaded from the Affymetrix website (http://www.affymetrix.com). For Agilent and Illumina arrays, we obtained the probe sequences from the Agilent website (http://www.agilent.com) and NCBI GEO database (http://www.ncbi.nlm.nih.gov/gds), respectively. The probe sequences were mapped against mm10 genome using blat, and the bed format annotations of the best hits were generated. Secondly, the bed format annotations of probes were intersected with lncRNA annotations and coding gene annotations to obtain lncRNA probes and coding gene probes, respectively. BedTools  were utilized to achieve this end. To avoid hybridizations, the probes that were mapped to multiple lncRNA annotations or coding gene annotations were removed. The summary information of probe reannotation result for each array platform is shown in Additional file 2. As a result, 3988 unique lncRNAs were obtained. The detailed reannotations of all probes of the six platforms for lncRNAs are shown in Additional file 3. The detailed reannotations of all probes of the six platforms for coding genes are shown in Additional file 6.
Determination of transcriptional start sites (TSSs)
We used CAGE  and nanoCAGE  TSS-seq to determine genome-wide TSSs for mouse genome as described elsewhere . To obtain full annotation of TSSs for mouse genome, we collected all the available TSS-seq from DBTSS  and NCBI SRA . The mm9 bed files of TSS-Seq sequences were downloaded from DBTSS (ftp://ftp.hgc.jp/pub/hgc/db/dbtss/dbtss_ver8), and then were converted from mm9 to mm10 using the UCSC liftOver tools (http://genome.ucsc.edu/cgi-bin/hgLiftOver). The fastq files of mouse TSS-Seq sequences (GSE49459 and GSE39849) were downloaded from NCBI SRA using SRA toolkit. Then the TSS-seq sequences were mapped to mm10 genome using bwa . A perl script was written to integrate all the TSS-Seq to obtain the TSS regions. Briefly, the 5′ end position of each TSS-Seq read was extracted as TSS. TSSs closer than 20 bp and derived from the same strand were clustered. Clusters within 400 bp of each other and on the same strand were further grouped as a TSS region. The TSS regions with less than 20 tags supported were discarded, thus 160116 TSS regions were retained.
Filter lncRNAs by TSS evidence
We associated the lncRNAs reannotated from arrays to the 160116 TSS regions using BEDTools . The lncRNA region plus 30 Kb upstream/downstream regions were used to scan for the TSS regions. As a result, 25100 TSS regions were found to locate nearby the 3575 lncRNAs that have determined TSSs. ChIP-seq raw reads for H3K4me3, H3K4me1 and H3K27Ac histone modifications and RNA PolII in unstimulated/LPS BMDMs were downloaded from NCBI GEO database (http://www.ncbi.nlm.nih.gov/gds/) (Additional file 4). The raw reads were aligned to mm10 mouse genome using bowtie 1.0.1  with the –m reporting option set to 2. The peaks of histone modifications and PolII were called using MACS  following published parameters . To further refine the TSS regions for lncRNAs, we integrated chromatin signatures such as histone modifications and PolII occupancy nearby lncRNAs to obtain a list of active and reliable TSS regions. The candidate TSS regions for lncRNAs were examined for the peaks of H3K4me3, H3K4me1 and PolII. Only those TSS regions with at least one peak of them were retained, which resulted in 7474 TSS regions associated with 2629 lncRNAs. To further refine the TSS regions for the lncRNAs, we excluded the ambiguous TSS regions which may be overlapped with the neighboring coding gene TSS regions, resulting in 1503 TSS regions for 994 lncRNAs, as listed in Additional file 5.
The lncRNA classification method was adopted from a previous study . The lncRNAs were classified according to their relation with neighbor coding genes. The neighbor coding genes of lncRNAs were selected on the basis of either the nearest distance to the lncRNA or the longest overlapping regions. The lncRNAs with distance to their neighbor coding gens shorter than 1 kb and with different orientation as their neighbor coding genes were categorized as bidirectional. The lncRNAs that have not any overlap with the neighbor coding genes and not belong to bidirectional were categorized as intergenic. The lncRNAs overlapping with their neighbor coding genes were categorized as genic. The genic lncRNAs where were further classified as sense or antisense according to the orientation relation with neighbor coding genes. The classification of all lncRNAs in this study is shown in Additional file 7.
Expression analysis of LPS stimulated BMDM
We collected expression array datasets from the studies on the investigation of transcriptional profile in mouse BMDMs, which resulted in 12 arrays from six array platforms (Additional file 1). The expression datasets were downloaded from NCBI GEO repository . For the Affymetrix and Illumina array datasets, we downloaded the probe-level preprocessed expression matrix file directly. For the Agilent array datasets, we downloaded the raw data, and used the R package “Agi4x44Preprocess”  to preprocess the raw data. KNN method  was used to fill the missing values for each preprocessed expression matrix. Then the probe-level expression matrix was transformed to gene/lncRNA level using the reannotated information as shown in Additional files 3, 4, 5 and 6. The average expression was taken if multiple probes were mapped to the same transcript. For each transcript, the average expression for unstimulated group and LPS stimulated group were calculated separately. Then the log2 fold change between the two groups for each transcript was calculated. The expression profiles in the 12 studies for all the lncRNAs are shown in Additional file 8. The expression profiles in the 12 studies for all the coding genes are shown in Additional file 10. We employed a recently published robust rank aggregation algorithm  to integrate these 12 expression profiles in an unbiased manner. A P value was obtained for each transcript to represent the upregulation and downregulation under LPS stimulation, respectively. The bonferroni-adjusted p value cutoff 0.05 was used to select the significantly changed lncRNAs/coding genes.
Preparation of BMDMs
Bone marrow cells were harvested from the femurs and tibias of 8-week-old C57BL/6 mice. BMDMs were generated by culture of bone marrow cells in RPMI medium containing 10% of FBS and10 ng/ml of recombinant M-CSF (R&D Systems, cat. no. 416-ML-010) for 7 days. Differentiated BMDMs were then stimulated with different concentrations of LPS (Sigma, cat. no. L3024) for 0, 3 and 6 hours in RPMI medium. All the mice were raised in a specific pathogen–free environment at the University of Chicago, and experiments were performed in accordance with the guidelines of the Institutional Animal Care and Use Committee.
RNA isolation and qRT-PCR
Total RNA from different time points of LPS stimulated BMDM was prepared with TRIZOL (Invitrogen, cat. no. 15596026), according to the manufacture’s instruction. The cDNA was synthesized from total RNA using SuperScript First-Strand Synthesis System (Invitrogen, cat. no. 11904018). Q-PCR was performed using SYBR Advantage Premix (Clontech, cat. no. 639676) in Strategene Mx3500 thermocycler. The corresponding primers were listed in Additional file 16.
Determining chromatin signatures at lncRNA TSSs
We did a quantity assessment for the enrichment of H3K4me3, H3K4me1, H3K27Ac and PolII around each lncRNA TSS region using in-house R script utilizing Rsamtools  in R. The relative enrichment of H3K4me3 and H3K4me1 surrounding the transcription start sites of the lncRNAs (−2 to 2 Kb) was calculated to define elncRNA and plncRNA as previously described . Heatmaps of the elncRNA and plncRNA histone modification profiles were generated using heatmap.2 function in R package “gplots” .
Association of lncRNA loci with transcription factor binding sites
ChIP-seq raw reads for transcription factors p65, IRF3, JunB, cJun and Bcl6 were downloaded from NCBI GEO database (http://www.ncbi.nlm.nih.gov/gds/) (Additional file 11). The raw reads were aligned to the mm10 mouse genome build using bowtie 1.0.1  with the –m reporting option set to 2. The bedgraphs of ChIP-seq were generated using HOMER, where the total number of aligned reads was normalized to 10 million. The peaks of transcription factors were called using MACS  following published parameters . The transcription factor binding sites were associated to lncRNA promoter-proximal region (−10 kb to 10 kb from TSS) using BEDtools .
Pearson product–moment correlation coefficient was used to measure the linear correlation. Student’s t test was used to evaluate the significance of difference for distance to neighboring gene between elncRNAs and plncRNAs. Kolmogorov-Smirnov (K-S) test was performed to evaluate the significance of difference between ecdf curves. All the statistical analyses were performed in R using the built-in packages.
This work was supported by grants from the National Science Foundation of China (No. 81170362 and No. 81370508).
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