Redefining the transcriptional regulatory dynamics of classically and alternatively activated macrophages by deepCAGE transcriptomics

Abstract

Classically or alternatively activated macrophages (M1 and M2, respectively) play distinct and important roles for microbiocidal activity, regulation of inflammation and tissue homeostasis. Despite this, their transcriptional regulatory dynamics are poorly understood. Using promoter-level expression profiling by non-biased deepCAGE we have studied the transcriptional dynamics of classically and alternatively activated macrophages. Transcription factor (TF) binding motif activity analysis revealed four motifs, NFKB1_REL_RELA, IRF1,2, IRF7 and TBP that are commonly activated but have distinct activity dynamics in M1 and M2 activation. We observe matching changes in the expression profiles of the corresponding TFs and show that only a restricted set of TFs change expression. There is an overall drastic and transient up-regulation in M1 and a weaker and more sustainable up-regulation in M2. Novel TFs, such as Thap6, Maff, (M1) and Hivep1, Nfil3, Prdm1, (M2) among others, were suggested to be involved in the activation processes. Additionally, 52 (M1) and 67 (M2) novel differentially expressed genes and, for the first time, several differentially expressed long non-coding RNA (lncRNA) transcriptome markers were identified. In conclusion, the finding of novel motifs, TFs and protein-coding and lncRNA genes is an important step forward to fully understand the transcriptional machinery of macrophage activation.

Figure 1.

Experimental design and quality control. (A) Schematic representation of the preparation and stimulation of BMDMs from BALB/c mice. After 10 days of differentiation, BMDMs were stimulated with IFNγ or IL-4/IL-13. At 2, 4, 6, 12 and 24 h post-stimulation, total RNA was collected followed by non-amplified deepCAGE analysis. Zero hour non-stimulated BMDMs were used as control. Three independent biological experiments were analyzed to obtain the promoter activity. (B) Biological replicates were plotted to obtain the relative Pearson correlation coefficients among the replicates. (C) Principal component analysis (PCA) was performed using IFNγ-stimulated macrophages (M1), IL-4/IL-13-stimulated macrophages (M2) and non-stimulated macrophages and the separation of M1, M2 and non-stimulation based on component 1 and component 2 is shown (see Material and Methods). Each number in the plot represents the average expression (triplicates) of each sample in one time point. Each of the stimulations has a different color. (D) Promoter expression profiles of classical activation marker gene Nos2, and alternative activation marker gene Arg1, are shown. The expression profiles of promoters are represented by tags per million (TPM). The data was obtained from three biological experiments and was plotted as mean expression.

Figure 2.

Motif activity response analysis of M(IFNγ) and M(IL-4/IL-13). Motif activity response analysis was performed using promoter activity profiles of M(IFNγ) and M(IL-4/IL-13), obtained from CAGE data. The identified top 5 motif activities with high activity change (z-acore >3 and delta motif activity change > 0.15) are shown in (A) NFKB1_REL_RELA, (B) IRF1,2, (C) IRF7, (D) TBP and (E) FOS_FOS{B,L1}_JUN{B,D}. The data is obtained from three independent biological experiments and plotted as mean ± SEM. The motif activity is calculated as relative value at each time point where summation of values for each stimulation series becomes zero.

Figure 3.

Expression profiles of transcription factors associated with top five motif activities. Expression of the associated transcription factor genes is shown as tags per million (TPM). Error bars were calculated based on the standard error of three replicates. (A) Transcription factors Rel, Rela and Nfκb1 associated with NFKB1_REL_RELA motif activity. (B) Transcription factors Irf1 and Irf2 associated with IRF1,2 motif activity. (C) Transcription factor Irf7 associated with IRF7 motif activity. (D) Tbp and Tbpl1 associated with TBP motif activity. (E) Fos, Fosl1, FosB, JunB and JunD associated FOS_FOS{B,L1}_JUN{B,D} motif activity. Fos and JunB are shown whereas FosB,Fosl1 and JunD are not shown because the expression remains close to the detection limit throughout the time course in M(IFNγ) and M(IL-4/IL-13).

Figure 4.

Transcription factors involved in M(IFNγ) and M(IL-4/IL-13). (A) Box plot analysis of the expression log fold-changes of all differentially up-regulated transcription factors in classically and alternatively activated macrophages over time (left and right panels, respectively). Boxes show median and interquartile ranges and whiskers show the 10th and 90th percentile values. (B) The Venn diagram shows that M(IFNγ) and M(IL-4/IL-13) up-regulate 26 and 22 (left) and down-regulate 9 and 6 (right) distinct transcription factor genes.