Repression of the expression of proinflammatory genes by mitochondrial transcription factor A is linked to its alternative splicing regulation in human lung epithelial cells

Abstract

Background: Mitochondrial transcription factor A (TFAM) is associated with a number of neurodegenerative diseases and also with asthma. TFAM deficiency-induced mitochondrial DNA stress primes the antiviral innate immune response in mouse embryonic fibroblasts. However, the role of TFAM in asthma related inflammation remains obscure. The purpose of this study was to investigate the regulatory mechanism of TFAM in asthma.

Results: In this study, we overexpressed TFAM in human lung epithelial cells (A549), then obtained the TFAM-regulated transcriptome by Illumina sequencing technology. Transcriptome analysis revealed that TFAM overexpression down-regulated and up-regulated the expression of 642 and 169 differentially expressed genes (DEGs), respectively. The TFAM-repressed genes were strongly enriched in cytokine-mediated signaling pathway, type I interferon- and INF-γ-mediated signaling pathways, and viral response pathways. We also revealed that 2563 alternative splicing events in 1796 alternative splicing genes (ASGs) were de-regulated upon TFAM overexpression. These TFAM-responding ASGs were enriched in DNA repair, nerve growth factor receptor signaling pathway, and also transcription regulation. Further analysis revealed that the promoters of TFAM-repressed DEGs were enriched by DNA binding motifs of transcription factors whose alternative splicing was regulated by TFAM.

Conclusions: These findings suggest that TFAM regulates not only immune response gene expression in human lung epithelial cells, but also pre-mRNA alternative splicing which may mediate transcriptional regulation; this TFAM-centered gene regulation network could be targeted in developing therapies against various diseases.

Keywords: A549; Alternative splicing; TFAM; Transcription factors; Viral response pathway gene expression.

Fig. 1

TFAM selectively represses gene expression in human lung epithelial A549 cells. a Enforced expression of TFAM in A549 cells detected by western blot experiment, gels and blots were cropped. b Heat map display of the hierarchically clustered Pearson’s correlation matrix resulting from comparing the expression level of each gene in the control and TFAM-overexpression transcriptomes. c Volcano plot shows the expression change of genes between the control and TFAM-overexpression transcriptomes, leading to the identified DEGs. Upregulated genes (FC ≥ 2; FDR < 0.05) are labeled in red and downregulated genes (FC ≤  − 2; FDR < 0.05) are labeled in blue. d Hierarchical clustering of the expression levels of all the identified DEGs in control and TFAM-overexpression samples. FPKM values are log2-transformed and then median-centered by each gene. e The top 10 GO biological processes of TFAM-up and down-regulated genes. f The top 10 KEGG pathways of TFAM-up and down-regulated genes. g Hierarchical clustering of the expression levels ISGs in the control and TFAM-overexpression A549 cells; these ISGs were negatively regulated in the Tfam-deficiency mouse murine embryonic fibroblast (MEF) cells [47]

Fig. 2

Validation of the TFAM-regulated gene expression in A549 cells. The expression levels of the two upregulated and five representative down-regulated genes were analyzed by RT-qPCR experiment. The quantification results from RNA sequencing data and RT-qPCR were showed in parallel. FPKM values were calculated as that has been explained in Materials and Methods. Error bars represent mean ± SEM. ***p < 0.001

Fig. 3

Identification and functional clustering of the TFAM-regulated alternative splicing events. a Classification of the TFAM-regulated alternative splicing events (RASEs). b Plots of several representative RASEs regulated by TFAM. The schematic diagrams depict the structures of ASEs. Alternative splicing (AS) junctions, purple line; model splicing (Model) junctions, green line. The exon sequences are denoted by boxes and intron sequences by the horizontal line (top panel). RNA-seq quantification validation of ASEs are shown in the bottom panel. Error bars represent mean ± SEM. *p < 0.05, **p < 0.01. The altered ratio of AS events in RNA-seq were calculated using the formula: AS junction reads/(AS junction reads + Model junction reads). Plots of the top 10 GO biological processes (c) and KEGG (d) enriched by TFAM-regulated RASGs. e Venn diagram shows the overlap between the TFAM-regulated DEGs and TFAM-regulated RASGs; at least one TFAM-regulated RASE was present in each RASG. f The GO biological processes and KEGG pathways enriched by genes regulated by TFAM at both the transcriptional and alternative splicing levels

Fig. 4

Validation of TFAM-regulated RASEs. The RNA-seq read distribution profiles of the RASEs subjected to RT-qPCR were depicted on the left, with the read density map and the numbers of alternative and model junction reads being shown. The black lines represent the splice junctions composing ASEs. The schematic diagrams depict the structures of ASEs are shown on the right upper panel, which are similar as in Fig. 3B. The results of the RNA-seq and RT-qPCR quantification of the change of the ratio between the alternative and model splicing products are shown on the right lower panel. Error bars represent mean ± SEM. *p < 0.05, **p < 0.01

Fig. 5

TFAM-regulated alternative splicing of the transcript factors are strongly linked to the TFAM-repressed gene expression. a Venn diagram shows the overlap of the DNA motifs enriched in the promoter regions of the TFAM-regulated genes. The overlap was performed among the motifs over-represented in the DNA regions that were 1 K, 2 K, and 3 K upstream and downstream from the transcription start sites. b Overlap of the DNA binding motifs of TFAM-regulated TFs (RASGs) with the DNA motifs enriched in the promoter regions of the TFAM-repressed genes (DEGs). c Venn diagram shows the overlap among the down-DEGs containing the DNA binding motifs for the top four TFs (NFIB, USF2, TEAD2, and NFE2L1) that were ranked by the number of down-DEGs harboring their DNA binding sites. d Overlap of genes that engage multiple functions. e Networks between the TFAM-regulated TFs and their targeted down-DEGs involved in the interferon/cytokine-mediated signaling pathways, immune/innate immune response, response/defense response to virus. The down-DEGs were grouped based on whether they are specific to each of the three classes of functions