Whole transcriptome analysis reveals an 8-oxoguanine DNA glycosylase-1-driven DNA repair-dependent gene expression linked to essential biological processes

Abstract

Reactive oxygen species inflict oxidative modifications on various biological molecules, including DNA. One of the most abundant DNA base lesions, 8-oxo-7,8-dihydroguanine (8-oxoG) is repaired by 8-oxoguanine DNA glycosylase-1 (OGG1) during DNA base excision repair (OGG1-BER). 8-OxoG accumulation in DNA has been associated with various pathological and aging processes, although its role is unclear. The lack of OGG1-BER in Ogg1(-/-) mice resulted in decreased inflammatory responses and increased susceptibility to infections and metabolic disorders. Therefore, we proposed that OGG1 and/or 8-oxoG base may have a role in immune and homeostatic processes. To test our hypothesis, we challenged mouse lungs with OGG1-BER product 8-oxoG base and changes in gene expression were determined by RNA sequencing and data were analyzed by Gene Ontology and statistical tools. RNA-Seq analysis identified 1592 differentially expressed (≥ 3-fold change) transcripts. The upregulated mRNAs were related to biological processes, including homeostatic, immune-system, macrophage activation, regulation of liquid-surface tension, and response to stimulus. These processes were mediated by chemokines, cytokines, gonadotropin-releasing hormone receptor, integrin, and interleukin signaling pathways. Taken together, these findings point to a new paradigm showing that OGG1-BER plays a role in various biological processes that may benefit the host, but when in excess could be implicated in disease and/or aging processes.

Keywords: 8-Oxoguanine; Biological processes; Gene expression; OGG1-BER.

Fig. 1

Whole-transcriptome profile induced by 8-oxoG challenge. (A) Graphical depiction of results from hierarchical clustering showing whole-transcriptome profile. Total RNA isolated from challenged lungs at 30, 60 and 120 min were subjected to RNA-Seq, heat maps were generated using GENE-E (http://www.broadinstitute.org/cancer/software/GENE-E/), and transcripts were clustered according to their expression patterns. Transcript levels (RPKM) were normalized to control (time 0 min). Rows represent identified transcripts and columns at time points after 8-oxoG challenge. The intensity of the red or green colors shows the degree of upregulation or downregulation, respectively. (B) The total number of upregulated (≥3-fold) transcripts (red bars) and downregulated (≤3-fold) transcripts (green bars) in each cluster. (C) Number of unique and shared transcripts altered by 8-oxoG challenge. Venn diagrams were constructed by using online software Venny (Materials and Methods). (D) Kinetic changes in transcript levels induced by 8-oxoG challenge.

Fig. 2

Gene ontology categories represented by the upregulated genes induced after introduction of the OGG1-BER product 8-oxoG into mouse lungs. Overrepresentation test of the genes upregulated (≥ 3 fold) at 60 min was performed by using PANTHER (http://www.pantherdb.org). (A) Biological process. (B) Protein class. (C) Cellular component. (D) Molecular function. X axis represent −log(P value).

Fig. 3

Signaling pathways induced by OGG1-BER product, 8-oxoG in mouse lungs. Pie chart was generated by using PANTHER (Materials and Methods). Pathway analysis included only those genes with expression levels ≥ 3-fold at 60 min.

Fig. 4

Validation of RNA-Seq data. (A) Fold changes in RPKM of selected genes as determined by RNA-Seq analysis. RNA was isolated from lungs at 60 min post challenge and pooled (n = 5) or kept separately. One μg of pooled RNA was subjected to RNA-Seq as in Materials and Methods. (B) qRT-PCR confirmation of RNA-Seq data. One μg RNA isolated from individual control and challenged mice (n = 5) was reverse transcribed, and cDNAs were amplified by using specific primer pairs as described in Materials and Methods.

Fig. 5

OGG1-BER alters expression of RAS signature genes. Effect of 8-oxoG challenge on the expression of genes reported to be up-regulated (A) or down regulated (B) by RAS signaling. (C) Differentially expressed RAS signature genes with ≥ 3-fold expression levels. Upregulated genes are shown in red bars and down regulated ones in green bars. (D) Activation of RAS-GTPase by 8-oxoG challenge in lungs (upper panel) and dependency on OGG1 expression in 8-oxoG-exposed lungs. (E) Down-regulation of Ogg1 in mouse airway epithelium by RNAi. (F) Decreased expression of Cxcl1 and Cxcl2 in Ogg1-depleted airways. **p < 0.01, ***p < 0.001.