A transcriptional factor B paralog functions as an activator to DNA damage-responsive expression in archaea

Abstract

Previously it was shown that UV irradiation induces a strong upregulation of tfb3 coding for a paralog of the archaeal transcriptional factor B (TFB) in Sulfolobus solfataricus, a crenarchaea. To investigate the function of this gene in DNA damage response (DDR), tfb3 was inactivated by gene deletion in Sulfolobus islandicus and the resulting Δtfb3 was more sensitive to DNA damage agents than the original strain. Transcriptome analysis revealed that a large set of genes show TFB3-dependent activation, including genes of the ups operon and ced system. Furthermore, the TFB3 protein was found to be associated with DDR gene promoters and functional dissection of TFB3 showed that the conserved Zn-ribbon and coiled-coil motif are essential for the activation. Together, the results indicated that TFB3 activates the expression of DDR genes by interaction with other transcriptional factors at the promoter regions of DDR genes to facilitate the formation of transcription initiation complex. Strikingly, TFB3 and Ced systems are present in a wide range of crenarchaea, suggesting that the Ced system function as a primary DNA damage repair mechanism in Crenarchaeota. Our findings further suggest that TFB3 and the concurrent TFB1 form a TFB3-dependent DNA damage-responsive circuit with their target genes, which is evolutionarily conserved in the major lineage of Archaea.

Figure 1.

NQO-induced upregulation of tfb3 in Sulfolobus islandicus. (A) qRT-PCR analysis of tfb3 mRNA in NQO-treated and untreated S. islandicus cells. Data were normalized to the level of 16S rRNA. Error bars represent the standard derivation values of three independent replicates. (B) Western blot analysis of TFB3 protein in the cell extracts of NQO-treated and untreated S. islandicus using PCNA as a reference S. islandicus E233S1 (WT) strain was grown in the absence (−NQO) or presence of the 2μM NQO (+NQO) for 9 h (hours post-treatment, hpt) during which cell samples were taken at the indicated time points and used for mRNA extraction (for qPCR) and cell extracts preparation (western analysis).

Figure 2.

Construction of a Δtfb3 in-frame deletion mutant of S. islandicus. (A) Schematic of PCR identification of the WT tfb3 gene and Δtfb3 alleles in S. islandicus strains. (B) Verification of the Δtfb3 genotype by PCR amplification of the tfb3 gene alleles (PCR). Primer set of F1/R1 was designed to check the gene deletion whereas primer set of F2/R2 was designed to amplify an internal DNA fragment in the tfb3 gene. (C) Western blot analysis of TFB3 protein in NQO-treated cultures of S. islandicus. Immunoblotting was conducted with the TFB3 antibody raised against the S. solfataricus TFB3 protein (α-TFB3). WT: S. islandicus E233S1, the genetic host; Δtfb3: tfb3 deletion mutant constructed with E233S1.

Figure 3.

Survival rates of the wild-type S. islandicus and Δtfb3 upon NQO treatment. Cell viability was estimated for S. islandicus E233S1 (WT) strain and Δtfb3 mutant at 24 h post-NQO addition by determination of CFUs. CFU of WT-0 μM and Δtfb3-0μM was assigned to 100% for each strain, with which the survived fractions of cells in other cultures were calculated individually.

Figure 4.

S. islandicus Δtfb3 mutant lost the capability of forming NQO-induced cell aggregation. (A) Microscopy of cells and cell aggregates in samples taken from the cultures of the WT strain (E233S1) and Δtfb3 mutant. Each strain was grown in the absence (−NQO) or presence (+NQO) of NQO for 24 h, and aliquots of cultures were placed on glass slides, covered with coverslips and directly observed under a phase-contrast microscope. 12 and 15 h: hours after NQO supplementation. (B) Quantification data of cell aggregation in the cell samples shown in panel A. At least 500 cells were analyzed for each cell sample.

Figure 5.

Enrichment of TFB3 association with promoters of upregulated DDR genes. S. islandicus E233S1 (WT) was grown in SCV in the presence (+NQO, 2 μM) or absence of the drug (−NQO) for 3 h. Cell mass was then collected from cultures of E233S1 from which cell extracts were prepared and used for CHIP analysis using the TFB3 antisera. The folds enrichment refers to the CHIP signal from the TFB3 antibody relative to the signal from the sheep serum. The promoter regions of three TFB3-dependent upregulated genes (upsE, cedB and herA1 (SiRe_1715)), one TFB3-independent downregulated gene, cdvB1, (SiRe_1550 encoding ESCRTIII-1) and cmrβ operon, together with intragenic region of 16S rRNA gene, were selected for CHIP analysis with the TFB3 antibody. Folds of enrichment were calculated with the data from three independent replicates with the error bars indicating the standard derivation values.

Figure 6.

Functional characterization of TFB3 by mutagenesis. (A) Schematic of the WT TFB3 and its mutant derivatives. A CC region was predicted by Coils server (38). CoilM1 and CoilM2 refer to the mutation of R145AK146A and L148AK149AL151A. The first and fourth conserved cysteine in Zn ribbon of TFB3 was replaced with the corresponding ones in TFB1 (SiRe_1555). (B) Western blot analysis of the total cell extracts of the strains carrying different mutated TFB3 after NQO treatment. Sample was taken 3 h after 2 μM NQO treatment. (C) Quantitative analysis of the expression levels of upsX and cedB in the strains encoding different isoform of TFB3. Sample was taken 3 h after 2 μM NQO treatment.

Figure 7.

Co-evolution of TFB3 and Ced system in Archaea DNA sequences of 16S rRNA genes of different crenarchaeal species were retrieved from the GenBank database and used for multiple sequence alignment using Cluster X and for construction of phylogenetic tree. The resulting tree was visualized and annotated using iTOL (Interactive Tree Of Life) (41). The presence or absence of a truncated version of TFB (TFB3) is indicated by the filled or empty rectangles, respectively; the presence or absence of the Ced system is shown as the filled or empty triangles individually, whereas the canonical and noncanonical TFB1 proteins are notified as the green and dark yellow circles, respectively.