Hyphal differentiation induced via a DNA damage checkpoint-dependent pathway engaged in crosstalk with nutrient stress signaling in Schizosaccharomyces japonicus

Abstract

DNA damage response includes DNA repair, nucleotide metabolism and even a control of cell fates including differentiation, cell death pathway or some combination of these. The responses to DNA damage differ from species to species. Here we aim to delineate the checkpoint pathway in the dimorphic fission yeast Schizosaccharomyces japonicus, where DNA damage can trigger a differentiation pathway that is a switch from a bidirectional yeast growth mode to an apical hyphal growth mode, and the switching is regulated via a checkpoint kinase, Chk1. This Chk1-dependent switch to hyphal growth is activated with even low doses of agents that damage DNA; therefore, we reasoned that this switch may depend on other genes orthologous to the components of the classical Sz. pombe Chk1-dependent DNA checkpoint pathway. As an initial test of this hypothesis, we assessed the effects of mutations in Sz. japonicus orthologs of Sz. pombe checkpoint genes on this switch from bidirectional to hyphal growth. The same set of DNA checkpoint genes was confirmed in Sz. japonicus. We tested the effect of each DNA checkpoint mutants on hyphal differentiation by DNA damage. We found that the Sz. japonicus hyphal differentiation pathway was dependent on Sz. japonicus orthologs of Sz. pombe checkpoint genes-(SP)rad3, (SP)rad26, (SP)rad9, (SP)rad1, (SP)rad24, (SP)rad25, (SP)crb2, and (SP)chk1-that function in the DNA damage checkpoint pathway, but was not dependent on orthologs of two Sz. pombe genes-(SP)cds1 or (SP)mrc1-that function in the DNA replication checkpoint pathway. These findings indicated that although the role of each component of the DNA damage checkpoint and DNA replication checkpoint is mostly same between the two fission yeasts, the DNA damage checkpoint was the only pathway that governed DNA damage-dependent hyphal growth. We also examined whether DNA damage checkpoint signaling engaged in functional crosstalk with other hyphal differentiation pathways because hyphal differentiation can also be triggered by nutritional stress. Here, we discovered genetic interactions that indicated that the cAMP pathway engaged in crosstalk with Chk1-dependent signaling.

Fig. 1

Requirement of DNA damage checkpoint genes for the DNA damage-stress-dependent hypha formation. cds1::nat and mrc1::nat colonies, but not other checkpoint mutant colonies, can present hypha when growing on YE agar media that contains camptothecin (CPT, 0.2 μM). Colonies were grown for 4 days and the photographed on the 4th day. The phenotypes of single mutant strains were summarized shown in Table 1

Fig. 2

Requirement of DNA damage checkpoint genes, rad24 or rad25, for the DNA damage-stress-dependent hypha formation. a Wild-type, rad24::nat, or rad25::nat cells were cultured in liquid YE media that contained CPT (0.2 μM) to assess hyphal induction. Cells were incubated at 30 °C for 6 h. Scale bar; 10 μm. b chk1-hyp and chk1-hyp rad24::nat colonies were grown at 30 °C on YE agar media to activate the chk1-hyp gene, which carried gain-of-function mutation

Fig. 3

Analysis of epistatic interactions among mutants of DNA damage checkpoint genes in Sz. japonicus. Growth of colonies was compared under camptothecin (CPT) or hydroxyurea (HU) exposure. The leftmost spot contained approximately 6,000 cells when spotted; the spots to the right each represent tenfold serial dilutions; all cells were grown on YE plate that contained indicated reagents. The colonies were grown at 30 °C and photographed at 4th day. The growth of, a wild-type (WT) vs. crb2::kanMX6, chk1::kanMX6, chk1::kanMX6 crb2::kanMX6 cells, b WT vs. cds1::nat, mrc1::nat, cds1::nat mrc1::nat cells, c WT vs. chk1::kanMX6, cds1::nat chk1::kanMX6, cds1::nat mrc1::nat and rad3::kanMX6 cells, d WT vs. chk1::kanMX6, rad3::kanMX6 and rad26::kanMX6 cells, and e WT vs. chk1::kanMX6 and rad9::kanMX6 cells

Fig. 4

Ectopic activation of hyphal pathway in mutants of checkpoint genes. a Prolonged exposure to hydroxyurea HU induces hypha in wild-type colonies (WT), and this HU-mediated induction was diminished in chk1::kanMX6 colonies. Cells were spread onto YE plates containing 10 mM of HU and then incubated at 30 °C for 3 days. b cds1::nat colonies present hypha when incubated on media containing a low concentration of HU (2 mM), but WT colonies did not. c Growth was compared between cells incubated on camptothecin (CPT) vs. on hydroxyurea (HU). WT, chk1::kanMX6, and tel1::kanMX6 cells were compared. d tel1::kanMX6 colonies present hypha when incubated on media containing a low concentration of HU (5 mM), but WT colonies did not

Fig. 5

The nutrient stress signal could affect DNA damage-dependent hypha. a Hyphal formation in chk1-hyp mutants was compared between cells grown on YE vs. EMM-2 media. On YE agar media, chk1-hyp induced hypha at 30 °C, but on EMM-2 agar media, the transgene induced hypha at 33 °C. The colonies were grown at the indicated temperature for 3 days and then photographed on the 3rd day. b cAMP inhibited CPT-induced hypha formation, but did not affect chk1-hyp dependent hypha on agar media (bar; 5 mm) c cAMP (50 mM) inhibited CPT-induced hypha formation in wild-type cells grown in liquid media (bar; 10 μm). 0.2 μM of CPT was used

Fig. 6

The checkpoint-dependent hyphal pathway. The diagram summarizes the functional relationship between genes involved in stress response and hyphal induction in Sz. japonicus and DNA damage checkpoint in Sz. pombe. The groups of proteins involved in hyphal induction are shaded in gray and the pathways to activate hypha are indicated by black arrows. The genes indicated in bold have been shown to be required in DNA damage-dependent hyphal induction (this study or Furuya and Niki 2010). Those orthologous genes involved in DNA damage hypha are required in DNA damage checkpoint in Sz. pombe