DNA Damage Checkpoints Govern Global Gene Transcription and Exhibit Species-Specific Regulation on HOF1 in Candida albicans

Abstract

DNA damage checkpoints are essential for coordinating cell cycle arrest and gene transcription during DNA damage response. Exploring the targets of checkpoint kinases in Saccharomyces cerevisiae and other fungi has expanded our comprehension of the downstream pathways involved in DNA damage response. While the function of checkpoint kinases, specifically Rad53, is well documented in the fungal pathogen Candida albicans, their targets remain poorly understood. In this study, we explored the impact of deleting RAD53 on the global transcription profiles and observed alterations in genes associated with ribosome biogenesis, DNA replication, and cell cycle. However, the deletion of RAD53 only affected a limited number of known DNA damage-responsive genes, including MRV6 and HMX1. Unlike S. cerevisiae, the downregulation of HOF1 transcription in C. albicans under the influence of Methyl Methanesulfonate (MMS) did not depend on Dun1 but still relied on Rad53 and Rad9. In addition, the transcription factor Mcm1 was identified as a regulator of HOF1 transcription, with evidence of dynamic binding to its promoter region; however, this dynamic binding was interrupted following the deletion of RAD53. Furthermore, Rad53 was observed to directly interact with the promoter region of HOF1, thus suggesting a potential role in governing its transcription. Overall, checkpoints regulate global gene transcription in C. albicans and show species-specific regulation on HOF1; these discoveries improve our understanding of the signaling pathway related to checkpoints in this pathogen.

Keywords: Candida albicans; DNA damage response; Hof1; RNA-seq; Rad53; methyl methanesulfonate.

Figure 1

Functional characterization of checkpoint kinases responding to genotoxic stresses in C. albicans. (A) Phenotypic assay of the RAD53 deletion, the RAD9 deletion, and the DUN1 deletion strains under genotoxic stresses. Two independent mutants for each strain were used for phenotypic assays, thus showing consistent results. (B) Nuclei separation of the wild type (SN148), the RAD53 deletion, the RAD9 deletion, and the DUN1 deletion strains. The log phase cells were treated with 0.02% MMS for 120 min and then stained with DAPI. Cells with buds containing different types of nuclei were divided into three groups as indicated. The result was averaged from two independent experiments. (C) Filamentous growth of the DUN1 strain induced by genotoxic stress. The wild-type and the DUN1 deletion cells were treated with 0.02% MMS or 40 mM HU for the indicated time. The cell morphology was checked and imaged (400×). (D) The long bud of the DUN1 deletion cells induced by 40 mM HU for 6 h was measured using Image J software 1.42. Over 30 cells were checked for each strain. The difference was compared using a paired t test with GraphPad Prism 8.0.1 software. ** represents p < 0.01.

Figure 2

Overview of RAD53-related transcriptome under MMS stress in C. albicans. (A) Volcano plot showing the global transcriptional changes affected by Rad53 under the stress of MMS. (B) Top-20 KEGG terms of differed genes in response to MMS by deleting RAD53. Cell cycle-involved genes (C), DNA replication-involved genes (D), and ribosome biogenesis-involved genes (E) affected by Rad53 in C. albicans. The fold change for each gene under the normal condition (first column) or the MMS stress condition (second column) is shown after the gene name, with blanks indicating no significant change based on transcriptome data. Genes highlighted in red indicate consistent changes between normal and MMS stress conditions. (F) DNA damage repair genes were affected by deleting RAD53 under MMS stress conditions. The fold change for each gene is shown after the gene name.

Figure 3

Uncovering RAD53-dependent DNA damage responsive genes in C. albicans. (A) Overview of MMS-responsive genes affected by deleting RAD53. The number without brackets represents the defined MMS-responsive genes, while the number with brackets represents the putative MMS-responsive genes. (B) List of MMS-induced (left panel) or -repressed genes (right panel) affected by Rad53 in C. albicans. The fold change for each gene affected by MMS stress in wild-type strain (left column) or by deleting RAD53 upon exposure to MMS (right column) is listed after the gene name. Genes highlighted in red represent the defined MMS-responsive genes, and those genes in black represent the putative MMS-responsive genes. (C) Relative transcription of MMS-responsive genes affected by deleting RAD53 in MMS stress conditions. The transcription of indicated genes in wild-type strain under MMS stress was compared to the level in wild-type strain without MMS treatment, and the transcription of indicated genes in the RAD53 deletion strain was compared to the level in wild-type strain with or without MMS treatment. (D) Relative transcription levels of Rad53-regulated genes affected by deleting RAD9 or DUN1 in the MMS stress condition. The transcriptional levels of indicated genes in the RAD9 or DUN1 deletion strains were compared to those in wild-type strain under MMS treatment. The qRT-PCR assays for each strain were repeated at least 3 times. The difference between each group was compared using paired t test with GraphPad Prism 8.0.1 software. * represents p < 0.05, ** represents p < 0.01, *** represents p < 0.001, **** represents p < 0.0001. NS represents no significant difference.

Figure 4

Checkpoint kinases Rad53 and Rad9 regulate the transcription of HOF1 in C. albicans. (A) The transcription of HOF1 after deleting RAD9, RAD53, and DUN1 was checked by qRT-PCR. The wild-type strain (SN148), the RAD9 deletion strain, the RAD53 deletion strain, and the DUN1 deletion strain were treated with 0.015% MMS for 90 min before being harvested for RNA extraction. The transcription of HOF1 in each strain was compared to the level in wild type with no MMS stress. (B) The transcription of HOF1 after deleting DUN1 was checked by qRT-PCR. The wild-type strain (SN148) and the DUN1 deletion strain were treated with 40 mM HU for 90 min before being harvested for RNA extraction. The transcription of HOF1 in each strain was compared to the level in wild type without MMS stress. (C) The transcription of HOF1 after overexpressing DUN1. The wild-type strain or the RAD53 deletion strain with or without the DUN1 overexpression cassette was treated with MMS, as mentioned in panel A. The qRT-PCR assay for each strain was repeated at least 3 times. The transcription of HOF1 in each group was compared to the level in wild type without MMS stress. The difference between each group was compared using paired t test with GraphPad Prism 8.0.1 software. * represents p < 0.05, ** represents p < 0.01, and *** represents p < 0.001. NS represents no significant difference. (D) Phenotypic assay of the HOF1 RAD9 double deletion and the HOF1 DUN1 double deletion strains to MMS stress.

Figure 5

Transcription of HOF1 affected by Mcm1 and Fkh2. (A) The transcription of HOF1 was affected by deleting (left panel) or overexpressing FKH2 (right panel). The log phase cells of indicated strains were used for qRT-PCR assays. (B) The transcription of HOF1 was affected by repressing (left panel) or overexpressing MCM1 (right panel). The promoter of MCM1 was replaced by a MET3 promoter in SN148 background. The overnight culture of the indicated strains was inoculated into SC media plus Met/Cys (5 mM for each) or SC-Met/Cys for 4 h before being harvested for RNA extraction. The qRT-PCR assay for each strain was repeated at least 3 times. The transcription of indicated genes was compared to the level in wild type using a paired t test with GraphPad Prism 8.0.1 software. * represents p < 0.05, and ** represents p < 0.01.

Figure 6

Transcription factors Mcm1 and Fkh2 bind to the promoter of HOF1. (A,B) Detection of the binding of Mcm1 or Fkh2 to the promoter of HOF1 by ChIP analysis. The log phase wild-type cells (SN148) carrying Mcm1–HA or Fkh2–HA fusion, with or without 0.02% MMS treatment, were fixed with 1% formaldehyde. A wild-type strain without an HA tag was used as a control. Immunoprecipitated pellets were used as templates for PCR with the primer pairs HOF1–Chip-F and R. The intensity of the band was quantified using ImageJ software. Under no stress conditions, the ratio of the band in the ChIP group to the input group was normalized as 1. The result was averaged from two independent experiments. (C,D) The enrichment of HOF1 to Mcm1 and Fkh2 was checked by ChEC assay coupled with qPCR. Wild-type cells carrying Mcm1–Mnase or Fkh2–Mnase fusions were used. The extracted DNA fragments around 100 bp to 400 bp from cells with or without MMS treatment were applied for qPCR assays, and the GAPDH level was used as a control. The difference was compared using a paired t test with GraphPad Prism 8.0.1 software. ** represents p < 0.01. (E) Rad53 regulates the dynamic enrichment of the HOF1 promoter to Mcm1. The RAD53 deletion cells carrying Mcm1–Mnase fusions were used. The extracted DNA fragments around 100 bp to 400 bp from cells with or without MMS treatment were applied to check the signal of the HOF1 promoter with primers HOF1–pro-F/R, and the GAPDH level was used as a control. The difference was compared using a paired t test with GraphPad Prism 8.0.1 software. NS represents no significant difference.

Figure 7

Rad53 is involved in the direct regulation of HOF1. (A) Diagram of yeast one-hybrid assay. (B) The FHA2 region of Rad53 binds to the promoter of HOF1. The different domains of Rad53 were cloned into the pGADT7 plasmid, and various regions of the HOF1 promoter were cloned into the pHIS2 plasmid before being transformed into the yeast Y187 strain. The transformants were dissolved in distilled water and dropped onto SC–Trp–Leu–His plates containing different concentrations of 3-AT. The plates were kept at 30 °C for 2–3 days. (C) Checkpoint kinase Rad53 binds to the promoter of HOF1. The log phase wild-type cells carrying the Rad53–HA fusion, with or without 0.02% MMS treatment, were fixed with 1% formaldehyde. A wild-type strain without an HA tag was used as a control. Immunoprecipitated pellets were used as templates for PCR with the primer pairs HOF1–pro-F/R. The band intensity was quantified using ImageJ software. Under no stress conditions, the ratio of the band in the ChIP group to the input group was normalized as 1. The result was averaged from two independent experiments.

Figure 8

Overview of checkpoint-related regulation on HOF1 in response to MMS in C. albicans. (A) The dynamic binding of Rad53 and Mcm1/Fkh2 to the promoter of HOF1. Under normal conditions, Mcm1 and Fkh2 bind to the promoter of HOF1 and regulate the transcription of HOF1 to ensure regular and timely cytokinesis. Upon DNA damage stress, Mcm1 or Fkh2 dissociates from the promoter of HOF1 either by activated Rad53 or through a competition with activated Rad53, thereby subsequently diminishing the transcription of HOF1 to impede cytokinesis and giving enough time for cells to repair damaged DNA. (B) The binding region for Rad53 and Mcm1/Fkh2 in the HOF1 promoter. The red box represents the binding region of Rad53, and the green box represents the detected binding region for Mcm1 and Fkh2.