Editor's Pick Research Article | Molecular Biology and Physiology

High-Resolution Genome-Wide Occupancy in Candida spp. Using ChEC-seq

Faiza Tebbji, Inès Khemiri, Adnane Sellam
Aaron P. Mitchell, Editor
DOI: 10.1128/mSphere.00646-20

Article Figures & Data

Figures

  • FIG 1
    FIG 1

    ChEC-seq method in C. albicans and Candida spp. (A) Plasmid constructs for in vivo TR-MNase tagging and the construction of the “free MNase” control strains in C. albicans and C. auris. Structure of the MNase-tagging cassette consisting of a 3xFLAG epitope fused to the C. albicans codon-optimized MNase. The FLAG-MNase construct for the “free MNase” strain was constitutively expressed using the C. albicans ACT1 promoter (pACT1). CYC, CYC1 terminator; SV40, nuclear localization signal. (B) Phenotypic characterization of strains bearing the MNase-tagged Nsi1 and Snf2 TRs and the “free MNase” control constructs in C. albicans. WT, Snf2MNase, Nsi1MNase, the free MNase, and the control (empty vector) strains were serially diluted, spotted on YPD, and incubated for 1 day at different temperatures. Growth patterns at different temperatures of the C. auris WT, CauNsi1MNase, the free MNase and the control strains are also shown. (C) Schematic representation of the experimental setup of the ChEC-seq methodology. Candida species cells where a TR of interest is fused to MNase are permeabilized with digitonin prior to MNase activation with calcium. This will lead to the fragmentation of unprotected neighboring chromatin. The resulting fragmented DNA is purified and subjected to size selection prior to high-throughput sequencing. (D) Evaluation of genomic DNA fragmentation by agarose gel electrophoresis at 0, 0.5, 1, 2.5, 5, 10, 20, and 60 min of calcium exposure in the Snf2MNase, Nsi1MNase, and the free MNase strains in C. albicans. L, 100-bp DNA ladder.

  • FIG 2
    FIG 2

    Genome-wide occupancy of the transcription factor Nsi1 with ChEC-seq. (A) Temporal analysis of Nsi1 binding events. Venn diagram showing the overlap of Nsi1 binding events at three distinct MNase activation times (5, 20, and 60 min). (B) Motif scores for Nsi1-bound promoters at 5, 20, and 60 min after MNase activation. The motif logos were generated using MEME-ChIP software on the 1,000 high-scoring peaks. (C to F) Snapshots of genomic regions showing the ChEC-seq signal for Nsi1MNase and the free MNase strains at 5, 20, and 60 min after MNase activation. The positions of the Nsi1 motifs are indicated by the dashed lines. Nsi1 occupies the promoter of MLC1 (C), AIM7 (D), and ASM4 (E), in addition to many sites within the rDNA locus (F).

  • FIG 3
    FIG 3

    Genome-wide occupancy of the chromatin remodeler Snf2 with ChEC-seq. (A) Venn diagram showing the overlap of Snf2 promoter binding at 5, 20, and 60 min after MNase activation. (B) Comparison of SWI/SNF genomic occupancies by ChEC-seq and ChIP-chip method. Venn diagram of overlap between Snf2 ChEC-seq sites and Snf6 ChIP-chip using high-density tiling arrays. (C) Gene ontology of biological process associated with Snf2-bound promoters at 5 min after MNase activation. The P values were calculated using hypergeometric distribution as described in the GO Term Finder Tool website (http://candidagenome.org/cgi-bin/GO/goTermFinder). (D) Snapshot of genomic regions showing the ChEC-seq signal for Snf2MNase and the free MNase strains at 5, 20, and 60 min after MNase activation. Genome browser view of Snf2 ChEC-seq displaying promoter occupancies of carbohydrate metabolism genes, including galactolysis (GAL1 and GAL10) and hexose transport (SHA3, HGT2, and HGT1).

Supplemental Material

Back to top
Download PDF
Citation Tools
Print
Alerts
Email

KEYWORDS

Candida albicans
Candida spp.
ChEC-seq
genome-wide occupancy
transcriptional regulatory network

Related Articles

Cited By...