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. 2016 Oct 3;7:12916. doi: 10.1038/ncomms12916

Figure 6. Rep1 recruits Ngs1 to the promoters of GlcNAc catabolic genes.

Figure 6

(a) REP1 is required for growth on GlcNAc and galactose in C. albicans. Dilutions of the rep1Δ/rep1Δ mutant (YLO141), rep1Δ/rep1Δ::REP1 (YLO142), and the wild type (SC5314) cells were spotted onto YNB solid medium containing 2.5 mM of the indicated sugar or 5% glycerol. Cells were also tested for growth on SC solid medium in the absence or presence of 2.5 mM GlcNAc. Photographs were taken after 2 days of growth at 30 °C. (b) qRT-PCR analysis of NAG1 transcription upon GlcNAc induction. Cells of the above three strains were grown in liquid SC medium with 2.5 mM GlcNAc for 2 h at 30 °C for RNA extraction. (c) ChIP of Rep1 at the promoter of NAG1 and DAC1. The rep1 mutant strain expressing Rep1-Myc or untagged Rep1 was grown in SC medium with GlcNAc or glucose at 30 °C, and cells were collected at 30 min for the ChIP experiment. The ratio of NAG1-DAC1 promoter IP (bound/input) versus ACT1 promoter IP (bound/input) in rep1 mutant cells carrying untagged Rep1 in glucose medium was set to 1.00. (d) Ngs1 interacts with Rep1. Protein lysates from strains HLY4454 (FLAG-Ngs1, Rep1-Myc) and HLY4402 (FLAG-Ngs1) were subjected to immunoprecipitation with anti-Myc antibody, and the precipitated proteins were probed with anti-FLAG antibody. As an input control, cell lysates were analysed by Western blotting with the anti-FLAG antibody. (e) Ngs1 association at the NAG1-DAC1 promoter depends on Rep1. For ChIP of Ngs1 in the wild-type strain (HLY4459) or the rep1 mutant (HLY4453), cells were grown in SC GlcNAc medium at 30 °C for 30 min. The ratio of NAG1-DAC1 promoter IP (bound/input) versus ACT1 promoter IP (bound/input) in wild-type cells carrying untagged Ngs1 was set to 1.00. All data showed the average of three independent qPCR data with error bars representing the s.d.