Figure 3. Ngs1 binds to the promoter of GlcNAc catabolic genes.

(a) The ngs1 mutant strain expressing GFP-Ngs1 under the MAL2 promoter (HLY4397) was grown to log phase in liquid SC medium containing 50 mM maltose at 30 °C, and then treated with or without 50 mM GlcNAc for 30 min. Cell morphology and Ngs1 localization were observed by using DIC and fluorescence microscopy. An untagged control (SC5314) was included. For all images: Scale bar, 5 μm. (b) Ngs1 is present at the promoter for NAG1 and DAC1 in a GlcNAc-independent manner. Overnight cultures of ngs1 mutant cells carrying 3FLAG-Ngs1 (HLY4402) or untagged Ngs1 (HLY4395) from the NGS1 promoter were pelleted, washed three times in PBS, diluted 1:20 in SC medium with 10 mM GlcNAc or glucose at 30 °C, and cells were collected at 30 min for the ChIP experiment. ChIP DNA was quantitated as described⁶⁴ by qPCR with primers at the promoter region of NAG1 and DAC1. The ACT1 promoter region was used as a control. The enrichment is presented as a ratio of NAG1-DAC1 promoter IP (bound/input) versus ACT1 promoter IP (bound/input). The value in ngs1 mutant cells carrying untagged Ngs1 (HLY4395) in glucose medium was set to 1.00. The ChIP data showed the average of three independent qPCR data with error bars representing the s.d.