Figure 5.

Hir1 affects the chromatin density in the HWP1 and UME6 promoter. (A,B) Loss of HIR1 increases the histone density in the HWP1 promoter. Histone density was measured using histone H3 ChIP and qPCR at different HWP1 promoter regions. (C) HWP1 expression is reduced in hir1Δ/Δ cells. WT and hir1Δ/Δ cells were grown in YPD with 10% FCS at 37 °C. Cultures were collected at the indicated time points followed by RNA extraction. Gene expression of HWP1 was measured via RT-qPCR and transcript levels were normalized to the reference gene RIP1. (D) Representative illustration of transcription factor binding sites in the HWP1 upstream intergenic sequence. Efg1 (yellow) and Nrg1 (red) binding motifs were taken from ref. 59 and Tec1 (green) and Brg1 (blue) from ref. 13. Note that no putative Tec1 binding site was identified in our in silico scan. (E) The same as in (C), but for UME6. (F,G) Genetic removal of HIR1 alters histone occupancy at distinct UME6 promoter regions. Histone density was measured using histone H3 ChIP and qPCR at different UME6 promoter regions. (H) Putative transcription factor binding sites for the upstream intergenic region of UME6 are represented as in (D). Transcription factor sites with asterisk (*) indicate multiple sites for a given regulator within less than 100 bp. (I) HIR1-deficient cells require stronger signal intensity to initiate hyphal formation. WT and hir1Δ/Δ cells were spotted on YPD supplemented with a continuous GlcNAc concentration gradient of 0–10 mM. Colony morphology was inspected after growth for 3 days at 37 °C. (A,B,F,G) The qPCR signals from Input and IP were normalized to an intergenic region on chromosome R. The ratio of normalized Input/IP values is shown on the y-axis labeled as “H3 density”. (A–C,E–G): Data are presented as mean + SD of three independent experiments. For significance testing, hir1Δ/Δ cells were compared to WT cells. *P < 0.05, **P < 0.01 with Student’s t-test.