Journal: Cell Reports

Article Title: Transcription levels of a noncoding RNA orchestrate opposing regulatory and cell fate outcomes in yeast

doi: 10.1016/j.celrep.2020.108643

Figure Lengend Snippet: IRT2 transcription has a level-dependent effect on local chromatin and transcription states (A) Scheme for measuring histone H3 exchange rates. (B) Histone H3 exchange rate at the IRT1 promoter in the presence or absence of IRT2 transcription. A strain harboring differentially epitope-tagged histone H3, with one copy expressed from the endogenous promoter (Myc-H3) and the other expressed from a GAL1-10 inducible promoter ( pGAL-FLAG-H3 ) were used for the analysis. Constitutive low levels of IRT2 transcription are achieved using the u6bs Δ mutation (FW7880), whereas WT cells (FW7853) display no IRT2 transcription in rich medium (YP). Cells were grown till mid-log in YP raffinose and arrested in G1 with α factor, and FLAG-H3 was induced with galactose. The signals for H3 ChIP (Myc-H3 and FLAG-H3) were normalized to a telomere locus, and ratios for n = 3 (error bars represent ±SEM) are displayed. ∗ p < 0.05; ∗∗ p < 0.005; and ∗∗∗ p < 0.0005, on a two-way ANOVA followed by Fisher’s LSD test. The slopes of the linear regression equations (Y = [0.07105 ⋅ X] + 1.466 for control, and Y = [0.1258 ⋅ X] + 3.325 for u6bs Δ) are significantly different. (C) Relative expression of IRT2 , IRT1 , and IME1 in cells and grown as described in (B). qPCR signals were normalized to ACT1 . Error bars represent ±SEM; n = 2. ∗ p < 0.05; ∗∗ p < 0.005, on an unpaired Student’s t test. (D) Similar to (B), histone exchange at the PHO5 and PGK1 control promoters in strains described in (B). Error bars represent ±SEM; n = 3. The slopes of the linear regression equations for both loci are not significantly different. (E) Scheme for controlling different levels of IRT2 by LexA-ER (top). Multiple lexA operator (lexO) sequences were integrated in the IRT2 promoter at +96 bp relative to IRT2 start site (lexO(+96)). LexA-ER is activated by β-estradiol. IRT1 and IRT2 levels as quantified by northern blot normalized to SCR1 , in cells harboring 1, 2, 3, 4, and 8 lexO(+96) sites in the IRT2 promoter (FW6594, FW6599, FW6607, FW6611, and FW6619) at 1 h in SPO. The MAT a LexA-ER control strain (control, FW6560) was included. Cells were treated (+β-estradiol) or not (mock) for 3 h and shifted to SPO plus β-estradiol. The ratio of +β-estradiol/mock is displayed. n = 2 data points and a trend line representing second-degree polynomial fit are shown. (F) Chromatin structure at the IRT1 promoter in the presence of distinct levels of IRT2 transcription. Control cells ( MAT a LexA-ER, FW6560) or cells harboring 1 or 4 lexO(+96) sites (FW6594 or FW6611) were treated as described in (E). MNase-digested fragments were subject to qPCRs using primer pairs nested in IRT2 . The red arrows indicate the position of the Rme1 binding sites. The signals were normalized over a telomere locus. Error bars represent ±SEM; n = 3. ∗ p < 0.05; ∗∗∗ p < 0.0005; and ∗∗∗∗ p < 0.0001, on a two-way ANOVA followed by Fisher’s LSD test performed on lexO strains compared to control SPO for 3 h. (G) MAT a/α and MAT a/a diploid cells (FW1511 and FW15) and MAT a/a cells harboring pCUP-IRT2 (FW8923) were shifted to SPO and either treated (+Cu) or not (−Cu) with copper sulfate. DAPI masses were counted from cells fixed at 72 h in SPO. Cells harboring >2 DAPI masses were considered to have undergone meiotic divisions (MI + MII). Error bars represent ±SEM; n = 4 for the controls, and n = 5 for pCUP-IRT2 . ∗∗∗∗ p < 0.0001, two-way ANOVA followed by Fisher’s LSD test performed on MAT a/a strains with or without copper sulfate treatment.

Article Snippet: For the histone H3 turnover experiments described in and , ChIPs were performed as described above using antibodies against the Myc epitope (clone 9E11, Thermofisher) or against the FLAG epitope (M2 beads, Sigma).

Techniques: Mutagenesis, Control, Expressing, Northern Blot, Binding Assay