Paf1 mediates ARG1 repression independently of Arg80 but inhibits Gcn4 association with the ARG1 promoter. (A) Quantitation of Northern analysis of ARG1 transcript levels in wild-type (KY1699), paf1Δ (KY1702), paf1Δ arg80Δ (KY1720), and arg80Δ (KY1709) strains relative to a loading control, SCR1. Transcript levels were quantified and normalized to the levels detected in arg80Δ (KY1709) cells as described in Materials and Methods. (B) Quantitation of Northern analysis of relative ARG1 transcript levels in wild-type (KY1699), paf1Δ (KY1702), paf1Δ gcn4Δ (KY1719), and gcn4Δ (KY1708) strains. (C) ChIP analysis of Gcn4 occupancy at the ARG1 promoter in wild-type (KY1728), paf1Δ (KA116), paf1Δ arg80Δ (KY1730), and arg80Δ (KY1729) strains expressing HA-tagged Gcn4 and an untagged Gcn4 control strain (KA119). The y axis was truncated to facilitate comparison of lower values. The value for the paf1Δ arg80Δ strain is indicated above the bar. (D) ChIP analysis of Gcn4 occupancy at the HIS7, TRP2, and ARO4 promoters in wild-type (KY1728) and paf1Δ (KA116) strains expressing HA-tagged Gcn4 and an untagged Gcn4 control strain (KA119). (E) Strand-specific RT-PCR examining HIS7, TRP2, and ARO4 sense transcript levels in wild-type (KY1699) and paf1Δ (KY1700) cells. ACT1 is used as a positive control for RT-PCR experiments. A minus indicates a reaction mixture lacking reverse transcriptase. Results are representative of those obtained in two independent experiments. Relative to the ATG for each gene, the 5′ ends of cDNA synthesis primers map to HIS7 (+969), TRP2 (+1142), and ARO4 (+613). Primers used in PCR amplify HIS7 (+633 to +853), TRP2 (+745 to +967), or ARO4 (+327 to +573). WT, wild type.

Mutation of H3 acetylation sites partially restores ARG1 repression in the absence of Paf1. (A) Quantitation of Northern analysis of relative ARG1 expression in wild-type (KY205), paf1Δ (KY803), and paf1Δ gcn5Δ (KY1348) strains. (B) Quantitation of Northern analysis of ARG1 transcript levels in wild-type (KY1699), HHTS (hht1-hhf1)Δ (KA120), hhts-K9A (hht1-hhf1)Δ (KA122), hhts-K14A (hht1-hhf1)Δ (KA124), hhts-K4, 9, 14, 18A (hht1-hhf1)Δ (KA126), paf1Δ (KY1700), HHTS paf1Δ (hht1-hhf1)Δ (KA121), paf1Δ hhts-K9A (hht1-hhf1)Δ (KA123), paf1Δ hhts-K14A (hht1-hhf1)Δ (KA125), paf1Δ hhts-K4, 9, 14, 18A (hht1-hhf1)Δ (KA127), and paf1Δ gcn4Δ hhts-K4,9,14,18A (hht1-hhf1)Δ (KA175-179) strains. WT, wild type.

Changes in nucleosome occupancy accompany ARG1 transcription, and the H2B-K123R substitution leads to increased Gcn4 levels at the ARG1 promoter. (A) Top left, nucleosome scanning assay examining nucleosome occupancy at the ARG1 promoter and 5′ region in wild-type strains in repressing and inducing conditions. Wild-type (KY1699) cells were grown to log phase in SC-IV medium and mock treated with DMSO (WT) or treated with SM for 2 h (WT + SM) prior to cross-linking. Mononucleosomal DNA was prepared and subjected to real-time PCR analysis using primers that tile a chromosomal region containing the ARG1 promoter. The midpoint position of each PCR fragment relative to the translation start site is plotted on the x axis. Relative MNase protection is plotted on the y axis. Bottom, schematic representation of predicted nucleosome occupancy. Shaded circles represent nucleosomes. Top right, representative Northern blot examining ARG1 transcript levels in strains harvested for RNA prior to cross-linking. (B) Nucleosome occupancy at the ARG1 promoter region in wild-type (KY1699) and paf1Δ (KY1700 and KY1701) strains grown in YPD. Samples were processed and relative MNase protection calculated as in (A). Right: representative Northern analysis of ARG1 mRNA levels immediately prior to cross-linking. (C) ChIP analysis of Gcn4 occupancy at the ARG1 promoter in wild-type (OKA178), paf1Δ (KA116), (hta2-htb2)Δ (KY2188-89), and (hta2-htb2)Δ htb1-K123R (KY2022-2024) strains expressing HA-tagged Gcn4 and an untagged control strain (KY1699).

Paf1 mediates repression through the ARG1 promoter. (A) Representation of the ARG1p-HIS3 gene where the ARG1 coding region was replaced with that of the HIS3 gene. (B) Left, Northern analysis examining HIS3 mRNA levels when expressed from the endogenous HIS3 chromosomal location in wild-type (KY1699), paf1Δ (KY1700), and rtf1Δ (KY1703) cells and when the HIS3 coding region is fused to the ARG1 promoter (ARG1p-HIS3) in wild-type (KY1736), paf1Δ (KY1737), and rtf1Δ (KY2175) cells. Right, Northern analysis of ARG1 transcript levels in wild-type (KY1699), paf1Δ (KY1700), and rtf1Δ (KY1704) strains. The SCR1 gene serves as a loading control. Results shown are representative of at least three independent experiments. (C) Quantitation of HIS3 transcript levels in wild-type (KY1699), paf1Δ (KY1700), and rtf1Δ (KY1703) cells in which HIS3 is expressed from its normal chromosomal location. (D) Quantitation of HIS3 transcript levels in wild-type (KY1736), paf1Δ (KY1737), and rtf1Δ (KY2175) cells in which the HIS3 coding region is under the control of the ARG1 promoter (ARG1p-HIS3). (E) Quantitation of ARG1 transcript levels in wild-type (KY1699), paf1Δ (KY1700), and rtf1Δ (KY1704) strains. These quantified data were published previously (reprinted from Fig. 1B in reference ) and are shown here for clarity. (F) ChIP analysis of Gcn4 occupancy at the ARG1 promoter in wild-type (OKA178), paf1Δ (KA116), ARG1p-HIS3 (KY2182-84), and ARG1p-HIS3 paf1Δ (KY2185-87) strains expressing HA-tagged Gcn4 and an untagged control strain (KY1699). WT, wild type.

Paf1 inhibits antisense transcription across the ARG1 promoter. (A) Diagram showing the locations of primers used in cDNA synthesis (arrows) and the PCR amplicon (bar) used for strand-specific RT-PCR experiments. (B) Strand-specific RT-PCR examining ARG1 sense transcription in wild-type (KY1699) and paf1Δ (KY1700) strains. Results shown are representative of more than three independent experiments. (C) Strand-specific RT-PCR examining antisense transcription within the ARG1 transcribed region (primers E and F) in wild-type (KY1699) and paf1Δ (KY1700) strains. Results shown are representative of two independent experiments. (D) Strand-specific RT-PCR examining antisense transcription within the ARG1 promoter (primers A to D) in wild-type (KY1699) and paf1Δ (KY1700) strains. Results shown are representative of at least two independent experiments. A minus indicates control reaction mixtures lacking reverse transcriptase. (E) Real-time PCR analysis of strand-specific cDNA generated using primer B in wild-type (KY1699) and paf1Δ (KY1700) strains. Results shown are the averages from two independent experiments. Error bars represent the ranges in the data. WT, wild type.

The ARG1 3′ UTR is not required for antisense transcription. (A) Diagram of the ARG1 regions integrated at the LYP1 locus in the same orientation as the LYP1 gene. Strand-specific RT-PCR examining ARG1 sense transcription (B) and antisense transcription within the ARG1 open reading frame (C) and promoter (D) in wild-type (KY2112) and paf1Δ (KY2115) cells in which the ARG1 promoter and coding region are fused to the LYP1 3′ UTR and in wild-type (KY2118) and paf1Δ (KY2121) cells in which the ARG1 promoter, coding region, and 3′ UTR were integrated at the LYP1 locus. cDNA synthesis primers used to detect antisense transcription are noted and correspond to those shown in A. For analysis of antisense transcription with primer E, 6 μl of no-RT reaction mixtures (−) and 2-μl and 6-μl volumes of cDNA were used in PCRs to amplify the ARG1 gene. Results are representative of two independent experiments. (E) Locations of cDNA synthesis and PCR primers used to detect antisense transcription at the ARG1p-HIS3 promoter. (F) Strand-specific RT-PCR analysis to detect antisense transcription traversing the ARG1p-HIS3 promoter in wild-type (KY1736) and paf1Δ (KY1737) cells (left). ACT1 is used as a positive control for RT-PCR experiments (right). A minus indicates a reaction mixture lacking reverse transcriptase. WT, wild type.

ARG1 sense transcription positively correlates with antisense transcription across the ARG1 promoter. (A) Strand-specific RT-PCR examining ARG1 sense transcription in wild-type (KY1699), paf1Δ (KY1702), paf1Δ gcn4Δ (KY1719), and gcn4Δ (KY1708) cells. The cDNA synthesis primer used to detect sense transcription is depicted in A. (B) Strand-specific RT-PCR examining antisense transcription across the ARG1 promoter in wild-type (KY1699), paf1Δ (KY1702), paf1Δ gcn4Δ (KY1719), and gcn4Δ (KY1708) cells. The cDNA synthesis primer (primer B) used to detect antisense transcription is depicted in A. Results shown are representative of two independent experiments. A minus indicates reaction mixtures lacking reverse transcriptase. (C) Real-time PCR analysis of strand-specific cDNA generated using primer B in wild-type (KY1699), paf1Δ (KY1702), paf1Δ gcn4Δ (KY1719), and gcn4Δ (KY1708) cells. Results shown are the averages from two independent experiments. Error bars represent the range in the data. Strand-specific RT-PCR analysis of ARG1 sense (D) and antisense (E) transcription in wild-type (KY1699) cells grown in SC-IV medium and mock treated with DMSO (WT) or treated with SM for 2 h (WT + SM). Results are representative of two independent experiments. WT, wild type.

Paf1 restricts antisense transcription and Gcn4 occupancy at the promoters of other Paf1-repressed genes. (A) Diagram of the locations of primers used for cDNA synthesis and PCR for strand-specific RT-PCR analysis of sense and antisense transcription at the SNZ1 locus. (B) Strand-specific RT-PCR examining SNZ1 sense transcription in wild-type (KY1699) and paf1Δ (KY1700) strains. Results are representative of two independent experiments. (C) Strand-specific RT-PCR examining antisense transcription across the SNZ1 promoter in wild-type (KY1699) and paf1Δ (KY1700) strains. Results are representative of two independent experiments. (D) Diagram of the locations of primers used for cDNA synthesis and PCR for strand-specific RT-PCR analysis of sense and antisense transcription at the ARG3 locus. (E) Strand-specific RT-PCR examining ARG3 sense transcription in wild-type (KY1699) and paf1Δ (KY1700) strains. The data shown are representative of two independent experiments. (F) Strand-specific RT-PCR examining antisense transcription across the ARG3 promoter in wild-type (KY1699) and paf1Δ (KY1700) strains. Results are representative of two independent experiments. (G) ChIP analysis of HA-Gcn4 occupancy at the ARG3 promoter in wild-type (OKA178) and paf1Δ (KA116) cells expressing HA-Gcn4 and a strain expressing untagged Gcn4 (KA119). WT, wild type.