Hmo1 binds preferentially to the Pol I-transcribed region of the rDNA. ChIP experiments were performed using strain HMO1-TAP (OGP126-1a) or the untagged BMA64-1a control strain, followed by a two-step purification. (A) Schematic representation of an rDNA unit. Sequence elements within the rDNA (nontranscribed spacers 1 and 2 [NTS1 and -2], the 5S RNA Pol III transcript, and the 35S RNA Pol I transcript) are noted. The oligonucleotides used to analyze the immunoprecipitates via quantitative PCR are indicated by arrows (1 to 20). (B) Hmo1 ChIP. Relative values of immunoprecipitated DNA and the whole-cell extract (Input) are shown. The amount of immunoprecipitated DNA was expressed as a value relative to that for input DNA, where 1 unit represents 0.005% of input DNA. DNAs immunoprecipitated from the Hmo1-TAP strain (OGP126-1a) and from the untagged BMA64-1a control strain are shown in gray and in black, respectively. Standard deviations for three different measurements are indicated.

Hmo1 is a nonessential activator of RP gene expression. (A) Ifh1 depletion is lethal in the absence of Hmo1. IFH1 was placed under the control of a tetracycline-regulatable promoter (P_tet-IFH1). Growth was scored without (0 μg/ml), with mild (2.5 μg/ml), or with strong (20 μg/ml) repression of IFH1 transcription. Ifh1 depletion was performed in wild-type (+; YAB103-1a), hmo1Δ (YAB103-1a cured of pRS316-HMO1), and fpr1Δ (YAB106-1a cured of pRS316-FPR1) strains. Tenfold serial dilution series were spotted on rich medium in the presence of increasing concentrations of doxycycline. Growth was observed after 5 days of incubation at 30°C. (B) Transcriptome analysis of hmo1 deletion and of Hmo1 depletion. Total RNA was extracted and reverse transcribed from a wild-type strain (BY4741) or from an hmo1-Δ strain (Y06969). Depletion of Hmo1 (Hmo1-depleted) was performed for 6 h by adding 5 μg/ml doxycycline to YAB108-1a; untreated cells were used as a control (WT). The histograms represent the percentages of total transcript (black) and RP gene mRNAs (gray) with given variations compared to the wild-type levels (x axis).

Hmo1 is involved in TORC1-dependent coregulation of rDNA and RP gene transcription. (A) The CARA mutant results in an up-regulation of RP genes upon TORC1 inhibition. (B) Hmo1 contributes to the coupling of Pol I and RP gene expression. The histograms represent the percentages of total transcript (black) and RP gene mRNAs (gray) with given variations compared to the wild-type (WT) levels (x axis). Total RNA was extracted and reverse transcribed from strains YPH500, CARA, YPH500/hmo1Δ (YAB72), and CARA/hmo1Δ (YAB73) treated for 60 min with 400 ng/ml rapamycin.

Genetic interactions of Hmo1. (A) Flow diagram of the GID screen. (B) Schematic representation of identified genetic interactors with HMO1. The interactors can be grouped roughly into three different classes of function that are partially overlapping: (i) transcription, (ii) ribosome, and (iii) stress response. (C) Growth comparison of wild-type, single-mutant, and double-deletion strains. The single-deletion hmo1Δ strain (YAB2-2a) and double-deletion hmo1Δ/net1Δc (YAB4-1a), hmo1Δ/rps23aΔ (YAB5-1a), and hmo1Δ/nsc2Δ (YAB8-1a) strains containing plasmid pGID-HMO1 were transformed with pFL36-HMO1 (left; WT) or an empty vector (right; hmo1Δ). Tenfold serial dilution series were spotted on 5-FOA-containing plates. Growth was scored after 4 days at 30°C. On 5-FOA medium, the pGID-HMO1 plasmid is lost. In the left panel, HMO1 is still present on plasmid pFL-HMO1, scoring the growth of the single-deletion mutant; in the right panel, HMO1 is absent, scoring the growth of the double-deletion mutant hmo1Δ plus the indicated deletion.

Inhibition of the TORC1-pathway is lethal in an hmo1Δ background. (A) An hmo1Δ mutant is hypersensitive to rapamycin. BY4741 (WT), hmo1Δ (Y16969), and hmo1Δ TOR1-1 strains were spread homogenously onto yeast extract-peptone-dextrose plates by using glass beads. TOR1-1 is generated by a dominant mutation in the TORC1-kinase TOR1, making TORC1 rapamycin insensitive. Sterile Whatman 3MM filter paper containing different amounts of rapamycin (1, 0.01 μg; 2, 0.02 μg; 3, 0.05 μg; 4, 2 μg) were deposited on top of these plates. The halo around each filter is indicative of the growth inhibition. (B) Hmo1 is genetically linked to the TOR pathway. KOG1 was placed under the control of a tetracycline-regulatable promoter (P_tet-KOG1). Growth was scored without (0 μg/ml), with mild (2.5 μg/ml) or with strong (20 μg/ml) repression of KOG1 transcription. Kog1 depletion was performed in wild-type (+; YAB102-1a), hmo1Δ (YAB102-1a cured of pRS316-HMO1), and fpr1Δ (YAB107-1a cured of pRS316-FPR1) strains. Tenfold serial dilution series were spotted on rich medium in the presence of increasing concentrations of doxycycline. Growth was observed after 5 days of incubation at 30°C.

Hmo1 is binding to specific RP gene promoters. (A) Effect of an hmo1Δ mutation on the steady-state mRNA levels of RPS5, RPL16B, RPS20, and RPL5. Quantitative PCR was performed on reverse-transcribed total mRNA extracted from hmo1Δ (Y16969) and BY4741 strains. mRNA levels were normalized against ACT1 transcript levels. Log₂ values for ratios of mutant versus wild-type levels are represented with standard errors for three independent experiments. (B) Hmo1 binds specifically to the promoter regions of RPS5 and RPL16B. A ChIP experiment was performed using strain HMO1-TAP (OGP126-1a), followed by a two-step purification (see Materials and Methods). Amplicons targeting the promoter of the indicated genes (Prom) contain the putative Fhl1 binding site. Amplicons marked “Trans” target the transcribed regions of the indicated gene.

Hmo1 is required for TORC1-dependent inhibition of RP gene expression. (A) The Hmo1 protein steady-state level is unaffected after 60 min of TORC1 inhibition. Total proteins were extracted from strain HMO1-TAP (OGP126-1a) without or after treatment for 60 min with 400 ng/ml rapamycin. Actin was used as a loading control. (B) Hmo1 DNA binding is rapamycin dependent. ChIP experiments were performed using strain HMO1-TAP (OGP126-1a) without (left) or after treatment with rapamycin (A). (C) RP genes are up-regulated in an hmo1Δ strain compared to wild-type (WT) levels upon TORC1 inhibition. The histogram represents the percentages of total transcript (black) and RP gene mRNAs (gray) with given variations compared to the wild-type levels (x axis). Total RNA was extracted and reverse transcribed from strains YPH500 hmo1Δ (YAB72) and YPH500 treated for 60 min with 400 ng/ml rapamycin.