Screening to identify cryptic-Tx⁺ mutants and relate the phenotype to the degree of co-transcriptional disruption. A, schematic shows each cryptic-Tx⁺ reporter (YFG_pr.HA₃-FLO8::HIS3). HIS3 is out of frame with the FLO8₊₁ ATG such that transcription must initiate from an internal TATA (FLO8₊₁₆₂₆) to allow growth in the absence of histidine (8). B, relative output of ∼80% of the yeast RNA polymerase II transcriptome (5065 of ∼6000 genes) is shown (19). For this study the promoter upstream of FLO8::HIS3 was retained (FLO8_pr), deleted (Δ_pr), or replaced by one of seven alternates. C, degree of cryptic transcription is strongly influenced by the strength of the upstream promoter. Reporter arrays were pinned onto synthetic complete SC medium lacking histidine (GAL1-S_pr with 2%/1% galactose/raffinose; TDH3_pr/TEF1_pr with 2% glucose), incubated at 30 °C, and photographed on days indicated. Panels of the indicated mutants (pinned in duplicate) are size-standardized to facilitate cross-comparison. D, mutants in most transcription-related factors that support cryptic-Tx⁺ primarily do so when the cryptic promoter is located in a highly transcribed region. Factors are grouped by complex or pathway. The transcriptional strength of each promoter (e.g. TDH3_pr) upstream of the cryptic promoter is arranged by decreasing output (left to right). The color within each box indicates when HIS⁺ colonies were visible above the relevant plate background (days 1–7 after pinning on media lacking histidine). This measures the strength of cryptic-Tx⁺ in each case. Δ indicates a complete gene deletion; ^D, a DAmP allele; *, as for set2Δ and yjl169wΔ, the deletion of adjacent overlapping genes. BTFs, basal transcription factors; CompAsS, complex associated with Set1; CTDK, C-terminal domain kinase.

Reduced histone dosage in spt10Δ cells leads to profound cryptic-Tx⁺. A, duplicated genes encoding each core histone (H2A, H2B, H3, and H4) are located in four “head-to-head” loci. Arrows indicate the direction of transcription. Roman numeral is the chromosome number. NEG, negative regulatory element required for recruitment of the HIR complex (62, 63). B, promoter fidelity is differentially dependent on individual histone loci. Spots are 10-fold dilutions onto SC medium (2%/1% galactose/raffinose ± histidine) followed by incubation at 30 °C for days indicated. C and D, protein level of all four core histones is reduced in cells lacking Spt10. Whole cell extracts (WCE) were isolated from mid-log (A₆₀₀ ∼ 1) asynchronous cultures, resolved by SDS-PAGE, and immunoblotted as indicated. Rpn8 (proteasome subunit) is a loading control. D, each immunoblot was quantified (ImageJ), and the intensity of each histone in WT or spt10Δ was divided by the respective Rpn8 control (all 2.5-μl lanes). For clarity, the relative abundance of each histone in WT was normalized to 100% and relevant histone level in spt10Δ compared with this. E, cryptic-Tx⁺ in spt10Δ is independent of co-transcriptional disruption (i.e. HIS⁺ growth is comparable from all tested reporters). Spots are 10-fold dilutions onto SC medium (2% glucose ± histidine) followed by incubation at 30 °C for days indicated. YFG_Pr, your favorite gene. F, slow growth and cryptic-Tx⁺ of spt10Δ are complemented by SPT10 or increasing the dosage of all four core histones. For histone complementation the indicated WT or spt10Δ reporter strains were transformed with an empty high copy plasmid (2 μm) or same containing all four histones (HTA1-HTB1 and HHT1-HHF1), or the indicated individual loci (see supplemental Table 3). Spots are 10-fold dilutions onto SC medium (2% glucose ± histidine) followed by incubation at 30 °C for days indicated.

Rtt106 function depends on its ability to bind dsDNA and H3-H4. A, schematic depicts domain structure of Rtt106. Indicated alleles were C-terminally tagged with HA₃ in a low copy plasmid vector (i.e. pRS315.rtt106.HA₃). AP, acidic patch; C-term, C terminus; FL, full-length; MUT, point-mutated; PH, Plekstrin homology. B, single domain deletions have no impact on rtt106 protein stability. In contrast, rtt106-APΔ/CΔ (deleted of the acidic patch and acidic C terminus) is hypomorphic. Each rtt106.HA₃ was determined by immunoblotting with anti-HA. WT is a C-terminal HA₃ tag of genomic RTT106 for comparison. Rpn10 (proteasome subunit) is a loading control. C, both PH domains of Rtt106 are required to suppress cryptic transcription. Spots are 10-fold dilutions onto SC media (2%/1% galactose/raffinose ± histidine) followed by incubation at 30 °C for days indicated. D, indicated point mutants have no impact on rtt106 protein stability. Alleles were C-terminally tagged with HA₃ in a low copy plasmid vector (i.e. pRS315.rtt106.HA₃) and expression determined by immunoblotting with anti-HA. Rpn10 is a loading control. E, ability of Rtt106 to bind dsDNA and H3-H4 is required to suppress cryptic transcription. Spots are 10-fold dilutions onto SC media (2%/1% galactose/raffinose ± histidine) followed by incubation at 30 °C for days indicated. F, ability of Rtt106 to bind dsDNA and H3-H4 is required to ChIP at HTA1-HTB1. The enrichment of each rtt106.HA₃ allele at regions A and C (inset depicts ∼1.6-kB source location) is expressed relative to its occupancy at a subtelomeric gene-free region (GFR) from chromosome V (see “Experimental Procedures”). Error bars represent S.D. from at least three replicate quantitative PCRs. U, untagged; RK, RK_86–87AA; KK, KK_245–246AA; ITRLT, ITRLT_264–268AAAAA.

Mutants in replication-independent histone chaperones show extensive cryptic-Tx⁺. Schematic depicts each factor relative to Asf1, a H3-H4 donor to CAF (RDD pathway), HIR (RID pathway), and various acetyltransferases (e.g. SAS, Rtt109, Hat1). The cryptic-Tx⁺ in each mutant from this network is depicted (scored as in Fig. 1D). The similarity of the phenotype in deletions of ASF1, HIR, and RTT106 strongly suggests a common functional relationship. Δ, complete gene deletion; ^D, DAmP allele; *, as for mrc1Δ and ycl060cΔ, the deletion of adjacent overlapping genes.

Mutants in replication-independent histone chaperones show extensive cryptic-Tx⁺. A, indicated point mutants have no impact on asf1 protein stability. The tested alleles selectively abrogate binding to either HIR (HD_36–37AA or D37R, E39R) or H3-H4 (D54A or V94R) (50, 51, 53). Alleles were C-terminally tagged with HA₃ in a low copy plasmid vector (i.e. pRS315.asf1.HA₃) and expression determined by immunoblotting with anti-HA. H2B is a loading control. B, ability of Asf1 to bind H3-H4 is required to ChIP Rtt106 at HTA1-HTB1. The enrichment of Rtt106.HA₃ at region C in the indicated asf1 backgrounds is expressed relative to a gene-free region (GFR) from chromosome V (as in Fig. 3F). Error bars represent S.D. from at least three replicate quantitative PCRs. U, untagged; HD, HD_36–37AA; DE, D37R/E39R. C, ability of Asf1 to bind H3-H4 is required to maintain promoter fidelity. Spots are 10-fold dilutions onto SC media (2% glucose ± uracil) followed by incubation at 30 °C for days indicated. D, model depicts specific interactions required to recruit HIR, Asf1, and Rtt106 to HTA1-HTB1. HIR complex binds a specific DNA element (the NEG box) and directly interacts with Asf1, with both factors required to recruit Rtt106 (44). Rtt106 associates with Asf1 via H3-H4 and also binds dsDNA, possibly to stabilize its recruitment. E, hir1Δ/rtt106Δ cells do not support increased cryptic transcription relative to hir1Δ or rtt106Δ alone. The transcriptional activity of TDH3_Pr and ADH1_Pr upstream of FLO8::HIS3 is as in Fig. 1B. Spots are 10-fold dilutions onto SC media (2% glucose ± histidine) followed by incubation at 30 °C for days indicated. F, cryptic-Tx⁺ of hir1Δ, rtt106Δ, or asf1Δ cells is rescued by histone overexpression. Strains were transformed with an empty high copy plasmid (2 μm) or same encoding all four core histones (as in Fig. 2F). Spots are 10-fold dilutions onto SC media (2% glucose ± histidine) followed by incubation at 30 °C for days indicated.