Nap1 interacts directly with Yra1. (A) Whole-cell lysates from a Nap1-TAP (right panel) and an untagged strain (left panel) were incubated with IgG Sepharose. Interacting proteins were eluted with the indicated MgCl₂ concentrations. Yra1 and Nap1-TAP were detected by Western blotting (WB). (B) Whole-cell lysates from a Yra1-FLAG-tagged (right panel) and an untagged strain (left panel) were incubated with anti-FLAG resin. Bound proteins were analyzed by Western blotting with the indicated antibodies. (C) Yra1-FLAG was immunoisolated from whole-cell extract made from formaldehyde-fixed cells. Coprecipitating Nap1 was detected by Western blotting. (D) Immobilized FLAG-GFP or Yra1-FLAG (250 nM) was incubated with Nap1 (250 nM). Complexes were analyzed by SDS-PAGE and Western blotting with FLAG and Nap1 antibodies. (E) Strains of the indicated genotype were equalized, spotted at 10-fold serial dilutions, and grown on YPD plates at 30°C for 2 days. (F) Strains of the indicated genotype were stained with calcofluor white (Cal White) and visualized by fluorescence and differential interference contrast microscopy. (G) The frequency of long and hyperelongated buds relative to normal buds in asynchronous populations of cells was determined. The length of long buds was approximately greater than the width, but not did not exceed the mother cell diameter. The length of hyperelongated buds was approximately greater than both the bud width and mother cell diameter. A minimum of 175 cells were characterized.

NAP1 functionally interacts with MEX67 and the THO complex. (A) Strains of the indicated genotype were equalized, spotted at 10-fold serial dilutions, and grown on YPD at 30°C for 2 days. (B) The indicated cell types were stained with calcofluor white (Cal White) and visualized by differential interference contrast (DIC) and fluorescence microscopy. (C) The frequency of long and hyperelongated buds relative to normal buds in asynchronous populations of the indicated cells was determined as described in the legend to Fig. 1. A minimum of 225 or 160 cells were characterized for the upper or lower tables, respectively.

Nap1 recruitment to sites of active transcription is Yra1 dependent. (A) Chromatin was isolated from a Δyra1 Δnap1 strain expressing plasmids encoding Yra1-FLAG and HA-Nap1. Yra1 and Nap1 occupancy at the PMA1 promoter and ORF was determined by ChIP assay with conventional PCR using oligonucleotide pairs to the indicated regions of PMA1. The assay was also performed without antibody as a negative control. (B) yra1-1 was coimmunoprecipitated from whole-cell extracts with Nap1-3HA and detected by Western blotting. (C) Nap1 recruitment to the ACT1 and ADH1 ORFs and intergenic regions was tested by ChIP assay using the indicated oligonucleotide pairs. (D) Nap1 ChIP assays quantified by real-time PCR were performed with a Δyra1 Δnap1 strain expressing yra1-1 and HA-Nap1. Cells were grown for 2 h at 25°C or 39°C prior to fixation as indicated. Occupancy is expressed as the log₂ difference (n-fold) between specific and background IP signals normalized to input, displayed as the mean and average standard deviation from two independent experiments. Oligo, oligonucleotide. (E) Nap1 ChIP assays quantified by real-time PCR were performed exactly as for panel D with a YRA1 Δnap1 strain expressing HA-Nap1. (F) RNAP II (Pol II) ChIP assays quantified by real-time PCR as for panel D were performed from a Δyra1 strain expressing yra1-1 exogenously. Means and standard deviations are indicated.

Model of Nap1 function during transcription. The TREX complex component Yra1 directly recruits Nap1 to a site of active transcription. (A) Nap1 facilitates the reincorporation of H2A/H2B dimers and octamer reassembly on the open template following RNAP II (Pol II) progression. (B) Nap1 may also function in H2A/H2B disassembly preceding RNAP II. (C) Nap1 also mediates histone mobilization at other regions, such as the promoter in Yra1-independent mechanisms.

Nap1 is not essential for Yra1 nuclear import and Yra1-mediated mRNA export. (A) pYra1-GFP₂ or pH2A_1-46-GFP₂ was expressed in wild-type or Δnap1 cells cotransformed with pRS425GPD or pRS425GPD-NAP1 and visualized by fluorescence microscopy. The coincident Hoechst images are shown. (B) Wild-type or Δnap1 cells were transformed with pRS425GPD or pRS425GPD-NAP1. mRNA localization was determined by poly(A) FISH using cells fixed at 30°C. The coincident Hoechst images are shown. (C) mRNA was localized by poly(A) FISH as described above, using strains of the indicated genotypes, after incubation at the indicated temperatures.

nap1 mutants are sensitive to transcriptional stress and DNA damage. Strains of the indicated genotype were equalized and spotted at 10-fold serial dilutions and grown (A) on plates lacking uracil (−Ura) with or without 100 μg/ml 6-azauracil (6-AU) and incubated for 3 days at 30°C, (B) as described for panel A except the plates were incubated for 4 days at 25°C, and (C) on CSM plates with or without 1 μM 4-nitroquinoline 1-oxide (4-NQO) for 4 days at 25°C.

The histone chaperone Nap1 regulates PHO5 transcription by mediating nucleosome mobilization. (A) Nap1 histone chaperone activity in the presence of Yra1 was tested by a plasmid supercoiling assay using recombinant Nap1 (500 nM) and GST or GST-Yra1 (500 nM). Topo I, topoisomerase I; C.E., chicken erythrocyte. (B) Yra1 and Nap1 occupancy at the PHO5 promoter, ORF, and a telomeric region was determined by ChIP assay with real-time PCR using the indicated primer pairs. Chromatin was prepared from a Δyra1Δnap1 strain expressing Yra1-FLAG and HA-Nap1 exogenously. Cells were grown in CSM-histidine at 30°C to an OD₆₀₀ of ≈0.8 and were transferred to synthetic medium lacking phosphate for 4 h prior to fixation. Yra1 (C) or Nap1 (D) occupancy was determined as before. +phosphate, CSM-histidine with phosphate; −phosphate, CSM-histidine lacking phosphate; Oligo, oligonucleotide. (E) RNAP II (Pol II), Yra1, and Nap1 recruitment to the GAL1 UAS was tested by ChIP assays using real-time PCR performed with the cells used for panels C and D. Cells were grown to log phase in 2% raffinose or 2% raffinose followed by 2 h in 2% galactose. Data are expressed as changes (n-fold) in occupancy following galactose induction. Means and standard deviations from two independent experiments are shown. (F) RNA was isolated from wild-type and Δnap1 cells, and the expression of PHO5 transcript relative to ACT1 transcript was determined by reverse transcription real-time PCR. (G) Acid phosphatase activities of wild-type (BY4741), Δnap1, and Δgcn5 cells were measured following phosphate deprivation. Statistically significant differences (P < 0.05) relative to wild-type activity are indicated by an asterisk. H2B occupancy at the PHO5 promoter (H) and within the ORF (I) was determined by ChIP assay with real-time PCR. Chromatin was isolated from FLAG-H2B and FLAG-H2BΔnap1 cells prior to and during phosphate starvation (CSM, no phosphate) and 5 and 10 min after phosphate reintroduction (5′ CSM, 10′ CSM). Occupancy is expressed as the log₂ difference (n-fold) between specific IP signal (UAS or ORF) and background normalized to input and TEL06R and displayed as the average and standard deviation from two independent experiments.