Repair-independent chromatin assembly onto active ribosomal genes in yeast after UV irradiation

Abstract

Chromatin rearrangements occur during repair of cyclobutane pyrimidine dimers (CPDs) by nucleotide excision repair (NER). Thereafter, the original structure must be restored to retain normal genomic functions. How NER proceeds through nonnucleosomal chromatin and how open chromatin is reestablished after repair are unknown. We analyzed NER in ribosomal genes (rDNA), which are present in multiple copies but only a fraction are actively transcribed and nonnucleosomal. We show that removal of CPDs is fast in the active rDNA and that chromatin reorganization occurs during NER. Furthermore, chromatin assembles on nonnucleosomal rDNA during the early events of NER but in the absence of DNA repair. The resumption of transcription after removal of CPDs correlates with the reappearance of nonnucleosomal chromatin. To date, only the passage of replication machinery was thought to package ribosomal genes in nucleosomes. In this report, we show that early events after formation of UV photoproducts in DNA also promote chromatin assembly.

FIG. 1.

(A) Experimental design. (B) Map of the yeast 35S rRNA gene. The rRNA gene, 5′ and 3′ ends, and direction of transcription (wavy arrow) are shown. The short black box represents the probe (∼140 bp) used in this work. The E₁ to E₇ positions mark the seven EcoRI restriction sites, N₁ and N₂ indicate the positions of the two NheI restriction sites, and P indicates the position of the PvuII sites.

FIG. 2.

Separation of active and inactive ribosomal gene chromatin. (A) Nuclei were isolated from nonirradiated (lane 5) and irradiated (lanes 6 to 11) cells that were harvested after different repair times. These nuclei were digested with EcoRI before psoralen cross-linking (lanes 5 to 11). The isolated DNA was then digested with NheI, separated on a 1% native agarose gel, blotted, and hybridized with a random primer-labeled probe (Fig. 1B). As controls, genomic DNA was isolated from non-cross-linked cells and digested with either EcoRI (lanes 1 and 15) or NheI (lanes 3 and 13). The presence of active and inactive rDNA chromatin was monitored by digesting nuclei with EcoRI or NheI before psoralen cross-linking and by redigesting the isolated DNA with EcoRI and NheI, respectively (lanes 2 and 14 and lanes 4 and 12). Labels indicate active rDNA (filled circle) and inactive rDNA (open circle). The major bands N₁-N₂ (NheI; 4,417 bp) and E₄-E₅ (EcoRI; 2,846 bp) are indicated, together with the products of partial digestions: E₃-N₂ (4,196 bp), N₁-E₆ (4,098 bp), E₃-E₆ (3,877 bp), N₁-E₅ (3,731 bp), E₄-N₂ (3,532 bp), E₃-E₅ (3,510 bp), and E₄-E₆ (3,213 bp). (B) Quantification of band intensities of active and inactive rDNA chromatin. Intensities for the N₁-N₂ (NheI) bands were divided by intensities for the E₄-E₅ (EcoRI) bands. Data show the average of two independent experiments. (Note that since the .25-h time point measurement was taken only once, it was not included.)

FIG. 3.

(A) Repair of NTS of active (EcoRI) and inactive (NheI) rDNA in wt cells. Yeast cells were irradiated with 180 J/m² UV and harvested at the times indicated. DNA was isolated from EcoRI-treated nuclei and digested with NheI. DNA samples were mock treated (−) or treated with T4 endo V (+); −UV indicates nuclei from nonirradiated cells, and 0 to 4 denotes nuclei from irradiated cells harvested after the indicated repair times (in hours). Samples were separated on a 1% alkaline agarose gel, blotted, and hybridized with strand-specific riboprobes (Fig. 1B). Dots point to the partial digestion products described in the legend of Fig. 2. (B) Repair of TS of active (EcoRI) and inactive (NheI) rDNA in wt cells. Experimental procedure and figure legends are as described for panel A. Dots point to the partial digestion products described in the legend of Fig. 2. (C) Repair of both strands of total rDNA in rad1Δ and rad14Δ mutant cells. Following different repair times, total DNA was isolated from rad1Δ and rad14Δ cells and digested with EcoRI. After separation on alkaline agarose gels and blotting as described for panel A, filter membranes were hybridized with random primer-labeled probe (Fig. 1B). Figure legends are as described for panel A. (D) Quantification of phosphorimages. DNA repair is expressed as the percentage of CPDs removed as a function of repair time. For wt cells data are from active rDNA (EcoRI, circles; wt-a) and inactive rDNA (NheI, triangles; wt-i). Solid and open symbols represent data from the TS and NTS, respectively. Data are the means ± 1 SD of three independent experiments. For both rad1Δ and rad14Δ cells, data are from total rDNA (diamonds) and represent the mean of two independent experiments each.

FIG. 4.

(A) Chromatin structure of ribosomal genes during NER in wt cells. Nuclei were isolated from nonirradiated (lane 2) or irradiated (lanes 3 to 7) cells, before (lane 3) and during NER (lanes 4 to 7). After cross-linking with psoralen, DNA was extracted from nuclei, digested with EcoRI, and separated on 1% native agarose gels. As a control (C), DNA was isolated from non-cross-linked nuclei and digested with EcoRI (lanes 1 and 8). After blotting, the filter membranes were hybridized with the random primer-labeled probe (Fig. 1B). Labels at right denote active rDNA (filled circle) and inactive rDNA (open circle). (B) Chromatin structure of ribosomal genes during NER in rad1Δ and rad14Δ strains. Experimental procedures and lanes are as described for panel A. (C) Scan profiles of gels shown in panels A and B.

FIG. 5.

Transcription of ribosomal genes during NER in wt, rad1Δ, and rad14Δ mutant strains. Nuclei were isolated from nonirradiated (−UV) cells and from UV-irradiated cultures at different repair times. After incubation under TRO reaction conditions, the purified radiolabeled RNA was used as a probe to hybridize the membrane-bound rDNA. Membranes were then exposed to phosphorimager screens, and the rDNA signals were normalized to the corresponding signals for the external standard (13). Transcription of the nonirradiated cultures is given an arbitrary value of 1, and transcription of other samples is expressed relative to this value. Black bars, wt strain; gray bars, rad1Δ strain; hatched bars, rad14Δ strain. Data are the means ± 1 SD of three independent experiments for wt and the average of two independent experiments for rad1Δ and rad14Δ cells.

FIG. 6.

Transcription and chromatin structure of ribosomal genes during cell growth. (A) Cells were grown in YEPD medium and samples for nuclei isolation were harvested during exponential (E) growth (optical density at 600 nm [OD₆₀₀] of 0.4 × 10⁷ to ∼1.2 × 10⁷ cells/ml), at early stationary phase (ES; 6 h after exponential phase) and at stationary phase (S; 24 h after exponential phase). (B) Chromatin structure of ribosomal genes during cell growth phases. At top, nuclei isolated from cells collected at selected growth stages, as described for panel A, were cross-linked with psoralen. DNA was extracted from nuclei, digested with EcoRI, and separated on 1% native agarose gels (lanes E, ES, and S). As a control (C), DNA was isolated from non-cross-linked nuclei and digested with EcoRI. After blotting, the filter membranes were hybridized with the random primer-labeled probe (Fig. 1B). Labels at left denote active rDNA (filled circle) and inactive rDNA (open circle). Below the blot are three scan profiles of the gels. (C) Transcription of ribosomal genes at different cell growth phases. Nuclei isolated from cells collected at selected growth stages as described for panel A were used for TRO reactions. The purified radiolabeled RNA was employed as a probe to hybridize the membrane-bound rDNA. Membranes were then exposed to phosphorimager screens, and the rDNA signals were normalized to the corresponding signals for the external standard. Transcription of the exponential phase culture is given an arbitrary value of 1, and transcription of the other samples is expressed relative to this value. Data are from the average of three independent experiments.

FIG. 7.

EcoRI accessibility to rDNA chromatin during NER and during cell growth. (A) Chromatin structure of ribosomal genes during NER in wt cells (top gel). Nuclei were isolated from nonirradiated (−UV; lane 2) or irradiated (lanes 3 to 7) cells, before (lane 3) and during (lanes 4 to 7) NER. After EcoRI digestion, DNA was extracted from nuclei and separated on 1% native agarose gels. As a control (C), DNA was isolated from nuclei and digested with EcoRI (lane 8). After being blotted, the filter membranes were hybridized with the random primer-labeled probe (Fig. 1B) to obtain the major band E₄-E₅ (EcoRI; 2,846 bp). The bands E₃-E₇ (4,468 bp), E₃-E₆ (3,877 bp), E₄-E₇ (3,804 bp), E₃-E₅ (3,510 bp) and E₄-E₆ (3,213 bp) are products of partial digestions (Fig. 1B). DNA size-markers (lane M): 5,090, 4,072, 3,054 and 2,036 bp. To correct for gel loading, the amount of total rDNA present in each sample was quantified by spotting aliquots of DNA in triplicate (bottom gel). The membranes were hybridized with the probe shown in Fig. 1B and exposed to phosphorimager screens. The average of each triplicate was used to correct for loading. (B) Chromatin structure of ribosomal genes during NER in rad1Δ strains. Experimental procedure and lanes are as described for panel A. (C) For wt cells, signals for the E₄-E₅ (EcoRI) bands were quantified, and, after corrections for DNA loading, the rDNA signals were normalized to the −UV samples (given an arbitrary value of 1). The EcoRI sensitivity of the other samples is expressed relative to this value (data are the means ± 1 SD of three experiments). Similar analyses were performed with gels from rad1Δ and rad14 Δ cells. Data show the average of two independent experiments each. (D) Chromatin structure of ribosomal genes during cell growth. Nuclei isolated from cells collected at selected growth stages (Fig. 6A) were digested with EcoRI. DNA was extracted and separated on 1% native agarose gels. As a control (C), DNA was isolated from nuclei and digested with EcoRI. After being blotted, membranes were hybridized with the random primer-labeled probe (Fig. 1B). Lanes are as described for Fig. 6A, and quantification of the gel (right panel) was performed as described for panel C. Data are the means ± 1 SD of three independent experiments.

FIG. 8.

MNase accessibility of rDNA and GAL chromatin during NER in wt cells. (A) Map of the GAl10-GAL1 locus. Thick arrows represent the GAL10 and GAL1 genes and directions of transcription. The solid bar denotes the location of the probe used in this work. The positions of the two EcoRI restriction sites (E) and the two PvuII restriction sites (P) are also shown. (B) MNase sensitivity of the GAL10-GAL1 region and ribosomal genes during NER in wt cells. Nuclei were isolated from nonirradiated (−UV, lane 1) or irradiated (lanes 2 to 6) cells, before (lane 2) and during (lanes 3 to 6) NER. After MNase treatment, DNA was extracted from nuclei, digested with EcoRI and PvuII, and separated on 1% native agarose gels. After being blotted, membranes were first hybridized with a random primer-labeled probe specific for the GAL locus (8A) to obtain the E-P band (∼1.66 kb). Membranes were rehybridized with probe specific for rDNA to obtain the P-E₅ band (∼1.96 kb) shown in Fig. 1B. (C) Quantification of intensities of rDNA and GAL bands. Histogram shows the ratio of signals for the rDNA and GAL bands at different repair times. The rDNA/GAL ratios during NER were normalized to the −UV samples (given an arbitrary value of 1). Data are the average of two independent experiments.