Analyses of rRNA gene chromatin in cell cycle arrested Saccharomyces cerevisiae cells

The existence of two chromatin structures in the rDNA locus was previously demonstrated for a large variety of organisms, ranging from yeast to human. In yeast there are about 150–200 rRNA genes organized in tandem repeats. Almost half of them are transcribed and largely depleted of nucleosomes (active/open), the other half is not transcribed and is assembled in regular arrays of nucleosomes (inactive/closed). It is proposed that RNA polymerase-I (RNAPI) transcription-elongation removes nucleosomes from closed rRNA genes (opening), and that soon after DNA replication there is deposition of nucleosomes on the open rRNA genes (closing). In G1 arrested cells, nearly all rRNA genes are depleted of nucleosomes, but most of them are not transcribed (inactive/open). In relation to the research article by Charton et al. (Mutat. Res.), the data presented here are on the hydroxyurea concentration-dependent inhibition of yeast culture growth, on cell cycle arrest before completion of genome replication, and on the opening of rRNA gene chromatin. As comparison, data are presented for yeast arrested in the G1-phase of the cell cycle by the pheromone α-factor.


Data
Hydroxyurea (HU) arrests cell division by inhibiting DNA replication [1,2]. The related research article describes that 200 mM HU arrests yeast (Saccharomyces cerevisiae) in the G1/early S-phase, and that the arrested cells have most rRNA genes in the open (non-nucleosomal) conformation [3]. Those results support a model proposing that there is deposition of nucleosomes on rRNA genes (closing) only after the passage of the replication fork through the rDNA locus [3 and references therein]. The data presented here describe the outcomes of treatments using lower concentrations of HU; 50 and 100 mM. Data are shown for the inhibition of culture growth, for cell cycle arrest and for the partition of rRNA genes between the two forms of chromatin. As comparison, data are also presented for yeast that are arrested in the G1-phase by the pheromone a-factor (a-F) [4].
Yeast cultures were mock treated or incubated with three different concentrations of HU. Cell growth was monitored over a period of 24 h by automated recording of optical densities [5,6] (Fig. 1A). Measurements show that 50 and 100 mM HU significantly impair, but do not fully inhibit, culture growth. In contrast, even after 24 h, no significant increase in the optical density is observed for yeast culture that are incubated with 200 mM HU. Similarly, yeast cultures that were incubated in the presence of 50 ng/ml of the pheromone a-F do not grow for up to 24 h (Fig. 1B).
Cell cycle arrest of yeast was monitored by flow cytometry (FACS). After 1e2 h incubation in the presence of 200 or 100 mM HU, most cells are synchronized at the G1/early S-phase ( Fig. 2A; compare grey and black curves). Similar cell synchronization is observed during the first 2 h in the presence of 50 mM HU ( Fig. 2B; compare grey and black curves). However, after 4 h incubation about 40% of the cell population appears to have duplicated at least part of the genomic DNA ( Fig. 2B; stripped area). As comparison, yeast cells were synchronized by adding a-F to the media (Fig. 2C).

Subject area
Biology More specific subject area Genetics, genomics, molecular biology (General) Type of data Graphs, figures How data was acquired Yeast treatments with HU or a-factor, cellular and biochemical analyses were done using standard techniques.

Data format
Raw data and analyzed data Experimental factors Saccharomyces cerevisiae strains JS311, JS306-A190MNbar1D.

Experimental features
Hydroxyurea and a-factor added to yeast cultures. Yeast growth curves obtained with a PowerWave Microplate scanning spectrophotometer. Cell cycle arrest followed by flow cytometry with Cytoflex B3-R1-V0. Psoralen photo-crosslinking of isolated nuclei from JS311 and JS306-A190MNbar1D. Southern blots of total DNA isolated from JS311 and JS306-A190MNbar1D strains, and digested with restriction enzymes.

Value of the data
Yeast is an appropriate model organism to investigate the biochemistry of cell responses to genotoxic and cytotoxic compounds, like the chemotherapeutic agent hydroxyurea (HU). The data presented here on the effects of increasing doses of HU on yeast cultures will be useful to studies on the HU cellular responses.
There are nucleosomes on non-transcribed rRNA genes whereas there are few, if any, nucleosomes on transcribed rRNA genes. In both, G1 arrested cells by a-factor and early-S arrested cells by HU most rRNA genes are largely depleted of nucleosomes, despite not being transcribed. These data could help identifying the mechanisms that shape the chromatin of rRNA genes. Because opening of rRNA gene chromatin and RNAPI transcription are coupled, the biochemical assays reported here could be applied to investigate the regulation of rRNA gene expression in the context of chromatin. Psoralen photo-crosslinking is employed to analyze the proportion of closed to open rRNA genes [7]. In the following set of experiments the psoralen assay was applied to track the segregation of the two structures of rRNA genes in HU and a-F treated cells, or in control cells (No a-F) (Fig. 3). Yeast incubated with 100 mM HU for up to 8 h show a gradual shift in the proportion of the two forms of chromatin,    3. Psoralen photo-crosslinking of rRNA gene chromatin. Nuclei suspensions prepared from cells that were exposed to 50, 100 and 200 mM HU, to 50 ng/ml a-F or that were untreated (No a-F), were photo-crosslinked with psoralen. After DNA isolation and EcoRI digestion, the fragmented DNA was separated by native agarose gel electrophoresis. (A) Map of the coding region for the 18S, 5.8S and 25S mature rRNAs; IS corresponds to the intergenic spacer between rRNA genes that is largely covered with nucleosomes [8,9]. The arrow points to initiation and direction of transcription, E 1 to E 4 show the approximate positions of the 4 EcoRI restriction sites; 'a', 'd' and 'g' represent the positions of end-labeled oligonucleotides that were used as probes (their sequences are specified in Tab. S2 of the related research article [3]). (B) Representative Southern blot illustrating the rRNA gene chromatin in 100 mM HU treated cells. After addition of HU, yeast cultures were incubated for 0e8 h. The respective nuclei suspensions were crosslinked with psoralen (þ). As control, nuclei from the 0 and 8 h samples were not photo-crosslinked (À). After blotting, the filter membranes were hybridized with probe 'g'.  Fig. 3B; 25S). Similar data were found for yeast cultured in media containing 200 mM HU [3]. Opening of rRNA gene chromatin also takes place during the first 2 h in yeast treated with 50 mM HU, namely; 54% and 58% of the rRNA genes are open after 1 and 2 h incubation, respectively (Fig. 3C, 50 mM HU). However, in yeast treated with 50 mM HU closing of rRNA genes starts at 4 h incubation (50% closed and 50% open). As control, yeast were treated with 200 mM HU for 4 h, because at this treating condition yeast have a large proportion of open rRNA genes [3] (~85%: Fig. 3C, 200 mM HU; 25S). Noteworthy, the intergenic spacer is covered with nucleosomes since it is largely not transcribed (Fig. 3A, IS) and, consequently, its structure does not change when cells are incubated with 50 or 200 mM HU ( Fig. 3C; IS, c). For comparison, changes in the proportion of the two forms of rRNA gene chromatin were tracked in cells synchronized by a-F. Like for yeast arrested in 200 mM HU for over 2 h [3], a-F treated yeast have most rRNA genes in the open form (63e73%: Fig. 3D; 25S), as previously described [8].

HU treatments, and monitoring of yeast culture growths by automatic measurements of optical densities
The JS311 and JS306-A190MNbar1D strains [3,10] were cultured in yeast extract-peptonedextrose supplemented with adenine (Y A PD). Each well of a 96-well microplate (non-coated polystyrene) was prepared with 95 ml of Y A PD with 0, 50, 100, 200 mM HU or 50 ng/ml a-F. Subsequently, 5 ml of yeast cultures (~8.10 6 cells/ml) were added to each well and cell growth was monitored with a PowerWave microplate scanning spectrophotometer (Bio-Tek). The optical densities were automatically recorded using KC4 microplate data analysis software (Bio-Tek) [5,6,11], with readings taken every 10 min for 24 h.