Histone reassembly during replication (hydroxyurea synchronization) 88

Apparently replication imposes an obstacle for PHO promoter chromatin remodeling. We set out to substantiate this interpretation by testing the according mechanistic prediction that replication resets

a nucleosomal structure at the PHO5 promoter even if it was already partially or fully open. We used yeast strains mutated in pho80 that showed a constitutively induced PHO5 promoter structure and synchronized the cells. We wanted to know what happens with regard to histone occupancy at the promoter after release into the cell cycle. Would we see promoter chromatin resetting during or after replication in S phase? Would the promoter structure change to the closed nucleosomal pattern or would it stay open? Importantly, answers to these questions could tell us how an open promoter struc- ture is inherited or re-established upon passage through S phase.

Logarithmically growing pho80 cells were synchronized with hydroxyurea which arrested the cells in G1/S phase. Hydroxyurea affects DNA synthesis by reducing the production of deoxyribonuc- leotides by inhibiting the ribonucleotide reductase (Alvino et al. 2007). Washing the cells in water and resuspending them into fresh medium released the synchronized population collectively into the cell cycle. Samples were taken every 15 min to monitor synchrony by FACS analysis and histone occu- pancy by H3 ChIP (Fig. 46A). To directly compare any changes at the histone level that occurred dur- ing the passage through the cell cycle we used pho80 and wild type cells, which represented induced and repressed PHO chromatin states, respectively. Both strains showed a characteristic FACS profile of cycling cells with a G1 (1n DNA content) and a G2 (2n DNA content) peak framing the cells of the S phase plateau. pho80 cells were exposed longer to hydroxyurea than wild type cells due to a slower growth rate. As pho80 cells had a longer generation time it took longer till the whole cell population arrived at the cell cycle block. Both showed G1 arrested cells after hydroxyurea arrest of 4 h and 3 h, respectively. Upon release into the cell cycle the cell population represented by the G1 peak in the FACS profiles moved on to S phase. pho80 cells entered S phase after 30 min and wild type cells after 45 min release (Fig. 46B). FACS analysis of subsequent time points showed a prominent G2 peak and soon afterwards the profile of cycling cells (Fig. 46B). Parallel sample preparation for FACS analysis and ChIP ensured good correlation of the observed stages of the cell cycle with the histone occupancy signals. The H3 ChIP corresponding to the described FACS analysis showed a very prominent signal of histone reassembly at the PHO5 and PHO8 promoters at the time point of S phase after 30 min release (Fig. 46C) for pho80 cells. This increase was more than fourfold in the case of the PHO5 pro- moter and more than threefold at the PHO8 promoter compared to the point of release. Time points before and after S phase did not show any increase in histone signal speaking for an otherwise consti- tutively open promoter chromatin structure.

The telomere (TEL) control locus showed no histone reassembly during the analysis and served as control region that is not regulated by the phosphate signaling pathway (Fig. 46).We observed this transient peak of histone H3 during S phase repeatedly. One more example is given in Supp. Fig. 4. A comparable H3 ChIP analysis with wild type cells did not lead to a histone peak in S phase after 45 min (Fig. 46). For unknown reasons this population started with a higher ChIP signal of cycling cells. The larger graphs represent H3 ChIP signals that were normalized to the input DNA, to the control locus actin (ACT1) and to the time point of release.

Fig. 46 Hydroxyurea synchronized pho80 cells showed histone reassembly during replication, in contrast

to wild type cells. (A) Experimental scheme: logarithmically growing cells were arrested with hydroxyurea

(HU) in G1/S phase. Arrest and release into the cell cycle was monitored by FACS and H3 ChIP in 15 min inter- vals. (B) FACS analysis of the DNA content of cycling pho80 and wt cells, cells that were arrested with HU and cells that were released into the cell cycle after HU arrest. (C) Histone H3 ChIP kinetics of histone occupancy of HU synchronized pho80 cells (upper ChIP profile) and wt cells (lower ChIP profile). The H3 ChIP signal was normalized to input DNA, to the control locus ACT1 and the time point of release. The inset shows an alternative analysis plotting the ChIP signal normalized to % input and the time point of release for wt cells. Legend in the graph shows analyzed loci: PHO5 (closed squares), PHO8 (closed circles), TEL (open diamonds).

The inset in Fig. 46C represents an alternative analysis normalizing the ChIP signal only to % in- put and to the time point of release. In the following ChIP experiments we switched to this kind of normalization as it included the individual input DNA of every sample. To have a second negative control in addition to wild type cells we also made use of a pho4 strain. This mutant is not able to in- duce PHO5 as it lacks the transcription factor Pho4. Here we observed a slight increase in the ChIP

signal during S phase, which was apparently due to the normalization to the control locus actin that showed rather low levels of histone occupancy. Anyhow, this slight increase was not significant as the parallel analysis using the normalization to % input did not display a histone peak in S phase (compare larger graph vs. inset Supp. Fig. 4).

In summary, we observed a transient peak of histone H3 occupancy during S phase at the PHO5

and PHO8 promoters in pho80 cells that were synchronized by hydroxyurea. Wild type cells or pho4

cells did not show histone reassembly during any cell cycle stage. This argues for promoter chromatin resetting during replication and against a continuously open promoter. However, a drawback in this system is the use of hydroxyurea as cell cycle arresting drug as it only slows progression through S phase and does not arrest the cells in a defined G1/S block (Santocanale and Diffley 1998). Hydroxyu- rea-induced dNTP depletion affects probably only one type of replication origin. Early firing origins of replication will therefore be activated while the replication forks continue to move very slowly dur- ing hydroxyurea arrest in early S phase and only late replicating origins will be stalled and await reac- tivation upon release (Santocanale and Diffley 1998). Analyzing the proximity of our regions of inter- est to origins of replication showed that both promoters could be part of early replication events (Sac- charomyces Genome Database, http://www.yeastgenome.org/ (25.03.2010), www.oridb.org). A differ- ent group even suggested hydroxyurea had the same effect on all active origins and does not differen- tiate between early and late firing but results in overall progression in slow motion through S phase (Alvino et al. 2007). Therefore we were not sure if the PHO5 and the PHO8 promoters were already replicated during hydroxyurea treatment starting from nearby origins of replication. We decided to verify our preliminary observations of histone reassembly during S phase using a different approach to synchronize the cells.