Histone Acetylation Regulates the Time of Replication Origin Firing Maria Vogelauer, Liudmilla Rubbi, Isabelle Lucas, Bonita J Brewer, Michael Grunstein  Molecular Cell  Volume 10, Issue 5, Pages 1223-1233 (November 2002) DOI: 10.1016/S1097-2765(02)00702-5

Figure 1 Global Histone Deacetylation by Rpd3p around Replication Origins Chr-IP using an antibody against acetylated K12 of histone H4 was performed on asynchronously growing cells. Recovery of specific genomic regions surrounding origins was determined by semiquantitative PCR. The graphs show the relative intensity of the individual fragments after normalization to a telomere-specific band and the input DNA. Open squares represent the K12 acetylation levels in the wt strain; filled squares show the same for the rpd3Δ strain. The map under each graph shows the location of genes in the analyzed chromosomal region. The bar graph at the bottom shows the mean increase of histone acetylation in the rpd3Δ mutant in a 3 kb region surrounding each origin. Molecular Cell 2002 10, 1223-1233DOI: (10.1016/S1097-2765(02)00702-5)

Figure 2 Effect of RPD3 Deletion on Cell Cycle Progression Wild-type and rpd3Δ strains were synchronized by α factor. After the release from α factor arrest, samples were taken at indicated times and analyzed by flow cytometry. Molecular Cell 2002 10, 1223-1233DOI: (10.1016/S1097-2765(02)00702-5)

Figure 3 Time of Origin Firing in Wild-Type and rpd3Δ Strains Determined by 2D Gel Electrophoresis Wild-type and rpd3Δ cells were arrested with α factor, and samples were taken at different times after the removal of α factor. Two dimensional gel analysis (A) was performed to examine the time of origin activation of the early origins ARS306 (B) and ARS1 (C), the internal late origin ARS603 (D), and the subtelomeric late origin ARS609 (E). Molecular Cell 2002 10, 1223-1233DOI: (10.1016/S1097-2765(02)00702-5)

Figure 4 Replication Times of Eight Origins in Wild-Type and rpd3Δ Strains Determined by Density Transfer Experiment (A) The replication times (Trep) are minutes after release from the α factor block. Time of budding (Tbud) is also indicated as a reference. The values correspond to the mean Trep and mean Tbud of three independent experiments. (B) For each locus tested, the mean Trep is plotted with its standard deviation. The values for the wt experiments are plotted in gray. The black bars show the results for the rpd3Δ experiments. (C) The mean values of increased histone acetylation in the rpd3Δ strain (see also bar graph in Figure 1) are plotted against the mean advancement of replication time (ΔTrep) in this mutant. The curve is not based on a particular theoretical expectation, but is based on the observation that after a certain increase of acetylation, no further increase of the advancement of the firing is observed. Molecular Cell 2002 10, 1223-1233DOI: (10.1016/S1097-2765(02)00702-5)

Figure 5 Cdc45p Binding to Origins in Wild-Type and rpd3Δ Strains Wild-type and rpd3Δ cells were released from α factor arrest at 16°C, and samples for FACS analyses and Chr-IP were taken at the indicated times. (A) FACS analysis shows the progression through the cell cycle of wt and rpd3Δ strains. (B) Chr-IP of Cdc45-3FLAGp was performed with α-FLAG antibodies and analyzed by PCR using primer pairs specific for the indicated ARS elements. (C) Graphical representation of Cdc45p Chr-IP showing the relative intensity of ARS-specific fragments after normalization to the telomeric loading control fragment (Tel) and the input DNA. Molecular Cell 2002 10, 1223-1233DOI: (10.1016/S1097-2765(02)00702-5)

Figure 6 Targeting of Histone Acetylation to the Late Firing Origin, ARS1412 (A) Chr-IP using an antibody against acetylated K18 of histone H3 was performed for strains MMY021 (wt ARS1412; lanes 1–4), MMY023 (ARS1412-Ac; lanes 5–8), and MMY024 (ARS1412-Ac, Δgcn5; lanes 9–12). Samples were analyzed by PCR, using primer pairs recognizing regions located at −930 bp (lanes 1, 5, and 9), −510 bp (lanes 2, 6, and 10), +70 bp (lanes 3, 7, and 11), and +630 bp (lanes 4, 8, and 12) from the ACS. Gcn4-BS refers to the presence of Gcn4p binding sites, and Gcn5p refers to the GCN5 and gcn5Δ strains. (B) Level of K18 acetylation relative to the telomere fragment (Tel) and normalized to the input DNA. (C) 2D gel analysis of MMY021 (ARS1412), MMY023 (ARS1412-Ac), MMY022 (ARS1412, gcn5Δ) at indicated times, and MMY024 (ARS1412-Ac, gcn5Δ) after release from α factor arrest at 30°C. Molecular Cell 2002 10, 1223-1233DOI: (10.1016/S1097-2765(02)00702-5)

Figure 7 Cdc45p Binding to ARS1412 and ARS1412-Ac FACS analysis (A) and Chr-IPs (B) were performed on MMY033 (wt ARS1412) and MMY034 (ARS1412-Ac) after release from α factor arrest at 16°C. Chr-IP of Cdc45-3FLAGp was performed with α-FLAG antibodies and analyzed by PCR using primer pairs specific for the indicated ARS elements. The graphs show the level of Cdc45p binding relative to a telomeric loading control and the input DNA. Open squares represent Cdc45p binding in MMY033 containing the wt ARS1412; filled squares show the result for MMY034, containing ARS1412-Ac. Molecular Cell 2002 10, 1223-1233DOI: (10.1016/S1097-2765(02)00702-5)