Establishment of histone modifications after chromatin assembly

Abstract

Every cell has to duplicate its entire genome during S-phase of the cell cycle. After replication, the newly synthesized DNA is rapidly assembled into chromatin. The newly assembled chromatin 'matures' and adopts a variety of different conformations. This differential packaging of DNA plays an important role for the maintenance of gene expression patterns and has to be reliably copied in each cell division. Posttranslational histone modifications are prime candidates for the regulation of the chromatin structure. In order to understand the maintenance of chromatin structures, it is crucial to understand the replication of histone modification patterns. To study the kinetics of histone modifications in vivo, we have pulse-labeled synchronized cells with an isotopically labeled arginine ((15)N(4)) that is 4 Da heavier than the naturally occurring (14)N(4) isoform. As most of the histone synthesis is coupled with replication, the cells were arrested at the G1/S boundary, released into S-phase and simultaneously incubated in the medium containing heavy arginine, thus labeling all newly synthesized proteins. This method allows a comparison of modification patterns on parental versus newly deposited histones. Experiments using various pulse/chase times show that particular modifications have considerably different kinetics until they have acquired a modification pattern indistinguishable from the parental histones.

Figure 1.

pSILAC can be used to distinguish old from newly synthesized histones. (A) Schematic experimental overview. HeLa cells were synchronized using a double thymidine block. Cells were released into S-phase and simultaneously grown in the R⁴ SILAC medium in order to label all newly synthesized proteins. The cells were harvested at indicated time points. (B) DNA content of synchronized HeLa cells after different time points of release into S-phase using FACS analysis. The fluorescence intensity (DNA content) is depicted on the abscissa. (C) mRNA amount of H3.2 for asynchronic and synchronic cells detected by reverse transcriptase PCR. 18S serves as a loading control. M, marker. (D) Labeling efficiency when using the SILAC medium (R⁴). Two peptides H3 aa 64–69 and H4 aa 68–78 are analyzed by MALDI-TOF from 0 h to 16 h after release into S-phase. Error bars indicate the standard error of the mean (SEM) from three independent biological replicates.

Figure 2.

Deacetylation of H4 peptide 4–17 happens fast. (A) Scheme of pulse-chase experiments including 10 mM sodium butyrate treatment. Sodium butyrate (NaBu) was added either at the time of release for 6 h or for a shorter period of time (2 h) during S-phase. (B) FACS analysis of synchronized HeLa cells treated with sodium butyrate harvested at 0, 2, 4, 6 and 8 h after release. (C) Acetylation patterns of H4 peptide 4–17 (GKGGKGLGKGGAKR) of ‘old’ (R⁰) and ‘new’ (R⁴) histones after 6 h after a release into G1/S-phase. Error bars indicate the SEM from three independent biological experiments. 1ac, monoacetylation; 2ac, diacetylation; 3ac, triacetylation. (D) Comparison of acetylation patterns of ‘old’ and ‘new’ histones after 6 h of NaBu treatment. Left: quantification; right: MALDI-TOF spectrum; asterisk indicates peaks of ‘new’ histones. (E) Comparing acetylation patterns of ‘old’ and ‘new’ histones when treating for 2 h with NaBu without additional chase or with an additional 2 h chase (F).

Figure 3.

Differences in methylation patterns of ‘old’ and ‘new’ histones 6 h after release in G1/S-phase. (A) Methylation patterns of H4 peptide 20–23 of ‘old’ and ‘new’ histones analyzed by MALDI-TOF. 1ac, monoacetylation; me1, monomethylation; me2, dimethylation; me3, trimethylation. (B) H3 73–83 (C) H3 27–40 (D) H3 9–17. Error bars indicate the SEM of three independent biological replicates. The P-values are calculated using Student's unpaired t-test. The significance of the differences in methylation levels were assessed by unpaired Student's t-tests and indicated on the top of the bars **P < 0.01: a highly significant difference; *P < 0.05: a significant difference; and no bracket means no significant difference.

Figure 4.

Double labeling using different SILAC media allows to follow histones for a period longer than one cell cycle. (A) Experimental scheme. After synchronization, HeLa cells are released into S-phase under the R⁴-medium conditions. After 6 h, the cells were transferred into a medium containing R¹⁰ and then harvested at indicated time points. time (h); time after release. (B) Incorporation efficiency of double-labeled histones for R⁴ and R ¹⁰ showing H3 peptide aa 64–69. Error bars indicate the SEM of three independent biological replicates. (C) MALDI-TOF spectrum of H3 aa 64–69 from HeLa cells that were R⁴ labeled for 6 h and afterward R¹⁰ labeled for additional 6 h.

Figure 5.

Establishment of posttranslational modification patterns of histones differ in their kinetics. Pulse-chase experiments as described in Figure 4A. Underneath the charts, a cell cycle scheme is depicted. time (h); time after release. Three independent biological experiments are shown and analyzed by MALDI-TOF. Error bars indicate standard deviation of a mean. Grey square indicates 20 h after release (A) top left: H4 peptide aa 20–23 is shown of the ‘old’ histones. Top right: H4 peptide aa 20–23 is shown of the ‘new’ histones. Bottom left: R⁰ (‘old’)-labeled histones showing H3 peptide 27–40. Bottom right: R⁴ (‘new’)-labeled histones showing H3 peptide 27–40. (B) Methylation patterns after 20-h release of H4 peptide 20–23 (left) and H3 peptide 27–40 (right) of ‘old’ and ‘new’ histones analyzed by MALDI-TOF. 1ac, monoacetylation; me1, monomethylation; me2, dimethylation; me3, trimethylation. Error bars indicate the SEM from three independent biological replicates.