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. 2010 Feb 5;6(2):e1000837.
doi: 10.1371/journal.pgen.1000837.

Replication and active demethylation represent partially overlapping mechanisms for erasure of H3K4me3 in budding yeast

Marta Radman-Livaja  1 , Chih Long Liu, Nir Friedman, Stuart L Schreiber, Oliver J Rando
Affiliations

"VSports app下载" Replication and active demethylation represent partially overlapping mechanisms for erasure of H3K4me3 in budding yeast

Marta Radman-Livaja et al. PLoS Genet. .

"VSports手机版" Abstract

Histone modifications affect DNA-templated processes ranging from transcription to genomic replication. In this study, we examine the cell cycle dynamics of the trimethylated form of histone H3 lysine 4 (H3K4me3), a mark of active chromatin that is viewed as "long-lived" and that is involved in memory during cell state inheritance in metazoans. We synchronized yeast using two different protocols, then followed H3K4me3 patterns as yeast passed through subsequent cell cycles VSports手机版. While most H3K4me3 patterns were conserved from one generation to the next, we found that methylation patterns induced by alpha factor or high temperature were erased within one cell cycle, during S phase. Early-replicating regions were erased before late-replicating regions, implicating replication in H3K4me3 loss. However, nearly complete H3K4me3 erasure occurred at the majority of loci even when replication was prevented, suggesting that most erasure results from an active process. Indeed, deletion of the demethylase Jhd2 slowed erasure at most loci. Together, these results indicate overlapping roles for passive dilution and active enzymatic demethylation in erasing ancestral histone methylation states in yeast. .

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Cell cycle variation in H3K4me3 patterns.
(A) Nucleosome occupancy (left) and H3K4me3 levels (right) were measured in yeast undergoing synchronous release from alpha factor arrest. Each row represents one of 2288 nucleosomes, and nucleosomes are ordered by previously measured replication time . Note that replication timing and our cell cycle are different experiments and thus the two time scales should not be considered identical. (B) Average nucleosome occupancy for four 10% bins of nucleosomes over the course of CCA. Early-replicating nucleosomes exhibit increased occupancy during S phase, while late-replicating nucleosomes show the converse pattern. (C) Average H3K4me3 levels as in (B). K4 methylation levels show an inverse pattern to occupancy profiles, consistent with newly-incorporated nucleosomes lacking H3K4me3.
Figure 2
Figure 2. Clustering of CCTS and CCA H3K4me3 patterns.
Microarray measurements of H3K4me3 for each nucleosome was centered to zero for each time course to emphasize relative variation in methylation levels, and concatenated data for both time courses was subject to k-means clustering with k = 6.
Figure 3
Figure 3. Nucleosomes methylated during cell cycle arrest lose H3K4me3 during the first S phase after release.
(A) Examples of genomic loci that have high early H3K4me3 that is largely lost during S phase in only one of the two cell cycles. FUS1 is an alpha-inducible gene, GRE1 is a stress response gene. (B) Nucleosomes that match Cluster 6 (r≥0.5) from Figure 2 in either of the two cell cycles (858 total), sorted according to the cell cycle where they best match the Cluster 6 profile. (C) Average heat shock induction levels (blue) or alpha factor induction levels (orange) for the three bins of nucleosomes as shown in (B). (D) Average H3K4me3 patterns over the cell cycle for nucleosomes matching Cluster 6 for CCTS (left) and CCA (right).
Figure 4
Figure 4. Kinetics of H3K4me3 loss during CCA.
(A) Cluster 6 nucleosomes are more methylated during alpha arrest than during midlog growth. Scatterplot of H3K4me3 levels per nucleosome during midlog growth (x axis) against levels during alpha arrest (y axis). Blue dots indicate the 473 Cluster 6 nucleosomes, grey dots indicate remaining nucleosomes. (B) Cluster 6 nucleosomes, midlog levels (x axis) versus levels during alpha arrest (y axis, blue dots), or two generations later (red dots). (C) Histogram of differences between midlog H3K4me3 levels and H3K4me3 levels at varying times during the CCA time course, for Cluster 6 nucleosomes. Most methylation has returned to midlog levels within one generation. (D) As in (C), for 1815 bulk (non-Cluster 6) nucleosomes.
Figure 5
Figure 5. Replication-related loss of H3K4me3.
Data for the 473 Cluster 6 nucleosomes from CCA, sorted by replication time from early (top) to late (bottom).
Figure 6
Figure 6. Replication is not required for loss of H3K4 methylation.
(A) Schematic of experiment. cdc7ts yeast were arrested for four hours with alpha factor, then were released from alpha arrest to grow either at the cdc7 permissive (24°C) or restrictive (37°C) temperature. H3K4me3 mapping was carried out on custom (B) and whole-genome (C) tiling microarrays. (B) FUS1 methylation loss does not require genomic replication. Data from two time independent courses of alpha factor release are shown for 29 nucleosomes surrounding the FUS1 gene. Time after release is shown in minutes above each time course. (C) Loci that lose methylation at the permissive temperature generally also lose methylation at the restrictive temperature. Probes with a methylation loss of over 40% from alpha factor arrest to 90 minutes release at the permissive temperature were selected. The extent of methylation change is histogrammed for permissive and restrictive temperature release (positive values indicate demethylation from arrest to the release time point)—x axis values are log(2). Probes that lose methylation at the permissive temperature generally also lose methylation at the restrictive temperature, albeit to a slightly lesser extent. We note that much of the variance at the permissive temperature can be ascribed to the heat shock of the restrictive temperature – most of the probes at the left of the histogram (ie not demethylated, or even overmethylated, at 37°C) are heat shock-induced genes, while many of the probes that lose methylation more at 37°C than at 24°C are associated with genes repressed during heat shock (analysis not shown). (D) Schematic of alternative system for replication-independent erasure. bar1Δ yeast were grown continuously in galactose, then arrested with alpha factor. Galactose-regulated genes were then shut off by shifting cells to dextrose, and were concomitantly either released from alpha factor arrest or maintained in alpha factor. (E) Methylation erasure over GAL genes does not require replication. Data from whole-genome tiling arrays shows high levels of H3K4me3 at the 5′ ends of GAL1, GAL7 and GAL10 when cells are arrested in alpha factor with galactose. Shift to dextrose results in loss of methylation whether cells are maintained in alpha factor or allowed to re-enter the cell cycle. (F) Loci that lose methylation during cell cycle release into dextrose generally also lose methylation when maintained in alpha factor. Analysis is similar to that shown in (C), but probes exhibiting higher methylation in alpha factor+galactose than in midlog+galactose (log2>0.75 difference) were eliminated to exclude confounding effects of erasure of these genes during release from alpha factor arrest. Furthermore, only probes with methylation decreases of 75% or more after 75 min of release into dextrose are shown.
Figure 7
Figure 7. Delayed loss of H3K4me3 in jhd2Δ yeast.
(A) H3K4me3 data are shown for a sample set of genomic loci during a short time course following alpha factor release. Data from wild type yeast are shown on the left, jhd2Δ on the right. (B) Global delay in H3K4me3 loss in jhd2Δ. Averaged data for early-replicating Cluster 6 nucleosomes from CCA are shown for wild-type and jhd2Δ yeast. (C,D) Inefficient H3K4me3 loss in jhd2Δ yeast. Whole-genome tiling microarrays were used to assay H3K4me3 levels during alpha arrest and after 60 minute release. 5′ coding probes (C) and 3′ coding probes (D) were ordered by the extent to which they are methylated in alpha factor arrest relative to midlog growth (bottom colorbar is used in place of x axis labels since axis is nonlinear. Red = hypermethylated in alpha factor, green = hypomethylated). Extent of methylation erasure after 60 minutes of release was calculated (erasure is positive), and a 50-probe running window average in presented. At both genic regions wild-type yeast erased H3K4me3 more efficiently than did jhd2Δ yeast, although in both cases methylation levels shifted away from the alpha arrest state back towards midlog levels (ie probes hypermethylated in alpha factor were demethylated upon release, hypomethylated probes were remethylated). Interestingly, there was a global baseline methylation loss at the 3′ ends of genes and a global methylation gain at 5′ ends—this was the result of a global shift in methylation patterns during alpha factor arrest—see Figure S12.
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