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H3 k36 methylation helps determine the timing of cdc45 association with replication origins.

Pryde F, Jain D, Kerr A, Curley R, Mariotti FR, Vogelauer M - PLoS ONE (2009)

Bottom Line: Such timing is determined by the chromosomal context, which includes the activity of nearby genes, telomeric position effects and chromatin structure, such as the acetylation state of the surrounding chromatin.Furthermore, a decrease in H3 K36me3 and a concomitant increase in H3 K36me1 around the time of Cdc45 binding to replication origins suggests opposing functions for these two methylation states.Indeed, we find K36me3 depleted from early firing origins when compared to late origins genomewide, supporting a delaying effect of this histone modification for the association of replication factors with origins.

View Article: PubMed Central - PubMed

Affiliation: Wellcome Trust Centre for Cell Biology, Institute of Cell Biology, University of Edinburgh, Edinburgh, United Kingdom.

ABSTRACT

Background: Replication origins fire at different times during S-phase. Such timing is determined by the chromosomal context, which includes the activity of nearby genes, telomeric position effects and chromatin structure, such as the acetylation state of the surrounding chromatin. Activation of replication origins involves the conversion of a pre-replicative complex to a replicative complex. A pivotal step during this conversion is the binding of the replication factor Cdc45, which associates with replication origins at approximately their time of activation in a manner partially controlled by histone acetylation.

Methodology/principal findings: Here we identify histone H3 K36 methylation (H3 K36me) by Set2 as a novel regulator of the time of Cdc45 association with replication origins. Deletion of SET2 abolishes all forms of H3 K36 methylation. This causes a delay in Cdc45 binding to origins and renders the dynamics of this interaction insensitive to the state of histone acetylation of the surrounding chromosomal region. Furthermore, a decrease in H3 K36me3 and a concomitant increase in H3 K36me1 around the time of Cdc45 binding to replication origins suggests opposing functions for these two methylation states. Indeed, we find K36me3 depleted from early firing origins when compared to late origins genomewide, supporting a delaying effect of this histone modification for the association of replication factors with origins.

Conclusions/significance: We propose a model in which K36me1 together with histone acetylation advance, while K36me3 and histone deacetylation delay, the time of Cdc45 association with replication origins. The involvement of the transcriptionally induced H3 K36 methylation mark in regulating the timing of Cdc45 binding to replication origins provides a novel means of how gene expression may affect origin dynamics during S-phase.

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Δset2 does not decrease histone acetylation at origins.ChIP of MMY033 (WT), MVY51 (Δrpd3), MVY57 (Δset2) and MVY58 (Δrpd3Δset2) with antibodies specific for pan-acetylated histone H3 and H4 was performed. (A) Representative autoradiographs of PCR products using primer pairs specific for the indicated ARS elements and a telomeric loading control (TEL). (B) Graphic representation: Analysis was by semiquantitative PCR using primer pairs specific for indicated ARS elements and a telomeric loading control. The relative intensity of ARS specific fragments after normalization to the loading control and the input is presented. Errorbars refer to the standard deviation of the results of three independent experiments.
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pone-0005882-g003: Δset2 does not decrease histone acetylation at origins.ChIP of MMY033 (WT), MVY51 (Δrpd3), MVY57 (Δset2) and MVY58 (Δrpd3Δset2) with antibodies specific for pan-acetylated histone H3 and H4 was performed. (A) Representative autoradiographs of PCR products using primer pairs specific for the indicated ARS elements and a telomeric loading control (TEL). (B) Graphic representation: Analysis was by semiquantitative PCR using primer pairs specific for indicated ARS elements and a telomeric loading control. The relative intensity of ARS specific fragments after normalization to the loading control and the input is presented. Errorbars refer to the standard deviation of the results of three independent experiments.

Mentions: To rule out the possibility that the SET2 deletion reduces histone acetylation at origins and therefore indirectly affects the dynamics of Cdc45 binding we analysed the level of histone acetylation at ten origins in WT, Δrpd3, Δset2 and Δrpd3Δset2 strains. Antibodies against acetylated histones H3 or H4 were used for ChIP. Deletion of SET2 alone led to a slight increase in H3 and H4 acetylation at most origins when compared to the WT and was further increased in the absence of RPD3 (Fig. S4). This is expected, as H3 K36me3 is known to recruit the Rpd3S histone deacetylase complex. Two origins (ARS607 and ARS603) showed a 2–3 fold increase in H4 acetylation in Δset2 cells compared to the WT. Importantly, H3 and H4 acetylation levels in the Δrpd3 mutant were not diminished upon deletion of SET2 (Fig. 3 and Fig. S4). Δrpd3Δset2 cells showed a slight overall increase (up to ∼1.5 fold) in histone acetylation when compared to the Δrpd3 strain (Fig. 3B). We conclude that the absence of H3 K36me delays Cdc45 binding to origins even when the surrounding chromatin is hyperacetylated.


H3 k36 methylation helps determine the timing of cdc45 association with replication origins.

Pryde F, Jain D, Kerr A, Curley R, Mariotti FR, Vogelauer M - PLoS ONE (2009)

Δset2 does not decrease histone acetylation at origins.ChIP of MMY033 (WT), MVY51 (Δrpd3), MVY57 (Δset2) and MVY58 (Δrpd3Δset2) with antibodies specific for pan-acetylated histone H3 and H4 was performed. (A) Representative autoradiographs of PCR products using primer pairs specific for the indicated ARS elements and a telomeric loading control (TEL). (B) Graphic representation: Analysis was by semiquantitative PCR using primer pairs specific for indicated ARS elements and a telomeric loading control. The relative intensity of ARS specific fragments after normalization to the loading control and the input is presented. Errorbars refer to the standard deviation of the results of three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2690658&req=5

pone-0005882-g003: Δset2 does not decrease histone acetylation at origins.ChIP of MMY033 (WT), MVY51 (Δrpd3), MVY57 (Δset2) and MVY58 (Δrpd3Δset2) with antibodies specific for pan-acetylated histone H3 and H4 was performed. (A) Representative autoradiographs of PCR products using primer pairs specific for the indicated ARS elements and a telomeric loading control (TEL). (B) Graphic representation: Analysis was by semiquantitative PCR using primer pairs specific for indicated ARS elements and a telomeric loading control. The relative intensity of ARS specific fragments after normalization to the loading control and the input is presented. Errorbars refer to the standard deviation of the results of three independent experiments.
Mentions: To rule out the possibility that the SET2 deletion reduces histone acetylation at origins and therefore indirectly affects the dynamics of Cdc45 binding we analysed the level of histone acetylation at ten origins in WT, Δrpd3, Δset2 and Δrpd3Δset2 strains. Antibodies against acetylated histones H3 or H4 were used for ChIP. Deletion of SET2 alone led to a slight increase in H3 and H4 acetylation at most origins when compared to the WT and was further increased in the absence of RPD3 (Fig. S4). This is expected, as H3 K36me3 is known to recruit the Rpd3S histone deacetylase complex. Two origins (ARS607 and ARS603) showed a 2–3 fold increase in H4 acetylation in Δset2 cells compared to the WT. Importantly, H3 and H4 acetylation levels in the Δrpd3 mutant were not diminished upon deletion of SET2 (Fig. 3 and Fig. S4). Δrpd3Δset2 cells showed a slight overall increase (up to ∼1.5 fold) in histone acetylation when compared to the Δrpd3 strain (Fig. 3B). We conclude that the absence of H3 K36me delays Cdc45 binding to origins even when the surrounding chromatin is hyperacetylated.

Bottom Line: Such timing is determined by the chromosomal context, which includes the activity of nearby genes, telomeric position effects and chromatin structure, such as the acetylation state of the surrounding chromatin.Furthermore, a decrease in H3 K36me3 and a concomitant increase in H3 K36me1 around the time of Cdc45 binding to replication origins suggests opposing functions for these two methylation states.Indeed, we find K36me3 depleted from early firing origins when compared to late origins genomewide, supporting a delaying effect of this histone modification for the association of replication factors with origins.

View Article: PubMed Central - PubMed

Affiliation: Wellcome Trust Centre for Cell Biology, Institute of Cell Biology, University of Edinburgh, Edinburgh, United Kingdom.

ABSTRACT

Background: Replication origins fire at different times during S-phase. Such timing is determined by the chromosomal context, which includes the activity of nearby genes, telomeric position effects and chromatin structure, such as the acetylation state of the surrounding chromatin. Activation of replication origins involves the conversion of a pre-replicative complex to a replicative complex. A pivotal step during this conversion is the binding of the replication factor Cdc45, which associates with replication origins at approximately their time of activation in a manner partially controlled by histone acetylation.

Methodology/principal findings: Here we identify histone H3 K36 methylation (H3 K36me) by Set2 as a novel regulator of the time of Cdc45 association with replication origins. Deletion of SET2 abolishes all forms of H3 K36 methylation. This causes a delay in Cdc45 binding to origins and renders the dynamics of this interaction insensitive to the state of histone acetylation of the surrounding chromosomal region. Furthermore, a decrease in H3 K36me3 and a concomitant increase in H3 K36me1 around the time of Cdc45 binding to replication origins suggests opposing functions for these two methylation states. Indeed, we find K36me3 depleted from early firing origins when compared to late origins genomewide, supporting a delaying effect of this histone modification for the association of replication factors with origins.

Conclusions/significance: We propose a model in which K36me1 together with histone acetylation advance, while K36me3 and histone deacetylation delay, the time of Cdc45 association with replication origins. The involvement of the transcriptionally induced H3 K36 methylation mark in regulating the timing of Cdc45 binding to replication origins provides a novel means of how gene expression may affect origin dynamics during S-phase.

Show MeSH