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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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EAF3 and NTO1 act together to delay S-phase progression.(A) Strains MMY033 (WT), MVY51 (Δrpd3), MVY54 (Δeaf3) and MVY55 (Δeaf3Δrpd3) were arrested in G1 with α-factor and released into S-phase at 30°C. Samples were taken at indicated times and processed for FACS analysis. (B) as (A) with strains MMY001 (WT), MMY002 (Δrpd3), MMY118 (Δnto1) and MVY119 (Δrpd3Δnto1). (C) as (A) with strains MMY001 (WT), MMY002 (Δrpd3), MVY137 (Δeaf3Δnto1) and MVY138 (Δrpd3Δeaf3Δnto1). Grey bars indicate the estimated length of S-phase. (D) Cell budding was assessed by microscopy. Complete lines indicate WT (black) and Δrpd3 (grey); broken lines indicate Δeaf3Δnto1 (black) and Δrpd3Δeaf3Δnto1 (grey).
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pone-0005882-g006: EAF3 and NTO1 act together to delay S-phase progression.(A) Strains MMY033 (WT), MVY51 (Δrpd3), MVY54 (Δeaf3) and MVY55 (Δeaf3Δrpd3) were arrested in G1 with α-factor and released into S-phase at 30°C. Samples were taken at indicated times and processed for FACS analysis. (B) as (A) with strains MMY001 (WT), MMY002 (Δrpd3), MMY118 (Δnto1) and MVY119 (Δrpd3Δnto1). (C) as (A) with strains MMY001 (WT), MMY002 (Δrpd3), MVY137 (Δeaf3Δnto1) and MVY138 (Δrpd3Δeaf3Δnto1). Grey bars indicate the estimated length of S-phase. (D) Cell budding was assessed by microscopy. Complete lines indicate WT (black) and Δrpd3 (grey); broken lines indicate Δeaf3Δnto1 (black) and Δrpd3Δeaf3Δnto1 (grey).

Mentions: If K36me3 delays the association of Cdc45 with origins, deletion of proteins that bind this modification should result in a shortening of S-phase, similar to Δrpd3. Eaf3 and Nto1 are two factors that have been shown to bind H3 K36me3 [36]–[38], [42]. Eaf3 is a non essential subunit of the NuA4 HAT complex and part of the Rpd3(S) complex, while Nto1 is a subunit of the NuA3 HAT complex [44], [50], [51]. We therefore deleted each of these factors singly or in combination and analysed S-phase progression by FACS analysis. S-phase progression was not affected by the deletion of EAF3 or NTO1 alone, as the single mutants progressed through S-phase with similar kinetics to the WT (Fig. 6A and 6B). As expected, DNA replication occurred more rapidly in the Δrpd3 strain and was not further accelerated by additional deletion of EAF3 or NTO1. However, when both EAF3 and NTO1 were deleted S-phase was accelerated, similar to the Δrpd3 strain. The triple mutant Δeaf3Δnto1Δrpd3 was similar to the Δeaf3Δnto1 double mutant, indicating that S-phase could not be further shortened by the deletion of RPD3 (Fig. 6C). These data show that K36me3-binding proteins Eaf3 and Nto1 act redundantly to delay S-phase progression via a mechanism that is genetically dependent on RPD3.


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)

EAF3 and NTO1 act together to delay S-phase progression.(A) Strains MMY033 (WT), MVY51 (Δrpd3), MVY54 (Δeaf3) and MVY55 (Δeaf3Δrpd3) were arrested in G1 with α-factor and released into S-phase at 30°C. Samples were taken at indicated times and processed for FACS analysis. (B) as (A) with strains MMY001 (WT), MMY002 (Δrpd3), MMY118 (Δnto1) and MVY119 (Δrpd3Δnto1). (C) as (A) with strains MMY001 (WT), MMY002 (Δrpd3), MVY137 (Δeaf3Δnto1) and MVY138 (Δrpd3Δeaf3Δnto1). Grey bars indicate the estimated length of S-phase. (D) Cell budding was assessed by microscopy. Complete lines indicate WT (black) and Δrpd3 (grey); broken lines indicate Δeaf3Δnto1 (black) and Δrpd3Δeaf3Δnto1 (grey).
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pone-0005882-g006: EAF3 and NTO1 act together to delay S-phase progression.(A) Strains MMY033 (WT), MVY51 (Δrpd3), MVY54 (Δeaf3) and MVY55 (Δeaf3Δrpd3) were arrested in G1 with α-factor and released into S-phase at 30°C. Samples were taken at indicated times and processed for FACS analysis. (B) as (A) with strains MMY001 (WT), MMY002 (Δrpd3), MMY118 (Δnto1) and MVY119 (Δrpd3Δnto1). (C) as (A) with strains MMY001 (WT), MMY002 (Δrpd3), MVY137 (Δeaf3Δnto1) and MVY138 (Δrpd3Δeaf3Δnto1). Grey bars indicate the estimated length of S-phase. (D) Cell budding was assessed by microscopy. Complete lines indicate WT (black) and Δrpd3 (grey); broken lines indicate Δeaf3Δnto1 (black) and Δrpd3Δeaf3Δnto1 (grey).
Mentions: If K36me3 delays the association of Cdc45 with origins, deletion of proteins that bind this modification should result in a shortening of S-phase, similar to Δrpd3. Eaf3 and Nto1 are two factors that have been shown to bind H3 K36me3 [36]–[38], [42]. Eaf3 is a non essential subunit of the NuA4 HAT complex and part of the Rpd3(S) complex, while Nto1 is a subunit of the NuA3 HAT complex [44], [50], [51]. We therefore deleted each of these factors singly or in combination and analysed S-phase progression by FACS analysis. S-phase progression was not affected by the deletion of EAF3 or NTO1 alone, as the single mutants progressed through S-phase with similar kinetics to the WT (Fig. 6A and 6B). As expected, DNA replication occurred more rapidly in the Δrpd3 strain and was not further accelerated by additional deletion of EAF3 or NTO1. However, when both EAF3 and NTO1 were deleted S-phase was accelerated, similar to the Δrpd3 strain. The triple mutant Δeaf3Δnto1Δrpd3 was similar to the Δeaf3Δnto1 double mutant, indicating that S-phase could not be further shortened by the deletion of RPD3 (Fig. 6C). These data show that K36me3-binding proteins Eaf3 and Nto1 act redundantly to delay S-phase progression via a mechanism that is genetically dependent on RPD3.

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