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Proteomic and genomic characterization of chromatin complexes at a boundary.

Tackett AJ, Dilworth DJ, Davey MJ, O'Donnell M, Aitchison JD, Rout MP, Chait BT - J. Cell Biol. (2005)

Bottom Line: We have dissected specialized assemblies on the Saccharomyces cerevisiae genome that help define and preserve the boundaries that separate silent and active chromatin.The complexes consist of at least 15 chromatin-associated proteins, including DNA pol epsilon, the Isw2-Itc1 and Top2 chromatin remodeling proteins, the Sas3-Spt16 chromatin modifying complex, and Yta7, a bromodomain-containing AAA ATPase.We show that these complexes are important for the faithful maintenance of an established boundary, as disruption of the complexes results in specific, anomalous alterations of the silent and active epigenetic states.

View Article: PubMed Central - PubMed

Affiliation: Rockefeller University, New York, NY 10021, USA.

ABSTRACT
We have dissected specialized assemblies on the Saccharomyces cerevisiae genome that help define and preserve the boundaries that separate silent and active chromatin. These assemblies contain characteristic stretches of DNA that flank particular regions of silent chromatin, as well as five distinctively modified histones and a set of protein complexes. The complexes consist of at least 15 chromatin-associated proteins, including DNA pol epsilon, the Isw2-Itc1 and Top2 chromatin remodeling proteins, the Sas3-Spt16 chromatin modifying complex, and Yta7, a bromodomain-containing AAA ATPase. We show that these complexes are important for the faithful maintenance of an established boundary, as disruption of the complexes results in specific, anomalous alterations of the silent and active epigenetic states.

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Pol ɛ associates with chromatin during DNA replication. (A) Pol2-PrA or Dpb4-PrA complexes were affinity purified at different cell cycle blocks, and their proteins were resolved by Coomassie blue–stained SDS-PAGE. White lines indicate that intervening lanes have been spliced out. (B) Site-specific levels of acetylation on histone H4 peptide 4–17 from the Pol2-PrA–associated histones from global (open), hydroxyurea block (dark gray), and cdc20Δ block (light gray).
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fig7: Pol ɛ associates with chromatin during DNA replication. (A) Pol2-PrA or Dpb4-PrA complexes were affinity purified at different cell cycle blocks, and their proteins were resolved by Coomassie blue–stained SDS-PAGE. White lines indicate that intervening lanes have been spliced out. (B) Site-specific levels of acetylation on histone H4 peptide 4–17 from the Pol2-PrA–associated histones from global (open), hydroxyurea block (dark gray), and cdc20Δ block (light gray).

Mentions: Because pol ɛ is localized to boundary chromatin (Fig. 5), yet is an active DNA polymerase (Fig. 3 A) known to have a role in S phase (Ohya et al., 2002), we tested if this chromatin association was cell cycle dependent (Fig. 7 A). Although the four pol ɛ subunits maintained stable interactions with each other at each block, pol ɛ was associated with core histones only during times of DNA replication and segregation, indicating a cell cycle–dependent association of pol ɛ with chromatin. We also analyzed the acetylation state of the NH2-terminal tail of histone H4 at the replication and mitosis blocks (Fig. 7 B). We observed that pol ɛ remained associated with the same unique type of modified histones during both blocks, displaying hypoacetylation on K5, K8, and K12 and relatively higher levels of acetylation on K16. This pattern is almost identical with that observed for pol ɛ–associated histones from the asynchronous cultures (Fig. 4 C), suggesting that pol ɛ is targeted to the same unique state of chromatin in a cell cycle–dependent manner. In contrast to the pol ɛ targeting, the Dpb4-containing chromatin remodeling complex remained associated with chromatin before and during times of DNA replication (Fig. 7 A), indicating a decoupling of the Dpb4-containing pol ɛ and chromatin remodeling complexes.


Proteomic and genomic characterization of chromatin complexes at a boundary.

Tackett AJ, Dilworth DJ, Davey MJ, O'Donnell M, Aitchison JD, Rout MP, Chait BT - J. Cell Biol. (2005)

Pol ɛ associates with chromatin during DNA replication. (A) Pol2-PrA or Dpb4-PrA complexes were affinity purified at different cell cycle blocks, and their proteins were resolved by Coomassie blue–stained SDS-PAGE. White lines indicate that intervening lanes have been spliced out. (B) Site-specific levels of acetylation on histone H4 peptide 4–17 from the Pol2-PrA–associated histones from global (open), hydroxyurea block (dark gray), and cdc20Δ block (light gray).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2171912&req=5

fig7: Pol ɛ associates with chromatin during DNA replication. (A) Pol2-PrA or Dpb4-PrA complexes were affinity purified at different cell cycle blocks, and their proteins were resolved by Coomassie blue–stained SDS-PAGE. White lines indicate that intervening lanes have been spliced out. (B) Site-specific levels of acetylation on histone H4 peptide 4–17 from the Pol2-PrA–associated histones from global (open), hydroxyurea block (dark gray), and cdc20Δ block (light gray).
Mentions: Because pol ɛ is localized to boundary chromatin (Fig. 5), yet is an active DNA polymerase (Fig. 3 A) known to have a role in S phase (Ohya et al., 2002), we tested if this chromatin association was cell cycle dependent (Fig. 7 A). Although the four pol ɛ subunits maintained stable interactions with each other at each block, pol ɛ was associated with core histones only during times of DNA replication and segregation, indicating a cell cycle–dependent association of pol ɛ with chromatin. We also analyzed the acetylation state of the NH2-terminal tail of histone H4 at the replication and mitosis blocks (Fig. 7 B). We observed that pol ɛ remained associated with the same unique type of modified histones during both blocks, displaying hypoacetylation on K5, K8, and K12 and relatively higher levels of acetylation on K16. This pattern is almost identical with that observed for pol ɛ–associated histones from the asynchronous cultures (Fig. 4 C), suggesting that pol ɛ is targeted to the same unique state of chromatin in a cell cycle–dependent manner. In contrast to the pol ɛ targeting, the Dpb4-containing chromatin remodeling complex remained associated with chromatin before and during times of DNA replication (Fig. 7 A), indicating a decoupling of the Dpb4-containing pol ɛ and chromatin remodeling complexes.

Bottom Line: We have dissected specialized assemblies on the Saccharomyces cerevisiae genome that help define and preserve the boundaries that separate silent and active chromatin.The complexes consist of at least 15 chromatin-associated proteins, including DNA pol epsilon, the Isw2-Itc1 and Top2 chromatin remodeling proteins, the Sas3-Spt16 chromatin modifying complex, and Yta7, a bromodomain-containing AAA ATPase.We show that these complexes are important for the faithful maintenance of an established boundary, as disruption of the complexes results in specific, anomalous alterations of the silent and active epigenetic states.

View Article: PubMed Central - PubMed

Affiliation: Rockefeller University, New York, NY 10021, USA.

ABSTRACT
We have dissected specialized assemblies on the Saccharomyces cerevisiae genome that help define and preserve the boundaries that separate silent and active chromatin. These assemblies contain characteristic stretches of DNA that flank particular regions of silent chromatin, as well as five distinctively modified histones and a set of protein complexes. The complexes consist of at least 15 chromatin-associated proteins, including DNA pol epsilon, the Isw2-Itc1 and Top2 chromatin remodeling proteins, the Sas3-Spt16 chromatin modifying complex, and Yta7, a bromodomain-containing AAA ATPase. We show that these complexes are important for the faithful maintenance of an established boundary, as disruption of the complexes results in specific, anomalous alterations of the silent and active epigenetic states.

Show MeSH
Related in: MedlinePlus