Limits...
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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Elucidating the architecture of chromatin-associated complexes. (A) Dpb3 or Dpb4 was separately deleted in combination with the various PrA-tagged pol ɛ components. PrA-tagged components and their interacting proteins were isolated and visualized as for Fig. 1 A. Gel positions shown with asterisks, corresponding to the migration position of the proteins identified in Fig. 1, were excised and screened for the presence of these proteins by MS. (B) Proposed assembly pathway for chromatin complexes containing Dpb4.
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fig2: Elucidating the architecture of chromatin-associated complexes. (A) Dpb3 or Dpb4 was separately deleted in combination with the various PrA-tagged pol ɛ components. PrA-tagged components and their interacting proteins were isolated and visualized as for Fig. 1 A. Gel positions shown with asterisks, corresponding to the migration position of the proteins identified in Fig. 1, were excised and screened for the presence of these proteins by MS. (B) Proposed assembly pathway for chromatin complexes containing Dpb4.

Mentions: To dissect these nucleosome-bound subcomplexes, we used a strategy whereby we isolated each pol ɛ subunit from cells in which either DPB3 or DPB4 had been deleted, reasoning that removal of a given component of pol ɛ could make the other interacting partners less stable (and so in principle provide information on the overall assembly of pol ɛ and associated complexes). To do this, we used a rapid “hypothesis-driven” mass spectrometric strategy to look for all major proteins detected in Fig. 1 (Kalkum et al., 2003). This strategy allowed us to determine the presence or absence of a protein with high sensitivity (Fig. 2 A).


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)

Elucidating the architecture of chromatin-associated complexes. (A) Dpb3 or Dpb4 was separately deleted in combination with the various PrA-tagged pol ɛ components. PrA-tagged components and their interacting proteins were isolated and visualized as for Fig. 1 A. Gel positions shown with asterisks, corresponding to the migration position of the proteins identified in Fig. 1, were excised and screened for the presence of these proteins by MS. (B) Proposed assembly pathway for chromatin complexes containing Dpb4.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2171912&req=5

fig2: Elucidating the architecture of chromatin-associated complexes. (A) Dpb3 or Dpb4 was separately deleted in combination with the various PrA-tagged pol ɛ components. PrA-tagged components and their interacting proteins were isolated and visualized as for Fig. 1 A. Gel positions shown with asterisks, corresponding to the migration position of the proteins identified in Fig. 1, were excised and screened for the presence of these proteins by MS. (B) Proposed assembly pathway for chromatin complexes containing Dpb4.
Mentions: To dissect these nucleosome-bound subcomplexes, we used a strategy whereby we isolated each pol ɛ subunit from cells in which either DPB3 or DPB4 had been deleted, reasoning that removal of a given component of pol ɛ could make the other interacting partners less stable (and so in principle provide information on the overall assembly of pol ɛ and associated complexes). To do this, we used a rapid “hypothesis-driven” mass spectrometric strategy to look for all major proteins detected in Fig. 1 (Kalkum et al., 2003). This strategy allowed us to determine the presence or absence of a protein with high sensitivity (Fig. 2 A).

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