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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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Isolation of chromatin-associated protein complexes with their cognate DNA and histones. Complexes containing Dpb4-PrA were immunoisolated and eluted under nondenaturing conditions. (A) The eluate was loaded on an anion exchange column and fractions were collected from a 0–1 M [NaCl] gradient and visualized by Coomassie blue–stained SDS-PAGE. DNA polymerase activity was assayed by dTMP incorporation into calf thymus DNA. The presence of copurifying DNA was assayed by real-time PCR. Lanes containing three unique complexes are shown expanded, and the protein components identified by MS are labeled. Bands labeled with an asterisk may represent a breakdown product or alternate form of the protein. White lines indicate that intervening lanes have been spliced out. (B) The three unique complexes from A were further resolved by gel filtration. Fraction proteins were visualized by silver-stained SDS-PAGE.
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fig3: Isolation of chromatin-associated protein complexes with their cognate DNA and histones. Complexes containing Dpb4-PrA were immunoisolated and eluted under nondenaturing conditions. (A) The eluate was loaded on an anion exchange column and fractions were collected from a 0–1 M [NaCl] gradient and visualized by Coomassie blue–stained SDS-PAGE. DNA polymerase activity was assayed by dTMP incorporation into calf thymus DNA. The presence of copurifying DNA was assayed by real-time PCR. Lanes containing three unique complexes are shown expanded, and the protein components identified by MS are labeled. Bands labeled with an asterisk may represent a breakdown product or alternate form of the protein. White lines indicate that intervening lanes have been spliced out. (B) The three unique complexes from A were further resolved by gel filtration. Fraction proteins were visualized by silver-stained SDS-PAGE.

Mentions: To further study the Dpb4-containing subcomplexes, we developed a semi-preparative purification strategy. This strategy uses PrA-affinity purification of the Dpb4-PrA complexes (∼75 μg), which after native elution from the affinity resin were separated by anion exchange chromatography. Three distinct Dpb4-containing fractions were resolved (Fig. 3 A). The first (fraction No. 26, 0.34 M [NaCl] eluate) contained the Dpb4-chromatin remodeling complex comprised of Itc1, Isw2, Dpb4-PrA, and Dls1. The second nearby eluting fraction (fraction No. 28, 0.38 M [NaCl]) contained the pol ɛ proteins Pol2, Dpb2, Dpb3, and Dpb4-PrA. Proteins in the 0.34 M [NaCl] eluate tailed into those in the 0.38 M [NaCl] eluate, although the compositions of these fractions appear to be distinct. The third fraction (fraction No. 40, 0.63 M [NaCl]) contained Yta7, Itc1, Isw2, Dpb4, and the histones. Thus, the Dpb4-chromatin remodeling/histone complex identified in Fig. 1 was further resolved into the two salt-stable fractions at 0.34 and 0.63 M [NaCl]. The sub-stoichiometric levels of Dpb4 and Itc1/Isw2 with the Yta7-histone fraction are consistent with the low stoichiometry of Dpb4 in the Yta7-PrA pullout (Fig. 1, A and B). The individual fractions from the anion exchange separation were assayed for polymerase activity in vitro, yielding a significant peak of activity in the fractions containing the Pol2 catalytic subunit of pol ɛ (Fig. 3 A). Given that we also identified clamp-loader proteins in association with the pol ɛ subunits (Fig. 1 A), we surmise that the pol ɛ holoenzyme is an active DNA polymerase in vivo.


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)

Isolation of chromatin-associated protein complexes with their cognate DNA and histones. Complexes containing Dpb4-PrA were immunoisolated and eluted under nondenaturing conditions. (A) The eluate was loaded on an anion exchange column and fractions were collected from a 0–1 M [NaCl] gradient and visualized by Coomassie blue–stained SDS-PAGE. DNA polymerase activity was assayed by dTMP incorporation into calf thymus DNA. The presence of copurifying DNA was assayed by real-time PCR. Lanes containing three unique complexes are shown expanded, and the protein components identified by MS are labeled. Bands labeled with an asterisk may represent a breakdown product or alternate form of the protein. White lines indicate that intervening lanes have been spliced out. (B) The three unique complexes from A were further resolved by gel filtration. Fraction proteins were visualized by silver-stained SDS-PAGE.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2171912&req=5

fig3: Isolation of chromatin-associated protein complexes with their cognate DNA and histones. Complexes containing Dpb4-PrA were immunoisolated and eluted under nondenaturing conditions. (A) The eluate was loaded on an anion exchange column and fractions were collected from a 0–1 M [NaCl] gradient and visualized by Coomassie blue–stained SDS-PAGE. DNA polymerase activity was assayed by dTMP incorporation into calf thymus DNA. The presence of copurifying DNA was assayed by real-time PCR. Lanes containing three unique complexes are shown expanded, and the protein components identified by MS are labeled. Bands labeled with an asterisk may represent a breakdown product or alternate form of the protein. White lines indicate that intervening lanes have been spliced out. (B) The three unique complexes from A were further resolved by gel filtration. Fraction proteins were visualized by silver-stained SDS-PAGE.
Mentions: To further study the Dpb4-containing subcomplexes, we developed a semi-preparative purification strategy. This strategy uses PrA-affinity purification of the Dpb4-PrA complexes (∼75 μg), which after native elution from the affinity resin were separated by anion exchange chromatography. Three distinct Dpb4-containing fractions were resolved (Fig. 3 A). The first (fraction No. 26, 0.34 M [NaCl] eluate) contained the Dpb4-chromatin remodeling complex comprised of Itc1, Isw2, Dpb4-PrA, and Dls1. The second nearby eluting fraction (fraction No. 28, 0.38 M [NaCl]) contained the pol ɛ proteins Pol2, Dpb2, Dpb3, and Dpb4-PrA. Proteins in the 0.34 M [NaCl] eluate tailed into those in the 0.38 M [NaCl] eluate, although the compositions of these fractions appear to be distinct. The third fraction (fraction No. 40, 0.63 M [NaCl]) contained Yta7, Itc1, Isw2, Dpb4, and the histones. Thus, the Dpb4-chromatin remodeling/histone complex identified in Fig. 1 was further resolved into the two salt-stable fractions at 0.34 and 0.63 M [NaCl]. The sub-stoichiometric levels of Dpb4 and Itc1/Isw2 with the Yta7-histone fraction are consistent with the low stoichiometry of Dpb4 in the Yta7-PrA pullout (Fig. 1, A and B). The individual fractions from the anion exchange separation were assayed for polymerase activity in vitro, yielding a significant peak of activity in the fractions containing the Pol2 catalytic subunit of pol ɛ (Fig. 3 A). Given that we also identified clamp-loader proteins in association with the pol ɛ subunits (Fig. 1 A), we surmise that the pol ɛ holoenzyme is an active DNA polymerase in vivo.

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