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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Dpb4 forms multiple stable complexes with chromatin. (A) Complexes were isolated via a PrA tag under conditions that copurified interacting proteins. These proteins were resolved by SDS-PAGE, visualized by Coomassie blue staining, and excised for MS identification. Labels show specific interactions with the PrA-tagged proteins. Reciprocal purifications of tagged members of the complexes demonstrated coisolation of Dpb4 with the exception of tagged Yta7. (B) Coimmunoprecipitation experiments with Yta7 and Dpb4 indicated that they associate in vivo. Note lane 7, which controls for post-lysis association of Yta7 and Dpb4 in vitro.
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fig1: Dpb4 forms multiple stable complexes with chromatin. (A) Complexes were isolated via a PrA tag under conditions that copurified interacting proteins. These proteins were resolved by SDS-PAGE, visualized by Coomassie blue staining, and excised for MS identification. Labels show specific interactions with the PrA-tagged proteins. Reciprocal purifications of tagged members of the complexes demonstrated coisolation of Dpb4 with the exception of tagged Yta7. (B) Coimmunoprecipitation experiments with Yta7 and Dpb4 indicated that they associate in vivo. Note lane 7, which controls for post-lysis association of Yta7 and Dpb4 in vitro.

Mentions: During this broad screen (Fig. S1 A), we discovered a discrete set of chromatin-bound proteins comprising overlapping complexes with the common component, Dpb4 (Fig. 1, Fig. S1 B, and Table S1, available at http://www.jcb.org/cgi/content/full/jcb.200502104/DC1). This set contained the DNA pol ɛ holoenzyme (Pol2, Dpb2, Dpb3, Dpb4) (Hamatake et al., 1990; Araki et al., 1991; Navas et al., 1995; Dua et al., 1998; Ohya et al., 2000; Hubscher et al., 2002; Ohya et al., 2002; Osborn et al., 2002; Chilkova et al., 2003; Edwards et al., 2003; Iida and Araki, 2004), as well as the clamp loader subunits Ctf18 and Rfc1 (Hanna et al., 2001; Mayer et al., 2001; Jeruzalmi et al., 2002; Edwards et al., 2003). The set also contained several other chromatin-associated proteins: the chromatin remodeling proteins Itc1, Isw2, and Dls1 (as expected [Iida and Araki, 2004; McConnell et al., 2004]), as well as the AAA ATPase Yta7, the histone acetyltransferase Sas3 and its partner Spt16, the DNA topoisomerase Top2, an RNA polymerase core subunit Rpb2, an uncharacterized protein Ylr455 (which by virtue of its PWWP domain is also likely to bind DNA), the core histones (H2A, H2B, H3, and H4), and the histone variant Htz1 (H2A.Z; Goto et al., 1984; Myer and Young, 1998; John et al., 2000; Stec et al., 2000; Frohlich, 2001; Howe et al., 2001; Kent et al., 2001; Kassabov et al., 2002; Qiu et al., 2002; Fazzio and Tsukiyama, 2003; Meneghini et al., 2003). In summary, these isolations yielded at least two Dpb4-containing complexes, the 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)

Dpb4 forms multiple stable complexes with chromatin. (A) Complexes were isolated via a PrA tag under conditions that copurified interacting proteins. These proteins were resolved by SDS-PAGE, visualized by Coomassie blue staining, and excised for MS identification. Labels show specific interactions with the PrA-tagged proteins. Reciprocal purifications of tagged members of the complexes demonstrated coisolation of Dpb4 with the exception of tagged Yta7. (B) Coimmunoprecipitation experiments with Yta7 and Dpb4 indicated that they associate in vivo. Note lane 7, which controls for post-lysis association of Yta7 and Dpb4 in vitro.
© Copyright Policy
Related In: Results  -  Collection

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

fig1: Dpb4 forms multiple stable complexes with chromatin. (A) Complexes were isolated via a PrA tag under conditions that copurified interacting proteins. These proteins were resolved by SDS-PAGE, visualized by Coomassie blue staining, and excised for MS identification. Labels show specific interactions with the PrA-tagged proteins. Reciprocal purifications of tagged members of the complexes demonstrated coisolation of Dpb4 with the exception of tagged Yta7. (B) Coimmunoprecipitation experiments with Yta7 and Dpb4 indicated that they associate in vivo. Note lane 7, which controls for post-lysis association of Yta7 and Dpb4 in vitro.
Mentions: During this broad screen (Fig. S1 A), we discovered a discrete set of chromatin-bound proteins comprising overlapping complexes with the common component, Dpb4 (Fig. 1, Fig. S1 B, and Table S1, available at http://www.jcb.org/cgi/content/full/jcb.200502104/DC1). This set contained the DNA pol ɛ holoenzyme (Pol2, Dpb2, Dpb3, Dpb4) (Hamatake et al., 1990; Araki et al., 1991; Navas et al., 1995; Dua et al., 1998; Ohya et al., 2000; Hubscher et al., 2002; Ohya et al., 2002; Osborn et al., 2002; Chilkova et al., 2003; Edwards et al., 2003; Iida and Araki, 2004), as well as the clamp loader subunits Ctf18 and Rfc1 (Hanna et al., 2001; Mayer et al., 2001; Jeruzalmi et al., 2002; Edwards et al., 2003). The set also contained several other chromatin-associated proteins: the chromatin remodeling proteins Itc1, Isw2, and Dls1 (as expected [Iida and Araki, 2004; McConnell et al., 2004]), as well as the AAA ATPase Yta7, the histone acetyltransferase Sas3 and its partner Spt16, the DNA topoisomerase Top2, an RNA polymerase core subunit Rpb2, an uncharacterized protein Ylr455 (which by virtue of its PWWP domain is also likely to bind DNA), the core histones (H2A, H2B, H3, and H4), and the histone variant Htz1 (H2A.Z; Goto et al., 1984; Myer and Young, 1998; John et al., 2000; Stec et al., 2000; Frohlich, 2001; Howe et al., 2001; Kent et al., 2001; Kassabov et al., 2002; Qiu et al., 2002; Fazzio and Tsukiyama, 2003; Meneghini et al., 2003). In summary, these isolations yielded at least two Dpb4-containing complexes, the 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