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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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Dpb4-containing chromatin complexes regulate an HMR boundary. (A) Strains carrying deletions of DPB4, DPB3, YTA7, SAS3, ITC1, ISW2, DLS1, and HTZ1 were assayed for the expression of a URA3 reporter gene placed in the following three locations: in the Sir-silenced region of HMR (∼640 bp to the right of HMR-E), at the left-hand boundary (∼475 bp to the left of HMR-E), and upstream of the boundary region (∼2,840 bp to the left of HMR-E, and within the YCR095C gene) (Donze et al., 1999). Decreased silencing of the reporter (i.e., increased transcription) results in increased cell death on FOA. Strains were assayed either without or with a Sir3-expressing plasmid. (B) Semiquantitative relative measure of the dilution-adjusted colony density seen in A. (C) Transcription levels of YCR095C (light gray) and GIT1 (dark gray), genes proximal to HMR, were measured by real-time PCR. Fold transcription is relative to wild type. Measurements less than onefold indicate repression of transcription, whereas measurements greater than onefold indicate above normal transcription. Error bars show the SD of the mean for triplicate measurements.
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fig6: Dpb4-containing chromatin complexes regulate an HMR boundary. (A) Strains carrying deletions of DPB4, DPB3, YTA7, SAS3, ITC1, ISW2, DLS1, and HTZ1 were assayed for the expression of a URA3 reporter gene placed in the following three locations: in the Sir-silenced region of HMR (∼640 bp to the right of HMR-E), at the left-hand boundary (∼475 bp to the left of HMR-E), and upstream of the boundary region (∼2,840 bp to the left of HMR-E, and within the YCR095C gene) (Donze et al., 1999). Decreased silencing of the reporter (i.e., increased transcription) results in increased cell death on FOA. Strains were assayed either without or with a Sir3-expressing plasmid. (B) Semiquantitative relative measure of the dilution-adjusted colony density seen in A. (C) Transcription levels of YCR095C (light gray) and GIT1 (dark gray), genes proximal to HMR, were measured by real-time PCR. Fold transcription is relative to wild type. Measurements less than onefold indicate repression of transcription, whereas measurements greater than onefold indicate above normal transcription. Error bars show the SD of the mean for triplicate measurements.

Mentions: To investigate the functional consequences of the association between the Dpb4-histone complexes and the boundary elements, we tested whether particular components of either pol ɛ holoenzyme (i.e., Dpb4, Dpb3) or the Dpb4-chromatin remodeling complex (i.e., Dpb4, Yta7, Sas3, Itc1, Isw2, and Dls1) play a role in the epigenetic regulation of the HMR locus. Specifically, we assayed strains carrying deletions of these proteins, as well as HTZ1 and the control SIR3, for the expression of a URA3 reporter gene placed in the following three locations: in the Sir-silenced region of HMR, at the left-hand boundary of HMR, and upstream of the boundary region (Donze et al., 1999). We assayed for silencing by duplicate plating of the strains on FOA and −Ura. Consistent results were observed under both conditions; hence, for simplicity, we show only the FOA results in which an increased level of survival on FOA measures an increased level of URA3 silencing (Fig. 6, A and B). As expected, removal of SIR3 completely abolishes silencing (Donze et al., 1999; unpublished data). We evaluated how the deletions affected boundary maintenance by assaying the reporter strains both with and without SIR3 plasmid.


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-containing chromatin complexes regulate an HMR boundary. (A) Strains carrying deletions of DPB4, DPB3, YTA7, SAS3, ITC1, ISW2, DLS1, and HTZ1 were assayed for the expression of a URA3 reporter gene placed in the following three locations: in the Sir-silenced region of HMR (∼640 bp to the right of HMR-E), at the left-hand boundary (∼475 bp to the left of HMR-E), and upstream of the boundary region (∼2,840 bp to the left of HMR-E, and within the YCR095C gene) (Donze et al., 1999). Decreased silencing of the reporter (i.e., increased transcription) results in increased cell death on FOA. Strains were assayed either without or with a Sir3-expressing plasmid. (B) Semiquantitative relative measure of the dilution-adjusted colony density seen in A. (C) Transcription levels of YCR095C (light gray) and GIT1 (dark gray), genes proximal to HMR, were measured by real-time PCR. Fold transcription is relative to wild type. Measurements less than onefold indicate repression of transcription, whereas measurements greater than onefold indicate above normal transcription. Error bars show the SD of the mean for triplicate measurements.
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Related In: Results  -  Collection

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fig6: Dpb4-containing chromatin complexes regulate an HMR boundary. (A) Strains carrying deletions of DPB4, DPB3, YTA7, SAS3, ITC1, ISW2, DLS1, and HTZ1 were assayed for the expression of a URA3 reporter gene placed in the following three locations: in the Sir-silenced region of HMR (∼640 bp to the right of HMR-E), at the left-hand boundary (∼475 bp to the left of HMR-E), and upstream of the boundary region (∼2,840 bp to the left of HMR-E, and within the YCR095C gene) (Donze et al., 1999). Decreased silencing of the reporter (i.e., increased transcription) results in increased cell death on FOA. Strains were assayed either without or with a Sir3-expressing plasmid. (B) Semiquantitative relative measure of the dilution-adjusted colony density seen in A. (C) Transcription levels of YCR095C (light gray) and GIT1 (dark gray), genes proximal to HMR, were measured by real-time PCR. Fold transcription is relative to wild type. Measurements less than onefold indicate repression of transcription, whereas measurements greater than onefold indicate above normal transcription. Error bars show the SD of the mean for triplicate measurements.
Mentions: To investigate the functional consequences of the association between the Dpb4-histone complexes and the boundary elements, we tested whether particular components of either pol ɛ holoenzyme (i.e., Dpb4, Dpb3) or the Dpb4-chromatin remodeling complex (i.e., Dpb4, Yta7, Sas3, Itc1, Isw2, and Dls1) play a role in the epigenetic regulation of the HMR locus. Specifically, we assayed strains carrying deletions of these proteins, as well as HTZ1 and the control SIR3, for the expression of a URA3 reporter gene placed in the following three locations: in the Sir-silenced region of HMR, at the left-hand boundary of HMR, and upstream of the boundary region (Donze et al., 1999). We assayed for silencing by duplicate plating of the strains on FOA and −Ura. Consistent results were observed under both conditions; hence, for simplicity, we show only the FOA results in which an increased level of survival on FOA measures an increased level of URA3 silencing (Fig. 6, A and B). As expected, removal of SIR3 completely abolishes silencing (Donze et al., 1999; unpublished data). We evaluated how the deletions affected boundary maintenance by assaying the reporter strains both with and without SIR3 plasmid.

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