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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Genome-wide localization of Dpb4-containing complexes. Protected DNA from the purified fraction of Dpb4-associated histones in Fig. 3 A was hybridized to an intergenic DNA microarray covering the S. cerevisiae genome. (A) The sequences of chromosomes I–XVI are depicted as horizontal black lines with the centromeres denoted by circles. Vertical ticks indicate the position of sequences enriched with the Dpb4-histone complexes. The positions of two silenced gene clusters (HM and FLO) are indicated, showing their close apposition to the Dpb4-enriched sequences. (B) A histogram showing the number of intergenic regions enriched with the Dpb4-histone complexes is plotted as a function of distance from the chromosome ends. (C) Dpb4-histone complex DNA is enriched at boundaries to the silent, mating loci on chromosome III. Positions of enriched sequences are shown as horizontal black lines above the annotated chromosome with increasing line thickness indicating increasing enrichment. The inset shows high resolution mapping of Dpb4-enriched sequences from semiquantitative PCR analysis. The asterisk marks a region that was not covered by the microarray analysis.
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fig5: Genome-wide localization of Dpb4-containing complexes. Protected DNA from the purified fraction of Dpb4-associated histones in Fig. 3 A was hybridized to an intergenic DNA microarray covering the S. cerevisiae genome. (A) The sequences of chromosomes I–XVI are depicted as horizontal black lines with the centromeres denoted by circles. Vertical ticks indicate the position of sequences enriched with the Dpb4-histone complexes. The positions of two silenced gene clusters (HM and FLO) are indicated, showing their close apposition to the Dpb4-enriched sequences. (B) A histogram showing the number of intergenic regions enriched with the Dpb4-histone complexes is plotted as a function of distance from the chromosome ends. (C) Dpb4-histone complex DNA is enriched at boundaries to the silent, mating loci on chromosome III. Positions of enriched sequences are shown as horizontal black lines above the annotated chromosome with increasing line thickness indicating increasing enrichment. The inset shows high resolution mapping of Dpb4-enriched sequences from semiquantitative PCR analysis. The asterisk marks a region that was not covered by the microarray analysis.

Mentions: We localized the genomic positions of the Dpb4-histone–associated DNA by hybridization of the Dpb4-associated DNA to an intergenic DNA microarray of the S. cerevisiae genome. We observed enrichments at numerous discrete sites along the yeast chromosomes, with a tendency for clustering (summarized in Fig. 5 A; for the full data set of all the chromosomes see Table S2). Notably, the Dpb4-associated DNA was found to be particularly enriched toward most chromosome ends (proximal to the telomeres; Fig. 5, A and B). Several functionally related genes families are found in these telomere proximal regions. We observed a high number of Dpb4-binding sites immediately adjacent to certain of these functionally related genes. For example, binding was found adjacent to all seven related members of the flocculation (FLO) gene family (FLO1, FLO5, FLO8, FLO9, FLO10, FLO11, FIG2; Fig. 5 A; Halme et al., 2004; Verstrepen et al., 2004). The Dpb4 complexes also flank other known stress response genes such as the PAU loci (anaerobic stress; Rachidi et al., 2000). It seems significant that the Dpb4 complex binding occurs next to families of genes that are known to be heterochromatically silenced and epigenetically controlled. In this context, we note that sites adjacent to the silent mating loci (HML and HMR) on chromosome III, canonical silent regions surrounded by active chromatin, were among the strongest binding sites for Dpb4 complexes (Fig. 5 C). Specifically, the complexes were preferentially associated with the well-defined right hand boundary element of HMR at (tT(AGU)C) as well as the less well-defined left hand boundary element (overlapping with or close to YCR095C) of HMR, which are areas of chromatin that prevent the heterochromatin covering HMRA1 and HMRA2 from spreading into surrounding euchromatic regions (Donze et al., 1999; Donze and Kamakaka, 2001; Lieb et al., 2001). This preferential enrichment with the boundary elements was confirmed with high resolution PCR mapping at HMR (Fig. 5 C) as well as with conventional ChIP-chip analysis (Fig. S3, available at http://www.jcb.org/cgi/content/full/jcb.200502104/DC1). At the HML locus, we observe the Dpb4 complexes to be preferentially associated with DNA spanning the region between HML-I and CHA1 and (to a lesser extent) on the telomere side of YCL073C (Bi et al., 1999; Lieb et al., 2001). We conclude from these results that the Dpb4-associated complexes bind preferentially to particular chromatin regions, some of which are associated with known boundaries or with variegated epigenetic states.


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)

Genome-wide localization of Dpb4-containing complexes. Protected DNA from the purified fraction of Dpb4-associated histones in Fig. 3 A was hybridized to an intergenic DNA microarray covering the S. cerevisiae genome. (A) The sequences of chromosomes I–XVI are depicted as horizontal black lines with the centromeres denoted by circles. Vertical ticks indicate the position of sequences enriched with the Dpb4-histone complexes. The positions of two silenced gene clusters (HM and FLO) are indicated, showing their close apposition to the Dpb4-enriched sequences. (B) A histogram showing the number of intergenic regions enriched with the Dpb4-histone complexes is plotted as a function of distance from the chromosome ends. (C) Dpb4-histone complex DNA is enriched at boundaries to the silent, mating loci on chromosome III. Positions of enriched sequences are shown as horizontal black lines above the annotated chromosome with increasing line thickness indicating increasing enrichment. The inset shows high resolution mapping of Dpb4-enriched sequences from semiquantitative PCR analysis. The asterisk marks a region that was not covered by the microarray analysis.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2171912&req=5

fig5: Genome-wide localization of Dpb4-containing complexes. Protected DNA from the purified fraction of Dpb4-associated histones in Fig. 3 A was hybridized to an intergenic DNA microarray covering the S. cerevisiae genome. (A) The sequences of chromosomes I–XVI are depicted as horizontal black lines with the centromeres denoted by circles. Vertical ticks indicate the position of sequences enriched with the Dpb4-histone complexes. The positions of two silenced gene clusters (HM and FLO) are indicated, showing their close apposition to the Dpb4-enriched sequences. (B) A histogram showing the number of intergenic regions enriched with the Dpb4-histone complexes is plotted as a function of distance from the chromosome ends. (C) Dpb4-histone complex DNA is enriched at boundaries to the silent, mating loci on chromosome III. Positions of enriched sequences are shown as horizontal black lines above the annotated chromosome with increasing line thickness indicating increasing enrichment. The inset shows high resolution mapping of Dpb4-enriched sequences from semiquantitative PCR analysis. The asterisk marks a region that was not covered by the microarray analysis.
Mentions: We localized the genomic positions of the Dpb4-histone–associated DNA by hybridization of the Dpb4-associated DNA to an intergenic DNA microarray of the S. cerevisiae genome. We observed enrichments at numerous discrete sites along the yeast chromosomes, with a tendency for clustering (summarized in Fig. 5 A; for the full data set of all the chromosomes see Table S2). Notably, the Dpb4-associated DNA was found to be particularly enriched toward most chromosome ends (proximal to the telomeres; Fig. 5, A and B). Several functionally related genes families are found in these telomere proximal regions. We observed a high number of Dpb4-binding sites immediately adjacent to certain of these functionally related genes. For example, binding was found adjacent to all seven related members of the flocculation (FLO) gene family (FLO1, FLO5, FLO8, FLO9, FLO10, FLO11, FIG2; Fig. 5 A; Halme et al., 2004; Verstrepen et al., 2004). The Dpb4 complexes also flank other known stress response genes such as the PAU loci (anaerobic stress; Rachidi et al., 2000). It seems significant that the Dpb4 complex binding occurs next to families of genes that are known to be heterochromatically silenced and epigenetically controlled. In this context, we note that sites adjacent to the silent mating loci (HML and HMR) on chromosome III, canonical silent regions surrounded by active chromatin, were among the strongest binding sites for Dpb4 complexes (Fig. 5 C). Specifically, the complexes were preferentially associated with the well-defined right hand boundary element of HMR at (tT(AGU)C) as well as the less well-defined left hand boundary element (overlapping with or close to YCR095C) of HMR, which are areas of chromatin that prevent the heterochromatin covering HMRA1 and HMRA2 from spreading into surrounding euchromatic regions (Donze et al., 1999; Donze and Kamakaka, 2001; Lieb et al., 2001). This preferential enrichment with the boundary elements was confirmed with high resolution PCR mapping at HMR (Fig. 5 C) as well as with conventional ChIP-chip analysis (Fig. S3, available at http://www.jcb.org/cgi/content/full/jcb.200502104/DC1). At the HML locus, we observe the Dpb4 complexes to be preferentially associated with DNA spanning the region between HML-I and CHA1 and (to a lesser extent) on the telomere side of YCL073C (Bi et al., 1999; Lieb et al., 2001). We conclude from these results that the Dpb4-associated complexes bind preferentially to particular chromatin regions, some of which are associated with known boundaries or with variegated epigenetic states.

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