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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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Pol ɛ and the chromatin-remodeling complexes are associated with a specific epigenetic state of chromatin. Histones from the Dpb4-chromatin remodeling complex and from the Dpb4–pol ɛ complex were PrA-affinity purified from DPB4-PrA dpb3Δ and DPB3-PrA strains, respectively. Histone H4 was modified in-gel with D6-acetic anhydride to convert free lysines to D3-acetyl lysines and then digested with trypsin. (A) MALDI-QqTOF mass spectrum of the tryptic peptide encompassing amino acid residues 4–17 of the NH2-terminal tail of histone H4. All five possible acetylation states are labeled. (B) Each peptide ion species observed in A was fragmented in a MALDI-ion trap MS. The resulting heavy and light fragment ion intensities were used to determine the levels of acetylation on specific lysines. Shown is a representative fragmentation spectrum of peptide 4–17 containing two heavy (Acd) and two light (Ac) acetylations. The b- and y-fragment ions used to quantitate the levels of acetylation on each of the four lysines are shown expanded. (C) Site-specific levels of acetylation on histone H4 peptide 4–17 from global histones (gray), Dpb4-chromatin remodeling complex-associated histones (light blue), pol ɛ–associated histones from Dpb3-PrA immunoprecipitation (medium blue), and pol ɛ–associated histones from Pol2-PrA immunoprecipitation (dark blue). (D) Site-specific levels of acetylation on histone H4 peptide 4–17 from immunoprecipitation of Pol2-PrA (dark blue) and Pol2-PrA in dpb3Δ strain (green).
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fig4: Pol ɛ and the chromatin-remodeling complexes are associated with a specific epigenetic state of chromatin. Histones from the Dpb4-chromatin remodeling complex and from the Dpb4–pol ɛ complex were PrA-affinity purified from DPB4-PrA dpb3Δ and DPB3-PrA strains, respectively. Histone H4 was modified in-gel with D6-acetic anhydride to convert free lysines to D3-acetyl lysines and then digested with trypsin. (A) MALDI-QqTOF mass spectrum of the tryptic peptide encompassing amino acid residues 4–17 of the NH2-terminal tail of histone H4. All five possible acetylation states are labeled. (B) Each peptide ion species observed in A was fragmented in a MALDI-ion trap MS. The resulting heavy and light fragment ion intensities were used to determine the levels of acetylation on specific lysines. Shown is a representative fragmentation spectrum of peptide 4–17 containing two heavy (Acd) and two light (Ac) acetylations. The b- and y-fragment ions used to quantitate the levels of acetylation on each of the four lysines are shown expanded. (C) Site-specific levels of acetylation on histone H4 peptide 4–17 from global histones (gray), Dpb4-chromatin remodeling complex-associated histones (light blue), pol ɛ–associated histones from Dpb3-PrA immunoprecipitation (medium blue), and pol ɛ–associated histones from Pol2-PrA immunoprecipitation (dark blue). (D) Site-specific levels of acetylation on histone H4 peptide 4–17 from immunoprecipitation of Pol2-PrA (dark blue) and Pol2-PrA in dpb3Δ strain (green).

Mentions: After tryptic digestion, the four potential sites of histone H4 acetylation (K5, K8, K12, and K16) were all found in a peptide stretching from amino acid residues 4 to 17, which we term the 4–17 peptide. The 4–17 peptides from both pol ɛ and the chromatin remodeling complex were mostly either completely unacetylated or just singly acetylated, although low levels of doubly, triply, and quadruply acetylated peptide were also present (Fig. 4 A). To determine the degree of acetylation of the individual sites in each of these five peptide species, we collected MS2 data (Fig. 4 B). Both pools of histone H4 were hypoacetylated at K5, K8, and K12 relative to global histone H4, whereas the levels of acetylation at K16 were more comparable to those observed in global histone H4 (Fig. 4 C). We found the same pattern of acetylation on histone H4 regardless of whether the pol ɛ–histone complex was isolated with Pol2-PrA or Dpb3-PrA (Fig. 4 C).


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)

Pol ɛ and the chromatin-remodeling complexes are associated with a specific epigenetic state of chromatin. Histones from the Dpb4-chromatin remodeling complex and from the Dpb4–pol ɛ complex were PrA-affinity purified from DPB4-PrA dpb3Δ and DPB3-PrA strains, respectively. Histone H4 was modified in-gel with D6-acetic anhydride to convert free lysines to D3-acetyl lysines and then digested with trypsin. (A) MALDI-QqTOF mass spectrum of the tryptic peptide encompassing amino acid residues 4–17 of the NH2-terminal tail of histone H4. All five possible acetylation states are labeled. (B) Each peptide ion species observed in A was fragmented in a MALDI-ion trap MS. The resulting heavy and light fragment ion intensities were used to determine the levels of acetylation on specific lysines. Shown is a representative fragmentation spectrum of peptide 4–17 containing two heavy (Acd) and two light (Ac) acetylations. The b- and y-fragment ions used to quantitate the levels of acetylation on each of the four lysines are shown expanded. (C) Site-specific levels of acetylation on histone H4 peptide 4–17 from global histones (gray), Dpb4-chromatin remodeling complex-associated histones (light blue), pol ɛ–associated histones from Dpb3-PrA immunoprecipitation (medium blue), and pol ɛ–associated histones from Pol2-PrA immunoprecipitation (dark blue). (D) Site-specific levels of acetylation on histone H4 peptide 4–17 from immunoprecipitation of Pol2-PrA (dark blue) and Pol2-PrA in dpb3Δ strain (green).
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Related In: Results  -  Collection

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fig4: Pol ɛ and the chromatin-remodeling complexes are associated with a specific epigenetic state of chromatin. Histones from the Dpb4-chromatin remodeling complex and from the Dpb4–pol ɛ complex were PrA-affinity purified from DPB4-PrA dpb3Δ and DPB3-PrA strains, respectively. Histone H4 was modified in-gel with D6-acetic anhydride to convert free lysines to D3-acetyl lysines and then digested with trypsin. (A) MALDI-QqTOF mass spectrum of the tryptic peptide encompassing amino acid residues 4–17 of the NH2-terminal tail of histone H4. All five possible acetylation states are labeled. (B) Each peptide ion species observed in A was fragmented in a MALDI-ion trap MS. The resulting heavy and light fragment ion intensities were used to determine the levels of acetylation on specific lysines. Shown is a representative fragmentation spectrum of peptide 4–17 containing two heavy (Acd) and two light (Ac) acetylations. The b- and y-fragment ions used to quantitate the levels of acetylation on each of the four lysines are shown expanded. (C) Site-specific levels of acetylation on histone H4 peptide 4–17 from global histones (gray), Dpb4-chromatin remodeling complex-associated histones (light blue), pol ɛ–associated histones from Dpb3-PrA immunoprecipitation (medium blue), and pol ɛ–associated histones from Pol2-PrA immunoprecipitation (dark blue). (D) Site-specific levels of acetylation on histone H4 peptide 4–17 from immunoprecipitation of Pol2-PrA (dark blue) and Pol2-PrA in dpb3Δ strain (green).
Mentions: After tryptic digestion, the four potential sites of histone H4 acetylation (K5, K8, K12, and K16) were all found in a peptide stretching from amino acid residues 4 to 17, which we term the 4–17 peptide. The 4–17 peptides from both pol ɛ and the chromatin remodeling complex were mostly either completely unacetylated or just singly acetylated, although low levels of doubly, triply, and quadruply acetylated peptide were also present (Fig. 4 A). To determine the degree of acetylation of the individual sites in each of these five peptide species, we collected MS2 data (Fig. 4 B). Both pools of histone H4 were hypoacetylated at K5, K8, and K12 relative to global histone H4, whereas the levels of acetylation at K16 were more comparable to those observed in global histone H4 (Fig. 4 C). We found the same pattern of acetylation on histone H4 regardless of whether the pol ɛ–histone complex was isolated with Pol2-PrA or Dpb3-PrA (Fig. 4 C).

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