Targeted Histone Peptides: Insights into the Spatial Regulation of the Methyltransferase PRC2 by using a Surrogate of Heterotypic Chromatin

Abstract

Eukaryotic genomes are dynamically regulated through a host of epigenetic stimuli. The substrate for these epigenetic transactions, chromatin, is a polymer of nucleosome building blocks. In native (i.e. cellular) chromatin, each nucleosome can differ from its neighbors through the localized installation of covalent modifications to both the genomic DNA and the histone packaging proteins. The heterotypic nature of chromatin presents a formidable obstacle to biochemical studies seeking to understand the role of context on epigenetic regulation and that, as a consequence, must employ compositionally defined chromatin substrates. Here, we introduce a chemical approach to the production of heterotypic ‘designer’ chromatin that can be used in such studies. Our method involves attachment of a user-defined modified histone peptide to a designated nucleosome within the polymer by using a Peptide Nucleic Acid (PNA) targeting compound. We apply this strategy to dissect the role of chromatin context on both the activation and inhibition of the histone methyltransferase, PRC2, which methylates Lys 27 of histone H3 (H3K27). Our studies show that PRC2 can be stimulated to produce de novo H3K27 methylation from a defined nucleation site. More generally, this technology promises to facilitate biochemical studies that require the use of heterotypic chromatin substrates.

S -adenosyl methionine is necessary for inhibition of the methyltransferase G9a by the lysine 9 to methionine mutation on histone H3

“…Unmodified H3 [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20] peptide (23 μM) was incubated with 25 nM G9a-SET in the presence of 50 μM SAM (radioactive: nonradioactive molar ratio = 0.016) in a buffer containing 50 mM Hepes (pH 7.9), 0.5 mM DTT, 0.25 mM PMSF, and 2 mM MgCl 2 . Where applicable, different H3 1-20 K9X peptides (Tufts University Peptide Synthesis Core) were present in the reaction.…”

Traceless Synthesis of Asymmetrically Modified Bivalent Nucleosomes

“…Previous methods to generate PTM asymmetry were based on statistical histone octamer assembly from histone mixtures. [3,9] To extend chromatin analysis to the bivalent state, novel methods are required that allow direct chemical control over the supramolecular assembly of nucleosomes while leaving no unnatural peptides or other traces in the final histones. [3,8] Owing to the preparative difficulty,only nucleosomes containing single-modified histones were assembled.…”

“…[3] By contrast, pre-existing H3K27me3 was found to stimulate the catalytic activity of the PRC2 complex. [3,9] To extend chromatin analysis to the bivalent state, novel methods are required that allow direct chemical control over the supramolecular assembly of nucleosomes while leaving no unnatural peptides or other traces in the final histones. Herein, we report at raceless and modular method to synthesize chemically pure asymmetrically modified nucleosomes containing arbitrary combinations of PTMs.W ethen applied this approach to generate an ucleosome library to probe the ability of PRC2 to modify and maintain bivalent chromatin.…”

Chemistry Can Make Strict and Fuzzy Controls for Bio-Systems: DNA Nanoarchitectonics and Cell-Macromolecular Nanoarchitectonics

“…233 A predetermined chromatin was selectively and stably bound to the corresponding enzyme (methyltransferase) by using a linker involving two PNA strands of 10-base sequence. As the result, "spreading" of post-translational methylation from a chromosome to others was analyzed in detail.…”