Facile synthesis of site-specifically acetylated and methylated histone proteins: Reagents for evaluation of the histone code hypothesis

Shu He, David Bauman, Jamaine S. Davis, Alejandra Loyola, Kenichi Nishioka, Jennifer L. Gronlund, Danny Reinberg, Fanyu Meng, Neil Kelleher, Dewey G. McCafferty*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

96 Scopus citations

Abstract

The functional capacity of genetically encoded histone proteins can be powerfully expanded by posttranslational modification. A growing body of biochemical and genetic evidence clearly links the unique combinatorial patterning of side chain acetylation, methylation, and phosphorylation mainly within the highly conserved N termini of histones H2A, H2B, H3, and H4 with the regulation of gene expression and chromatin assembly and remodeling, in effect constituting a "histone code" for epigenetic signaling. Deconvoluting this code has proved challenging given the inherent posttranslational heterogeneity of histone proteins isolated from biological sources. Here we describe the application of native chemical ligation to the preparation of full-length histone proteins containing site-specific acetylation and methylation modifications. Peptide thioesters corresponding to histone N termini were prepared by solid phase peptide synthesis using an acid labile Boc/HF assembly strategy, then subsequently ligated to recombinantly produced histone C-terminal globular domains containing an engineered N-terminal cysteine residue. The ligation site is then rendered traceless by hydrogenolytic desulfurization, generating a native histone protein sequence. Synthetic histones generated by this method are fully functional, as evidenced by their self-assembly into a higher order H3/H4 heterotetramer, their deposition into nucleosomes by human ISWI-containing (Imitation of Switch) factor RSF (Remodeling and Spacing factor), and by enzymatic modification by human Sirt1 deacetylase and G9a methyltransferase. Site-specifically modified histone proteins generated by this method will prove invaluable as novel reagents for the evaluation of the histone code hypothesis and analysis of epigenetic signaling mechanisms.

Original languageEnglish (US)
Pages (from-to)12033-12038
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume100
Issue number21
DOIs
StatePublished - Oct 14 2003

ASJC Scopus subject areas

  • General

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