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

Publication ,  Journal Article
He, S; Bauman, D; Davis, JS; Loyola, A; Nishioka, K; Gronlund, JL; Reinberg, D; Meng, F; Kelleher, N; McCafferty, DG
Published in: Proceedings of the National Academy of Sciences of the United States of America
October 2003

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.

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Published In

Proceedings of the National Academy of Sciences of the United States of America

DOI

EISSN

1091-6490

ISSN

0027-8424

Publication Date

October 2003

Volume

100

Issue

21

Start / End Page

12033 / 12038

Related Subject Headings

  • Xenopus laevis
  • Recombinant Fusion Proteins
  • Protein Structure, Quaternary
  • Molecular Structure
  • Molecular Sequence Data
  • Models, Biological
  • Methylation
  • Indicators and Reagents
  • In Vitro Techniques
  • Humans
 

Citation

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He, S., Bauman, D., Davis, J. S., Loyola, A., Nishioka, K., Gronlund, J. L., … McCafferty, D. G. (2003). Facile synthesis of site-specifically acetylated and methylated histone proteins: reagents for evaluation of the histone code hypothesis. Proceedings of the National Academy of Sciences of the United States of America, 100(21), 12033–12038. https://doi.org/10.1073/pnas.2035256100
He, Shu, David Bauman, Jamaine S. Davis, Alejandra Loyola, Kenichi Nishioka, Jennifer L. Gronlund, Danny Reinberg, Fanyu Meng, Neil Kelleher, and Dewey G. McCafferty. “Facile synthesis of site-specifically acetylated and methylated histone proteins: reagents for evaluation of the histone code hypothesis.Proceedings of the National Academy of Sciences of the United States of America 100, no. 21 (October 2003): 12033–38. https://doi.org/10.1073/pnas.2035256100.
He S, Bauman D, Davis JS, Loyola A, Nishioka K, Gronlund JL, et al. Facile synthesis of site-specifically acetylated and methylated histone proteins: reagents for evaluation of the histone code hypothesis. Proceedings of the National Academy of Sciences of the United States of America. 2003 Oct;100(21):12033–8.
He, Shu, et al. “Facile synthesis of site-specifically acetylated and methylated histone proteins: reagents for evaluation of the histone code hypothesis.Proceedings of the National Academy of Sciences of the United States of America, vol. 100, no. 21, Oct. 2003, pp. 12033–38. Epmc, doi:10.1073/pnas.2035256100.
He S, Bauman D, Davis JS, Loyola A, Nishioka K, Gronlund JL, Reinberg D, Meng F, Kelleher N, McCafferty DG. Facile synthesis of site-specifically acetylated and methylated histone proteins: reagents for evaluation of the histone code hypothesis. Proceedings of the National Academy of Sciences of the United States of America. 2003 Oct;100(21):12033–12038.
Journal cover image

Published In

Proceedings of the National Academy of Sciences of the United States of America

DOI

EISSN

1091-6490

ISSN

0027-8424

Publication Date

October 2003

Volume

100

Issue

21

Start / End Page

12033 / 12038

Related Subject Headings

  • Xenopus laevis
  • Recombinant Fusion Proteins
  • Protein Structure, Quaternary
  • Molecular Structure
  • Molecular Sequence Data
  • Models, Biological
  • Methylation
  • Indicators and Reagents
  • In Vitro Techniques
  • Humans