Long-distance combinatorial linkage between methylation and acetylation on histone H3 N termini

Laboratories of *Chromatin Biology and
^§Mass Spectrometry and Gaseous Ion Chemistry, The Rockefeller University, New York, NY 10021;
^†Department of Chemistry, University of Virginia, Charlottesville, VA 22904; and
^‖Department of Pathology, Health Sciences Center, University of Virginia, Charlottesville, VA 22908

Contributed by C. David Allis, December 14, 2006 (received for review November 2, 2006)

Abstract

Individual posttranslational modifications (PTMs) on histones have well established roles in certain biological processes, notably transcriptional programming. Recent genomewide studies describe patterns of covalent modifications, such as H3 methylation and acetylation at promoters of specific target genes, or “bivalent domains,” in stem cells, suggestive of a possible combinatorial interplay between PTMs on the same histone. However, detection of long-range PTM associations is often problematic in antibody-based or traditional mass spectrometric-based analyses. Here, histone H3 from a ciliate model was analyzed as an enriched source of transcriptionally active chromatin. Using a recently developed mass spectrometric approach, combinatorial modification states on single, long N-terminal H3 fragments (residues 1–50) were determined. The entire modification status of intact N termini was obtained and indicated correlations between K4 methylation and H3 acetylation. In addition, K4 and K27 methylation were identified concurrently on one H3 species. This methodology is applicable to other histones and larger polypeptides and will likely be a valuable tool in understanding the roles of combinatorial patterns of PTMs.

Footnotes

**To whom correspondence may be addressed: E-mail: dfh{at}virginia.edu or alliscd{at}rockefeller.edu
Author contributions: S.D.T. and B.M.U. contributed equally to this work; S.D.T., B.M.U., B.T.C., D.F.H., and C.D.A. designed research; S.D.T., B.M.U., Y.L., A.J.T., and R.L.D. performed research; B.T.C., D.F.H., and C.D.A. contributed new reagents/analytic tools; S.D.T., B.M.U., J.S., D.F.H., and C.D.A. analyzed data; and S.D.T., B.M.U., B.T.C., D.F.H., and C.D.A. wrote the paper.
↵ ^‡Present address: Laboratory of Mass Spectrometry and Gaseous Ion Chemistry, The Rockefeller University, New York, NY 10021.
↵ ^¶Present address: Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, AR 72205.
The authors declare no conflict of interest.
This article contains supporting information online at www.pnas.org/cgi/content/full/0610993104/DC1.
Abbreviations:

PTM,

posttranslational modification;

ETD,

electron transfer dissociation;

PTR,

proton transfer charge reduction;

MAC,

macronucleus;

MIC,

micronucleus;

FTMS,

Fourier transform mass spectrometry.