Long-distance combinatorial linkage between methylation and acetylation on histone H3 N termini.
Journal: 2007/May - Proceedings of the National Academy of Sciences of the United States of America
ISSN: 0027-8424
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.
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Proc Natl Acad Sci U S A 104(7): 2086-2091

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
**To whom correspondence may be addressed: E-mail: ude.ainigriv@hfd or ude.rellefekcor@dcsilla

Contributed by C. David Allis, December 14, 2006

.

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.
Received 2006 Nov 2

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.

Keywords: bivalent domain, electron transfer dissociation, mass spectrometry, posttranslational modifications, Tetrahymena
Abstract

In eukaryotes, DNA is wrapped around octameric cores of the histone proteins H3, H4, H2A, and H2B, forming nucleosomes, the fundamental subunit of chromatin (1, 2). Flexible “histone tails” that extend from nucleosomes are subject to a wealth of posttranslational modifications (PTMs) including methylation, acetylation, and ubiquitination on lysines, methylation on arginines, and phosphorylation on serine or threonine residues (14). The complexity and nonrandom distribution of known PTMs on the N terminus of histone H3 alone (Fig. 1A) suggests functional implications for unique combinations of modifications, several of which are under active investigation (57).

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Analysis of somatic chromatin suggests long-range connectivity among modifications on histone H3. (A) Schematic of PTMs observed on the Glu-C generated N-terminal peptide of histone H3 [for the entire sequence of Tetrahymena H3.2, see supporting information (SI) Fig. 4]. Conventional mass spectrometry protocols often require digestion into much smaller pieces such that connectivity between modifications spaced by many amino acids as may occur in bivalent domains (open arrows on K4 and K27) is poorly understood. (B) Highly purified Tetrahymena MIC and MAC histones were resolved by SDS/PAGE and detected by Western blot analysis with antibodies recognizing monomethylated, dimethylated, or trimethylated species of H3K4. (C) Histones were purified and resolved as in B; however, Western blot analysis was performed with antibodies recognizing monomethylated, dimethylated, or trimethylated species of H3K27. A MIC-specific H3 isoform that is proteolytically processed endogenously is indicated by ∗. (D) MAC histones were acid-extracted and resolved by acid-urea gel to separate positively and negatively charged histone H3 species into six rungs representing H3 isoforms that range from relatively hypoacetylated (0) to hyperacetylated (5). Equivalent Ponceau staining was used as a loading control.

Emerging studies suggest that neighboring histone PTMs may function together on the same histone tail in a combinatorial fashion, sometimes reducing or enhancing the ability for the targeted residue to become modified in what is referred to as modification “cross-talk” (810). Furthermore, immunogen-based approaches such as ChIP or ChIP on Chip suggest unique groupings of histone PTMs are involved in demarcating chromatin domains as either active or repressed, in an epigenetically stable fashion. In somatic cell lineages, for example, H3K4me3 and H3 hyperacetylation are consistently colocalized at 5′ regions of transcriptionally active genes in euchromatin (11, 12). In contrast, transcriptionally silent heterochromatin, such as the inactive X chromosome (Xi) in mammalian females, is often characterized by H3K27me, H3K9me, and H3 hypoacetylation (13). Interestingly, undifferentiated embryonic stem cells were recently suggested to have “bivalent domains,” regions of the genome enriched for both H3K4me3 and H3K27me3 on a nucleosomal scale (see ref. 14 and downward arrows in Fig. 1A), which, during differentiation, might be resolved into either a repressive H3K27me3-enriched environment or a transcriptionally favorable H3K4me3-enriched region. However, while these reports are suggestive of functional interplay among histone PTMs, direct determination of their interdependence remains poorly understood, in part because the analytical techniques available for the study of long-distance combinatorial patterns on the same histone tail (or protein) are limited.

Mass spectrometry has become an enabling method for mapping PTMs on histones (reviewed in refs. 15 and 16), without some of the caveats of antibody-based analyses that include antibody production, unwanted cross-reactivity, epitope occlusion, and the inability to detect multiple modifications (patterns) or novel modification sites. However, a drawback of conventional mass spectrometry approaches is that histones are typically digested into short peptides to make sequence and PTM analysis possible (17, 18), referred to as “bottom-up” analysis. With short peptides, it becomes difficult to determine which peptides originated from the same molecule, and information about interdependence of modifications on a given histone molecule is often lost (see ref. 19).

A newer mass spectrometry technique, electron capture dissociation (ECD), allows sequencing of intact proteins (20). This “top-down” analysis (21) has been successfully used to determine histone PTMs. Using ECD, Kelleher and coworkers (2225) characterized PTMs on all four human core histones, Burlingame and colleagues (26) characterized Tetrahymena H2B variants, and Zhang and Freitas (27) characterized histone H4 from calf thymus. Recently, an ion/ion analogue of ECD, termed electron transfer dissociation (ETD) (28), was developed. This technique can sequence highly charged, longer peptides (>20 aa), and in combination with a second ion/ion reaction, termed proton transfer charge reduction (PTR) (29, 30), can determine the N- and C-terminal sequence of intact proteins on a chromatographic time scale (31, 32). Using this technique, we examined the PTM status of the N-terminal H3 peptide residues 1–50 (H31–50) from macronuclei isolated from the ciliated protozoan Tetrahymena thermophila to determine long-range combinations of histone PTMs (i.e., PTMs separated by 20 or more residues) associated with a transcriptionally active state.

The genome of T. thermophila is functionally separated into two nuclei that can be easily purified: a “somatic” macronucleus (MAC) from which all transcription occurs, and a transcriptionally silent “germ-line” micronucleus (MIC) (33). To determine PTM correlations on H31–50, we purified MAC H3 based on charge and modification state and used the combination of ETD/PTR and accurate mass measurements to determine patterns of histone PTMs. We observed a strong correlation on the same histone molecule between H3K4me3 and increased acetylation occupancy at distinct lysine residues, as well as coexistence of H3K4me1 with H3K27me1 or H3K27me2. These long-range histone PTM associations point to an underlying hierarchical mechanism of establishing histone modification patterns and suggest a unique configuration of transcriptionally poised H3 modifications that resemble the bivalent domains recently reported for embryonic stem cells (14).

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Acknowledgments

We thank UBI/Millipore (formerly UBI Antibodies) for antibody donations and members of D.F.H.'s and C.D.A.'s laboratories for helpful discussions, especially J. Syka, J. Coon, M. Lachner, J. Tanny, and I. Cristea for critical readings of this manuscript. This work was supported by National Institutes of Health Grants GM53512 (to S.D.T.), GM63959 (to C.D.A.), RR00862 and RR022220 (to B.T.C.), and GM37537 (to D.F.H.) and The Rockefeller University (S.D.T., C.D.A., A.J.T., and B.T.C.).

Acknowledgments

Abbreviations

PTMposttranslational modification
ETDelectron transfer dissociation
PTRproton transfer charge reduction
MACmacronucleus
MICmicronucleus
FTMSFourier transform mass spectrometry.
Abbreviations

Note Added in Proof:

Garcia et al. (56) recently reported additional histone H3 PTMs that are not included in Fig. 1A.

Note Added in Proof:

Footnotes

The authors declare no conflict of interest.

This article contains supporting information online at www.pnas.org/cgi/content/full/0610993104/DC1.

Footnotes

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