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Published in final edited form as: Mol Cell. 2025 Oct 2;85(19):3554–3561. doi: 10.1016/j.molcel.2025.08.029

A Needed Nomenclature for Nucleosomes

Michael-Christopher Keogh 1, Genevieve Almouzni 2, Andrew J Andrews 3, Karim-Jean Armache 4, Cheryl H Arrowsmith 5, Sung Hee Baek 6, Mark T Bedford 7, Emily Bernstein 8, Jacques Côté 9, Yael David 10, John M Denu 11, Beat Fierz 12, Benjamin A Garcia 13, Karen C Glass 14, Or Gozani 15, Kristian Helin 16, Steve Henikoff 17, Ole N Jensen 18, Steven Z Josefowicz 19, Neil L Kelleher 20, Tatiana G Kutateladze 21, Herbert Lindner 22, Chao Lu 23, Karolin Luger 24, Parag Mallick 25, Catherine A Musselman 26, Tom W Muir 27, Ljiljana Paša-Tolić 28, Robert Schneider 29, Xiaobing Shi 30, Yang Shi 31, Simone Sidoli 32, Lloyd M Smith 33, Jessica K Tyler 34, Cynthia Wolberger 35, Jerry L Workman 36, Brian D Strahl 37, Nicolas L Young 38,*
PMCID: PMC12778995  NIHMSID: NIHMS2113709  PMID: 41043390

Abstract

Histone post-translational modifications (PTMs) are crucial to eukaryotic genome regulation, with a range of reported functions and mechanisms of action. Though often studied individually, it has long been recognized that the modifications function by combinatorial synergy or antagonism. Interplay may involve PTMs on the same histone, within the same nucleosome (containing a histone octamer), or between nucleosomes in higher-order chromatin. Given this, the field must distinguish ever greater complexity, and the context in which it is studied, with brevity and precision. The proteoform was introduced to define individual forms of a protein by sequence and PTMs, followed by nucleoform to describe the particular gathering of histones within an individual nucleosome. There is now a need to define specific forms of these entities in prose, while providing space for experimental nuance. To this end, we introduce a nomenclature that can express discrete PTMs, proteoforms, nucleoforms, or situations where defined PTMs exist in an uncertain context. Though specifically designed for the chromatin field, adaptions of the framework could be used to describe, and thus dissect, how proteoforms are configured in functionally distinct complexes across biology.

INTRODUCTION

The core histones (H2A, H2B, H3 and H4) package the genome into nucleosomes, and are a central component of chromatin biology.1 Histone function is regulated by diverse post-translational modifications (PTMs) that operate in combination to transduce signals and recruit additional enzymatic activities; thus impacting DNA replication, transcription, and repair.2 Discussions of this biology have been enabled by the 2005 Brno nomenclature3 that communicates histone, residue - position - modification type (e.g., H3K4me3 or H3K9ac): an intuitive clarity only slightly undermined by core histone proteins being canonically numbered from the second residue since initiator methionine is absent in the mature polypeptide (e.g., human H3.1 (UniProtKB P68431): A1RTKQTARK …). We have an ever-growing capability to study function in the histone, nucleosome or chromatin context, so the field now needs an expanded nomenclature to describe each element and any potential limits of related experimental datasets.

The nature of chromatin complexity (and how it is studied)

‘Proteoform’ was coined in 2012 to concisely describe individual protein molecules; and can encompass the differential products of transcriptional initiation or splicing, mutation, post-translational processing or modification, etc.4,5 Thus, the physiological state of each ‘protein’ is an ensemble of ‘proteoforms’, with distinct forms potentially mediating distinct functions. Collated proteoforms in tabular formats are useful for mass spectrometry (MS)-focused efforts6, but do not work well to communicate biological function or mechanism, and thus enable hypothesis driven research.7 The need for a concise and consistent proteoform notation is particularly urgent in chromatin biology. Histone proteoforms are often hypermodified (Figure 1). Thus, substrate engagement for reading, writing, erasing or clipping most often occurs in the context of high heterogeneity.1 The consideration of histone mutants (e.g., H3K27M associated with pediatric gliomas8), variants (e.g., centromere defining CenH39), and variant specific PTMs (e.g., γH2A.X associated with DNA double-strand breaks10) provides additional needs for clarity. Finally, this diversity is further encompassed within nucleosomes (each a core histone octamer wrapped by 147bp DNA11), necessitating description of the ‘nucleoform’.12

Figure 1.

Figure 1.

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Most studies of histone PTMs and their role in genome regulation address the modifications as an attribute of a molecule, without knowledge of any other attributes. H3K4me3 is enriched at transcriptionally active gene promoters but found in many proteoforms; each with implications for PTM function. Despite this, most experimental approaches do not speak to the proteoform. Immunoblots or CUT&RUN use antibodies that detect a single PTM, and almost never its relationship with even adjacent modifications. Bottom-up proteomics can capture proximal PTMs on the same peptide, but generally loses any connections to distal PTMs. Top-down MS methods can unambiguously determine the full proteoform, including variant identity and PTM co-occurrence. The new nomenclature addresses these concepts and any related experimental ambiguities.

Top- and middle-down proteomics allow the quantitative study of intact proteoforms, and can describe how PTMs co-occur on a single histone (in cis)13–22. Advances in engineering fully defined histones and nucleosomes have enabled the dissection of PTM interplay in cis or trans (latter within the same nucleosome but on different histones)15,23–25, or between PTMs and other nucleosome surfaces (e.g., the ‘acidic patch’ or wrapping DNA).26–29 Emerging technologies, such as NanoPore protein sequencing30 or Nuc-MS12, promise (but cannot yet routinely deliver) the complete characterization of native histones or even nucleosomes. The production of fully defined nucleosome arrays is already at a practical stage and stepping towards synthetic chromatin. Further inquiry along these many axes will accelerate as novel reagents and technologies become more widely available.

Elucidating chromatin biology with nucleoform-level information would be ideal. This is rarely possible, so any methodological limits on the gathered data should be considered. As an example, ChIP-seq and CUT&RUN are regularly used to map the genomic localization of individual PTMs in cell populations (Figure 2). The overlapping genomic localization of two PTMs in parallel experiments could indicate co-localization (whether co-incident or co-regulated) at the histone or nucleosome level, or mutually independent populations. The correct interpretation is essential to understand the related biology. While bottom-up MS can inform on PTM co-localization on the same molecule, this is limited to the length of the proteolytic peptide and often does not resolve histone variant identity. Top-down MS can describe PTM co-localization in cis, but not relationships in trans. Being able to specify the extent of knowledge about a peptide or protein, and to define knowns and ambiguities for an experimental dataset, is an essential aspect of the proposed notation.

Figure 2.

Figure 2.

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The configuration of histone PTMs and variants within the nucleoform can be essential to function, but requires a means to effectively communicate molecular detail and data. In this example a native nucleosome sample (Blue, H3; Green, H4; Red, H2A; Yellow, H2B) contains H3K4me3 and H3K27me3, but this could represent very different configurations (e.g., the two PTMs together in trans form the canonical ‘bivalent’ signature22). Current methods to interrogate bulk samples are either unable to distinguish (e.g., immunoblot, ChIP-seq, CUT&RUN); can discriminate some possibilities (e.g., sequential ChIP, middle-down MS); or have the potential to be definitive (e.g., combinatorial enrichment, Nuc-MS12,45). The proposed nomenclature is designed to express the (un)certainty of the starting options, and what the experimental data can(not) discriminate. Of note, it is now possible to engineer fully-defined proteoforms and nucleoforms to support biochemical and genomic studies with unparalleled mechanistic insight.25,26,46,47 Such reagents also need to be concisely described in prose.

Combinatorial mechanisms in chromatin biology are most often realized through the nucleosome.11 This extends regulatory potential (and descriptive need) beyond the proteoform. In cis histone mechanisms indicate where two or more attributes co-occur and mediate function on the same molecule within the same nucleosome. In trans mechanisms are where two or more attributes on different molecules work in concert within a single nucleosome. Further increasing complexity, two different proteoforms of the same histone can co-occupy a ‘heterotypic’ (aka ‘asymmetric’) nucleosome – entities of great interest for the regulatory potential they provide31–33. Of note homo- / hetero- typic is our preferred designation over a- / symmetric, since it follows the histone variant literature34,35 and avoids confusion with hemi- / symmetric- DNA methylation. Finally trans-nucleosomal mechanisms are where attributes from neighboring or spatially co-localized (such as by ‘looping’) nucleosomes function together. It is now possible to synthesize fully defined histones, nucleosomes or even nucleosome arrays, and study the regulatory potential at each level.

The proposed nomenclature

A nomenclature that can communicate all the above concepts defines proteoforms (Figure 1), their combination in a nucleoform (Figure 2), and the various nucleoforms in chromatin arrays (Figure 3). Further it conveys effectively in prose, has intuitive biological meaning, can accommodate experimental imprecision, and enables a nuanced discussion of mechanism and hypotheses. Finally it does all this while encompassing the Brno nomenclature3 that efficiently describes histone PTMs. Below, we provide some examples of the nomenclature in practice, but for more detail see Table 1.

Figure 3.

Figure 3.

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An array of semi-synthetic nucleosomes with defined properties. Nucleosome #1 (Blue, H3; Green, H4; Red, H2A; Yellow, H2B) is homotypic for both H2A.Z and H3.3 (one copy as K4me1 only; the second K4me3, K9ac, K14ac, K18ac and K23ac). Nucleosome #2 is heterotypic for H3.1 and H3.3, with each modified at K14ac. Nucleosome #3 is homotypic for H3.1 containing K14ac, K23ac and K36me1. Finally, nucleosome #4 is homotypic for H3.3K36me3. In each nucleosome the other core histones are not noted, and can thus be considered unmodified major histones. This complexity can be expressed in prose with precision as beneath the figure. Importantly, a computer readable derivative of the nomenclature could be parsed for downstream applications such as figure generation.

Table 1.

Nomenclature examples.

Type of Information Delimiters/Operators Example Example description
A defined proteoform (semi-synthetic or top-down proteomics). Absence of a residue communicates not modified. [ ] [H3.2K27me3] A fully defined histone H3.2 containing K27me3. All other sites of variable modification not noted are unmodified.
[H3.2K14acK23ac] A fully defined H3.2 containing only K14ac and K23ac.
[H3.2] A fully defined and completely unmodified H3.2.
Discrete PTM (usually native material, bottom-up proteomics, or marginalized top-down data). Absence of a residue communicates ambiguity. None or { } H3K27me3
{H3K27me3}		 A set of H3 molecules of unspecified isoform that contain K27me3. All other sites of variable modification on the same molecules are undefined.
H3K27un
{H3K27un}		 A set of H3 molecules that are unmodified at K27.
H3.2
{H3.2}		 A set of H3.2 molecules in a completely undefined modification state.
H3.2K14acK23ac
{H3.2K14acK23ac}		 A set of H3.2 molecules that containK14ac and K23ac.
H2AXS139phY142ph
{H2AXS139phY142ph}		 A set of H2AX molecules phosphorylated at both S139 and Y142.
A nucleosome. ( ) or ( • ) ([H3.2K14acK18acK23ac]2) A fully defined nucleosome homotypic for histone H3.2 acetylated at K14, K18 and K23. The other histones are major and unmodified.
Semi-synthetic and fully defined. Absence of a histone family communicates major and unmodified. ([H3.2]•[H3.2K14acK18acK23ac]]) A defined nucleosome heterotypic for H3.2. One molecule is unmodified. The other is acetylated at K14, K18 and K23.
([H3.2K14ac]•[H3.3K14ac]) A defined nucleosome heterotypic for H3. One molecule is major H3.2. The other is variant H3.3. Both are acetylated at K14.
‘Native’ material. Absence of a histone family communicates ambiguity. ({H3K14ac}) A set of nucleosomes with at least one H3K14ac. All other sites of variable modification on H3 are undefined. The other histones are undefined.
({H3K9me3K36me3}) A set of nucleosomes containing H3K9me3 and H3K36me3. The PTMs could be in cis or trans. Such material could be enriched by sequential immunoprecipitation or with a combinatorial PTM reader.50
Nucleosome arrays ( • ) ( • ) ([H3.3K4me3]2)
([H3.2K14ac]2)
([H3.2]•[H3.3])		 A defined tri-nucleosome with unspecified DNA linker length. The most 5’ nucleosome is a [H3.3K4me3] homotypic. The second nucleosome is a [H3.2K14ac] homotypic. The most 3’ nucleosome is heterotypic for unmodified H3.2 and H3.3.
([H3.3]2)50([H2AZ.1]2) A defined di-Nucleosome separated by 50bp linker DNA. The 5’ nucleosome is homotypic for H3.3 (with all others in the octamer major histones as default). The 3’ nucleosome is homotypic for H2AZ.1 (with all others in the octamer major histones as default).
Not ! {H3.2!K27me3} A set of H3.2 molecules that do not contain K27me3.
{H3.2!K27me3K36me3} A set of H3.2 molecules that contain K36me3 but not K27me3.
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Square brackets ‘[H3K4me3]’ indicate a fully defined proteoform, as in a semi-synthetic histone15,25 where other sites of potential modification not denoted can be understood as definitively unmodified. Braces ‘{H3K4me3}’ indicate a partially understood native proteoform where one PTM is experimentally known to exist (as by immunoprecipitating H3K4me3 from a cell extract), but other sites of potential modification not denoted can be understood of undefined status: the general situation with native material. However, if braces are not used it should be assumed as referring to discrete PTMs (e.g., H3K4me3 represents {H3K4me3}) since this is historical practice. If identifiable, the histone variant carrying the PTM is denoted immediately within the open bracket or braces: e.g., [H3.3K4me3]. If the species is a histone peptide, such as a synthetic reagent or measured by bottom-up proteomics, this is indicated by subscripted brackets identifying the start and end separated by a hyphen: e.g., H3[1–12]K4me3.

Parentheses ‘( )’ are used to group proteoforms in a nucleosome, with ‘•’ separating any distinguishing histones in alphabetical and numerical order. It is now possible to make semi-synthetic nucleosomes as fully defined homo- or hetero- typics15,25,36, with ([H3K27me3]2) a homotypic and the ([H3]•[H3K4me3K14ac]) or ([H3K4me3]•[H3K14ac]) heterotypics respectively distributing the same PTM complement in cis or trans. The situation with native material (such as enriched by a PTM-specific antibody) is more complex, and so ({H3K27me3}) does not inform on the homo- / hetero- typic state since these generally cannot be distinguished. For the advanced user, chromatin arrays can be expressed as a series of individual nucleosomes (optionally with linker DNA information: Table 1) and follow the 5’ to 3’ standard from left to right (Figure 3).

Discussing Hypotheses and Mechanisms

The capacity to concisely define histones and nucleosomes with rigor also allows the effective communication of nuanced hypotheses. The nomenclature readily supports the use of relational operators (>, <, or =) for comparing abundances or activities. For example, H3K4me3 is most commonly found with four or five cis acetyllysines and rarely in their absence,15 so the abundance of [H3K4me3K9acK14acK18acK23ac] >> [H3K4me3]. Figure 4 depicts the experimental formats used to resolve the multivalent nature of ZMYND11 (BS69) : histone binding37. While such graphical models are informative, there remains a need to concisely present distinct observations in prose. In this particular case, observed experimental binding of the ZMYND11 (Bromo-ZnF-PWWP) domain triplet is: H3.1[19–42]K36me3 < H3.3[19–42]K36me3 <<< ([H3.3K36me3]2).37,38 This communicates that ZMYND11 binds K36me3 on histone variant H3.3 (containing S31 vs. the A31 of H3.1), but such binding is dramatically enhanced in the nucleosome vs. peptide context (since the PWWP domain synergistically engages DNA).

Figure 4.

Figure 4.

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Molecular context impacts ZMYND11 (BD-ZnF-PWWP) binding. Blue: H3; Green: H4; Red: H2A; Yellow: H2B. Weak engagement is observed with H3.3[19–42]K36me3 over H3.1[19–42]K36me3 peptides. However, binding is dramatically enhanced on ([H3.3K36me3]2) nucleosomes. On a mechanistic level ZMYND11 PWWP is co-operatively engaging H3.3S31un, the K36me3 PTM and nucleosomal DNA.37 The still further complexity; that this binding is inhibited by S31 phosphorylation, as in ({H3.3S31phK36me3})48,49, is not depicted.

DISCUSSION

The proposed nomenclature is consistent with current usage and requires little to no change for most common applications, yet provides clarity and brevity for increasingly complex data and concepts. It can effectively describe a specific histone proteoform [H3K14acK18acK23acK27me3]. But it can also describe classes of proteoforms with shared attributes {H3K14acK27me3} that might represent a functional unit found in multiple proteoforms, including [H3K14acK18acK23acK27me3]. It can readily distinguish nucleosomes: homotypic ([H3K4me3K27me3]2) and cis or trans heterotypic ([H3]•[H3K4me3K27me3]) vs. ([H3K4me3]•[H3K27me3]). It can express functional relationships, such as [H4K20me2] is the preferred substrate of acetyltransferases writing H4K16ac, with the primary acetylated product [H4K16acK20me2]14 and subsequent observed states including [H4K12acK16acK20me2] and [H4K8acK12acK16acK20me2].

The new nomenclature can communicate experimental nuance (and potential limitations): H3K4un vs. H3K4me3 vs. H3[1–12]K4me3 vs. [H3.2K4me3] vs. ({H3K4me3}) vs. ([H3.2K4me3]2) vs. ([H3.2]•[H3.2K4me3]): an unmodified amino acid, a PTM, a modified peptide, a fully defined proteoform, a native nucleosome containing a PTM (such as enriched via same), a homotypic nucleoform and a heterotypic nucleoform. The language of histone PTMs has always been considered to operate by combinatorial means,2,26 and this nomenclature enables the user to posit hypotheses for such investigations, which can then be experimentally tested with defined proteoforms and nucleoforms. Ultimately, the nomenclature provides a clear path to even greater molecular specificity, such as additions to communicate species, linker length, linker histones, DNA modifications, non-canonical nucleosomes (e.g., tetrasomes or hexasomes39,40) (Table 1). However, the initial version is purposely focused on the immediate need to accurately describe core histone proteoforms, variants, oncomutants, PTMs and nucleoforms in chromatin, and thus enable the nuanced discussion of complex concepts and experimental data.

Finally, combinatorial modifications and differential associations are central to normal function and its dysregulation, from EGFR in cell growth and cancer, to Tau in axonal transport and neurodegeneration.6,41–44 The facile and expandable framework of our chromatin-focused nomenclature will provide a basis for colleagues in other fields to describe, and thus dissect, how proteoforms could be configured in functionally distinct complexes across biology.

Acknowledgment

Figures by Patrick Lane, ScEYEnceStudios.

ABBREVIATIONS

ChIP

Chromatin ImmunoPrecipitation

CUT&RUN

Cleavage Under Targets & Release Using Nuclease

MS

Mass spectrometry

PTM

Post-Translational Modification

Footnotes

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Statement of Interest

M.-C.K., M.T.B., O.G., and B.D.S. are board members of EpiCypher Inc.; a commercial developer of reagents and platforms for chromatin studies. All authors declare no conflicts of interest.

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