Unraveling the histone's potential

Abstract

Post translational modification (PTM) of histones has long been associated with epigenetic regulation. Although genomic approaches have established correlation between a handful of histone PTMs and transcriptional states, only recently have advancements in proteomics provided the tools necessary to study histone proteins and their relevant modifications in this context. Using mass spectrometry, researchers have demonstrated the ability to determine the full repertoire of histone PTMs, their residue specific location, the combinations in which they exist, and the proteins that interact with these combinations. Moving forward it will be imperative to develop novel approaches that combine proteomic and genomic technologies to determine the functional significance of these combinations of modifications. Assays with increased specificity will resolve more focused biological questions and determine to what extent, and by what mechanisms, histones influence transcription.

Over the last decade a variety of research has established a firm link between chromatin and transcription.^1-5 One such example is the extensive work on various histone post translational modifications (PTMs), which correlate strongly with dynamic transcriptional activity.^6-8 Since different histone PTMs associate with specific biological states in development and disease, untangling the role of a given modification or pattern of modifications is of critical importance to both fundamental science and medicine.^7,9-12 At this juncture, however, the mechanisms underlying this potential form of epigenetic regulation remain poorly understood—that is, how does a particular mark elicit a change in transcription on a molecular level or is histone modficiation merely a byproduct of other regulatory mechanisms? To answer these and related questions about the potential regulatory consequences of histone modifications it is imperative to develop new technologies and to combine genomic and proteomic approaches. Here, we offer our views on both state-of-the-art and emerging technologies with an emphasis on proteomics-based approaches for unraveling the role of histones in transcription.

Although it is difficult to establish direct causal links between histone modification and gene transcription, a number of key PTMs generally correlate with transcriptional activation or repression. For example, acetylation is largely associated with actively transcribed genes while methylation of H3K27 appear to localize largely to the promoters of repressed genes.^6,10,13,14 For a more exhaustive review, see Berger et al.¹⁵ These generalizations must be treated with care, however, since some modifications can exert different transcriptional outcomes depending on the surrounding microenvironment.^15-17 Considering the nucleosome alone, histone N-terminal tails are subject to myriad modifications—e.g., over 3 million combinations are possible on the histone H4 tail—and it is unclear how the crosstalk between different combinations of these modifications impact transcription. For example, what happens if both an activating and repressive mark are found on the same histone tail? The added complexity of histone variants, the extent to which DNA is packaged (i.e., is DNA fully saturated with histone?), and the dynamic nature of histone modifications convolute the situation. Historically, antibodies were used to identify histone modifications and, when used in conjunction with expression profiling, can determine a modifications' possible effects on transcription. Genomic approaches like chromatin immunoprecipitation (ChIP) have provided valuable insight into functionality associated with a given mark. That said, antibody-based approaches require a priori knowledge of a modification, can suffer from cross reactivity and epitope occlusion, and rarely detect more than a single PTM on a histone tail. Imagine histone modification patterns as complex sentences. Antibodies can perhaps recognize a few letters or a word, but this isolated piece of information lacks the context needed to interpret the sentence. To fully understand the complex regulatory consequences of a particular histone modification, we must also take the surrounding modifications into account.

Mass spectrometry (MS) is an effective tool for examining histone modifications because it (1) does not require a priori knowledge of a modification, (2) can provide quantitative information and (3) can unambiguously identify combinatorial modification patterns (i.e., multiple PTMs on a single molecule).^18-32 The position of a given PTM is determined via tandem MS (MS/MS), since it provides direct identification of a protein's primary sequence and the presence of a PTM alters the residue mass of an associated amino acid in a predictable way (i.e., phosphorylation adds 80 Daltons to serine, threonine or tyrosine).^20,33-38 A number of groups have employed proteolytic digestion of histone proteins to produce peptides that are sufficiently short for sequencing via collision activated dissociation (CAD) MS/MS.^30,39-42 The availability of high mass accuracy mass spectrometers aided this approach and allowed for the distinction between PTMs of similar mass, such as acetylation and trimethylation of lysines which differ by a mere 0.03638 Daltons.^21,42-45 These enzyme-based approaches allowed researchers to catalog sites of modifications on histones from a number of species but provided little information regarding the combinations of modifications present on a given histone molecule; that information is largely lost upon proteolytic digestion. Some groups omitted enzymatic digestion and tandem mass spectrometry altogether and relied on high mass accuracy detection of intact histone proteins to infer the modifications present.⁴⁶ Although this approach provides information on the presence of all PTMs on a given histone molecule, it can rarely be used to determine which residues are modified. The introduction of electron-based fragmentation methods—electron capture dissociation (ECD) and electron transfer dissociation (ETD)—now allow researchers to sequence large, basic and highly modified proteins like histones.^{23,26,28,33,47-50} By employing these fragmentation methods to large, n-terminal, tail peptides or even entire proteins it is possible to study the complete combinatorial space of histone modifications.

A handful of groups have used ECD and ETD mass spectrometry to investigate specific patterns of histone modifications. In 2004, Kellher et al. sequenced histone H4 proteins using a top down approach (i.e., without enzymatic digestion prior to analysis).⁴⁹ A high mass accuracy Fourier transform-ion cyclotron resonance mass spectrometer (FT-ICR MS) capable of ECD enabled the group to unambiguously assign ten discrete modification patterns that span the entire H4 tail. Not only did this work place multiple H4 modifications in context of one another, but it also pioneered a method for relative quantification of each H4 isoform. Thus, although this work identified only a modest number of isoforms, it served as an important starting point for further studies. Building upon this work, our own group and others have focused on the entire n-terminal tails of histones, where the majority of the modifications are present. Since a majority of the PTMs of interest are located within the first 20 amino acids of H4 or first 37 amino acids of H3, proteases were used to specifically cleave the N-terminal portion of these proteins. Hunt et al. pioneered this approach in 2005 on histone H3 from HeLa cells and later used it to identify more than fifty H3 isoforms in Tetrahymena thermophila.^48,51 A year later Garcia et al. identified over 150 n-terminal tail isoforms in HeLa cells using a similar approach.⁵² And recently, our own study yielded 74 distinct H4 isoforms in human embryonic stem cells.¹⁹

Closer inspection of these data sets revealed interesting patterns of histone modification. For instance, H3K4me3 and H3 hyperacetylation, both of which are commonly associated with transcriptional activation, are often found together in HeLa cells. Alternatively, H4R3 methylation was only observed in the presence of multiply methylated H4K20, an interesting observation since these marks are thought to have competing effects.¹⁹ One explanation for this is that opposing marks temper the transcriptional output of either mark alone. On the other hand, di- or tri- methylation of H4K20 may be prerequisite for the recruitment of factors that modify H4R3. The exact nature of these relationships and the subsequent transcriptional consequences remain to be determined; however, such findings provide evidence that histones are modified in a concerted and purposeful manner and beg the question: do specific patterns of modifications exhibit distinct function? Moving forward, it will be critical to seek out cross-disciplinary work that will apply the current technology to answer relevant biological questions like that posed above.

Investigation of histone isoforms in various biological samples has allowed for correlation with cell states. For example, Kelleher et al. used MS to examine the effects of cell cycle on histone isoforms in HeLa cells.^50,53 The group found that newly synthesized histone H4 is progressively methylated at lysine 20 as cells cycle and that this process is largely unaffected by acetylation. Meanwhile, our own group has quantitatively tracked histone H4 tail modifications as human embryonic stem (ES) cells differentiate.¹⁹ We found that acetylation on H4 tails decreases as cells progress through differentiation, while di-methyl isoforms increase steadily and unmethylated H4 drops precipitously. Note that these changes happen on the same time scale as ES cell commitment. Each of these experiments provide interesting ties between specific patterns of histone PTMs and biological states; however, none of these studies claim a casual relationship between the identified marks and the biology.

Correlative studies are a good foundation for answering biological questions; however, once a histone modification pattern is identified, more in depth studies will be necessary to better understand how these modifications elicit changes in transcription. There are different ways that modification of histones can alter gene expression. It is believed, for example, that addition or removal of acetylation alters chromatin structure, making it more or less accessible to transcriptional machinery.^54-56 Also, histones and their respective modifications may serve as binding partners for transcriptional regulators that, upon recognizing a certain mark, then exert their function at that specific location.^2,4,57-59 Several recent publications have examined the latter possibility in more depth. For example, Heo et al. overexpressed a tagged portion of histone H3 (residues 1-40), pulled down the tagged histone and its associated protein complexes, and analyzed the interacting proteins via mass spectrometry. The group observed a number of relevant proteins, including PRMT5, an arginine methyl-transferase, and several constituents of the histone deacetylase complex.⁶⁰ Unfortunately, however, since the group had no knowledge of the recombinant histones' modifications, it was not possible to determine which histone PTMs correspond to the interacting proteins. Using a more directed approach, Wysocka et al. synthesized biotin-linked histone H3 tails with known modifications. These tails were then incubated with cell lysate and, following washing and elution, the resulting samples were analyzed via mass spectrometry to characterize proteins that bind the respective H3 isoforms. From this study, the group identified WDR5, a protein that binds with high specificity to hypermethylated forms of H3K4.⁶⁰ Interestingly, WDR5 appears to be involved in activation of certain HOX genes and injection of a WDR5 morpholino into Xenopus embryos resulted in developmental defects.⁵⁷ By characterizing a set of known histone modification-specific interactions and examining the binding partners' cellular functions, more direct connections can be made between a given histone mark and the resulting biological function. Once this is established, it is possible to gain even more information by applying quantitative mass spectrometry to compare lysates from two different biological treatments or conditions (i.e., are particular binding partners increased in disease vs. normal tissue?).

A major problem with the techniques described above is the global nature inherent in the experiments. More simply put, the entire population of proteins, microenvironments and genomic loci within the cell are sampled together, making it difficult to assign functional meaning to the results. ChIP is a notable exception and the genome-specific information obtained from this kind of analysis is certainly one reason that the technique is so powerful. However, even ChIP is limited since typically, only a single histone modification can be probed at a time. Once again, this limits analysis since the modification's context in relation to the surrounding PTMs is unkown. The next step in this field may be finding a way to perform proteomics on the level of a single genomic locus. This would open the door to numerous meaningful experiments, whether considering histone modification patterns at insulator regions or examining histone binding proteins at intron-exon junctions. As analytical instrumentation improves, these experiments are becoming more feasible and we inch closer to unraveling the histone regulatory role. More specifically, improved technology and cross-disciplinary work should help to determine whether particular histone modifications exert specific transcriptional regulation or simply correlate with gene expression. It seems likely that both occur and we must be cognizant that a given modification's function may change based on cell line, the cellular environment, cell cycle, etc. Improving technology, together with combined genomic and proteomic approaches will help to fill in these gaps.