In vivo assays to study histone ubiquitylation
Introduction
Once thought to provide merely a structural role in compacting chromatin, nucleosomes are now recognized as important players in all processes that occur on eukaryotic chromosomes [1], [2]. Chromatin is in fact highly dynamic, undergoing global changes in folding during mitosis and local changes in folding during gene expression. Posttranslational modifications of histones, the protein constituents of nucleosomes, contribute to the dynamic nature of chromatin [3], [4]. These modifications, which include acetylation, phosphorylation, and methylation, are generally targeted to the flexible N-terminal tails of the four core histones, where their presence leads to chromatin states that are either “open” or “closed” with respect to transcriptional activity [5], [6], [7]. The histones H2A, H2B, H3, H2A.Z, and H1 are also modified by ubiquitylation, in which the 76-amino-acid protein ubiquitin is attached via an isopeptide linkage between its terminal glycine residue and an ε amino group of lysine in the histone protein [8], [9], [10], [11], [12], [13], [14], [15]. Acceptor lysines have been identified in the C-terminal tails of histones H2A (lysine 119) and H2B (lysine 120 in vertebrates and lysine 123 of yeast), but the sites of ubiquitin attachment in other histones remain unknown [13], [16], [17], [18]. Ubiquitylated histones are found in a wide range of organisms and cell types, with estimates of their abundance ranging from ∼1% to 15% of unmodified histones [8], [11], [13], [14], [19]. Like acetylation, ubiquitylation is a highly dynamic and reversible histone modification. In cycling vertebrate cells ubiquitylated histones have been found to turn over rapidly, and during mitosis there is a global cycle of histone deubiquitylation [11], [12], [20], [21], [22], [23], [24]. Deubiquitylation may be accomplished by a family of ubiquitin-specific proteases or Ubps, some of which may target histones [25], [26], [27].
Although protein ubiquitylation is a widely used mechanism to target proteins for degradation, histone ubiquitylation appears to play a different role [26], [28], [29], [30]. Histones are predominantly monoubiquitylated in vivo, a modification that, unlike polyubiquitylation, is not associated with protein turnover. Moreover, ubiquitylated histones have been identified as stable constituents of nucleosomes in chromatin isolated from both fly and vertebrate cells [31], [32], [33], [34]. The effect of histone ubiquitylation on chromatin structure is largely unknown [35]. Mononucleosomes reconstituted in vitro from ubiquitylated H2A or H2B (uH2A or uH2B) appear similar in many parameters to nucleosomes containing unmodified histones [36]. Because ubiquitin is a bulky moiety, it has been postulated that its attachment to histones could disrupt chromatin folding [35], [37]. This argument tends to be supported by the observation that metazoan histones are globally deubiquitylated at metaphase, when chromatin becomes highly compacted [11], [21]. However, in the absence of linker histones, nucleosome arrays reconstituted in vitro from ubiquitylated H2A show degrees of folding similar to those of arrays formed from unmodified H2A [38]. Thus, histone ubiquitylation could form part of the so-called “histone code,” acting as a tag or recognition element to direct proteins such as chromatin remodeling factors, transcription factors, and repair and replication factors to specific chromosomal domains [39]. The notion that ubiquitylated histones serve as a mark to direct the localization of specific factors to chromatin is supported by recent studies showing that yeast uH2B has differential effects on the methylation of adjacent lysine residues in the H3 N tail by several SET domain proteins [40], [41], [42], [43]. These results are not consistent with a model in which histone ubiquitylation merely serves a structural role to open chromatin.
Ubiquitylation of proteins destined for degradation occurs through a concerted series of enzymatic reactions initiated by a ubiquitin-activating enzyme [28], [29], [30]. Activated ubiquitin is then attached via a thiolester linkage to a cysteine residue in one of many different ubiquitin-conjugating enzymes (Ubcs) or E2s. Ubiquitin is finally transferred to a lysine residue in target proteins through the activity of a family of ubiquitin ligases or E3s, which have either catalytic or regulatory roles. These latter factors confer specificity by targeting ubiquitin attachment to particular substrates. Until recently, the factors involved in histone ubiquitylation were unknown. The most likely candidate for a histone-specific Ubc was Rad6, an evolutionarily conserved ubiquitin-conjugating enzyme that is able to ubiquitylate free histones in vitro [44], [45], [46], [47], [48]. Rad6 was shown to be required for monoubiquitylation of yeast H2B in vivo, although it is not known if it performs a similar role in other eukaryotic cells [13], [49]. In vitro, Rad6 ubiquitylates histones in the absence of an E3 [44], [45], [48]. Three E3s that interact directly or indirectly with Rad6 (Rad18, Rad5, and Ubr1) in vivo are dispensable for H2B ubiquitylation in yeast [41]. While this suggests that Rad6 ubiquitylates cellular H2B in the absence of an E3, the dynamic changes in the levels of ubiquitylated histones during transcription and other cellular processes argues for the existence of specificity factors [8], [13], [33], [49], [50], [51], [52]. Support for this view comes from two recent reports that have identified the yeast Bre1 protein as an E3 that appears to target Rad6 to H2B at a constitutively transcribed gene [53], [54].
The biological roles of ubiquitylated histones appear to be wide and varied. Both uH2A and uH2B have been localized to nucleosomes that flank transcriptionally active genes [31], [33], [50]. This has led to the hypothesis that histone ubiquitylation might be a prerequisite for activated transcription, although transcription itself might also create a chromatin environment that is permissive for ubiquitylation [55], [56]. Recent studies have also implicated uH2B in both gene repression and heterochromatic gene silencing [40], [41], [42], [43], [57]. uH2A, uH2B, and uH3 have each been connected to meiosis, and uH2A has been shown to be concentrated in replication foci in some transformed cell types [8], [13], [49], [52]. In addition, the global cycle of histone deubiquitylation/ubiquitylation that occurs during metazoan mitoses implicates this histone modification in aspects of chromosome segregation [11]. Finally, yeast H2B has shown to have a regulatory role with respect to the methylation of specific lysine residues in histone H3 [40]. However, it is important to point out that in no case is it known how ubiquitylated histones function. As discussed above, this function could be structural or regulatory, or some combination of both mechanisms.
For researchers interested in histone modifications, there are a number of questions relating to the presence of ubiquitylated histones in eukaryotic cells. Which histones are ubiquitylated? How abundant are these modified histones? How is histone ubiquitylation regulated and distributed in vivo? What are the biological roles of ubiquitylated histones? Key to answering many of these questions is a sensitive method to detect ubiquitylated histones in the cell. Antibodies against specific acetylated lysine residues in histones have been instrumental in unraveling the regulation and biological roles of these particular histone modifications. However, only a few antibodies have been described that specifically recognize ubiquitylated histones and only antibodies against ubiquitylated H2A have become commercially available [52]. Nonetheless, detection of ubiquitylated histones is possible using antibodies that recognize free ubiquitin and ubiquitin–protein conjugates, as well as through genetic approaches that use epitope-tagged histones and ubiquitin. In the following section, several methods are outlined to detect ubiquitylated histones in vivo in two eukaryotic organisms. Emphasis is placed on detection of these modified histones in the budding yeast, Saccharomyces cerevisiae, where genetic approaches have provided sensitive assays for the regulation and biological roles of ubiquitylated histones [13].
Section snippets
Identification of ubiquitylated histones in budding yeast
The low histone gene copy number in yeast, coupled with the genetic tractability of this organism, has made it possible to replace chromosomal histone genes with epitope-tagged versions of these genes. Yeast strains have been constructed that contain a single H2B gene with a Flag epitope at its amino terminus, which does not appear to interfere with the function of H2B in cell growth or other measurable in vivo processes [56]. The Flag epitope provides a convenient immunological tag to identify
Concluding remarks
Much progress has been made in understanding the regulation, distribution, and cellular roles of acetylated histones because of the existence of antibodies against histones modified on specific lysine residues. In the absence of widely available antibodies against ubiquitylated histones, other immunological approaches outlined in this article can be effective in detecting these forms of modified histones. Theoretically, these approaches should be sensitive enough to detect ubiquitylated
Acknowledgements
Judith Recht and Kenneth Robzyk are thanked for their seminal contributions to the detection and identification of uH2B in yeast. Zu-Wen Sun is gratefully acknowledged for providing the protocol for the immunoprecipitation and elution of Flag-H2B from yeast chromatin, Pamela Meluh is thanked for her modifications of yeast lysate preparations, and Rosa Bermudez is thanked for technical information. The work in this article from the authors’ laboratory was supported by Grant GM40118 from the NIH.
References (66)
- et al.
Curr. Opin. Genet. Dev.
(2001) - et al.
Trends Biochem. Sci.
(2000) - et al.
Gene
(1999) - et al.
J. Biol. Chem.
(1998) - et al.
J. Biol. Chem.
(1975) - et al.
Biochem. Biophys. Res. Commun.
(1975) - et al.
J. Biol. Chem.
(1985) - et al.
Biochem. Biophys. Res. Commun.
(1977) - et al.
Cell Differ.
(1986) - et al.
Biochem. Biophys. Res. Commun.
(1980)
J. Biol. Chem.
Curr. Opin. Cell Biol.
Trends Biochem. Sci.
J. Biol. Chem.
Cell
Biochim. Biophys. Acta
J. Biol. Chem.
J. Biol. Chem.
J. Biol. Chem.
J. Biol. Chem.
J. Biol. Chem.
Dev. Biol.
Mol. Cell.
Mol. Cell.
Biochem. Biophys. Res. Commun.
J. Biol. Chem.
Methods
Mol. Cell.
J. Biol. Chem.
Anal. Biochem.
Crit. Rev. Eukaryot. Gene Expr.
Bioessays
Cold Spring Harbor Symp. Quant. Biol.
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