Despite the fact that histone H2A ubiquitination affects about 10-15% of this histone in most eukaryotic cells, histone ubiquitination is among one of the less-well-characterized post-translational histone modifications. Nevertheless, some important observations have been made in recent years. Whilst several enzymes had been known to ubiquitinate histones in vitro, recent studies in yeast have led to the unequivocal identification of the enzyme responsible for this post-translational modification in this organism. A strong functional co-relation to meiosis and spermiogenesis has also now been well documented, although its participation in other functional aspects of chromatin metabolism, such as transcription or DNA repair, still remains rather speculative and controversial. Because of its nature, histone ubiquitination represents the most bulky structural change to histones and as such it would be expected to exert an important effect on chromatin structure. Past and recent structural studies, however, indicate a surprising lack of effect of (H2A/H2B) ubiquitination on nucleosome architecture and of uH2A on chromatin folding. These results suggest that this modification may serve as a signal for recognition by functionally relevant trans-acting factors and/or operate synergistically in conjunction with other post-translational modifications such as for instance acetylation.

Histone H2A ubiquitination is a bulky posttranslational modification that occurs at the vicinity of the binding site for linker histones in the nucleosome. Therefore, we took several experimental approaches to investigate the role of ubiquitinated H2A (uH2A) in the binding of linker histones. Our results showed that uH2A was present in situ in histone H1-containing nucleosomes. Notably in vitro experiments using nucleosomes reconstituted onto 167-bp random sequence and 208-bp (5 S rRNA gene) DNA fragments showed that ubiquitination of H2A did not prevent binding of histone H1 but it rather enhanced the binding of this histone to the nucleosome. We also showed that ubiquitination of H2A did not affect the positioning of the histone octamer in the nucleosome in either the absence or the presence of linker histones.Despite the renewed interest in histone H2A/H2B ubiquitination (1-3), the functional role of uH2A 1 still remains controversial. This is in contrast to ubiquitinated H2B where a strong correlation with transcriptional activation has long been established (2), and even a molecular mechanism involving a transhistone regulatory pathway has been shown to be involved in this process (4, 5). With uH2A, there is almost as much experimental evidence for its association to actively transcribing chromatin as there is to the opposite. For instance, it was shown that nucleosomes from the transcriptionally poised hsp70 and copia genes from Drosophila contain 50% uH2A (6). Also nucleosomes containing mono-and diubiquitinated H2A were found to preferentially occur near the 5Ј-end of the transcribed mouse dihydrofolate reductase gene (7). On the other hand, early studies with rat liver nuclei linked the disappearance of uH2A to increased transcription (8), and it was shown that Drosophila nucleosomes consisting of non-transcribed 1.705 satellite DNA showed an enrichment in uH2A (9). In support of these early data, a role for histone uH2A in Polycomb silencing has been demonstrated recently (10). Furthermore uH2A has been shown to be concentrated in the heterochromatic inactive sex body in pachytene spermatocytes (11). Nevertheless the molecular mechanism(s) involved remains completely unknown.Despite the controversy, ubiquitinated histones have been associated with chromatin partially depleted of linker histones (12) and transcriptionally active or poised sequences (1, 2, 6, 7, 13) that are thought to be have a reduced linker histone content or have altered association with linker histones (for a review, see Ref. 14). However, there is evidence that suggests that histone H2A ubiquitination does not interfere with linker histone binding. Two independent studies have shown that H1 can be cross-linked to uH2A in vitro (15) and in vivo (16). Moreover, in the cross-linking studies performed in mouse cells, the molar ratio of H1-uH2A to H1-H2A was the same as the molar ratio of uH2A to H2A (16).Linker histones are required for the stabilization of well defined chromatin fibers (17-19), and interference with the binding of lin...

The MgCl 2 -induced folding of defined 12-mer nucleosomal arrays, in which ubiquitinated histone H2A (uH2A) replaced H2A, was analyzed by quantitative agarose gel electrophoresis and analytical centrifugation. Both types of analysis showed that uH2A arrays attained a degree of compaction similar to that of control arrays in 2 mM MgCl 2 . These results indicate that attachment of ubiquitin to H2A has little effect on the ability of nucleosomal arrays to form higher order folded structures in the ionic conditions tested. In contrast, uH2A arrays were found to oligomerize at lower MgCl 2 concentrations than control nucleosomal arrays, suggesting that histone ubiquitination may play a role in nucleosomal fiber association.Although for many years histones were thought to be merely structural components of nucleosomes, the primary level of DNA organization required to compact the genome in the nucleus, they are now recognized as important players in the mechanisms underlying gene expression. One of the keys to chromatin's dynamic nature is post-translational modification of the flexible histone tails. These modifications include acetylation, phosphorylation, methylation, and ubiquitination (1-3). Ubiquitin is a small, mainly globular and highly conserved protein consisting of 76 amino acids found, as its name implies, in most living organisms. Ubiquitin has been found to be conjugated in vivo to histones H2A, H2B, H3, and H1 (4 -7). Ubiquitin is reversibly attached to bovine H2A by means of an isopeptide bond between its terminal glycine and the ⑀ amino group of H2A lysine 119 (8), which lies in the trypsin-accessible region of the carboxyl-terminal tail (9). Histones are among the most abundant ubiquitin-protein conjugates in higher eukaryotes, where 5-15% of the total H2A is ubiquitinated (10). The function of histone ubiquitination remains unclear. Although ubiquitin has been shown to play an important role in the degradation of many short-lived proteins (for reviews see Refs. 11, 12), two independent studies have shown that ubiquitination does not tag histones for degradation (13,14). Nucleosomes can be reconstituted with two molecules of uH2A or uH2B 1 without obvious perturbation of the nucleosomal structure (15, 16). Although some studies have reported an enrichment of uH2A in transcriptionally poised or active chromatin (17, 18) others do not find this correlation (19 -21). Moreover, inhibition of transcription does not alter the levels of uH2A in a variety of cell lines (22-24), whereas inhibitors of hnRNA synthesis were found to cause a decrease in uH2B levels (23,24). Cell cycle studies have shown that, in cells undergoing mitosis, uH2A levels decrease progressively to non-detectable levels at metaphase but increase again in late anaphase (14,25,26). Based on these and other observations, several authors have proposed that H2A ubiquitination could perturb chromatin structure (e.g. Refs. 6, 17, 18), but until now this model has not been tested. In this report we analyze the folding of defined nucleosomal arr...

SummaryWe report the characterization of an Arabidopsis thaliana mutant, ups1, isolated on the basis of reduced expression of phosphoribosylanthranilate transferase, a tryptophan biosynthetic enzyme. ups1 also exhibits defects in a wide range of defence responses. After infection with Pseudomonas syringae or Botrytis cinerea, the expression of genes regulated by both the salicylic acid and jasmonic acid/ethylene pathways is reduced in ups1 compared with wild type. Camalexin accumulation in ups1 is greatly reduced after infection with these two pathogens, as well as after amino acid starvation or oxidative stress. Reactive oxygen species (ROS)-mediated gene expression is also compromised in ups1 indicating that this mutant is defective in signalling pathways activated in response to both biotic and abiotic stress. The fact that all three major defence signalling pathways are disrupted in ups1, together with the oxidative stress phenotype, leads us to suggest that UPS1 is involved in ROS signal transduction.

It is increasingly apparent that histone posttranslational modifications are important in chromatin structure and dynamics. However, histone ubiquitination has received little attention. Histones H1, H3, H2A, and H2B can be ubiquitinated in vivo, but the most prevalent are uH2A and uH2B. The size of this modification suggests some sort of structural impact. Physiological observations suggest that ubiquitinated histones may have multiple functions and structural effects. Ubiquitinated histones have been correlated with transcriptionally active DNA, implying that it may prevent chromatin folding or help maintain an open conformation. Also, in some organisms during spermiogenesis, a process involving extensive chromatin remodeling, uH2A levels increase just prior to histone replacement by protamines. Determination of chromatin's structural changes resulting from histone ubiquitination is therefore important. Recent work using reconstituted nucleosomes and chromatin fibers containing uH2A indicate that in the absence of linker histones, ubiquitination has little structural impact. DNase I digests and analytical ultracentrifugation of reconstituted ubiquitinated nucleosomes show no structural differences. Solubility assays using reconstituted chromatin fibers in the presence of divalent ions demonstrate that uH2A fibers are slightly more prone to aggregation than controls, and analytical ultracentrifugation results with different MgCl2 and NaCl concentrations determined that chromatin folding is not affected by this modification. Additional work to assess possible synergistic affects with histone acetylation also precludes any structural implications. Protamine displacement experiments concluded that the presence of uH2A does not significantly affect the ability of the protamines to displace histones. In addition, uH2A does not interfere with histone H1 binding to the nucleosome. While work with uH2B remains insufficient to come to any definitive conclusions about its structural impact, current work with uH-2A indicates that, contrary to predictions, this histone modification does not affect either nucleosome or chromatin structure. Consequently, the search for a structural role for ubiquitinated histones continues and their effect on and importance in chromatin dynamics remains elusive.