Identification of ubiquitinated histones 2A and 2B in Physarum polycephalum. Disappearance of these proteins at metaphase and reappearance at anaphase

Ubiquitinated histones uH2A.1, uH2A.Z, and uH2B have been identified in the basic nuclear proteins of the slime mold Physarum polycephalum by three methods: peptide mapping, cross-reaction with anti-ubiquitin antibody, and uH2A and uH2B isopeptidase cleavage. In microplasmodia, uH2A amounts to 7% of H2A and uH2B amounts to 6% of H2B. Detailed studies of mitosis in Physarum polycephalum macroplasmodia show that in early prophase, which last 15 min, the uH2As and uH2B are both strongly present, whereas minutes later in metaphase, which lasts 7 min, they disappear. When the nuclei enter anaphase, which lasts 3 min, both the uH2As and uH2B reappear. These precise studies suggest that cleavage of ubiquitin from the uH2As and uH2B is a very late, possibly final event in chromosome condensation to metaphase chromosomes and that ubiquitination is an early event in their decondensation. It is proposed that the uH2A and uH2B mark specific regions of the genome which have to be deubiquitinated prior to packaging into metaphase chromosomes; after metaphase these regions are the first to be decondensed and ubiquitinated. This modification, however, is not thought to be a general factor in chromosome condensation but labels a specific subcomponent of chromatin containing the expressed genes of a particular cell type or an important subset of these genes required by the cell to be available for activation, e.g. stress genes.     

The ubiquitinated histone species are enriched in histone H1-depleted chromatin regions

Bovine thymus and trout testis chromatin were fractionated into regions which differed in their micrococcal nuclease accessibility and solubility properties, and the distribution of the ubiquitinated histone species among these chromatin regions was elucidated. Ubiquitinated (u) species of histones H2A and H2B were enriched in the nuclease-sensitive, low-ionic-strength, soluble fraction of both chromatins. These results indicate that the presence of ubiquitinated histones may alter nucleosome-nucleosome interactions and destabilize higher-order chromatin structures. Bovine thymus chromatin was separated into aggregation-resistant, salt-soluble and aggregation-prone, salt-insoluble chromatin fractions. The aggregation-resistant chromatin fraction depleted in H1 histones was enriched in uH2A and uH2B, with uH2B showing the greater enrichment. The chromatin fragments were also stripped and reconstituted with the H1 histones prior to fractionation. The results were the same as above: uH2A and uH2B were preferentially localized in the aggregation-resistant. H1-depleted chromatin fraction, suggesting that chromatin regions enriched in ubiquitinated histone species have a reduced affinity for the H1 histones. Thus, ubiquitinated histone species may be one of the contributing factors in the differential assembly of various parts of the genome.     

Caspase-dependent deubiquitination of monoubiquitinated nucleosomal histone H2A induced by diverse apoptogenic stimuli.

Enzymatic deubiquitination of mono-ubiquitinated nucleosomal histone H2A (uH2A) and H2B (uH2B) is closely associated with mitotic chromatin condensation, although the function of this histone modification in cell division remains ambiguous. Here we show that rapid and extensive deubiquitination of nucleosomal uH2A occurs in Jurkat cells undergoing apoptosis initiated by anti-Fas activating antibody, staurosporine, etoposide, doxorubicin and the proteasome inhibitor, N-acetyl-leucyl-leucyl-norlucinal. These diverse apoptosis inducers also promoted the accumulation of slowly migrating, high molecular weight ubiquitinated proteins and depleted the cellular pool of unconjugated ubiquitin. In apoptotic cells, ubiquitin was cleaved from uH2A subsequent to the appearance of plasma membrane blebbing, and deubiquitination of uH2A closely coincided with the onset of nuclear pyknosis and chromatin condensation. Nucleosomal uH2A deubiquitination, poly (ADP-ribose)polymerase (PARP) cleavage and chromatin condensation were prevented in cells challenged with apoptosis inducers by pretreatment with the pan-caspase inhibitor, zVAD-fmk, or by over-expressing anti-apoptotic Bcl-xL protein. These results implicate a connection between caspase cascade activation and nucleosomal uH2A deubiquitination. Transient transfection of 293 cells with the gene encoding Ubp-M, a human deubiquitinating enzyme, promoted uH2A deubiquitination, while an inactive mutated Ubp-M enzyme did not. However, Ubp-M-promoted deubiquitination of uH2A was insufficient to initiate apoptosis in these cells. We conclude that uH2A deubiquitination is a down-stream consequence of procaspase activation and that unscheduled cleavage of ubiquitin from uH2A is a consistent feature of the execution phase of apoptosis rather than a determining or initiating apoptogenic event. Nucleosomal uH2A deubiquitination may function as a cellular sensor of stress in situations like apoptosis through which cells attempt to preserve genomic integrity.     

Human USP3 is a chromatin modifier required for S phase progression and genome stability

Protein ubiquitination is critical for numerous cellular functions, including DNA damage response pathways. Histones are the most abundant monoubiquitin conjugates in mammalian cells; however, the regulation and the function of monoubiquitinated H2A (uH2A) and H2B (uH2B) remain poorly understood. In particular, little is known about mammalian deubiquitinating enzymes (DUBs) that catalyze the removal of ubiquitin from uH2A/uH2B. Here we identify the ubiquitin-specific protease 3 USP3 as a deubiquitinating enzyme for uH2A and uH2B. USP3 dynamically associates with chromatin and deubiquitinates H2A/H2B in vivo. The ZnF-UBP domain of USP3 mediates uH2A-USP3 interaction. Functional ablation of USP3 by RNAi leads to delay of S phase progression and to accumulation of DNA breaks, with ensuing activation of DNA damage checkpoint pathways. In addition, we show that in response to ionizing radiation, (1) uH2A redistributes and colocalizes in gamma-H2AX DNA repair foci and (2) USP3 is required for full deubiquitination of ubiquitin-conjugates/uH2A and gamma-H2AX dephosphorylation. Our studies identify USP3 as a novel regulator of H2A and H2B ubiquitination, highlight its role in preventing replication stress, and suggest its involvement in the response to DNA double-strand breaks. Together, our results implicate USP3 as a novel chromatin modifier in the maintenance of genome integrity.     

Chromatin restoration following nucleotide excision repair involves the incorporation of ubiquitinated H2A at damaged genomic sites

Restoration of functionally intact chromatin structure following DNA damage processing is crucial for maintaining genetic and epigenetic information in human cells. Here, we show the UV-induced uH2A foci formation in cells lacking XPC, DDB2, CSA or CSB, but not in cells lacking XPA, XPG or XPF indicating that uH2A incorporation relied on successful damage repair occurring through either GGR or TCR sub-pathway. In contrast, XPA, XPG or XPF were not required for formation of γH2AX foci in asynchronous cells. Notably, the H2A ubiquitin ligase Ring1B, a component of Polycomb repressor complex 1, did not localize at DNA damage sites. However, histone chaperone CAF-1 showed distinct localization to the damage sites. Knockdown of CAF-1 p60 abolished CAF-1 as well as uH2A foci formation. CAF-1 p150 was found to associate with NER factors TFIIH, RPA p70 and PCNA in chromatin. These data demonstrate that successful NER of genomic lesions and prompt CAF-1-mediated chromatin restoration link uH2A incorporation at the sites of damage repair within chromatin.     

组蛋白H2B单泛素化参与DNA损伤修复的调控机制

作为一种重要的组蛋白修饰形式,H2B的单泛素化(uH2B)广泛地参与DNA复制、基因的表达与转录、DNA损伤修复及异染色质维持等生物学事件.在裂殖酵母中,H2B的单泛素化发生在其羧基端的119位赖氨酸(K119),并依赖于Rhp6/Bre1泛素连接酶复合体.研究表明,uH2B通过破坏H2A/H2B二聚体的结构促进mRNA在转录过程中的延伸,同时促进H3K4的三甲基化激活基因的表达及参与DNA损伤修复.本研究发现,Rhp6能够对核糖核苷酸还原酶抑制基因(Spd1)位点进行活跃的染色质修饰,促进H2B的单泛素化并抑制基因表达,从而促进dNTP的合成并调控DNA复制及损伤修复.重要的是,本研究发现,该过程不依赖于H3K4而决定于H3K9的三甲基化.同时uH2B直接在DNA双链断裂位点富集,通过改变染色质的结构参与DNA损伤修复,该过程中可能存在其他更为复杂的分子机制.     

Chicken erythrocyte polynucleosomes which are soluble at physiological ionic strength and contain linker histones are highly enriched in beta-globin gene sequences.

Mature chicken erythrocyte polynucleosomes which are soluble at physiological ionic strength are enriched in beta-globin DNA sequences. Vitellogenin chromatin, which is not expressed in this tissue, is found in aggregation prone, salt insoluble chromatin. There is a direct correlation between the size of soluble fragments and the degree of globin gene enrichment, with the largest fragments being most highly enriched. The highly globin enriched (about 50 fold) polynucleosomes contain significantly elevated levels of acetylated histones H4, H2A.Z, and H2B, and ubiquitinated (prefix "u") histones H2A and H2B (with a significant relative increase of uH2B over uH2A). These polynucleosomes were complexed with histones H1 and H5 but at a lower level than that found in unfractionated chromatin.     

Satellite DNA-correlated nucleosomal proteins in Drosophila virilis

Three major satellite DNAs comprise 40–45% of the genome of Drosophila virilis. Since these satellites are not substrates for most restriction enzymes, we were able to digest D. virilis nuclei with HaeIII and micrococcal nuclease and isolate chromatin fractions containing variable levels of satellite DNA. Electrophoretic analysis of these chromatin fractions revealed that the level of the acid-soluble chromosomal protein, cp17.3, was directly related to the percentage of satellite DNA in chromatin. The correlation between cp17.3 and satellite DNA abundance suggests that cp17.3 is involved in the heterochromatic condensation of satellite DNAs. cp17.3 occurs at a frequency of one molecule per 10–20 nucleosomes. It is detected in an electrophoretically distinguishable class of mononucleosomes, provisionally identified as MN1uH2A, which contains ubiquitinated histone H2A (uH2a) but lacks histone H1. It is not detected in MN1, a second class of mononucleosomes, which lacks uH2A and H1. Since cp17.3 is correlated with satellite DNAs and present in nucleosome cores, it might be a histone variant specifically associated with satellite DNAs.This work was supported by Grant GM22138 from the National Institutes of Health. G.A.V. was a predoctoral trainee supported by Grant GM07094 from the National Institutes of Health.     

Polycomb group proteins Ring1A/B link ubiquitylation of histone H2A to heritable gene silencing and X inactivation

In many higher organisms, 5%-15% of histone H2A is ubiquitylated at lysine 119 (uH2A). The function of this modification and the factors involved in its establishment, however, are unknown. Here we demonstrate that uH2A occurs on the inactive X chromosome in female mammals and that this correlates with recruitment of Polycomb group (PcG) proteins belonging to Polycomb repressor complex 1 (PRC1). Based on our observations, we tested the role of the PRC1 protein Ring1B and its closely related homolog Ring1A in H2A ubiquitylation. Analysis of Ring1B null embryonic stem (ES) cells revealed extensive depletion of global uH2A levels. On the inactive X chromosome, uH2A was maintained in Ring1A or Ring1B null cells, but not in double knockout cells, demonstrating an overlapping function for these proteins in development. These observations link H2A ubiquitylation, X inactivation, and PRC1 PcG function, suggesting an unanticipated and novel mechanism for chromatin-mediated heritable gene silencing.     

Structure of chromatin from the pigeon brain. I. Composition, electrophoretic motility and circular dichroism spectra of nucleosome particles

Composition and structural properties of pigeon brain hemisphere neuron chromatin have been studied. In these cells nucleosomal DNA repeat length is about 165 nucleotide pairs. The content of H1 and H2A histones was found to decrease by 18 and 30% respectively in comparison with the chromatin possessing the normal quantity of those histones. At the same time the content of protein uH2A (A-24), being the conjugate of H2A histone and ubiquitine, is increased. Mononucleosomes isolated from neuron chromatin was found to have relatively low electrophoretic mobility in polyacrylamide gel taking into account the size of their DNA fragment. Circular dichroism spectra of nucleosome particles show that the neuron mononucleosomes are more unfolded than the rat thymus ones. Data obtained allow to suggest that the short DNA repeat and accumulation of protein uH2A in neurons are the factors influencing the compactization of neuron chromatin.     

Structure of polyubiquitinated histone H2A

We have recently demonstrated that trout liver histones H2A, H2B, and H2A.Z can be polyubiquitinated [Davie, J.R., Delcuve, G.P., Nickel, B.E., Moyer, R., & Bailey, G. (1987) Cancer Res. 47, 5407-5410]. In the present study we determined the arrangement of the ubiquitin molecules in polyubiquitinated histone H2A. Trout liver chromatin fragments. which had histone H1 removed, were digested with Staphylococcus aureus (V8 strain) protease which cleaves specifically on the carboxyl side of glutamic acid residues under the conditions used. The V8 protease readily degraded histone H2A and ubiquitinated (u) H2A at equivalent rates. One site in H2A and uH2A, the peptide bond between Glu 121 and Lys 122, was cleaved, yielding protein species cH2A and cuH2A, respectively. None of the other nucleosomal histones (H2B, H2A.Z, H3, and H4) including uH2B and uH2A.Z were sensitive to digestion. Trout liver histones cleaved with either V8 protease, histone H2A specific protease, or cyanogen bromide were resolved by two-dimensional gel electrophoresis and ubiquitinated peptides detected with anti-ubiquitin IgG. The results suggest that the major arrangement of ubiquitin in polyubiquitinated H2A is a chain of ubiquitin molecules joined to each other by isopeptide bonds to a ubiquitin molecule that is attached to the epsilon-amino group of lysine 119 of histone H2A.     

A haploid affair: core histone transitions during spermatogenesis.

The process of meiosis reduces a diploid cell to four haploid gametes and is accompanied by extensive recombination. Thus, the dynamics of chromatin during meiosis are significantly different than in mitotic cells. As spermatogenesis progresses, there is a widespread reorganization of the haploid genome followed by extensive DNA compaction. It has become increasingly clear that the dynamic composition of chromatin plays a critical role in the activities of enzymes and processes that act upon it. Therefore, an analysis of the role of histone variants and modifications in these processes may shed light upon the mechanisms involved and the control of chromatin structure in general. Histone variants such as histone H3.3, H2AX, and macroH2A appear to play key roles in the various stages of spermiogenesis, in addition to the specifically modulated acetylation of histone H4 (acH4), ubiquitination of histones H2A and H2B (uH2A, uH2B), and phosphorylation of histone H3 (H3p). This review will examine recent discoveries concerning the role of histone modifications and variants during meiosis and spermatogenesis.     

Histone synthesis and deposition in the G1 and S phases of hepatoma tissue culture cells

Hepatoma tissue culture cells were synchronized in G1 and in S phase in order to examine the level of synthesis of different histone types and to determine the rate, timing, and location of their deposition onto DNA. We observe a basal level of synthesis in G1 (5% of that seen in S phase) for H2A.1, H2A.2, H3.2, H2B, and H4. The minor histone variants X and Z are synthesized at 30% of the rate observed in S cells. The rate of synthesis of the ubiquinated histones uH2A.1,2 is not as depressed in G1 cells as seen for H2A.1 and H2A.2. Histones synthesized in G1 are not deposited on the DNA of these cells at equivalent rates. Thus, histones H3.2 and H4 are not deposited significantly until S phase begins, at which time deposition occurs selectively on newly synthesized DNA. The deposition of H2A.1, H2A.2, H2B, X, and Z proceeds in G1; however, it occurs to a 2-4-fold lower extent than seen for the deposition of H1, HMG 14, and HMG 17. The deposition of all histones synthesized in S phase occurs rapidly, but there are variations in the sites of deposition. Thus, newly synthesized H3.1, H3.2, and H4 deposit primarily on newly replicated DNA whereas H2A.1, H2A.2, uH2A.1, 2, and H2B deposit only partially on new DNA (30%) and mostly on old. H1, HMG 14, and HMG 17 are deposited in an apparently fully random manner over the chromatin. To interpret these observations, we propose a model which includes a measure of histone exchange on the chromatin fiber. The model emphasizes the dynamics of histone-histone and histone-DNA interactions in regions of active genes and at replication forks.