CAF-1-induced oligomerization of histones H3/H4 and mutually exclusive interactions with Asf1 guide H3/H4 transitions among histone chaperones and DNA

Anti-silencing function 1 (Asf1) and Chromatin Assembly Factor 1 (CAF-1) chaperone histones H3/H4 during the assembly of nucleosomes on newly replicated DNA. To understand the mechanism of histone H3/H4 transfer among Asf1, CAF-1 and DNA from a thermodynamic perspective, we developed and employed biophysical approaches using full-length proteins in the budding yeast system. We find that the C-terminal tail of Asf1 enhances the interaction of Asf1 with CAF-1. Surprisingly, although H3/H4 also enhances the interaction of Asf1 with the CAF-1 subunit Cac2, H3/H4 forms a tight complex with CAF-1 exclusive of Asf1, with an affinity weaker than Asf1–H3/H4 or H3/H4–DNA interactions. Unlike Asf1, monomeric CAF-1 binds to multiple H3/H4 dimers, which ultimately promotes the formation of (H3/H4)₂ tetramers on DNA. Thus, transition of H3/H4 from the Asf1-associated dimer to the DNA-associated tetramer is promoted by CAF-1-induced H3/H4 oligomerization.     

Conservative segregation of tetrameric units of H3 and H4 histones during nucleosome replication

We have specifically investigated the behavior of H3 and H4 histones during the replication cycle of MH-134SC cells. Mononucleosomes obtained from cells density-labeled with IdU or dense amino acids in the presence of appropriate radiolabeled precursors were applied to sucrose gradients containing 0.3 M NaCl and 4 M urea for rate zonal centrifugation. This allowed the resolution of dense and normal subnucleosome particles composed of DNA and two molecules each of H3 and H4 without any measurable interparticle histone exchange. On labeling with dense amino acids and radiolabeled lysine, a distinct peak of radiolabeled dense particles was obtained. In contrast, pre-radiolabeled H3 and H4 remained in the normal subnucleosome peak region even after one generation time of culturing with dense amino acids. These data indicate the formation of (H3-H4)2 tetramers composed entirely of new H3 and H4 molecules as well as the conservation of pre-existing tetramers. Density labeling for 1 h with IdU in the presence of radiolabeled lysine yielded a distinct peak of radiolabeled dense particles, indicating the deposition of new tetramers on newly replicated DNA. Similar rate zonal analysis of subnucleosome particles obtained from cells prelabeled for 1 h with radiolabeled lysine followed by various IdU-labeling schedules in nonisotopic media yielded data suggesting that tetramers once deposited do not move about randomly during the replication cycle. A possible mode of nucleosome replication is discussed in the light of the present data.     

Polybromo-1-bromodomains bind histone H3 at specific acetyl-lysine positions

The human polybromo-1 protein is thought to localize the Polybromo, BRG1-associated factors chromatin-remodeling complex to kinetochores during mitosis via direct interaction of its six tandem bromodomains with acetylated nucleosomes. Bromodomains are acetyl-lysine binding modules roughly 100 amino acids in length originally found in chromatin associated proteins. Previous studies verified acetyl-histone binding by each bromodomain, but site-specificity, a central tenet of the histone code hypothesis, was not examined. Here, the acetylation site-dependence of bromodomain-histone interactions was examined using steady-state fluorescence anisotropy. Results indicate that single bromodomains bind specific acetyl-lysine sites within the histone tail with sub-micromolar affinity. Identification of duplicate target sites suggests that native Pb1 interacts with both copies of histone H3 upon nucleosome assembly. Quantitative analysis of single bromodomain-histone interactions can be used to develop hypotheses regarding the histone acetylation pattern that acts as the binding target of the native polybromo-1 protein.     

The interaction of histone H3 with histone H4 and with other histones studied by 19F nuclear magnetic resonance.

The behaviour, upon variations in ionic strength, pH and temperature of 19F nuclear nuclear magnetic resonance signals of the trifluoroacetonylated derivative of histone H3 is compared with those of the H3-H4 complex and of the Hv fraction (an equimolar mixture of H2A, H2B, H3 and h4). The line width of the 19F-labelled histone H3 signals increases with ionic strength or pH, an effect consistent with aggregation of the protein. In the case of H3-H4 complex or Hv the line width decreases at intermediate ionic strengths (0.1-0.25 M NaCl). This effect is interpreted as the consequence of the formation of a well defined structure with ionic strength. At high salt concentrations the line width increases as a consequence of the final rigid quaternary structure or of the formation of higher aggregates.     

ADP-ribosylation of pancreatic histone H1 and of other histones

Incubation of pancreatic nuclei with high NAD concentrations resulted in increased ADP-ribosylation of histone H1. Interaction of [3H]ADP-ribosylated histone H1 with chromatin was significantly different from unmodified histone H1. The presence of a protein which is eluted at a lower salt concentration and which is ADP-ribosylated was also noticed. Pancreatic histones were isolated by column chromatography and their degree of ADP-ribosylation evaluated both by gel electrophoresis and by chromatography: histone H1 was the main acceptor while the core histones H3, H2B, and H2A were lightly labelled. Histones H1 and H1(0) have a differential binding to pancreatic chromatin and histone H1(0) is not ADP-ribosylated.     

The Rtt109-Vps75 histone acetyltransferase complex acetylates non-nucleosomal histone H3

Acetylation of lysine 56 of histone H3 (H3-Lys-56) occurs in S phase and disappears during G(2)/M phase of the cell cycle. However, it is not clear how this modification is regulated during the progression of the cell cycle. We and others have shown that the histone acetyltransferase (HAT) Rtt109 is the primary HAT responsible for acetylating H3-Lys-56 in budding yeast. Here we show that Rtt109 forms a complex with Vps75 and that both recombinant Rtt109-Vps75 complexes and native complexes purified from yeast cells acetylate H3 present in H3/H4/H2A/H2B core histones but not other histones. In addition, both recombinant and native Rtt109-Vps75 HAT complexes exhibited no detectable activity toward nucleosomal H3, suggesting that H3-Lys-56 acetylation is at least in part regulated by the inability of Rtt109-Vps75 complexes to acetylate nucleosomal H3 during G(2)/M phase of the cell cycle. Further, Rtt109 bound mutant H3/H4 tetramers composed of histones lacking their N-terminal tail domains less efficiently than wild-type H3/H4 tetramers, and Rtt109-Vps75 complexes displayed reduced HAT activity toward these mutant H3/H4 tetramers. Thus, the N termini of H3/H4 tetramers are required for efficient acetylation of H3 by the Rtt109-Vps75 complex. Taken together, these studies provide insights into how H3-Lys-56 acetylation is regulated during the cell cycle.     

Specific degradation of histones H1 and H3

It is shown that acid treated histones H1 and H3 are susceptible to specific degradation by an associated acid resistant protease. Dialysis against distilled water (pH 5.5–6) of the acid treated histones enhances proteolysis. On the other hand, no degradation is observed in nucleohistone either in the presence of Ca⁺⁺ or Na⁺⁺ ions. The conditions required to avoid degradation during nucleohistone and histone manipulation are described.     

The histone H3 and H4 mRNAs are polyadenylated in maize

Northern blot analysis revealed that the histone H3 and H4 mRNAs are of unusual large size in germinating maize embryos. S1-mapping experiments show that the 3'-untranslated regions of the mRNAs transcribed from 3 H3 and 2 H4 maize genes previously described are much longer than in the non-polyadenylated histone mRNAs which represent a major class in animals. Moreover, oligo d(T) cellulose fractionation of RNAs isolated at different developmental stages indicates that more than 99% of the maize H3 and H4 mRNAs are polyadenylated. A putative polyadenylation signal is present in all five genes 17 to 27 nucleotides before the 3'-ends of the mRNAs.     

Organismal differences in post-translational modifications in histones H3 and H4

Post-translational modifications (PTMs) of histones play an important role in many cellular processes, notably gene regulation. Using a combination of mass spectrometric and immunobiochemical approaches, we show that the PTM profile of histone H3 differs significantly among the various model organisms examined. Unicellular eukaryotes, such as Saccharomyces cerevisiae (yeast) and Tetrahymena thermophila (Tet), for example, contain more activation than silencing marks as compared with mammalian cells (mouse and human), which are generally enriched in PTMs more often associated with gene silencing. Close examination reveals that many of the better-known modified lysines (Lys) can be either methylated or acetylated and that the overall modification patterns become more complex from unicellular eukaryotes to mammals. Additionally, novel species-specific H3 PTMs from wild-type asynchronously grown cells are also detected by mass spectrometry. Our results suggest that some PTMs are more conserved than previously thought, including H3K9me1 and H4K20me2 in yeast and H3K27me1, -me2, and -me3 in Tet. On histone H4, methylation at Lys-20 showed a similar pattern as H3 methylation at Lys-9, with mammals containing more methylation than the unicellular organisms. Additionally, modification profiles of H4 acetylation were very similar among the organisms examined.     

Cell-cycle-regulated control of VSG expression site silencing by histones and histone chaperones ASF1A and CAF-1b in Trypanosoma brucei

Antigenic variation in African trypanosomes involves monoallelic expression and reversible silencing of variant surface glycoprotein (VSG) genes found adjacent to telomeres in polycistronic expression sites (ESs). We assessed the impact on ES silencing of five candidate essential chromatin-associated factors that emerged from a genome-wide RNA interference viability screen. Using this approach, we demonstrate roles in VSG ES silencing for two histone chaperones. Defects in S-phase progression in cells depleted for histone H3, or either chaperone, highlight in particular the link between chromatin assembly and DNA replication control. S-phase checkpoint arrest was incomplete, however, allowing G₂/M-specific VSG ES derepression following knockdown of histone H3. In striking contrast, knockdown of anti-silencing factor 1A (ASF1A) allowed for derepression at all cell cycle stages, whereas knockdown of chromatin assembly factor 1b (CAF-1b) revealed derepression predominantly in S-phase and G₂/M. Our results support a central role for chromatin in maintaining VSG ES silencing. ASF1A and CAF-1b appear to play constitutive and DNA replication-dependent roles, respectively, in the recycling and assembly of chromatin. Defects in these functions typically lead to arrest in S-phase but defective cells can also progress through the cell cycle leading to nucleosome depletion and derepression of telomeric VSG ESs.     

Cross-talk between histone modifications in response to histone deacetylase inhibitors: MLL4 links histone H3 acetylation and histone H3K4 methylation

Histones are subject to a wide variety of post-translational modifications that play a central role in gene activation and silencing. We have used histone modification-specific antibodies to demonstrate that two histone modifications involved in gene activation, histone H3 acetylation and H3 lysine 4 methylation, are functionally linked. This interaction, in which the extent of histone H3 acetylation determines both the abundance and the "degree" of H3K4 methylation, plays a major role in the epigenetic response to histone deacetylase inhibitors. A combination of in vivo knockdown experiments and in vitro methyltransferase assays shows that the abundance of H3K4 methylation is regulated by the activities of two opposing enzyme activities, the methyltransferase MLL4, which is stimulated by acetylated substrates, and a novel and as yet unidentified H3K4me3 demethylase.     

The effect of poly(ADP-ribose) on interactions of DNA with histones H1, H3 and H4

The competition between poly(ADP-ribose) and DNA for binding of the histones H1, H3 and H4 was studied, using a membrane filter-binding test. Poly(ADP-ribose) differently affected the interaction between DNA and the individual histones. While poly(ADP-ribose) effectively competed with DNA for binding of histone H4, it equally competed with DNA for binding of histone H3 and only inefficiently competed with DNA for binding of histone H1. Moreover, preformed complexes were correspondingly affected by the addition of competing polynucleotides, thereby also indicating the reversibility of complex formation. The competition capacity of DNA for histone H4 binding did not depend on DNA size. Competition experiments with poly(A) also indicated that poly(ADP-ribose) preferentially affected DNA-histone H4 interaction. The significance of the differing binding properties is discussed with regard to the possible molecular function of poly(ADP-ribose), especially with regard to its potential effect on nucleosome structure.     

Sequential establishment of marks on soluble histones H3 and H4

Much progress has been made concerning histone function in the nucleus; however, following their synthesis, how their marking and subcellular trafficking are regulated remains to be explored. To gain an insight into these issues, we focused on soluble histones and analyzed endogenous and tagged H3 histones in parallel. We distinguished six complexes that we could place to account for maturation events occurring on histones H3 and H4 from their synthesis onward. In each complex, a different set of chaperones is involved, and we found specific post-translational modifications. Interestingly, we revealed that histones H3 and H4 are transiently poly(ADP-ribosylated). The impact of these marks in histone metabolism proved to be important as we found that acetylation of lysines 5 and 12 on histone H4 stimulated its nuclear translocation. Furthermore, we showed that, depending on particular histone H3 modifications, the balance in the presence of the different translocation complexes changes. Therefore, our results enabled us to propose a regulatory means of these marks for controlling cytoplasmic/nuclear shuttling and the establishment of early modification patterns.     

The activity of the histone chaperone yeast Asf1 in the assembly and disassembly of histone H3/H4-DNA complexes

The deposition of the histones H3/H4 onto DNA to give the tetrasome intermediate and the displacement of H3/H4 from DNA are thought to be the first and the last steps in nucleosome assembly and disassembly, respectively. Anti-silencing function 1 (Asf1) is a chaperone of the H3/H4 dimer that functions in both of these processes. However, little is known about the thermodynamics of chaperone–histone interactions or the direct role of Asf1 in the formation or disassembly of histone–DNA complexes. Here, we show that Saccharomyces cerevisiae Asf1 shields H3/H4 from unfavorable DNA interactions and aids the formation of favorable histone–DNA interactions through the formation of disomes. However, Asf1 was unable to disengage histones from DNA for tetrasomes formed with H3/H4 and strong nucleosome positioning DNA sequences or tetrasomes weakened by mutant (H3K56Q/H4) histones or non-positioning DNA sequences. Furthermore, Asf1 did not associate with preformed tetrasomes. These results are consistent with the measured affinity of Asf1 for H3/H4 dimers of 2.5 nM, which is weaker than the association of H3/H4 for DNA. These studies support a mechanism by which Asf1 aids H3/H4 deposition onto DNA but suggest that additional factors or post-translational modifications are required for Asf1 to remove H3/H4 from tetrasome intermediates in chromatin.