Modulation of the higher-order folding of chromatin by deletion of histone H3 and H4 terminal domains.
ABSTRACT: The 'tails' of histones H3 and H4 were removed by light in situ trypsin digestion of the nuclei. The alterations in the higher-order folding of chromatin resulting from this treatment were monitored by ethidium bromide titration. We found that DNA-intercalation of ethidium bromide under these conditions exhibited a complex concentration effect that was dependent on the extent of chromatin folding. This most likely reflects the structural transitions of chromatin during its folding as a result of the changes in the nucleosome linker twist [Woodcock, Grigoryev, Horowitz and Whitaker (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 9021-9025]. These results strongly suggest that the H3 and H4 terminal domains play a very important role in chromatin folding. We discuss the molecular basis of this phenomenon and propose a novel generalized model for the higher-order folding of chromatin.
Project description:Eukaryotic DNA is compacted in the form of chromatin, in a complex with histones and other non-histone proteins. The intimate association of DNA and histones in chromatin raises the possibility that DNA-interactive small molecules may bind to chromatin-associated proteins such as histones. Employing biophysical and biochemical techniques we have characterized the interaction of a classical intercalator, ethidium bromide (EB) and its structural analogue propidium iodide (PI) with hierarchical genomic components: long chromatin, chromatosome, core octamer and chromosomal DNA. Our studies show that EB and PI affect both chromatin structure and function, inducing chromatin compaction and disruption of the integrity of the chromatosome. Calorimetric studies and fluorescence measurements of the ligands demonstrated and characterized the association of these ligands with core histones and the intact octamer in absence of DNA. The ligands affect acetylation of histone H3 at lysine 9 and acetylation of histone H4 at lysine 5 and lysine 8 ex vivo. PI alters the post-translational modifications to a greater extent than EB. This is the first report showing the dual binding (chromosomal DNA and core histones) property of a classical intercalator, EB, and its longer analogue, PI, in the context of chromatin.
Project description:Studies of binding of ethidium bromide and quinacrine hydrochloride to native DNA at low ionic strength indicate that for both compounds the binding is selective, with about one binding site for about four nucleotides. Annealing of unfractionated histones to DNA by a salt-gradient dialysis method slightly decreases the binding of the dyes to DNA. Similar observations made with reconstituted preparations by using individual histone fractions reveal that the arginine-rich histones (histones H3 and H4) are most effective in decreasing the binding. The binding studies with ethidium bromide at high ionic strength and with denatured DNA show that strong dye binding to DNA is strongly dependent on the ionic strength and on the secondary structure of DNA. The histones are not effective in decreasing the dye binding under conditions of high ionic strength. The results are consistent with the observations [Oliver & Chalkley (1974) Biochemistry13, 5093-5098; Axel, Melchoir, Sollner-Web & Felsenfield (1974) Proc. Natl. Acad. Sci. U.S.A.71, 4101-4105] that histones form some kind of surface structures on DNA through non-specific interactions and [Kornberg & Thomas (1974) Science184, 865-868; Kornberg (1974) Science184, 868-871; D'Anna & Isenberg (1974) Biochemistry13, 4992-4997; Vandegrift, Serra, Marve & Wagner (1974) Biochemistry13, 5087-5092] that the tendency of arginine-rich histones to aggregate may be an important factor in determining the structure of chromatin.
Project description:Förster resonance energy transfer was used to monitor the dynamic conformations of mononucleosomes under different chromatin folding conditions to elucidate the role of the flexible N-terminal regions of H3 and H4 histones. The H3 tail was shown to partake in intranucleosomal interactions by restricting the DNA breathing motion and compacting the nucleosome. The H3 tail effects were mostly independent of the ionic strength and valency of the ions. The H4 tail was shown to not greatly affect the nucleosome conformation, but did slightly influence the relative population of the preferred conformation. The role of the H4 tail varied depending on the valency and ionic strength, suggesting that electrostatic forces play a primary role in H4 tail interactions. Interestingly, despite the H4 tail's lack of influence, when H3 and H4 tails were simultaneously clipped, a more dramatic effect was seen than when only H3 or H4 tails were clipped. The combinatorial effect of H3 and H4 tail truncation suggests a potential mechanism by which various combinations of histone tail modifications can be used to control accessibility of DNA-binding proteins to nucleosomal DNA.
Project description:In eukaryotes, DNA is packaged into chromatin by canonical histone proteins. The specialized histone H3 variant CENP-A provides an epigenetic and structural basis for chromosome segregation by replacing H3 at centromeres. Unlike exclusively octameric canonical H3 nucleosomes, CENP-A nucleosomes have been shown to exist as octamers, hexamers, and tetramers. An intriguing possibility reconciling these observations is that CENP-A nucleosomes cycle between octamers and tetramers in vivo. We tested this hypothesis by tracking CENP-A nucleosomal components, structure, chromatin folding, and covalent modifications across the human cell cycle. We report that CENP-A nucleosomes alter from tetramers to octamers before replication and revert to tetramers after replication. These structural transitions are accompanied by reversible chaperone binding, chromatin fiber folding changes, and previously undescribed modifications within the histone fold domains of CENP-A and H4. Our results reveal a cyclical nature to CENP-A nucleosome structure and have implications for the maintenance of epigenetic memory after centromere replication.
Project description:While specific posttranslational modification patterns within the H3 and H4 tail domains are associated with the S-phase, their actual functions in replication-dependent chromatin assembly have not yet been defined. Here we used incorporation of trace amounts of recombinant proteins into naturally synchronous macroplasmodia of Physarum polycephalum to examine the function of H3 and H4 tail domains in replication-coupled chromatin assembly. We found that the H3/H4 complex lacking the H4 tail domain was not efficiently recovered in nuclei, whereas depletion of the H3 tail domain did not impede nuclear import but chromatin assembly failed. Furthermore, our results revealed that the proper pattern of acetylation on the H4 tail domain is required for nuclear import and chromatin assembly. This is most likely due to binding of Hat1, as coimmunoprecipitation experiments showed Hat1 associated with predeposition histones in the cytoplasm and with replicating chromatin. These results suggest that the type B histone acetyltransferase assists in shuttling the H3/H4 complex from cytoplasm to the replication forks.
Project description:Histone methylation regulates chromatin function dependent on the site and degree of the modification. In addition to creating binding sites for proteins, methylated lysine residues are likely to influence chromatin structure directly. Here we present crystal structures of nucleosomes reconstituted with methylated histones and investigate the folding behavior of resulting arrays. We demonstrate that dimethylation of histone H3 at lysine residue 79 locally alters the nucleosomal surface, whereas trimethylation of H4 at lysine residue 20 affects higher-order structure.
Project description:Histone tail acetylation is a key epigenetic marker that tends to open chromatin folding and activate transcription. Despite intensive studies, precise roles of individual lysine acetylation in chromatin folding have only been poorly understood. Here, we revealed structural dynamics of tri-nucleosomes with several histone tail acetylation states and analyzed histone tail interactions with DNA by performing molecular simulations at an unprecedentedly high resolution. We found versatile acetylation-dependent landscapes of tri-nucleosome. The H4 and H2A tail acetylation reduced the contact between the first and third nucleosomes mediated by the histone tails. The H3 tail acetylation reduced its interaction with neighboring linker DNAs resulting in increase of the distance between consecutive nucleosomes. Notably, two copies of the same histone in a single nucleosome have markedly asymmetric interactions with DNAs, suggesting specific pattern of nucleosome docking albeit high inherent flexibility. Estimated transcription factor accessibility was significantly high for the H4 tail acetylated structures.
Project description:Anti-silencing function 1 (Asf1) is a highly conserved chaperone of histones H3/H4 that assembles or disassembles chromatin during transcription, replication, and repair. The structure of the globular domain of Asf1 bound to H3/H4 determined by X-ray crystallography to a resolution of 1.7 Angstroms shows how Asf1 binds the H3/H4 heterodimer, enveloping the C terminus of histone H3 and physically blocking formation of the H3/H4 heterotetramer. Unexpectedly, the C terminus of histone H4 that forms a mini-beta sheet with histone H2A in the nucleosome undergoes a major conformational change upon binding to Asf1 and adds a beta strand to the Asf1 beta sheet sandwich. Interactions with both H3 and H4 were required for Asf1 histone chaperone function in vivo and in vitro. The Asf1-H3/H4 structure suggests a "strand-capture" mechanism whereby the H4 tail acts as a lever to facilitate chromatin disassembly/assembly that may be used ubiquitously by histone chaperones.
Project description:Promoter chromatin disassembly is a widely used mechanism to regulate eukaryotic transcriptional induction. Delaying histone H3/H4 removal from the yeast PHO5 promoter also leads to delayed removal of histones H2A/H2B, suggesting a constant equilibrium of assembly and disassembly of H2A/H2B, whereas H3/H4 disassembly is the highly regulated step. Toward understanding how H3/H4 disassembly is regulated, we observe a drastic increase in the levels of histone H3 acetylated on lysine-56 (K56ac) during promoter chromatin disassembly. Indeed, promoter chromatin disassembly is driven by Rtt109 and Asf1-dependent acetylation of H3 K56. Conversely, promoter chromatin reassembly during transcriptional repression is accompanied by decreased levels of histone H3 acetylated on lysine-56, and a mutation that prevents K56 acetylation increases the rate of transcriptional repression. As such, H3 K56 acetylation drives chromatin toward the disassembled state during transcriptional activation, whereas loss of H3 K56 acetylation drives the chromatin toward the assembled state.
Project description:Eukaryotic chromatin is a highly dynamic structure with essential roles in virtually all DNA-dependent cellular processes. Nucleosomes are a barrier to DNA access, and during DNA replication, they are disassembled ahead of the replication machinery (the replisome) and reassembled following its passage. The Histone chaperone Chromatin Assembly Factor-1 (CAF-1) interacts with the replisome and deposits H3-H4 directly onto newly synthesized DNA. Therefore, CAF-1 is important for the establishment and propagation of chromatin structure. The molecular mechanism by which CAF-1 mediates H3-H4 deposition has remained unclear. However, recent studies have revealed new insights into the architecture and stoichiometry of the trimeric CAF-1 complex and how it interacts with and deposits H3-H4 onto substrate DNA. The CAF-1 trimer binds to a single H3-H4 dimer, which induces a conformational rearrangement in CAF-1 promoting its interaction with substrate DNA. Two CAF-1•H3-H4 complexes co-associate on nucleosome-free DNA depositing (H3-H4)2 tetramers in the first step of nucleosome assembly. Here, we review the progress made in our understanding of CAF-1 structure, mechanism of action, and how CAF-1 contributes to chromatin dynamics during DNA replication.