Histone modifications influence the action of Snf2 family remodelling enzymes by different mechanisms

Alteration of chromatin structure by chromatin modifying and remodelling activities is a key stage in the regulation of many nuclear processes. These activities are frequently interlinked, and many chromatin remodelling enzymes contain motifs that recognise modified histones. Here we adopt a peptide ligation strategy to generate specifically modified chromatin templates and used these to study the interaction of the Chd1, Isw2 and RSC remodelling complexes with differentially acetylated nucleosomes. Specific patterns of histone acetylation are found to alter the rate of chromatin remodelling in different ways. For example, histone H3 lysine 14 acetylation acts to increase recruitment of the RSC complex to nucleosomes. However, histone H4 tetra-acetylation alters the spectrum of remodelled products generated by increasing octamer transfer in trans. In contrast, histone H4 tetra-acetylation was also found to reduce the activity of the Chd1 and Isw2 remodelling enzymes by reducing catalytic turnover without affecting recruitment. These observations illustrate a range of different means by which modifications to histones can influence the action of remodelling enzymes.     

Mutations to the histone H3 αN region selectively alter the outcome of ATP-dependent nucleosome-remodelling reactions

Mutational analysis of the histone H3 N-terminal region has shown it to play an important role both in chromatin function in vivo and nucleosome dynamics in vitro. Here we use a library of mutations in the H3 N-terminal region to investigate the contribution of this region to the action of the ATP-dependent remodelling enzymes Chd1, RSC and SWI/SNF. All of the enzymes were affected differently by the mutations with Chd1 being affected the least and RSC being most sensitive. In addition to affecting the rate of remodelling by RSC, some mutations prevented RSC from moving nucleosomes to locations in which DNA was unravelled. These observations illustrate that the mechanisms by which different ATP-dependent remodelling enzymes act are sensitive to different features of nucleosome structure. They also show how alterations to histones can affect the products generated as a result of ATP-dependent remodelling reactions.     

The yeast RSC chromatin-remodeling complex is required for kinetochore function in chromosome segregation

The accurate segregation of chromosomes requires the kinetochore, a complex protein machine that assembles onto centromeric DNA to mediate attachment of replicated sister chromatids to the mitotic spindle apparatus. This study reveals an important role for the yeast RSC ATP-dependent chromatin-remodeling complex at the kinetochore in chromosome transmission. Mutations in genes encoding two core subunits of RSC, the ATPase Sth1p and the Snf5p homolog Sfh1p, interact genetically with mutations in genes encoding kinetochore proteins and with a mutation in centromeric DNA. RSC also interacts genetically and physically with the histone and histone variant components of centromeric chromatin. Importantly, RSC is localized to centromeric and centromere-proximal chromosomal regions, and its association with these loci is dependent on Sth1p. Both sth1 and sfh1 mutants exhibit altered centromeric and centromere-proximal chromatin structure and increased missegregation of authentic chromosomes. Finally, RSC is not required for centromeric deposition of the histone H3 variant Cse4p, suggesting that RSC plays a role in reconfiguring centromeric and flanking nucleosomes following Cse4p recruitment for proper chromosome transmission.     

Critical role for the histone H4 N terminus in nucleosome remodeling by ISWI

The ATPase ISWI can be considered the catalytic core of several multiprotein nucleosome remodeling machines. Alone or in the context of nucleosome remodeling factor, the chromatin accessibility complex (CHRAC), or ACF, ISWI catalyzes a number of ATP-dependent transitions of chromatin structure that are currently best explained by its ability to induce nucleosome sliding. In addition, ISWI can function as a nucleosome spacing factor during chromatin assembly, where it will trigger the ordering of newly assembled nucleosomes into regular arrays. Both nucleosome remodeling and nucleosome spacing reactions are mechanistically unexplained. As a step toward defining the interaction of ISWI with its substrate during nucleosome remodeling and chromatin assembly we generated a set of nucleosomes lacking individual histone N termini from recombinant histones. We found the conserved N termini (the N-terminal tails) of histone H4 essential to stimulate ISWI ATPase activity, in contrast to other histone tails. Remarkably, the H4 N terminus, but none of the other tails, was critical for CHRAC-induced nucleosome sliding and for the generation of regularity in nucleosomal arrays by ISWI. Direct nucleosome binding studies did not reflect a dependence on the H4 tail for ISWI-nucleosome interactions. We conclude that the H4 tail is critically required for nucleosome remodeling and spacing at a step subsequent to interaction with the substrate.     

30 nm chromatin fibre decompaction requires both H4-K16 acetylation and linker histone eviction

The mechanism by which chromatin is decondensed to permit access to DNA is largely unknown. Here, using a model nucleosome array reconstituted from recombinant histone octamers, we have defined the relative contribution of the individual histone octamer N-terminal tails as well as the effect of a targeted histone tail acetylation on the compaction state of the 30 nm chromatin fiber. This study goes beyond previous studies as it is based on a nucleosome array that is very long (61 nucleosomes) and contains a stoichiometric concentration of bound linker histone, which is essential for the formation of the 30 nm chromatin fiber. We find that compaction is regulated in two steps: Introduction of H4 acetylated to 30% on K16 inhibits compaction to a greater degree than deletion of the H4 N-terminal tail. Further decompaction is achieved by removal of the linker histone.     

Acetylation increases the alpha-helical content of the histone tails of the nucleosome

The nature of the structural changes induced by histone acetylation at the different levels of chromatin organization has been very elusive. At the histone level, it has been proposed on several occasions that acetylation may induce an alpha-helical conformation of their acetylated N-terminal domains (tails). In an attempt to provide experimental support for this hypothesis, we have purified and characterized the tail of histone H4 in its native and mono-, di-, tri-, and tetra- acetylated form. The circular dichroism analysis of these peptides shows conclusively that acetylation does increase their alpha-helical content. Furthermore, the same spectroscopic analysis shows that this is also true for both the acetylated nucleosome core particle and the whole histone octamer in solution. In contrast to the native tails in which the alpha-helical organization appears to be dependent upon interaction of these histone regions with DNA, the acetylated tails show an increase in alpha-helical content that does not depend on such an interaction.     

Chromatin remodeling through directional DNA translocation from an internal nucleosomal site

The RSC chromatin remodeler contains Sth1, an ATP-dependent DNA translocase. On DNA substrates, RSC/Sth1 tracks along one strand of the duplex with a 3' --> 5' polarity and a tracking requirement of one base, properties that may enable directional DNA translocation on nucleosomes. The binding of RSC or Sth1 elicits a DNase I-hypersensitive site approximately two DNA turns from the nucleosomal dyad, and the binding of Sth1 requires intact DNA at this location. Results with various nucleosome substrates suggest that RSC/Sth1 remains at a fixed position on the histone octamer and that Sth1 conducts directional DNA translocation from a location about two turns from the nucleosomal dyad, drawing in DNA from one side of the nucleosome and pumping it toward the other. These studies suggest that nucleosome mobilization involves directional DNA translocation initiating from a fixed internal site on the nucleosome.     

Histone acetylation reduces H1-mediated nucleosome interactions during chromatin assembly

During chromatin replication and nucleosome assembly, newly synthesized histone H4 is acetylated before it is deposited onto DNA, then deacetylated as assembly proceeds. In a previous study (Perry and Annunziato, Nucleic Acids Res. 17, 4275 [1989]) it was shown that when replication occurs in the presence of sodium butyrate (thereby inhibiting histone deacetylation), nascent chromatin fails to mature fully and instead remains preferentially sensitive to DNaseI, more soluble in magnesium, and depleted of histone H1 (relative to mature chromatin). In the following report the relationships between chromatin replication, histone acetylation, and H1-mediated nucleosome aggregation were further investigated. Chromatin was replicated in the presence or absence of sodium butyrate; isolated nucleosomes were stripped of linker histone, reconstituted with H1, and treated to produce Mg(2+)-soluble and Mg(2+)-insoluble chromatin fractions. Following the removal of H1, all solubility differences between chromatin replicated in sodium butyrate for 30 min (bu-chromatin) and control chromatin were lost. Reconstitution with H1 completely restored the preferential Mg(2+)-solubility of bu-chromatin, demonstrating that a reduced capacity for aggregation/condensation is an inherent feature of acetylated nascent nucleosomes; however, titration with excess H1 caused the solubility differences to be lost again. Moreover, when the core histone N-terminal "tails" (the sites of acetylation) were removed by trypsinization prior to reconstitution, H1 was unable to reestablish the altered solubility of chromatin replicated in butyrate. Thus, the core histone "tails," and the acetylation thereof, not only modulate H1-mediated nucleosome interactions in vitro, but also strongly influence the ability of H1 to differentiate between new and old nucleosomes. The data suggest a possible mechanism for the control of H1 deposition and/or chromatin folding during nucleosome assembly.