Histone H4 can be acetylated at N-terminal lysines K5, K8, K12, and K16, but newly synthesized H4 is diacetylated at K5͞K12 in diverse organisms. This pattern is widely thought to be important for histone deposition onto replicating DNA. To investigate the importance of K5͞K12 we have mutagenized these lysines in yeast and assayed for nucleosome assembly. Assaying was done in the absence of the histone H3 N terminus, which has functions redundant with those of H4 in histone deposition. Nucleosome assembly was assayed by three methods. Because nucleosome depletion may be lethal, we examined cell viability. We also analyzed nucleosome assembly in vivo and in vitro by examining plasmid superhelicity density in whole cells and supercoiling in yeast cell extracts. All three approaches demonstrate that mutagenizing K5 and K12 together does not prevent cell growth and histone deposition in vivo or in vitro. Therefore, K5͞K12 cannot be required for nucleosome assembly in yeast. It is only when the first three sites of acetylation-K5, K8, and K12-are mutagenized simultaneously that lethality occurs and assembly is most strongly decreased both in vivo and in vitro. These data argue for the redundancy of sites K5, K8, and K12 in the deposition of yeast histone H4.Nucleosome assembly involves the deposition of a tetramer of histones H3 and H4 onto DNA, followed by the association of two histone H2A͞H2B dimers. In this process, acetylation of histone H4 is likely to play a key role. Newly synthesized histone H4 was shown by pulse-labeling to be diacetylated at lysine residues 5 and 12 (K5͞K12), a conserved feature in Tetrahymena, flies, and humans (1-3). This distinct nonrandom pattern of acetylation among the four acetylatable lysines (K5, K8, K12, and K16) has led to the suggestion that K5͞K12 diacetylation serves a unique role in targeting newly synthesized histone H4 for assembly (4). Another argument for the importance of H4 acetylation in nucleosome assembly derives from the study of the human multiprotein complex (CAF-1, for chromatin assembly factor) that enables H3 and H4 assembly onto replicating DNA in a simian virus 40-based cell-free system (5-7). CAF-1 deposits newly synthesized histones but not those extracted from bulk chromatin onto DNA (7), a result which is consistent with the finding that CAF-1 associates preferentially with histone H4 acetylated in a specific manner (8). However, H4 associated with CAF-1 is not uniquely diacetylated at K5 and K12 but is heterogeneously acetylated at K5, K8, and K12. Moreover, some 33% of H4 in the complex is not acetylated. In addition, much of the H3 in the complex is monoacetylated, whereas some 60% of H3 is unacetylated (9).The yeast (Saccharomyces cerevisiae) histone H4 N-terminal sequence and the location of its acetylated lysines are extremely conserved in evolution. While it is not known whether newly synthesized yeast H4 is diacetylated or whether acetylated H4 is associated with a yeast chromatin assembly factor, we set out to ask whether K5͞K12 is requi...

A simple in vitro system that supports chromatin assembly was developed for Saccharomyces cerevisiae. The assembly reaction is ATP-dependent, uses soluble histones and assembly factors, and generates physiologically spaced nucleosomes. We analyze the pathway of histone recruitment into nucleosomes, using this system in combination with genetic methods for the manipulation of yeast. This analysis supports the model of sequential recruitment of H3͞H4 tetramers and H2A͞H2B dimers into nucleosomes. Using a similar approach, we show that DNA ligase I can play an important role in template repair during assembly. These studies demonstrate the utility of this system for the combined biochemical and genetic analysis of chromatin assembly in yeast.The nuclear DNA of eukaryotes is assembled with histones into chromatin, of which the basic building block is the nucleosome (reviewed in refs. 1 and 2). The assembly of chromatin has been intensively studied in a variety of biological systems, using many sophisticated experimental approaches. Studies in Saccharomyces cerevisiae have exploited powerful genetic methods and a broad spectrum of biochemical techniques. However one important approach developed in metazoans, the analysis of chromatin assembly in simple cell extracts, has not been available in yeast. In the present report we describe a system for yeast that supports this approach. This system uses a whole-cell extract previously shown to be competent for transcription by all three nuclear RNA polymerases (3, 4). The assembly reaction occurs under physiological salt conditions, is ATP-dependent, and uses soluble histones and chromatin assembly factors.Studies of complex biochemical processes such as replication and transcription have benefited from the advantages of combined biochemical and genetic analysis in yeast. We apply this approach to the study of chromatin assembly using the in vitro system described here. Strains expressing selected histones from an inducible promoter (5, 6) are used to characterize the pathway of histone recruitment during chromatin assembly in yeast, and a temperature-sensitive strain with a mutated DNA ligase I gene (7) is used to analyze template repair during assembly. These studies provide evidence for the expected stepwise recruitment of H3͞H4 tetramers and H2A͞ H2B dimers into nucleosomes, and they establish that DNA ligase can function to repair DNA in the context of the chromatin assembly reaction. Our work shows that in combination with available genetic techniques, the yeast in vitro assembly system should provide novel opportunities for analyzing the mechanism and regulation of chromatin assembly.

We describe a replication-independent, cell cycle-regulated chromatin assembly pathway in budding yeast. The activity of this pathway is low in S phase extracts but is very high in G 2 , M, and G 1 cell extracts, with peak activity in late M͞early G 1 . The cell cycle regulation of this pathway requires a specific pattern of posttranslational modification of histones H3 and͞or H4, which is distinct for H3͞H4 present in S phase versus M and G 1 phase cell extracts. Histone H3͞H4 modification is therefore important for the reciprocal control of replication-dependent and -independent chromatin assembly pathways during the cell cycle.Under physiological conditions nucleosome assembly is mediated by proteins called chromatin assembly factors (CAFs) (1-5). CAFs operate in two general pathways of nucleosome reconstitution in vivo, one coupled to DNA replication, and one that occurs independently of replication. The ''replicationdependent'' pathway is cell cycle regulated, being maximally active in S phase. Although this pathway accounts for the bulk of chromatin assembly in dividing cells, it seems likely that during normal cell division nucleosome deposition also occurs by a ''replication-independent'' mechanism. Chromatin remodeling associated with transcriptional regulation can involve nucleosome deposition that is independent of DNA synthesis (6) and therefore perhaps involves replicationindependent assembly factors. When nucleosomes are lost during G 2 , M, and G 1 as a result of histone degradation, new nucleosome deposition is likely to occur before the next S phase (7), and this reaction also potentially involves replication-independent CAFs. Although these observations suggest significant physiological functions for replication-independent CAFs in dividing cells, the biochemical properties of such factors, and the regulation of replication-independent assembly pathways in relation to global changes in cellular metabolism, are poorly understood. We are exploring the regulation of replicationindependent chromatin assembly by a biochemical approach in budding yeast (8, 9). Here we focus on the cell cycle regulation of replication-independent chromatin assembly in mitotically dividing cells. We have discovered a replication-independent assembly pathway that is cell cycle regulated in yeast. The activity pattern of this pathway mirrors that of the replicationdependent pathway responsible for bulk assembly in dividing cells, that is, replication-independent assembly is repressed in S phase, during which time bulk replication-coupled assembly is activated (2, 10). We show that posttranslational modification of histones H3 and͞or H4 plays an important role in the cell cycle control of assembly by this pathway. Posttranslational modification of histones H3 and H4 is also associated with the activation of replication-coupled chromatin assembly during S phase (2, 10). The regulation of histone H3 and͞or H4 is therefore an important component of a reciprocal control mechanism that represses replication-independent...

Transcription of nuclear genes usually involves trans-activators, whereas repression is exerted by chromatin. For several genes the transcription mediated by trans-activators and the repression mediated by chromatin depend on the CP complex, a recently described abundant yeast nuclear complex of the Pob3 and Cdc68/Spt16 proteins. We report that the N-terminal third of the Saccharomyces cerevisiae Cdc68 protein is dispensable for gene activation but necessary for the maintenance of chromatin repression. The absence of this 300-residue N-terminal domain also decreases the need for the Swi/Snf chromatin-remodeling complex in transcription and confers an Spt- effect characteristic of chromatin alterations. The repression domain, and indeed the entire Cdc68 protein, is highly conserved, as shown by the sequence of the Cdc68 functional homolog from the yeast Kluyveromyces lactis and by database searches. The repression-defective (truncated) form of Cdc68 is stable but less active at high temperatures, whereas the known point-mutant form of Cdc68, encoded by three independent mutant alleles, alters the N-terminal repression domain and destabilizes the mutant protein.