Anthony T. Annunziato

Split Decision: What Happens to Nucleosomes during DNA Replication?

The heritability of cell-specific gene regulation argues that chromatin structures must be propagated across cell generations (1–5). A corollary of this hypothesis is that specific histone-DNA interactions are reestablished during chromatin synthesis, and in fact nucleosomes are rapidly generated on newly replicated DNA (6–8). The histones required for nascent nucleosomes are derived from two sources: parental histones (dispersively segregated to both arms of the fork) and new histones, especially new H3/H4, that are deposited during de novo nucleosome assembly. (Note: new H2A/ H2B dimers are not uniquely targeted to nascent DNA but are also deposited onto non-replicating chromatin (9, 10).) Replicationcoupled nucleosome assembly occurs in a stepwise fashion; first histones H3 and H4 are deposited and then H2A and H2B (11, 12). The deposition of H3/H4 onto new DNA is mediated by the assembly factor CAF-1; the H3/H4-escort protein Asf1 appears to assist CAF-1 in this process (8, 13–15). Not all histone synthesis occurs in conjunction with DNA replication. Distinct non-allelic histone variants are synthesized at basal levels throughout the cell cycle and can be incorporated into chromatin in a replication-independent manner during G1 and G2 (16–19). These “basal histone variants” are found in virtually all eukaryotes and contain conserved amino acid substitutions that differentiate them from replication-dependent subtypes (20). For example, in mammals the replication-independent variant H3.3 differs by only 4–5 amino acids from the major replication-dependent H3 isoform, H3.1 (20, 21). There is evidence that H3.3 is incorporated into chromatin during transcription (22, 23). Moreover, in a recent paper by Tagami et al. (24) it was shown that the replication-independent deposition of H3.3 into chromatin is mediated by a specialized assembly factor, HIRA, in agreement with previous findings that HIRA can mediate replication-independent nucleosome assembly (25). Thus, there is evidence that replicationdependent and replication-independent histone deposition pathways utilize two separate assembly factors, CAF-1 and HIRA, respectively (15, 26, 27). Newly synthesized H3 and H4 are associated with each other prior to their assembly into chromatin (reviewed in Ref. 8) (28–30). Until recently there has been little evidence to indicate whether the coordinate deposition of H3 and H4 occurs in the form of dimers or tetramers. However, in the aforementioned paper by Tagami et al. (24) it was shown that purified chromatin assembly complexes containing either CAF-1 or HIRA are likely to contain H3/H4 dimers, not tetramers. The observation of pre-deposition complexes containing H3/H4 dimers has prompted the rethinking of how histones are assembled onto DNA (31). It also has sparked new interest in the question of how parental, pre-fork histones are distributed during replication. Extrapolating from their findings, one of the models proposed by Tagami et al. (24) is that parental H3/H4 tetramers dissociate into heterotypic dimers, which are segregated to both arms of the replication fork (Fig. 1). It was further proposed that segregated dimers are then converted to tetramers by the deposition of newly synthesized H3/H4 dimers, as mediated by CAF-1 (24). Other chromatin assembly models were also proposed by Tagami et al. (24), including the cooperative action of two CAF-1 complexes (each carrying one nascent H3/H4 dimer). Nevertheless, questions surrounding the “splitting tetramer” model have refocused attention on the manner in which old histones are segregated to new DNA. There is a large and varied literature on this topic, embodying experiments that reach back to some of the first analyses of chromatin replication and assembly (reviewed in Ref. 6). In the following article, experiments dealing with the fate of pre-replicative nucleosomes are examined in an effort to better evaluate possible models of chromatin replication and assembly in vivo.

Modifications of H3 and H4 during chromatin replication, nucleosome assembly, and histone exchange.

Histone posttranslational modifications that accompany DNA replication, nucleosome assembly, and H2A/H2B exchange were examined in human tissue culture cells. Through microsequencing analysis and chromatin immunoprecipitation, it was found that a subset of newly synthesized H3.2/H3.3 is modified by acetylation and methylation at sites that correlate with transcriptional competence. Immunoprecipitation experiments suggest that cytosolic predeposition complexes purified from cells expressing FLAG-H4 contain H3/H4 dimers, not tetramers. Studies of the deposition of newly synthesized H2A/H2B onto replicating and nonreplicating chromatin demonstrated that H2A/H2B exchange takes place in chromatin regions that contain acetylated H4; however, there is no single pattern of H4 acetylation that accompanies exchange. H2A/H2B exchange is also largely independent of the deposition of replacement histone variant, H3.3. Finally, immunoprecipitation of nucleosomes replicated in the absence of de novo nucleosome assembly showed that histone modifications do not prevent the transfer of parental histones to newly replicated DNA and thus have the potential to serve as means of epigenetic inheritance. Our experiments provide an in-depth analysis of the “histone code” associated with chromatin replication and dynamic histone exchange in human cells.

Generation and characterization of novel antibodies highly selective for phosphorylated linked histone H1 in Tetrahymena and HeLa cells

Phosphorylated forms of Tetrahymena macronuclear histone H1 were separated from each other and from dephosphorylated H1 by cation-exchange HPLC. A homogeneous fraction of hyperphosphorylated macronuclear H1 was then used to generate novel polyclonal antibodies highly selective for phosphorylated H1 in Tetrahymena and in human cells. These antibodies fail to recognize dephosphorylated forms of H1 in both organisms and are not reactive with most other nuclear or cytoplasmic phosphoproteins including those induced during mitosis. The selectivity of these antibodies for phosphorylated forms of H1 in Tetrahymena and in HeLa argues strongly that these antibodies recognize highly conserved phosphorylated epitopes found in most H1s and from this standpoint Tetrahymena H1 is not atypical. Using these antibodies in indirect immunofluorescence analyses, we find that a significant fraction of interphase mammalian cells display a strikingly punctate pattern of nuclear fluorescence. As cells enter S-phase, nuclear staining becomes more diffuse, increases significantly, and continues to increase as cells enter mitosis. As cells exit from mitosis, staining with the anti-phosphorylated H1 antibodies is rapidly lost presumably owing to the dephosphorylation of H1. These immunofluorescent data document precisely the cell cycle changes in the level of H1 phosphorylation determined by earlier biochemical studies and suggest that these antibodies represent a powerful new tool to probe the functions(s) of H1 phosphorylation in a wide variety of eukaryotic systems.

Properties of the type B histone acetyltransferase HAT1: H4 tail interaction, site preference, and involvement in DNA repair

The Hat1 histone acetyltransferase catalyzes the acetylation of H4 at lysines 5 and 12, the same sites that are acetylated in newly synthesized histone H4. By performing histone acetyltransferase (HAT) assays on various synthetic H4 N-terminal peptides, we have examined the interactions between Hat1 and the H4 tail domain. It was found that acetylation requires the presence of positively charged amino acids at positions 8 and 16 of H4, positions that are normally occupied by lysine; however, lysine per se is not essential and can be replaced by arginine. In contrast, replacing Lys-8 and -16 of H4 with glutamines reduces acetylation to background levels. Similarly, phosphorylation of Ser-1 of the H4 tail depresses acetylation by both yeast Hat1p and the human HAT-B complex. These results strongly support the model proposed by Ramakrishnan and colleagues for the interaction between Hat1 and the H4 tail (Dutnall, R. N., Tafrov, S. T., Sternglanz, R., and Ramakrishnan, V. (1998) Cell 94, 427–438) and may have implications for the genetic analysis of histone acetylation. It was also found that Lys-12 of H4 is preferentially acetylated by human HAT-B, in further agreement with the proposed model of H4 tail binding. Finally, we have demonstrated that deletion of the hat1 gene from the fission yeast Schizosaccharomyces pombe causes increased sensitivity to the DNA-damaging agent methyl methanesulfonate in the absence of any additional mutations. This is in contrast to results obtained with a Saccharomyces cerevisiae hat1Δ strain, which must also carry mutations of the acetylatable lysines of H3 for heightened methyl methanesulfonate sensitivity to be observed. Thus, although the role of Hat1 in DNA damage repair is evolutionarily conserved, the ability of H3 acetylation to compensate for Hat1 deletion appears to be more variable.

Treatment with sodium butyrate inhibits the complete condensation of interphase chromatin

The effects of histone hyperacetylation on chromatin fiber structure were studied using direct observations with the electron microscope. Histone hyperacetylation was induced in HeLa cells by treatment with sodium butyrate, and the ultrastructure of control and of acetylated chromatin fibers examined after fixation at different stages of compaction. No differences between control and acetylated chromatin were seen when the fibers were partially unfolded (10 mM NaCl, 20 mM NaCl, 50 mM NaCl), but in 100 mM NaCl, control chromatin showed further compaction to the “30 nm” fiber, while hyperacetylated chromatin failed to undergo this final compaction step. These results strongly suggest that histone acetylation causes a moderate “relaxation” rather than complete decondensation of interphase chromatin fibers. The relationship of these findings to the increased DNase I sensitivity of acetylated chromatin, and to transcription and replication, is discussed.

Histone acetyl transferase 1 is essential for mammalian development, genome stability, and the processing of newly synthesized histones H3 and H4.

Histone acetyltransferase 1 is an evolutionarily conserved type B histone acetyltransferase that is thought to be responsible for the diacetylation of newly synthesized histone H4 on lysines 5 and 12 during chromatin assembly. To understand the function of this enzyme in a complex organism, we have constructed a conditional mouse knockout model of Hat1. Murine Hat1 is essential for viability, as homozygous deletion of Hat1 results in neonatal lethality. The lungs of embryos and pups genetically deficient in Hat1 were much less mature upon histological evaluation. The neonatal lethality is due to severe defects in lung development that result in less aeration and respiratory distress. Many of the Hat1−/− neonates also display significant craniofacial defects with abnormalities in the bones of the skull and jaw. Hat1−/− mouse embryonic fibroblasts (MEFs) are defective in cell proliferation and are sensitive to DNA damaging agents. In addition, the Hat1−/− MEFs display a marked increase in genome instability. Analysis of histone dynamics at sites of replication-coupled chromatin assembly demonstrates that Hat1 is not only responsible for the acetylation of newly synthesized histone H4 but is also required to maintain the acetylation of histone H3 on lysines 9, 18, and 27 during replication-coupled chromatin assembly.

Separation of Histone Variants and Post‐Translationally Modified Isoforms by Triton/Acetic Acid/Urea Polyacrylamide Gel Electrophoresis

Due to their similarities in size and charge, complete resolution of histones by electrophoresis poses a considerable challenge. The addition of nonionic detergents to the traditional acetic acid/urea (AU) polyacrylamide gel electrophoresis (PAGE) system has afforded an excellent method to separate not only the different modified forms of histones, but also the primary sequence variant subtypes of selected histone species; it is widely used to separate histones with varying levels of acetylation. This unit describes the use of gels containing the nonionic detergent Triton X-100, referred to as Triton/acetic acid/urea (TAU) polyacrylamide gels, for analysis of histones. Also included are support protocols detailing several accessory techniques: assembly of gel plates for the TAU gel, preparation of histones from isolated nuclei in a solubilized form amenable to electrophoresis, and electrophoretic transfer of proteins from these gels to PVDF membranes.

Schizosaccharomyces pombe Hat1 (Kat1) Is Associated with Mis16 and Is Required for Telomeric Silencing

ABSTRACT The Hat1 histone acetyltransferase has been implicated in the acetylation of histone H4 during chromatin assembly. In this study, we have characterized the Hat1 complex from the fission yeast Schizosaccharomyces pombe and have examined its role in telomeric silencing. Hat1 is found associated with the RbAp46 homologue Mis16, an essential protein. The Hat1 complex acetylates lysines 5 and 12 of histone H4, the sites that are acetylated in newly synthesized H4 in a wide range of eukaryotes. Deletion of hat1 in S. pombe is itself sufficient to cause the loss of silencing at telomeres. This is in contrast to results obtained with an S. cerevisiae hat1Δ strain, which must also carry mutations of specific acetylatable lysines in the H3 tail domain for loss of telomeric silencing to occur. Notably, deletion of hat1 from S. pombe resulted in an increase of acetylation of histone H4 in subtelomeric chromatin, concomitant with derepression of this region. A similar loss of telomeric silencing was also observed after growing cells in the presence of the deacetylase inhibitor trichostatin A. However, deleting hat1 did not cause loss of silencing at centromeres or the silent mating type locus. These results point to a direct link between Hat1, H4 acetylation, and the establishment of repressed telomeric chromatin in fission yeast.

Histone Acetylation During Chromatin Replication and Nucleosome Assembly

Publisher Summary The biochemical and genetic data indicate that the reversible acetylation of nascent H4 is dispensable for histone deposition and physiological nucleosome alignment. Of course, the genetic evidence obtained from yeast may in part reflect the peculiarities of this system, while the ability of acetylated H3 to substitute for H4Ac2 remains untested. These matters should be fertile areas for future research. Approaches for addressing these problems might include a conditional mutant for HATB, or a specific inhibitor of this enzyme. In vitro assembly systems, in which the acetylation of H4 can be manipulated, should also prove valuable. Given that heightened DNaseI sensitivity is a feature of acetylated chromatin in general, it is perhaps ironic that the only clearly identified assembly-related effect of histone acetylation is the prolonged DNaseI sensitivity of chromatin replicated in the presence of butyrate. Whether this represents an actual role for acetylation during nucleosome assembly in vivo, it does underscore the importance of timely deacetylation in the proper and complete formation of chromatin.

Preparation/analysis of chromatin replicated in vivo and in isolated nuclei

This article outlined biochemical methodologies for the labeling, detection, and analysis of newly replicated and newly assembled nucleosomes. The isolation of specific vertebrate factors that may be involved in chromatin assembly in vivo, such as nucleoplasmin, CAF-1, and NAP-1 and their counterparts in Drosophila and yeast add a further dimension to the study of nucleosome assembly in living cells. In particular, the ability to genetically manipulate the yeast system, together with the identification of yeast enzymes that acetylate newly synthesized H4, will certainly provide exciting new avenues for the investigation of chromatin assembly in vivo.