Heterochromatin dynamics in mouse cells: interaction between chromatin assembly factor 1 and HP1 proteins.

Mechanisms contributing to the maintenance of heterochromatin in proliferating cells are poorly understood. We demonstrate that chromatin assembly factor 1 (CAF-1) binds to mouse HP1 proteins via an N-terminal domain of its p150 subunit, a domain dispensable for nucleosome assembly during DNA replication. Mutations in p150 prevent association with HP1 in heterochromatin in cells that are not in S phase and the formation of CAF-1-HP1 complexes in nascent chromatin during DNA replication in vitro. We suggest that CAF-1 p150 has a heterochromatin-specific function distinct from its nucleosome assembly function during S phase. Just before mitosis, CAF-1 p150 and some HP1 progressively dissociate from heterochromatin concomitant with histone H3 phosphorylation. The HP1 proteins reassociate with chromatin at the end of mitosis, as histone H3 is dephosphorylated.     

Human CAF-1-dependent nucleosome assembly in a defined system

Replication-coupled nucleosome assembly is a critical step in packaging newly synthesized DNA into chromatin. Previous studies have defined the importance of the histone chaperones CAF-1 and ASF1A, the replicative clamp PCNA, and the clamp loader RFC for the assembly of nucleosomes during DNA replication. Despite significant progress in the field, replication-coupled nucleosome assembly is not well understood. One of the complications in elucidating the mechanisms of replication-coupled nucleosome assembly is the lack of a defined system that faithfully recapitulates this important biological process in vitro. We describe here a defined system that assembles nucleosomal arrays in a manner dependent on the presence of CAF-1, ASF1A-H3-H4, H2A-H2B, PCNA, RFC, NAP1L1, ATP, and strand breaks. The loss of CAF-1 p48 subunit causes a strong defect in packaging DNA into nucleosomes by this system. We also show that the defined system forms nucleosomes on nascent DNA synthesized by the replicative polymerase δ. Thus, the developed system reproduces several key features of replication-coupled nucleosome assembly.     

Stability of the conservative mode of nucleosome assembly.

The conservative assembly of nucleosome histone octamer cores has been confirmed by electrophoretic analysis of density labeled histones following equilibrium buoyant density centrifugation. After normal replication, crosslinked octamers are shown not to contain a mixture of new and old core histones. Moreover, when DNA synthesis is inhibited by ara-C nucleosome cores are still assembled exclusively from nascent histone. Similarly, after release from cycloheximide inhibition newly synthesized core histone is conservatively deposited. Thus, a conservative mechanism of histone octamer assembly occurs when nascent histone is present in the normal stoichiometry to nascent DNA and when chromatin is assembled in nascent histone or nascent DNA excess.     

Dispersive segregation of nucleosomes during replication of simian virus 40 chromosomes

The distribution of preformed ("old") histone octamers between the two arms of DNA replication forks was analyzed in simian virus 40(SV40)-infected cells following treatment with cycloheximide to prevent nucleosome assembly from nascent histones. Viral chromatin synthesized in the presence of cycloheximide was shown to be deficient in nucleosomes. Replicating SV40 DNA (wild-type 800 and capsid assembly mutant, tsB11) was radiolabeled in either intact cells or nuclear extracts supplemented with cytosol. Nascent nucleosomal monomers were then released by extensive digestion of isolated nuclei, nuclear extracts or isolated viral chromosomes with micrococcal nuclease. The labeled nucleosomal DNA was purified and found to hybridize to both strands of SV40 DNA restriction fragments taken from each side of the origin of DNA replication, whereas Okazaki fragments hybridized only to the strand representing the retrograde DNA template. In addition, isolated, replicating SV40 chromosomes were digested with two strand-specific exonucleases that excised nascent DNA from either the forward or the retrograde side of replication forks. Pretreatment of cells with cycloheximide did not result in an excess of prenucleosomal DNA on either side of replication forks, but did increase the amount of internucleosomal DNA. These data are consistent with a dispersive model for nucleosome segregation in which "old" histone octamers are distributed to both arms of DNA replication forks.     

New histone supply regulates replication fork speed and PCNA unloading

Correct duplication of DNA sequence and its organization into chromatin is central to genome function and stability. However, it remains unclear how cells coordinate DNA synthesis with provision of new histones for chromatin assembly to ensure chromosomal stability. In this paper, we show that replication fork speed is dependent on new histone supply and efficient nucleosome assembly. Inhibition of canonical histone biosynthesis impaired replication fork progression and reduced nucleosome occupancy on newly synthesized DNA. Replication forks initially remained stable without activation of conventional checkpoints, although prolonged histone deficiency generated DNA damage. PCNA accumulated on newly synthesized DNA in cells lacking new histones, possibly to maintain opportunity for CAF-1 recruitment and nucleosome assembly. Consistent with this, in vitro and in vivo analysis showed that PCNA unloading is delayed in the absence of nucleosome assembly. We propose that coupling of fork speed and PCNA unloading to nucleosome assembly provides a simple mechanism to adjust DNA replication and maintain chromatin integrity during transient histone shortage.     

Requirement of Cyclin/Cdk2 and protein phosphatase 1 activity for chromatin assembly factor 1-dependent chromatin assembly during DNA synthesis

The influence of reversible protein phosphorylation on nucleosome assembly during DNA replication was analyzed in extracts from human cells. Inhibitor studies and add-back experiments indicated requirements of cyclin A/Cdk2, cyclin E/Cdk2, and protein phosphatase type 1 (PP1) activities for nucleosome assembly during DNA synthesis by chromatin assembly factor 1 (CAF-1). The p60 subunit of CAF-1 is a molecular target for reversible phosphorylation by cyclin/Cdk complexes and PP1 during nucleosome assembly and DNA synthesis in vitro. Purified p60 can be directly phosphorylated by purified cyclin A/Cdk2, cyclin E/Cdk2, and cyclin B1/Cdk1, but not by cyclin D/Cdk4 complexes in vitro. Cyclin B1/Cdk1 triggers hyperphosphorylation of p60 in the presence of additional cytosolic factors. CAF-1 containing hyperphosphorylated p60 prepared from mitotic cells is inactive in nucleosome assembly and becomes activated by dephosphorylation in vitro. These data provide functional evidence for a requirement of the cell cycle machinery for nucleosome assembly by CAF-1 during DNA replication.     

Preincubation of T antigen with DNA overcomes repression of SV40 DNA replication by nucleosome assembly.

Circular duplex DNA containing the SV40 replication origin was assembled into chromosomes in vitro with core histones and nucleosome assembly factor from HeLa cells. Their ability to serve as a template for replication was examined by incubating them with SV40 T antigen and HeLa cell extract. Nucleosome assembly of the template prevented DNA replication. Replication of chromosomes was severely inhibited at more than two-thirds of physiological nucleosome density. When the DNA was preincubated with T antigen and then assembled into chromosomes, however, inhibition of DNA replication was greatly reduced. These results suggest that nucleosome assembly of the template inhibits initiation of SV40 DNA replication and that the inhibition can be overcome by formation of an initiation complex before nucleosome assembly.     

Chromatin assembled in the presence of cytosine arabinoside has a short nucleosome repeat.

Incubation of MSB cells with cytosine arabinoside (1-beta-D-arabinofuranosylcytosine, ara-C) inhibits 3H-thymidine incorporation into nascent DNA while nucleosome core histone synthesis proceeds in molar stoichiometry at about 20% of control rates. The excess nascent histone is incorporated into chromatin and nucleosome cores are assembled normally on the small amount of DNA which is synthesized at submaximal levels of ara-C. This DNA becomes packaged into a shortened nucleosome repeat, however. These results indicate that the nucleosome core is a strongly conserved unit of chromatin replication and suggest that the stoichiometry of nascent histone to DNA may be one factor influencing the establishment of the nucleosome repeat length. It cannot be the only factor, however, since the closely packed nucleosomes made in the presence of ara-C begin to return to their normal spacing within six hours after reversal.     

Asf1 is required for viability and chromatin assembly during DNA replication in vertebrate cells

Asf1 (anti-silencing function 1), a well conserved protein from yeast to humans, acts as a histone chaperone and is predicted to participate in a variety of chromatin-mediated cellular processes. To investigate the physiological role of vertebrate Asf1 in vivo, we generated a conditional Asf1-deficient mutant from chicken DT40 cells. Induction of Asf1 depletion resulted in the accumulation of cells in S phase with decreased DNA replication and increased mitotic aberrancy forming multipolar spindles, leading to cell death. In addition, nascent chromatin in Asf1-depleted cells showed increased nuclease sensitivity, indicating impaired nucleosome assembly during DNA replication. Complementation analyses revealed that the functional domain of Asf1 for cell viability was confined to the N-terminal core domain (amino acids 1-155) that is a binding platform for histones H3/H4, CAF-1p60, and HIRA, whereas Asf1 mutant proteins, abolishing binding abilities with both p60 and HIRA, exhibit no effect on viability. These results together indicate that the vertebrate Asf1 plays a crucial role in replication-coupled chromatin assembly, cell cycle progression, and cellular viability and provide a clue of a possible role in a CAF-1- and HIRA-independent chromatin-modulating process for cell proliferation.     

DNA sequence templates adjacent nucleosome and ORC sites at gene amplification origins in Drosophila

Eukaryotic origins of DNA replication are bound by the origin recognition complex (ORC), which scaffolds assembly of a pre-replicative complex (pre-RC) that is then activated to initiate replication. Both pre-RC assembly and activation are strongly influenced by developmental changes to the epigenome, but molecular mechanisms remain incompletely defined. We have been examining the activation of origins responsible for developmental gene amplification in Drosophila. At a specific time in oogenesis, somatic follicle cells transition from genomic replication to a locus-specific replication from six amplicon origins. Previous evidence indicated that these amplicon origins are activated by nucleosome acetylation, but how this affects origin chromatin is unknown. Here, we examine nucleosome position in follicle cells using micrococcal nuclease digestion with Ilumina sequencing. The results indicate that ORC binding sites and other essential origin sequences are nucleosome-depleted regions (NDRs). Nucleosome position at the amplicons was highly similar among developmental stages during which ORC is or is not bound, indicating that being an NDR is not sufficient to specify ORC binding. Importantly, the data suggest that nucleosomes and ORC have opposite preferences for DNA sequence and structure. We propose that nucleosome hyperacetylation promotes pre-RC assembly onto adjacent DNA sequences that are disfavored by nucleosomes but favored by ORC.     

Nuclear accumulation of HMG1 protein is correlated to DNA synthesis

The subcellular localization of HMG1 protein was studied by immunoelectron microscopy during growth of CV1 cells in culture and in confluent CV1 cells subsequently lytically infected with SV40. HMG1 was always detected in the cytoplasm of both non-infected and infected cells. On the other hand, this protein displayed a nuclear localization only in those cells active in cellular and/or viral DNA replication, that is, in actively dividing non-infected cells and in confluent cells following SV40 infection. The combination of electron microscope immunocytochemistry and autoradiography revealed that during SV40 lytic infection, HMG1 accumulates at sites of active viral DNA replication. Since HMG1 is a single-stranded DNA binding protein and acts in vitro as a physiological nucleosome assembly factor, we suggest that its presence in the nucleus is related to its requirement in the DNA replication process.     

Monitoring the spatiotemporal dynamics of proteins at replication forks and in assembled chromatin using isolation of proteins on nascent DNA

Understanding the processes of DNA replication, chromatin assembly and maturation, and the replication stress response requires the ability to monitor protein dynamics at active and damaged replication forks. Detecting protein accumulation at replication forks or damaged sites has primarily relied on immunofluorescence imaging, which is limited in resolution and antibody sensitivity. Here we describe a procedure to isolate proteins on nascent DNA (iPOND) that permits a high-resolution spatiotemporal analysis of proteins at replication forks or on chromatin following DNA replication in cultured cells. iPOND relies on labeling of nascent DNA with the nucleoside analog 5-ethynyl-2'-deoxyuridine (EdU). Biotin conjugation to EdU-labeled DNA using click chemistry facilitates a single-step streptavidin purification of proteins bound to the nascent DNA. iPOND permits an interrogation of any cellular process linked to DNA synthesis using a 3- to 4-d protocol.     

Replication-guided nucleosome packing and nucleosome breathing expedite the formation of dense arrays

The first level of genome packaging in eukaryotic cells involves the formation of dense nucleosome arrays, with DNA coverage near 90% in yeasts. How cells achieve such high coverage within a short time, e.g. after DNA replication, remains poorly understood. It is known that random sequential adsorption of impenetrable particles on a line reaches high density extremely slowly, due to a jamming phenomenon. The nucleosome-shifting action of remodeling enzymes has been proposed as a mechanism to resolve such jams. Here, we suggest two biophysical mechanisms which assist rapid filling of DNA with nucleosomes, and we quantitatively characterize these mechanisms within mathematical models. First, we show that the ‘softness’ of nucleosomes, due to nucleosome breathing and stepwise nucleosome assembly, significantly alters the filling behavior, speeding up the process relative to ‘hard’ particles with fixed, mutually exclusive DNA footprints. Second, we explore model scenarios in which the progression of the replication fork could eliminate nucleosome jamming, either by rapid filling in its wake or via memory of the parental nucleosome positions. Taken together, our results suggest that biophysical effects promote rapid nucleosome filling, making the reassembly of densely packed nucleosomes after DNA replication a simpler task for cells than was previously thought.     

The kinetic landscape of nucleosome assembly: A coarse-grained molecular dynamics study

The organization of nucleosomes along the Eukaryotic genome is maintained over time despite disruptive events such as replication. During this complex process, histones and DNA can form a variety of non-canonical nucleosome conformations, but their precise molecular details and roles during nucleosome assembly remain unclear. In this study, employing coarse-grained molecular dynamics simulations and Markov state modeling, we characterized the complete kinetics of nucleosome assembly. On the nucleosome-positioning 601 DNA sequence, we observe a rich transition network among various canonical and non-canonical tetrasome, hexasome, and nucleosome conformations. A low salt environment makes nucleosomes stable, but the kinetic landscape becomes more rugged, so that the system is more likely to be trapped in off-pathway partially assembled intermediates. Finally, we find that the co-operativity between DNA bending and histone association enables positioning sequence motifs to direct the assembly process, with potential implications for the dynamic organization of nucleosomes on real genomic sequences.     

Temporal dynamics of cytomegalovirus chromatin assembly in productively infected human cells

The genomes of herpesviruses, including human cytomegalovirus (CMV), are double-stranded DNA molecules maintained as episomes during infection. The viral DNA lacks histones when encapsidated in the virion. However, it has been found histone associated inside infected cells, implying unidentified chromatin assembly mechanisms. Our results indicate that components of the host cell nucleosome deposition machinery target intranuclear CMV DNA, resulting in stepwise viral-chromatin assembly. CMV genomes undergo limited histone association and nucleosome assembly as early as 30 min after infection via DNA replication-independent mechanisms. Low average viral-genome chromatinization is maintained throughout the early stages of infection. The late phase of infection is characterized by a striking increase in average histone occupancy coupled with the process of viral-DNA replication. While the initial chromatinization affected all analyzed parts of the CMV chromosome, a subset of viral genomic regions, including the major immediate-early promoter, proved to be largely resistant to replication-dependent histone deposition. Finally, our results predict the likely requirement for an unanticipated chromatin disassembly process that enables packaging of histone-free DNA into progeny capsids.     

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.