Linker histones are mobilized during infection with herpes simplex virus type 1

Histones interact with herpes simplex virus type 1 (HSV-1) genomes and localize to replication compartments early during infections. However, HSV-1 genomes do not interact with histones in virions and are deposited in nuclear domains devoid of histones. Moreover, late viral replication compartments are also devoid of histones. The processes whereby histones come to interact with HSV-1 genomes, to be later displaced, remain unknown. However, they would involve the early movement of histones to the domains containing HSV-1 genomes and the later movement away from them. Histones unbind from chromatin, diffuse through the nucleoplasm, and rebind at different sites. Such mobility is upregulated by, for example, phosphorylation or acetylation. We evaluated whether HSV-1 infection modulates histone mobility, using fluorescence recovery after photobleaching. All somatic H1 variants were mobilized to different degrees. H1.2, the most mobilized, was mobilized at 4 h and further so at 7 h after infection, resulting in increases in its "free" pools. H1.2 was mobilized to a "basal" degree under conditions of little to no HSV-1 protein expression. This basal mobilization required nuclear native HSV-1 genomes but was independent of HSV-1 proteins and most likely due to cellular responses. Mobilization above this basal degree, and increases in H1.2 free pools, however, depended on immediate-early or early HSV-1 proteins, but not on HSV-1 genome replication or late proteins. Linker histone mobilization is a novel consequence of cell-virus interactions, which is consistent with the dynamic interactions between histones and HSV-1 genomes during lytic infection; it may also participate in the regulation of viral gene expression.     

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.     

Epigenetic asymmetry in the mammalian zygote and early embryo: relationship to lineage commitment?

Epigenetic asymmetry between parental genomes and embryonic lineages exists at the earliest stages of mammalian development. The maternal genome in the zygote is highly methylated in both its DNA and its histones and most imprinted genes have maternal germline methylation imprints. The paternal genome is rapidly remodelled with protamine removal, addition of acetylated histones, and rapid demethylation of DNA before replication. A minority of imprinted genes have paternal germline methylation imprints. Methylation and chromatin reprogramming continues during cleavage divisions, but at the blastocyst stage lineage commitment to inner cell mass (ICM) or trophectoderm (TE) fate is accompanied by a dramatic increase in DNA and histone methylation, predominantly in the ICM. This may set up major epigenetic differences between embryonic and extraembryonic tissues, including in X-chromosome inactivation and perhaps imprinting. Maintaining epigenetic asymmetry appears important for development as asymmetry is lost in cloned embryos, most of which have developmental defects, and in particular an imbalance between extraembryonic and embryonic tissue development.     

In and out: histone variant exchange in chromatin

Alterations in nucleosome structure affect the accessibility of the DNA and can generate specialized domains of chromatin in the genome. Such changes can be introduced by posttranslational modifications of histones, by chromatin remodeling, or by the incorporation of variants of H2A and H3 into nucleosomes. In contrast to the canonical histones, which are deposited behind the replication fork during S phase, histone variants are incorporated in a process that is independent of DNA replication. Recent studies have shown that distinct multiprotein complexes are responsible for the targeted deposition of histone variants at active genes, centromeres and silent loci. The incorporation of histone variants most probably has epigenetic consequences and contributes to architectural changes in chromosomes.     

The Differential Mobilization of Histones H3.1 and H3.3 by Herpes Simplex Virus 1 Relates Histone Dynamics to the Assembly of Viral Chromatin

During lytic infections, HSV-1 genomes are assembled into unstable nucleosomes. The histones required for HSV-1 chromatin assembly, however, are in the cellular chromatin. We have shown that linker (H1) and core (H2B and H4) histones are mobilized during HSV-1 infection, and proposed that the mobilized histones are available for assembly into viral chromatin. However, the actual relevance of histone mobilization remained unknown. We now show that canonical H3.1 and variant H3.3 are also mobilized during HSV-1 infection. Mobilization required no HSV-1 protein expression, although immediate early or early proteins enhanced it. We used the previously known differential association of H3.3 and H3.1 with HSV-1 DNA to test the relevance of histone mobilization. H3.3 binds to HSV-1 genomes first, whereas H3.1 only binds after HSV-1 DNA replication initiates. Consistently, H3.3 and H3.1 were differentially mobilized. H3.1 mobilization decreased with HSV-1 DNA replication, whereas H3.3 mobilization was largely unaffected by it. These results support a model in which previously mobilized H3.1 is immobilized by assembly into viral chromatin during HSV-1 DNA replication, whereas H3.3 is mobilized and assembled into HSV-1 chromatin throughout infection. The differential mobilizations of H3.3 and H3.1 are consistent with their differential assembly into viral chromatin. These data therefore relate nuclear histone dynamics to the composition of viral chromatin and provide the first evidence that histone mobilization relates to viral chromatin assembly.     

Epigenetic instability due to defective replication of structured DNA

The accurate propagation of histone marks during chromosomal replication is proposed to rely on the tight coupling of replication with the recycling of parental histones to the daughter strands. Here, we show in the avian cell line DT40 that REV1, a key regulator of DNA translesion synthesis at the replication fork, is required for the maintenance of repressive chromatin marks and gene silencing in the vicinity of DNA capable of forming G-quadruplex (G4) structures. We demonstrate a previously unappreciated requirement for REV1 in replication of G4 forming sequences and show that transplanting a G4 forming sequence into a silent locus leads to its derepression in REV1-deficient cells. Together, our observations support a model in which failure to maintain processive DNA replication at G4 DNA in REV1-deficient cells leads to uncoupling of DNA synthesis from histone recycling, resulting in localized loss of repressive chromatin through biased incorporation of newly synthesized histones.     

Replication stress,a source of epigenetic aberrations in cancer?

Cancer cells accumulate widespread local and global chromatin changes and the source of this instability remains a key question. Here we hypothesize that chromatin alterations including unscheduled silencing can arise as a consequence of perturbed histone dynamics in response to replication stress. Chromatin organization is transiently disrupted during DNA replication and maintenance of epigenetic information thus relies on faithful restoration of chromatin on the new daughter strands. Acute replication stress challenges proper chromatin restoration by deregulating histone H3 lysine 9 mono‐methylation on new histones and impairing parental histone recycling. This could facilitate stochastic epigenetic silencing by laying down repressive histone marks at sites of fork stalling. Deregulation of replication in response to oncogenes and other tumor‐promoting insults is recognized as a significant source of genome instability in cancer. We propose that replication stress not only presents a threat to genome stability, but also jeopardizes chromatin integrity and increases epigenetic plasticity during tumorigenesis.     

Temporal association of the herpes simplex virus genome with histone proteins during a lytic infection

Previous work has determined that there are nucleosomes on the herpes simplex virus (HSV) genome during a lytic infection but that they are not arranged in an equally spaced array like in cellular DNA. However, like in cellular DNA, the promoter regions of several viral genes have been shown to be associated with nucleosomes containing modified histone proteins that are generally found associated with actively transcribed genes. Furthermore, it has been found that the association of modified histones with the HSV genome can be detected at the earliest times postinfection (1 h postinfection) and increases up to 3 h postinfection. However from 3 h to 6 h postinfection (the late phase of the replication cycle), the association decreases. In this study we have examined histone association with promoter regions of all kinetic classes of genes. This was done over the time course of an infection in Sy5y cells using sucrose gradient sedimentation, bromodeoxyuridine labeling, chromatin immunoprecipitation assays, Western blot analysis, trypsin and DNase digestion, and quantitative real-time PCR. Because no histones were detected inside HSV type 1 capsids, the viral genome probably starts to associate with histones after being transported from infecting virions into the host nucleus. Promoter regions of all gene classes (immediate early, early, and late) bind with histone proteins at the start of viral gene expression. However, after viral DNA replication initiates, histones appear not to associate with newly synthesized viral genomes.     

Conservative assembly and segregation of nucleosomal histones

The assembly of new histones into nucleosomes and the segregation of old histones during replication were investigated using a density gradient, sedimentation equilibrium analysis of histones labeled in vivo with dense amino acids. After a 1 hr pulse of dense amino acids and 3H-lysine, nucleosomes were isolated from chick myoblast organ cultures, and the histones were cross-linked to octamers. The octamers were purified from DNA and then banded to equilibrium in cesium-formate guanidinium-HCI density gradients. The cross-linked dense octamers have the same density as the noncross-linked dense histones, and both were significantly heavier than histones synthesized in the presence of light amino acids. This experiment shows that new histone does not mix with old histone in the new nucleosomes, since the labeling protocol allows density labeling of only one histone for every seven preexisting unlabeled histones. Thus the assembly of new histone octamers is conservative. Using essentially the same experimental design, but varying the details of the labeling procedures, we also show that the dense histone octamer is stable over 3-4 generations, that neighboring octamers tend to be synthesized at the same time, and that old and new histone octamers segregate conservatively over 2-3 generations.