A bridging model for persistence of a polycomb group protein complex through DNA replication in vitro

Epigenetic regulation may involve heritable chromatin states, but how chromatin features can be inherited through DNA replication is incompletely understood. We address this question using cell-free replication of chromatin. Previously, we showed that a Polycomb group complex, PRC1, remains continuously associated with chromatin through DNA replication. Here we investigate the mechanism of persistence. We find that a single PRC1 subunit, Posterior sex combs (PSC), can reconstitute persistence through DNA replication. PSC binds nucleosomes and self-interacts, bridging nucleosomes into a stable, oligomeric structure. Within these structures, individual PSC-chromatin contacts are dynamic. Stable association of PSC with chromatin, including through DNA replication, depends on PSC-PSC interactions. Our data suggest that labile individual PSC-chromatin contacts allow passage of the DNA replication machinery while PSC-PSC interactions prevent PSC from dissociating, allowing it to rebind to replicated chromatin. This mechanism may allow inheritance of chromatin proteins including PRC1 through DNA replication to maintain chromatin states.     

Electron microscopic analysis of chromatin replication in the cellular blastoderm drosophila melanogaster embryo

Transmission electron microscopic techniques were used to study the spatial distribution of replicons and the ultrastructure of chromatin in the S phase genome of cellular blastoderm Drosophila melanogaster embryos. We observed chromatin exhibiting distinct bifurcations along each fiber during the initial 20 min of the first cell cycle of blastulation. We interpreted the “bubble-like” configurations produced by adjacent bifurcations as intermediate structures in chromatin replication (that is, replicons). We observed homologous ribonucleoprotein (RNP) fiber arrays on both chromatid arms within some replicons, implying DNA sequence homology and reinforcing the identification of such arms as daughter chromatid fibers. We did not observe replicon configurations on chromatin obtained from embryos staged at more than 20 min into cellular blastulation. Daughter chromatid fibers, however, were identified by the presence of identical RNP fiber arrays on chromatid strands arranged in parallel on the electron microscope grid.We examined the distribution of replicon structures on the cellular blastoderm genome and compared it with electron microscopic data on DNA replication in cleavage embryos (Blumenthal, Kriegstein and Hogness, 1973). S phase is completed in slightly < 4 min during cleavage, or approximately one fifth the time required for DNA synthesis in cellular blastoderm embryos. The mean distance separating adjacent replication origins at cellularization was estimated to be 10.6 kilobases (kb), a value 35% greater than the 7.9 kb inter-origin average determined for cleavage embryos. In contrast to the near-simultaneous activation of replication origins during cleavage replication, we observed that replication origins are not activated synchronously at cellular blastulation. We concluded that the marked increase in the duration of S phase is effected by a reduction in the frequency of replication activation events which occur asynchronously during genome replication at cellularization.We found that the ultrastructure of newly replicated chromatin exhibited a morphology indistinguishable from nucleosomal chromatin. Unreplicated chromatin fibers separating adjacent replicons also exhibit spherical subunits. We inferred that the spherical structures on replicating chromatin are nucleosomes and concluded that histones are not disassociated from the DNA significantly prior to DNA replication, and that a very rapid reassociation of nucleosomes occurs on both daughter DNA molecules following replication.     

Physical and functional crosstalk between Fanconi anemia core components and the GINS replication complex

Fanconi anemia (FA) is an inherited disease characterized by bone marrow failure, increased cancer risk and hypersensitivity to DNA cross-linking agents, implying a role for this pathway in the maintenance of genomic stability. The central player of the FA pathway is the multi-subunit E3 ubiquitin ligase complex activated through a replication- and DNA damage-dependent mechanism. A consequence of the activation of the complex is the monoubiquitylation of FANCD2 and FANCI, late term effectors in the maintenance of genome integrity. The details regarding the coordination of the FA-dependent response and the DNA replication process are still mostly unknown. We found, by yeast two-hybrid assay and co-immunoprecipitation in human cells, that the core complex subunit FANCF physically interacts with PSF2, a member of the GINS complex essential for both the initiation and elongation steps of DNA replication. In HeLa cells depleted for PSF2, we observed a decreased binding to chromatin of the FA core complex, suggesting that the GINS complex may have a role in either loading or stabilizing the FA core complex onto chromatin. Consistently, GINS and core complex bind chromatin contemporarily upon origin firing and PSF2 depletion sensitizes cells to DNA cross-linking agents. However, depletion of PSF2 is not sufficient to reduce monoubiquitylation of FANCD2 or its localization to nuclear foci following DNA damage. Our results suggest a novel crosstalk between DNA replication and the FA pathway.     

Chromatin elimination and the genetic organisation of the macronucleus in Tetrahymena thermophila

In exponentially growing Tretrahymena thermophila the DNA content of the following structures was determined by cytophotometry: macronuclei of sister cells immediately after division; micronuclei; extranuclear chromatin in dividing cells and postdividers. Further, the development of macro-nuclear DNA amount in successive cell generations was determined. It was found that chromatin elimination is a frequent process reducing DNA content by about 4% per fission. This chromatin disappears within 20 min after division. The quantity of DNA extruded is highly variable and is different from the micronuclear DNA amount or multiples of it. The frequency of generations with two replication rounds as well as those without replication is estimated to be in the range of 2% each. These findings together with the qualitative difference between micro- and macronuclear DNAs suggest that the macronucleus of Tetrahymena is not entirely composed of complete genomes and that parts of the genetic material must be treated specifically for different sequences either during extrusion or during replication.     

The structure of chromatin at meiotic prophase I

The qualitative and quantitative changes in molecular chromatin structures during the meiotic prophase I were studied. The following patterns were discovered: (1) unlike somatic cells, the syntheses of total histone and DNA and its integration into the chromatin occur independently and asynchronously: DNA replication is completed by the interphase, whereas the synthesis of histone and its integration into the chromatin continue to late meiotic prophase I, and (2) individual histone fractions are synthesized and integrated into the chromatin during meiotic prophase independently and asynchronously. Chromatin hydrolysis with nucleases DNI, STN, and SI demonstrated considerable differences in the hydrolysis products obtained at different stages of the meiotic prophase I; presumably, this reflects the differences between the structures of initial chromatin at different stages of the meiotic prophase I.     

The structure of chromatin at meiotic prophase I

The qualitative and quantitative changes in molecular chromatin structures during the meiotic prophase I were studied. The following patterns were discovered: (1) unlike somatic cells, the syntheses of total histone and DNA and its integration into the chromatin occur independently and asynchronously: DNA replication is completed by the interphase, whereas the synthesis of histone and its integration into the chromatin continue to late meiotic prophase I, and (2) individual histone fractions are synthesized and integrated into the chromatin during meiotic prophase independently and asynchronously. Chromatin hydrolysis with nucleases DNI, STN, and SI demonstrated considerable differences in the hydrolysis products obtained at different stages of the meiotic prophase I; presumably, this reflects the differences between the structures of initial chromatin at different stages of the meiotic prophase I.     

Replication of heterochromatin: insights into mechanisms of epigenetic inheritance

Heterochromatin is composed of tightly condensed chromatin in which the histones are deacetylated and methylated, and specific nonhistone proteins are bound. Additionally, in vertebrates and plants, the DNA within heterochromatin is methylated. As the heterochromatic state is stably inherited, replication of heterochromatin requires not only duplication of the DNA but also a reinstallment of the appropriate protein and DNA modifications. Thus replication of heterochromatin provides a framework for understanding mechanisms of epigenetic inheritance. In recent studies, roles have been identified for replication factors in reinstating heterochromatin, particularly functions for origin recognition complex, proliferating cell nuclear antigen, and chromatin-assembly factor 1 in recruiting the heterochromatin binding protein HP1, a histone methyltransferase, a DNA methyltransferase, and a chromatin remodeling complex. Potential mechanistic links between these factors are discussed. In some cells, replication of the heterochromatin is blocked, and in Drosophila this inhibition is mediated by a chromatin binding protein SuUR.     

Histone h3 lysine 4 methylation marks postreplicative human cytomegalovirus chromatin

In the nuclei of permissive cells, human cytomegalovirus genomes form nucleosomal structures initially resembling heterochromatin but gradually switching to a euchromatin-like state. This switch is characterized by a decrease in histone H3 K9 methylation and a marked increase in H3 tail acetylation and H3 K4 methylation across the viral genome. We used ganciclovir and a mutant virus encoding a reversibly destabilized DNA polymerase to examine the impact of DNA replication on histone modification dynamics at the viral chromatin. The changes in H3 tail acetylation and H3 K9 methylation proceeded in a DNA replication-independent fashion. In contrast, the increase in H3 K4 methylation proved to depend widely on viral DNA synthesis. Consistently, labeling of nascent DNA using "click chemistry" revealed preferential incorporation of methylated H3 K4 into viral (but not cellular) chromatin during or following DNA replication. This study demonstrates largely selective epigenetic tagging of postreplicative human cytomegalovirus chromatin.     

Fanconi anemia proteins stabilize replication forks

Fanconi anemia (FA) is a recessive genetic disorder characterized by hypersensitivity to crosslinking agents that has been attributed to defects in DNA repair and/or replication. FANCD2 and the FA core complex bind to chromatin during DNA replication; however, the role of FA proteins during replication is unknown. Using Xenopus cell-free extracts, we show that FANCL depletion results in defective DNA replication restart following treatment with camptothecin, a drug that results in DSBs during DNA replication. This defect is more pronounced following treatment with mitomycin C, presumably because of an additional role of the FA pathway in DNA crosslink repair. Moreover, we show that chromatin binding of FA core complex proteins during DNA replication follows origin assembly and origin firing and is dependent on the binding of RPA to ssDNA while FANCD2 additionally requires ATR, consistent with FA proteins acting at replication forks. Together, our data suggest that FA proteins play a role in replication restart at collapsed replication forks.     

The role of single-stranded DNA and polymerase alpha in establishing the ATR,Hus1 DNA replication checkpoint

Using a nucleus-free DNA replication system we have investigated the roles of Xenopus ATR (XATR) and Hus1 (Xhus1) as the DNA replication checkpoint sensors. Like XATR, Xhus1 is required for the checkpoint-dependent phosphorylation of Xchk1 and associates with chromatin in an initiation-dependent manner. While removal of replication protein A inhibits chromatin association of both XATR and Xhus1, removal of polymerase alpha only disrupts chromatin association of Xhus1. In addition, chromatin association of XATR and Xhus1 are independent of each other. Finally, like XATR, Xhus1 associates with chromatin in unperturbed S phase and dissociates from chromatin following completion of DNA replication.     

Chromatin binding of the fission yeast replication factor mcm4 occurs during anaphase and requires ORC and cdc18

We describe an in situ technique for studying the chromatin binding of proteins in the fission yeast Schizosaccharomyces pombe. After tagging the protein of interest with green fluorescent protein (GFP), chromatin-associated protein is detected by GFP fluorescence following cell permeabilization and washing with a non-ionic detergent. Cell morphology and nuclear structure are preserved in this procedure, allowing structures such as the mitotic spindle to be detected by indirect immunofluorescence. Cell cycle changes in the chromatin association of proteins can therefore be determined from individual cells in asynchronous cultures. We have applied this method to the DNA replication factor mcm4/cdc21, and find that chromatin association occurs during anaphase B, significantly earlier than is the case in budding yeast. Binding of mcm4 to chromatin requires orc1 and cdc18 (homologous to Cdc6 in budding yeast). Release of mcm4 from chromatin occurs during S phase and requires DNA replication. Upon overexpressing cdc18, we show that mcm4 is required for re-replication of the genome in the absence of mitosis and is associated with chromatin in cells undergoing re-replication.     

Innate structure of DNA foci restricts the mixing of DNA from different chromosome territories

The distribution of chromatin within the mammalian nucleus is constrained by its organization into chromosome territories (CTs). However, recent studies have suggested that promiscuous intra- and inter-chromosomal interactions play fundamental roles in regulating chromatin function and so might define the spatial integrity of CTs. In order to test the extent of DNA mixing between CTs, DNA foci of individual CTs were labeled in living cells following incorporation of Alexa-488 and Cy-3 conjugated replication precursor analogues during consecutive cell cycles. Uniquely labeled chromatin domains, resolved following random mitotic segregation, were visualized as discrete structures with defined borders. At the level of resolution analysed, evidence for mixing of chromatin from adjacent domains was only apparent within the surface volumes where neighboring CTs touched. However, while less than 1% of the nuclear volume represented domains of inter-chromosomal mixing, the dynamic plasticity of DNA foci within individual CTs allows continual transformation of CT structure so that different domains of chromatin mixing evolve over time. Notably, chromatin mixing at the boundaries of adjacent CTs had little impact on the innate structural properties of DNA foci. However, when TSA was used to alter the extent of histone acetylation changes in chromatin correlated with increased chromatin mixing. We propose that DNA foci maintain a structural integrity that restricts widespread mixing of DNA and discuss how the potential to dynamically remodel genome organization might alter during cell differentiation.