组蛋白修饰在染色质复制过程中的作用

真核生物中的DNA复制,不但要保证DNA编码的基因组信息高保真复制,也要保证染色质结构所蕴含的表观遗传组稳定传递,这个过程对于维持基因组的完整性和稳定性至关重要。时至今日,人们对DNA复制的机制已经有了深入的认识,但是对染色质复制以及表观遗传信息传递的了解才刚刚开始。组蛋白是染色质结构中最主要的蛋白组成部分,其上面丰富的转录后修饰是表观遗传调控的核心方式之一。从最近几年组蛋白的修饰研究进展入手,主要综述在DNA复制过程中组蛋白修饰如何参与染色质复制的调控。     

Coordinate regulation of histone mRNA metabolism and DNA replication

S phase is characterized by the replication of DNA and assembly of chromatin. This requires the synthesis of large amounts of histone proteins to package the newly replicated DNA. Histone mRNAs are the only mRNAs that do not have polyA tails, ending instead in a conserved stemloop sequence. The stemloop binding protein (SLBP) that binds the 3' end of histone mRNA is cell cycle regulated and SLBP is required in all steps of histone mRNA metabolism. Activation of cyclin E/cdk2 prior to entry into S phase is critical for initiation of DNA replication and histone mRNA accumulation. At the end of S phase SLBP is rapidly degraded as a result of phosphorylation of SLBP by cyclin A/cdk1 and CK2 effectively shutting off histone mRNA biosynthesis. E2F1, which is required for expression of many S-phase genes, is regulated in parallel with SLBP and its degradation also requires a cyclin binding site, suggesting that it may also be regulated by the same pathway. It is likely that activation of cyclin A/cdk1 so helps inhibit both DNA replication and histone mRNA accumulation, marking the end of S phase and entry into G2 phase.     

Chromatin assembly during DNA replication in somatic cells.

Newly replicated DNA is assembled into chromatin through two principle pathways. Firstly, parental nucleosomes segregate to replicated DNA, and are transferred directly to one of the two daughter strands during replication fork passage. Secondly, chromatin assembly factors mediate de-novo assembly of nucleosomes on replicating DNA using newly synthesized and acetylated histone proteins. In somatic cells, chromatin assembly factor 1 (CAF-1) appears to be a key player in assembling new nucleosomes during DNA replication. It provides a molecular connection between newly synthesized histones and components of the DNA replication machinery during the S phase of the cell division cycle.     

DNA replication stress in CHK1-depleted tumour cells triggers premature (S-phase) mitosis through inappropriate activation of Aurora kinase B

The disruption of DNA replication in cells triggers checkpoint responses that slow-down S-phase progression and protect replication fork integrity. These checkpoints are also determinants of cell fate and can help maintain cell viability or trigger cell death pathways. CHK1 has a pivotal role in such S-phase responses. It helps maintain fork integrity during replication stress and protects cells from several catastrophic fates including premature mitosis, premature chromosome condensation and apoptosis. Here we investigated the role of CHK1 in protecting cancer cells from premature mitosis and apoptosis. We show that premature mitosis (characterized by the induction of histone H3 phosphorylation, aberrant chromatin condensation, and persistent RPA foci in arrested S-phase cells) is induced in p53-deficient tumour cells depleted of CHK1 when DNA synthesis is disrupted. These events are accompanied by an activation of Aurora kinase B in S-phase cells that is essential for histone H3 Ser10 phosphorylation. Histone H3 phosphorylation precedes the induction of apoptosis in p53−/− tumour cell lines but does not appear to be required for this fate as an Aurora kinase inhibitor suppresses phosphorylation of both Aurora B and histone H3 but has little effect on cell death. In contrast, only a small fraction of p53+/+ tumour cells shows this premature mitotic response, although they undergo a more rapid and robust apoptotic response. Taken together, our results suggest a novel role for CHK1 in the control of Aurora B activation during DNA replication stress and support the idea that premature mitosis is a distinct cell fate triggered by the disruption of DNA replication when CHK1 function is suppressed.     

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.     

Cell cycle specific plasmid DNA replication in the nuclear extract of Saccharomyces cerevisiae: modulation by replication protein A and proliferating cell nuclear antigen

Plasmid DNA replication in nuclear extracts of Saccharomyces cerevisiae in vitro has been shown to be S-phase specific, similar to that observed in vivo. We report here a reconstituted in vitro system with partially purified replication proteins, purified replication protein A (RPA), and recombinant proliferating cell nuclear antigen (PCNA). Nuclear extracts from S-phase, G(1)-phase, and unsynchronized yeast cells were fractionated by phosphocellulose chromatography. Protein fraction (polymerase fraction) enriched with replication proteins, including DNA polymerases (alpha, delta, etc.), was isolated, which was not capable of in vitro replication of supercoiled plasmid DNA. However, when purified yeast RPA and recombinant PCNA together were added to the polymerase fraction obtained from S-phase synchronized cells, in vitro plasmid DNA replication was restored. In vitro plasmid DNA replication with polymerase fractions from unsynchronized and G(1)-phase cells could not be reconstituted upon addition of purified RPA and PCNA. RPA and PCNA isolated from various phases of the cell cycle complemented the S-phase polymerase pool to the same extent. Reconstituted systems with the S-phase polymerase pool, complemented with either the RPA- and PCNA-containing fraction or purified RPA and recombinant PCNA together, were able to produce replication intermediates (ranging in size from 50 to 1500 bp) similar to that observed with the S-phase nuclear extract. Results presented here demonstrate that both RPA and PCNA are cell cycle-independent in their ability to stimulate in vitro plasmid DNA replication, whereas replication factors in the polymerase fractions are strictly S-phase dependent.     

Assembling pieces of the centromere epigenetics puzzle

The centromere is a key region for cell division where the kinetochore assembles, recognizes and attaches to microtubules so that each sister chromatid can segregate to each daughter cell. The centromeric chromatin is a unique rigid chromatin state promoted by the presence of the histone H3 variant CENP-A, in which epigenetic histone modifications of both heterochromatin or euchromatin states and associated protein elements are present. Although DNA sequence is not regarded as important for the establishment of centromere chromatin, it has become clear that this structure is formed as a result of a highly regulated epigenetic event that leads to the recruitment and stability of kinetochore proteins. We describe an integrative model for epigenetic processes that conform regional chromatin interactions indispensable for the recruitment and stability of kinetochore proteins. If alterations of these chromatin regions occur, chromosomal instability is promoted, although segregation may still take place.     

Cell cycle regulation of chromatin at an origin of DNA replication

Selection and licensing of mammalian DNA replication origins may be regulated by epigenetic changes in chromatin structure. The Epstein-Barr virus (EBV) origin of plasmid replication (OriP) uses the cellular licensing machinery to regulate replication during latent infection of human cells. We found that the minimal replicator sequence of OriP, referred to as the dyad symmetry (DS), is flanked by nucleosomes. These nucleosomes were subject to cell cycle-dependent chromatin remodeling and histone modifications. Restriction enzyme accessibility assay indicated that the DS-bounded nucleosomes were remodeled in late G1. Remarkably, histone H3 acetylation of DS-bounded nucleosomes decreased during late G1, coinciding with nucleosome remodeling and MCM3 loading, and preceding the onset of DNA replication. The ATP-dependent chromatin-remodeling factor SNF2h was also recruited to DS in late G1, and formed a stable complex with HDAC2 at DS. siRNA depletion of SNF2h reduced G1-specific nucleosome remodeling, histone deacetylation, and MCM3 loading at DS. We conclude that an SNF2h-HDAC1/2 complex coordinates G1-specific chromatin remodeling and histone deacetylation with the DNA replication initiation process at OriP.