Spatio-temporal re-organization of replication foci accompanies replication domain consolidation during human pluripotent stem cell lineage specification

Lineage specification of both mouse and human pluripotent stem cells (PSCs) is accompanied by spatial consolidation of chromosome domains and temporal consolidation of their replication timing. Replication timing and chromatin organization are both established during G1 phase at the timing decision point (TDP). Here, we have developed live cell imaging tools to track spatio-temporal replication domain consolidation during differentiation. First, we demonstrate that the fluorescence ubiquitination cell cycle indicator (Fucci) system is incapable of demarcating G1/S or G2/M cell cycle transitions. Instead, we employ a combination of fluorescent PCNA to monitor S phase progression, cytokinesis to demarcate mitosis, and fluorescent nucleotides to label early and late replication foci and track their 3D organization into sub-nuclear chromatin compartments throughout all cell cycle transitions. We find that, as human PSCs differentiate, the length of S phase devoted to replication of spatially clustered replication foci increases, coincident with global compartmentalization of domains into temporally clustered blocks of chromatin. Importantly, re-localization and anchorage of domains was completed prior to the onset of S phase, even in the context of an abbreviated PSC G1 phase. This approach can also be employed to investigate cell fate transitions in single PSCs, which could be seen to differentiate preferentially from G1 phase. Together, our results establish real-time, live-cell imaging methods for tracking cell cycle transitions during human PSC differentiation that can be applied to study chromosome domain consolidation and other aspects of lineage specification.     

General and specific replication profiles are detected in normal human cells by genome-wide and single-locus molecular combing

Mammalian genomes are replicated under a flexible program, with random use of origins and variable fork rates, and many details of the process must be still unraveled. Molecular combing provides a set of direct data regarding the replication profile of eukaryotic cells: fork rates; organization of the replication clusters; proportion of unidirectional forks; and fork dynamics. In this study the replication profiles of different primary and immortalized non-cancer human cells (lymphocytes, lymphoblastoid cells, fibroblasts) were evaluated at the whole-genome level or within reference genomic regions harboring coding genes. It emerged that these different cell types are characterized by specific replication profiles. In primary fibroblasts, a remarkable fraction of the mammalian genome was found to be replicated by unidirectional forks, and interestingly, the proportion of unidirectional forks further increased in the replicating genome along the population divisions. A second difference concerned in the proportion of paused replication forks, again more frequent in primary fibroblasts than in PBL/lymphoblastoid cells. We concluded that these patterns, whose relevance could escape when genomic methods are applied, represent normal replication features. In single-locus analyses, unidirectional and paused replication forks were highly represented in all genomic regions considered with respect to the average estimates referring to the whole-genome. In addition, fork rates were significantly lower than whole-genome estimates. Instead, when considering the specificities of each genomic region investigated (early to late replication, normal or fragile site) no further differentiating features of replication profiles were detected. These data, representing the integration of genome-wide and single-locus analyses, highlight a large heterogeneity of replication profiles among cell types and within the genome, which should be considered for the correct use of replication datasets.     

A CAF-1 dependent pool of HP1 during heterochromatin duplication

To investigate how the complex organization of heterochromatin is reproduced at each replication cycle, we examined the fate of HP1-rich pericentric domains in mouse cells. We find that replication occurs mainly at the surface of these domains where both PCNA and chromatin assembly factor 1 (CAF-1) are located. Pulse-chase experiments combined with high-resolution analysis and 3D modeling show that within 90 min newly replicated DNA become internalized inside the domain. Remarkably, during this time period, a specific subset of HP1 molecules (alpha and gamma) coinciding with CAF-1 and replicative sites is resistant to RNase treatment. Furthermore, these replication-associated HP1 molecules are detected in Suv39 knockout cells, which otherwise lack stable HP1 staining at pericentric heterochromatin. This replicative pool of HP1 molecules disappears completely following p150CAF-1 siRNA treatment. We conclude that during replication, the interaction of HP1 with p150CAF-1 is essential to promote delivery of HP1 molecules to heterochromatic sites, where they are subsequently retained by further interactions with methylated H3-K9 and RNA.     

Rif1 regulates the replication timing domains on the human genome

DNA replication is spatially and temporally regulated during S-phase. DNA replication timing is established in early-G1-phase at a point referred to as timing decision point. However, how the genome-wide replication timing domains are established is unknown. Here, we show that Rif1 (Rap1-interacting-factor-1), originally identified as a telomere-binding factor in yeast, is a critical determinant of the replication timing programme in human cells. Depletion of Rif1 results in specific loss of mid-S replication foci profiles, stimulation of initiation events in early-S-phase and changes in long-range replication timing domain structures. Analyses of replication timing show replication of sequences normally replicating early is delayed, whereas that normally replicating late is advanced, suggesting that replication timing regulation is abrogated in the absence of Rif1. Rif1 tightly binds to nuclear-insoluble structures at late-M-to-early-G1 and regulates chromatin-loop sizes. Furthermore, Rif1 colocalizes specifically with the mid-S replication foci. Thus, Rif1 establishes the mid-S replication domains that are restrained from being activated at early-S-phase. Our results indicate that Rif1 plays crucial roles in determining the replication timing domain structures in human cells through regulating higher-order chromatin architecture.     

Mechanisms of single-stranded DNA-binding protein functioning in cellular DNA metabolism

This review deals with analysis of mechanisms involved in coordination of DNA replication and repair by SSB proteins; characteristics of eukaryotic, prokaryotic, and archaeal SSB proteins are considered, which made it possible to distinguish general mechanisms specific for functioning of proteins from organisms of different life domains. Mechanisms of SSB protein interactions with DNA during metabolism of the latter are studied; structural organization of the SSB protein complexes with DNA, as well as structural and functional peculiarities of different SSB proteins are analyzed.     

Review: The Dynamics of the Nuclear Lamins during the Cell Cycle— Relationship between Structure and Function

The nuclear lamins are members of the intermediate filament (IF) family of proteins. The lamins have an essential role in maintaining nuclear integrity, as do the other IF family members in the cytoplasm. Also like cytoplasmic IFs, the organization of lamins is dynamic. The lamins are found not only at the nuclear periphery but also in the interior of the nucleus, as distinct nucleoplasmic foci and possibly as a network throughout the nucleus. Nuclear processes such as DNA replication may be organized around these structures. In this review, we discuss changes in the structure and organization of the nuclear lamins during the cell cycle and during cell differentiation. These changes are correlated with changes in nuclear structure and function. For example, the interactions of lamins with chromatin and nuclear envelope components occur very early during nuclear assembly following mitosis. During S-phase, the lamins colocalize with markers of DNA replication, and proper lamin organization must be maintained for replication to proceed. When cells differentiate, the expression pattern of lamin isotypes changes. In addition, changes in lamin organization and expression patterns accompany the nuclear alterations observed in transformed cells. These lamin structures may modulate nuclear function in each of these processes.     

DNA replication and nuclear architecture

The model of in situ DNA replication provided by immunofluorescence and confocal imaging is compared with observations obtained by electron microscopic studies. Discrepancies between both types of observations call into question the replication focus as a persistent nuclear structure and as a replication entity where DNA replication takes place. Most electron microscopic analyses reveal that replication sites are confined to dispersed chromatin areas at the periphery of condensed chromatin, and the distribution of replication factors exhibits the same localization pattern. Moreover, rapid migration of newly synthesized DNA from the replication sites towards the interior of condensed chromatin regions obviously takes place during S-phase. It implies modifications of replication domains, hardly detectable by fluorescence microscopy. The confrontation of different observations carried out at light microscopic or electron microscopic levels of resolution lead to a conclusion that a combination of in vivo fluorescence analysis with a subsequent ultrastructural investigation performed on the same cells will represent an optimal approach in future studies of nuclear functions in situ.     

Mcm2, but not RPA, is a component of the mammalian early G1-phase prereplication complex.

Previous experiments in Xenopus egg extracts identified what appeared to be two independently assembled prereplication complexes (pre-RCs) for DNA replication: the stepwise assembly of ORC, Cdc6, and Mcm onto chromatin, and the FFA-1-mediated recruitment of RPA into foci on chromatin. We have investigated whether both of these pre-RCs can be detected in Chinese hamster ovary (CHO) cells. Early- and late-replicating chromosomal domains were pulse-labeled with halogenated nucleotides and prelabeled cells were synchronized at various times during the following G1-phase. The recruitment of Mcm2 and RPA to these domains was examined in relation to the formation of a nuclear envelope, specification of the dihydrofolate reductase (DHFR) replication origin and entry into S-phase. Mcm2 was loaded gradually and cumulatively onto both early- and late-replicating chromatin from late telophase throughout G1-phase. During S-phase, detectable Mcm2 was rapidly excluded from PCNA-containing active replication forks. By contrast, detergent-resistant RPA foci were undetectable until the onset of S-phase, when RPA joined only the earliest-firing replicons. During S-phase, RPA was present with PCNA specifically at active replication forks. Together, our data are consistent with a role for Mcm proteins, but not RPA, in the formation of mammalian pre-RCs during early G1-phase.     

Differential activation of intra-S-phase checkpoint in response to tripchlorolide and its effects on DNA replication

DNA replication is tightly regulated during the S phase of the cell cycle, and the activation of the intra-S-phase checkpoint due to DNA damage usually results in arrest of DNA synthesis. However, the molecular details about the correlation between the checkpoint and regulation of DNA replication are still unclear. To investigate the connections between DNA replication and DNA damage checkpoint, a DNA-damage reagent, tripchlorolide, was applied to CHO (Chinese ovary hamster) cells at early- or middle-stages of the S phase. The early-S-phase treatment with TC signifi-cantly delayed the progression of the S phase and caused the phosphorylation of the Chk 1 checkpoint protein, whereas the middle-S-phase treatment only slightly slowed down the progression of the S phase. Furthermore, the analysis of DNA replication patterns revealed that replication pattern II was greatly prolonged in the cells treated with the drug during the early-S phase, whereas the late-replication patterns of these cells were hardly detected, suggesting that the activation of the intra-S-phase checkpoint inhibits the late-origin firing of DNA replication. We conclude that cells at different stages of the S phase are differentially sensitive to the DNA-damage reagent, and the activation of the intra-S-phase checkpoint blocks the DNA replication progression in the late stage of S phase.     

Mapping mammalian replication domains using the ion torrent semiconductor sequencing platform

ABSTRACT

Here, we show that semiconductor-based sequencing technology can be used to map mammalian replication domains, chromosomal units with similar DNA replication timing. Replicating DNA purified from mammalian cells was successfully sequenced by the Ion Torrent platform. The resultant replication domain map of mouse embryonic stem cells is comparable to those obtained by the conventional microarray-based method.     

Isolation, characterization, and expression analysis of zebrafish large Mafs

Large Maf proteins, which are members of the basic leucine zipper (b-Zip) superfamily, are involved in the determination and control of cellular differentiation. The expression patterns of various vertebrate large Maf mRNAs were described previously. Here, we report the cloning of a novel zebrafish large Maf cDNA, SMaf1 (Somite Maf1), and other zebrafish large Mafs, the N-terminus domains of which possess transactivational activity. We also analyzed the expression patterns of SMaf1 and SMaf2 (Somite Maf2)/Krml2 as well as MafB/Val and c-Maf during zebrafish embryogenesis. In particular, the robust expression of the novel SMaf1 mRNA, which overlapped that of MyoD, in somitic cells during somitogenesis was noteworthy. In addition, the expression patterns of SMaf2 and MafB in the blood-forming regions, and those of c-Maf and MafB in the lens cells showed spatial redundancy, although the temporal appearance of these genes at these sites differed. These data indicate that SMafs may play important roles in somitogenesis, and that Maf proteins may have overlapping and yet specific functions as to the determination and differentiation of cell lineages.     

Dynamic elements of replication protein A at the crossroads of DNA replication,recombination, and repair

Abstract

The heterotrimeric eukaryotic Replication protein A (RPA) is a master regulator of numerous DNA metabolic processes. For a long time, it has been viewed as an inert protector of ssDNA and a platform for assembly of various genome maintenance and signaling machines. Later, the modular organization of the RPA DNA binding domains suggested a possibility for dynamic interaction with ssDNA. This modular organization has inspired several models for the RPA-ssDNA interaction that aimed to explain how RPA, the high-affinity ssDNA binding protein, is replaced by the downstream players in DNA replication, recombination, and repair that bind ssDNA with much lower affinity. Recent studies, and in particular single-molecule observations of RPA-ssDNA interactions, led to the development of a new model for the ssDNA handoff from RPA to a specific downstream factor where not only stability and structural rearrangements but also RPA conformational dynamics guide the ssDNA handoff. Here we will review the current knowledge of the RPA structure, its dynamic interaction with ssDNA, and how RPA conformational dynamics may be influenced by posttranslational modification and proteins that interact with RPA, as well as how RPA dynamics may be harnessed in cellular decision making.     

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。