Chromatin replication.

Just as the faithful replication of DNA is an essential process for the cell, chromatin structures of active and inactive genes have to be copied accurately. Under certain circumstances, however, the activity pattern has to be changed in specific ways. Although analysis of specific aspects of these complex processes, by means of model systems, has led to their further elucidation, the mechanisms of chromatin replication in vivo are still controversial and far from being understood completely. Progress has been achieved in understanding: 1. The initiation of chromatin replication, indicating that a nucleosome-free origin is necessary for the initiation of replication; 2. The segregation of the parental nucleosomes, where convincing data support the model of random distribution of the parental nucleosomes to the daughter strands; and 3. The assembly of histones on the newly synthesized strands, where growing evidence is emerging for a two-step mechanism of nucleosome assembly, starting with the deposition of H3/H4 tetramers onto the DNA, followed by H2A/H2B dimers.     

Chromatin assembly at kinetochores is uncoupled from DNA replication

The specification of metazoan centromeres does not depend strictly on centromeric DNA sequences, but also requires epigenetic factors. The mechanistic basis for establishing a centromeric "state" on the DNA remains unclear. In this work, we have directly examined replication timing of the prekinetochore domain of human chromosomes. Kinetochores were labeled by expression of epitope-tagged CENP-A, which stably marks prekinetochore domains in human cells. By immunoprecipitating CENP-A mononucleosomes from synchronized cells pulsed with [(3)H]thymidine we demonstrate that CENP-A-associated DNA is replicated in mid-to-late S phase. Cytological analysis of DNA replication further demonstrated that centromeres replicate asynchronously in parallel with numerous other genomic regions. In contrast, quantitative Western blot analysis demonstrates that CENP-A protein synthesis occurs later, in G2. Quantitative fluorescence microscopy and transient transfection in the presence of aphidicolin, an inhibitor of DNA replication, show that CENP-A can assemble into centromeres in the absence of DNA replication. Thus, unlike most genomic chromatin, histone synthesis and assembly are uncoupled from DNA replication at the kinetochore. Uncoupling DNA replication from CENP-A synthesis suggests that regulated chromatin assembly or remodeling could play a role in epigenetic centromere propagation.     

Chromatin replication and epigenome maintenance

Stability and function of eukaryotic genomes are closely linked to chromatin structure and organization. During cell division the entire genome must be accurately replicated and the chromatin landscape reproduced on new DNA. Chromatin and nuclear structure influence where and when DNA replication initiates, whereas the replication process itself disrupts chromatin and challenges established patterns of genome regulation. Specialized replication-coupled mechanisms assemble new DNA into chromatin, but epigenome maintenance is a continuous process taking place throughout the cell cycle. If DNA synthesis is perturbed, cells can suffer loss of both genome and epigenome integrity with severe consequences for the organism.     

Chromatin dynamics during spermiogenesis

The function of sperm is to safely transport the haploid paternal genome to the egg containing the maternal genome. The subsequent fertilization leads to transmission of a new unique diploid genome to the next generation. Before the sperm can set out on its adventurous journey, remarkable arrangements need to be made during the post-meiotic stages of spermatogenesis. Haploid spermatids undergo extensive morphological changes, including a striking reorganization and compaction of their chromatin. Thereby, the nucleosomal, histone-based structure is nearly completely substituted by a protamine-based structure. This replacement is likely facilitated by incorporation of histone variants, post-translational histone modifications, chromatin-remodeling complexes, as well as transient DNA strand breaks. The consequences of mutations have revealed that a protamine-based chromatin is essential for fertility in mice but not in Drosophila. Nevertheless, loss of protamines in Drosophila increases the sensitivity to X-rays and thus supports the hypothesis that protamines are necessary to protect the paternal genome. Pharmaceutical approaches have provided the first mechanistic insights and have shown that hyperacetylation of histones just before their displacement is vital for progress in chromatin reorganization but is clearly not the sole inducer. In this review, we highlight the current knowledge on post-meiotic chromatin reorganization and reveal for the first time intriguing parallels in this process in Drosophila and mammals. We conclude with a model that illustrates the possible mechanisms that lead from a histone-based chromatin to a mainly protamine-based structure during spermatid differentiation. This article is part of a Special Issue entitled: Chromatin and epigenetic regulation of animal development.     

3D chromatin conformation correlates with replication timing and is conserved in resting cells

Although chromatin folding is known to be of functional importance to control the gene expression program, less is known regarding its interplay with DNA replication. Here, using Circular Chromatin Conformation Capture combined with high-throughput sequencing, we identified megabase-sized self-interacting domains in the nucleus of a human lymphoblastoid cell line, as well as in cycling and resting peripheral blood mononuclear cells (PBMC). Strikingly, the boundaries of those domains coincide with early-initiation zones in every cell types. Preferential interactions have been observed between the consecutive early-initiation zones, but also between those separated by several tens of megabases. Thus, the 3D conformation of chromatin is strongly correlated with the replication timing along the whole chromosome. We furthermore provide direct clues that, in addition to the timing value per se, the shape of the timing profile at a given locus defines its set of genomic contacts. As this timing-related scheme of chromatin organization exists in lymphoblastoid cells, resting and cycling PBMC, this indicates that it is maintained several weeks or months after the previous S-phase. Lastly, our work highlights that the major chromatin changes accompanying PBMC entry into cell cycle occur while keeping largely unchanged the long-range chromatin contacts.     

Chromatin replication,reconstitution and assembly

Many previously held concepts about the replication of chromatin have recently been revised, or seriously challenged. For instance, within the last two years, evidence has accumulated to indicate that newly synthesized DNA is not the sole site of deposition of newly synthesized histones, and that histones are not only made, but are assembled into chromatin in the absence of DNA synthesis. Furthermore, segregation of parental histones to daughter DNA duplexes may be bidirectional, rather than the previously accepted unidirectional mechanism. The storage of histones prior to assembly apparently involves histone pairs rather than octamers, and similarly, histones associate with DNA in (apparent) pairs, rather than as pre-assembled octameric units. It is currently questioned whether or not nucleoplasmin is involved in either histone storage or nucleosome assembly. The onset of histone synthesis has recently been found to occur in late G1 rather than in S, and thus is independent of DNA synthesis; however, the cessation of histone synthesis is linked to that of DNA. Thus, there emerges from this newly accumulated data the conclusion that chromatin biosynthesis is not as straightforward as was believed just a few years ago. As we review the evidence on each of these subjects, we attempt to point out directions for future experimentation.     

Chromatin dynamics during DSB repair

We show that double strand breaks (DSBs) induced in chromatin of low as well as high density by exposure of human cells to gamma-rays are repaired in low-density chromatin. Extensive chromatin decondensation manifested in the vicinity of DSBs by decreased intensity of chromatin labelling, increased H4K5 acetylation, and decreased H3K9 dimethylation was observed already 15 min after irradiation. Only slight movement of sporadic DSB loci for short distances was noticed in living cells associated with chromatin decondensation around DSBs. This frequently resulted in their protrusion into the low-density chromatin domains. In these regions, the clustering (contact or fusion) of DSB foci was seen in vivo, and in situ after cell fixation. The majority of these clustered foci were repaired within 240 min, but some of them persisted in the nucleus for several days after irradiation, indicating damage that is not easily repaired. We propose that the repair of DSB in clustered foci might lead to misjoining of ends and, consequently, to exchange aberrations. On the other hand, the foci that persist for several days without being repaired could lead instead to cell death.     

Chromatin structure within sites of DNA replication

Conformational changes in chromatin structure are nowadays the object of intensive research due to its importance for proper regulation of intranuclear processes. The fine structure of chromatin within the DNA replication sites was studied in in situ fixed cells and cells permebilized by low ionic strength solutions in the presence of divalent cations. The latter method provides visualization of higher level chromatin structures such as globular chromomeres and chromonema fibres. Nascent DNA was detected immunochemically using anti-BrdU antibodies on the surface of ultrathin sections prepared from Epon-embedded material. It was shown that newly replicated DNA preferentially localized within the zones filled with globular and fibrillar elements with characteristic diameter of 30 nm, and not in chromonema fibres, while after replication had been completed DNA became embedded into as thick as 60-80 nm chromonema elements. The results obtained are discussed in the context of conception of hierarchical folding of chromatin fibers.     

Chromatin structure in sites of DNA replication

Conformational changes of in chromatin structure play a key role in the regulation of intranuclear processes and, therefore, are under advanced study. In the paper presented, the fine structure of chromatin in DNA replication sites was examined in cells fixed in situ and in cells permeabilized in low ionic strength solutions in the presence of divalent cations. The method provides the visualization of higher-level chromatin structures, globular chromomeres, and chromonema fibres. Nascent DNA was detected on the surface of ultrathin sections immunochemically using anti-BrdU antibodies. It was shown that newly replicated DNA preferentially localizes within the zones filled with globular and fibrillar elements 30 nm in diameter. DNA-completed replication became embedded in 60–100-nm-thick chromonema elements. The results are discussed in the context of the hierarchical folding of chromatin fibers.     

TmRNA is required for correct timing of DNA replication in Caulobacter crescentus

SsrA, or tmRNA, is a small RNA that interacts with selected translating ribosomes to target the nascent polypeptides for degradation. Here we report that SsrA activity is required for normal timing of the G(1)-to-S transition in Caulobacter crescentus. A deletion of the ssrA gene, or of the gene encoding SmpB, a protein required for SsrA activity, results in a specific delay in the cell cycle during the G(1)-to-S transition. The ssrA deletion phenotype is not due to accumulation of stalled ribosomes, because the deletion is not complemented by a mutated version of SsrA that releases ribosomes but does not target proteins for degradation. Degradation of the CtrA response regulator normally coincides with initiation of DNA replication, but in strains lacking SsrA activity there is a 40-min delay between the degradation of CtrA and replication initiation. This uncoupling of initiation of replication from CtrA degradation indicates that there is an SsrA-dependent pathway required for correct timing of DNA replication.     

Centromere identity in Drosophila is not determined in vivo by replication timing

Centromeric chromatin is uniquely marked by the centromere-specific histone CENP-A. For assembly of CENP-A into nucleosomes to occur without competition from H3 deposition, it was proposed that centromeres are among the first or last sequences to be replicated. In this study, centromere replication in Drosophila was studied in cell lines and in larval tissues that contain minichromosomes that have structurally defined centromeres. Two different nucleotide incorporation methods were used to evaluate replication timing of chromatin containing CID, a Drosophila homologue of CENP-A. Centromeres in Drosophila cell lines were replicated throughout S phase but primarily in mid S phase. However, endogenous centromeres and X-derived minichromosome centromeres in vivo were replicated asynchronously in mid to late S phase. Minichromosomes with structurally intact centromeres were replicated in late S phase, and those in which centric and surrounding heterochromatin were partially or fully deleted were replicated earlier in mid S phase. We provide the first in vivo evidence that centromeric chromatin is replicated at different times in S phase. These studies indicate that incorporation of CID/CENP-A into newly duplicated centromeres is independent of replication timing and argue against determination of centromere identity by temporal sequestration of centromeric chromatin replication relative to bulk genomic chromatin.     

Chromatin challenges during DNA replication and repair

Inheritance and maintenance of the DNA sequence and its organization into chromatin are central for eukaryotic life. To orchestrate DNA-replication and -repair processes in the context of chromatin is a challenge, both in terms of accessibility and maintenance of chromatin organization. To meet the challenge of maintenance, cells have evolved efficient nucleosome-assembly pathways and chromatin-maturation mechanisms that reproduce chromatin organization in the wake of DNA replication and repair. The aim of this Review is to describe how these pathways operate and to highlight how the epigenetic landscape may be stably maintained even in the face of dramatic changes in chromatin structure.     

Life history as a constraint on plasticity: developmental timing is correlated with phenotypic variation in birds

Understanding why organisms vary in developmental plasticity has implications for predicting population responses to changing environments and the maintenance of intraspecific variation. The epiphenotype hypothesis posits that the timing of development can constrain plasticity—the earlier alternate phenotypes begin to develop, the greater the difference that can result amongst the final traits. This research extends this idea by considering how life history timing shapes the opportunity for the environment to influence trait development. We test the prediction that the earlier an individual begins to actively interact with and explore their environment, the greater the opportunity for plasticity and thus variation in foraging traits. This research focuses on life history variation across four groups of birds using museum specimens and measurements from the literature. We reasoned that greater phenotypic plasticity, through either environmental effects or genotype-by-environment interactions in development, would be manifest in larger trait ranges (bills and tarsi) within species. Among shorebirds and ducks, we found that species with relatively shorter incubation times tended to show greater phenotypic variation. Across warblers and sparrows, we found little support linking timing of flight and trait variation. Overall, our results also suggest a pattern between body size and trait variation, consistent with constraints on egg size that might result in larger species having more environmental influences on development. Taken together, our results provide some support for the hypothesis that variation in life histories affects how the environment shapes development, through either the expression of plasticity or the release of cryptic genetic variation.     

Cellular senescence induces replication stress with almost no affect on DNA replication timing

Organismal aging entails a gradual decline of normal physiological functions and a major contributor to this decline is withdrawal of the cell cycle, known as senescence. Senescence can result from telomere diminution leading to a finite number of population doublings, known as replicative senescence (RS), or from oncogene overexpression, as a protective mechanism against cancer. Senescence is associated with large-scale chromatin re-organization and changes in gene expression. Replication stress is a complex phenomenon, defined as the slowing or stalling of replication fork progression and/or DNA synthesis, which has serious implications for genome stability, and consequently in human diseases. Aberrant replication fork structures activate the replication stress response leading to the activation of dormant origins, which is thought to be a safeguard mechanism to complete DNA replication on time. However, the relationship between replicative stress and the changes in the spatiotemporal program of DNA replication in senescence progression remains unclear.

Here, we studied the DNA replication program during senescence progression in proliferative and pre-senescent cells from donors of various ages by single DNA fiber combing of replicated DNA, origin mapping by sequencing short nascent strands and genome-wide profiling of replication timing (TRT).

We demonstrate that, progression into RS leads to reduced replication fork rates and activation of dormant origins, which are the hallmarks of replication stress. However, with the exception of a delay in RT of the CREB5 gene in all pre-senescent cells, RT was globally unaffected by replication stress during entry into either oncogene-induced or RS. Consequently, we conclude that RT alterations associated with physiological and accelerated aging, do not result from senescence progression. Our results clarify the interplay between senescence, aging and replication programs and demonstrate that RT is largely resistant to replication stress.