Impact of replication timing on non-CpG and CpG substitution rates in mammalian genomes.

Neutral nucleotide substitutions occur at varying rates along genomes, and it remains a major issue to unravel the mechanisms that cause these variations and to analyze their evolutionary consequences. Here, we study the role of replication in the neutral substitution pattern. We obtained a high-resolution replication timing profile of the whole human genome by massively parallel sequencing of nascent BrdU-labeled replicating DNA. These data were compared to the neutral substitution rates along the human genome, obtained by aligning human and chimpanzee genomes using macaque and orangutan as outgroups. All substitution rates increase monotonously with replication timing even after controlling for local or regional nucleotide composition, crossover rate, distance to telomeres, and chromatin compaction. The increase in non-CpG substitution rates might result from several mechanisms including the increase in mutation-prone activities or the decrease in efficiency of DNA repair during the S phase. In contrast, the rate of C --> T transitions in CpG dinucleotides increases in later-replicating regions due to increasing DNA methylation level that reflects a negative correlation between timing and gene expression. Similar results are observed in the mouse, which indicates that replication timing is a main factor affecting nucleotide substitution dynamics at non-CpG sites and constitutes a major neutral process driving mammalian genome evolution.

Replication fork polarity gradients revealed by megabase-sized U-shaped replication timing domains in human cell lines.

In higher eukaryotes, replication program specification in different cell types remains to be fully understood. We show for seven human cell lines that about half of the genome is divided in domains that display a characteristic U-shaped replication timing profile with early initiation zones at borders and late replication at centers. Significant overlap is observed between U-domains of different cell lines and also with germline replication domains exhibiting a N-shaped nucleotide compositional skew. From the demonstration that the average fork polarity is directly reflected by both the compositional skew and the derivative of the replication timing profile, we argue that the fact that this derivative displays a N-shape in U-domains sustains the existence of large-scale gradients of replication fork polarity in somatic and germline cells. Analysis of chromatin interaction (Hi-C) and chromatin marker data reveals that U-domains correspond to high-order chromatin structural units. We discuss possible models for replication origin activation within U/N-domains. The compartmentalization of the genome into replication U/N-domains provides new insights on the organization of the replication program in the human genome.

Replication-associated mutational asymmetry in the human genome.

During evolution, mutations occur at rates that can differ between the two DNA strands. In the human genome, nucleotide substitutions occur at different rates on the transcribed and non-transcribed strands that may result from transcription-coupled repair. These mutational asymmetries generate transcription-associated compositional skews. To date, the existence of such asymmetries associated with replication has not yet been established. Here, we compute the nucleotide substitution matrices around replication initiation zones identified as sharp peaks in replication timing profiles and associated with abrupt jumps in the compositional skew profile. We show that the substitution matrices computed in these regions fully explain the jumps in the compositional skew profile when crossing initiation zones. In intergenic regions, we observe mutational asymmetries measured as differences between complementary substitution rates; their sign changes when crossing initiation zones. These mutational asymmetries are unlikely to result from cryptic transcription but can be explained by a model based on replication errors and strand-biased repair. In transcribed regions, mutational asymmetries associated with replication superimpose on the previously described mutational asymmetries associated with transcription. We separate the substitution asymmetries associated with both mechanisms, which allows us to determine for the first time in eukaryotes, the mutational asymmetries associated with replication and to reevaluate those associated with transcription. Replication-associated mutational asymmetry may result from unequal rates of complementary base misincorporation by the DNA polymerases coupled with DNA mismatch repair (MMR) acting with different efficiencies on the leading and lagging strands. Replication, acting in germ line cells during long evolutionary times, contributed equally with transcription to produce the present abrupt jumps in the compositional skew. These results demonstrate that DNA replication is one of the major processes that shape human genome composition.

Evidence for sequential and increasing activation of replication origins along replication timing gradients in the human genome.

Genome-wide replication timing studies have suggested that mammalian chromosomes consist of megabase-scale domains of coordinated origin firing separated by large originless transition regions. Here, we report a quantitative genome-wide analysis of DNA replication kinetics in several human cell types that contradicts this view. DNA combing in HeLa cells sorted into four temporal compartments of S phase shows that replication origins are spaced at 40 kb intervals and fire as small clusters whose synchrony increases during S phase and that replication fork velocity (mean 0.7 kb/min, maximum 2.0 kb/min) remains constant and narrowly distributed through S phase. However, multi-scale analysis of a genome-wide replication timing profile shows a broad distribution of replication timing gradients with practically no regions larger than 100 kb replicating at less than 2 kb/min. Therefore, HeLa cells lack large regions of unidirectional fork progression. Temporal transition regions are replicated by sequential activation of origins at a rate that increases during S phase and replication timing gradients are set by the delay and the spacing between successive origin firings rather than by the velocity of single forks. Activation of internal origins in a specific temporal transition region is directly demonstrated by DNA combing of the IGH locus in HeLa cells. Analysis of published origin maps in HeLa cells and published replication timing and DNA combing data in several other cell types corroborate these findings, with the interesting exception of embryonic stem cells where regions of unidirectional fork progression seem more abundant. These results can be explained if origins fire independently of each other but under the control of long-range chromatin structure, or if replication forks progressing from early origins stimulate initiation in nearby unreplicated DNA. These findings shed a new light on the replication timing program of mammalian genomes and provide a general model for their replication kinetics.

Recruitment of Drosophila Polycomb group proteins to chromatin by DSP1.

Polycomb and trithorax group (PcG and trxG) proteins maintain silent and active transcriptional states, respectively, throughout development. In Drosophila, PcG and trxG proteins associate with DNA regions named Polycomb and trithorax response elements (PRE and TRE), but the mechanisms of recruitment are unknown. We previously characterized a minimal element from the regulatory region of the Abdominal-B gene, termed Ab-Fab. Ab-Fab contains a PRE and a TRE and is able to maintain repressed or active chromatin states during development. Here we show that the Dorsal switch protein 1 (DSP1), a Drosophila HMGB2 homologue, binds to a sequence present within Ab-Fab and in other characterized PREs. Addition of this motif to an artificial sequence containing Pleiohomeotic and GAGA factor consensus sites is sufficient for PcG protein recruitment in vivo. Mutations that abolish DSP1 binding to Ab-Fab and to a PRE from the engrailed locus lead to loss of PcG protein binding, loss of silencing, and switching of these PREs into constitutive TREs. The binding of DSP1 to PREs is therefore important for the recruitment of PcG proteins.

Multiscale analysis of genome-wide replication timing profiles using a wavelet-based signal-processing algorithm.

In this protocol, we describe the use of the LastWave open-source signal-processing command language (http://perso.ens-lyon.fr/benjamin.audit/LastWave/) for analyzing cellular DNA replication timing profiles. LastWave makes use of a multiscale, wavelet-based signal-processing algorithm that is based on a rigorous theoretical analysis linking timing profiles to fundamental features of the cell's DNA replication program, such as the average replication fork polarity and the difference between replication origin density and termination site density. We describe the flow of signal-processing operations to obtain interactive visual analyses of DNA replication timing profiles. We focus on procedures for exploring the space-scale map of apparent replication speeds to detect peaks in the replication timing profiles that represent preferential replication initiation zones, and for delimiting U-shaped domains in the replication timing profile. In comparison with the generally adopted approach that involves genome segmentation into regions of constant timing separated by timing transition regions, the present protocol enables the recognition of more complex patterns of the spatio-temporal replication program and has a broader range of applications. Completing the full procedure should not take more than 1 h, although learning the basics of the program can take a few hours and achieving full proficiency in the use of the software may take days.

From simple bacterial and archaeal replicons to replication N/U-domains.

The Replicon Theory proposed 50 years ago has proven to apply for replicons of the three domains of life. Here, we review our knowledge of genome organization into single and multiple replicons in bacteria, archaea and eukarya. Bacterial and archaeal replicator/initiator systems are quite specific and efficient, whereas eukaryotic replicons show degenerate specificity and efficiency, allowing for complex regulation of origin firing time. We expand on recent evidence that ~50% of the human genome is organized as ~1,500 megabase-sized replication domains with a characteristic parabolic (U-shaped) replication timing profile and linear (N-shaped) gradient of replication fork polarity. These N/U-domains correspond to self-interacting segments of the chromatin fiber bordered by open chromatin zones and replicate by cascades of origin firing initiating at their borders and propagating to their center, possibly by fork-stimulated initiation. The conserved occurrence of this replication pattern in the germline of mammals has resulted over evolutionary times in the formation of megabase-sized domains with an N-shaped nucleotide compositional skew profile due to replication-associated mutational asymmetries. Overall, these results reveal an evolutionarily conserved but developmentally plastic organization of replication that is driving mammalian genome evolution.

DSP1, a Drosophila HMG protein, is involved in spatiotemporal expression of the homoeotic gene Sex combs reduced.

BACKGROUND INFORMATION: The Pc-G (Polycomb group) and trx-G (trithorax group) genes play a key role in the regulation of the homoeotic genes. The homoeotic gene Scr (Sex combs reduced) contained in the Antennapedia complex specifies segmental identity of the labial and prothoracic segments in Drosophila. Regulation of Scr requires the action of different enhancer elements spread over several kilobases. We previously identified an HMGB (high mobility group)-like protein DSP1 (dorsal switch protein 1), which works like a trx-G protein for the normal Scr expression. RESULTS: In the present study, we attempted to characterize the regulatory sequences involved in the maintenance of the Scr activation by DSP1. We report here, using a transgenic line for the Scr10.0XbaI-regulatory element, that lack of DSP1 affects the function of a reporter gene in legs' imaginal discs but not in embryos. We show by immunolocalization that DSP1 is recruited on polytene chromosomes to the insertion site of the transgene. Moreover, using chromatin immunoprecipitation experiments, we identify two regions of 1 kb in Scr10.0XbaI as the main DSP1 targets. CONCLUSION: These results provide strong evidence that the Scr gene expression is influenced by direct interaction between DSP1 and two Scr regulation elements. In addition, our results show that this interaction undergoes dynamic changes during development.