Monitoring and quantifying replication fork dynamics with high-throughput methods.

Before each cell division, eukaryotic cells must replicate their chromosomes to ensure the accurate transmission of genetic information. Chromosome replication involves more than just DNA duplication; it also includes chromatin assembly, inheritance of epigenetic marks, and faithful resumption of all genomic functions after replication. Recent progress in quantitative technologies has revolutionized our understanding of the complexity and dynamics of DNA replication forks at both molecular and genomic scales. Here, we highlight the pivotal role of these novel methods in uncovering the principles and mechanisms of chromosome replication. These technologies have illuminated the regulation of genome replication programs, quantified the impact of DNA replication on genomic mutations and evolutionary processes, and elucidated the mechanisms of replication-coupled chromatin assembly and epigenome maintenance.

Genome-wide measurement of DNA replication fork directionality and quantification of DNA replication initiation and termination with Okazaki fragment sequencing.

Studying the dynamics of genome replication in mammalian cells has been historically challenging. To reveal the location of replication initiation and termination in the human genome, we developed Okazaki fragment sequencing (OK-seq), a quantitative approach based on the isolation and strand-specific sequencing of Okazaki fragments, the lagging strand replication intermediates. OK-seq quantitates the proportion of leftward- and rightward-oriented forks at every genomic locus and reveals the location and efficiency of replication initiation and termination events. Here we provide the detailed experimental procedures for performing OK-seq in unperturbed cultured human cells and budding yeast and the bioinformatics pipelines for data processing and computation of replication fork directionality. Furthermore, we present the analytical approach based on a hidden Markov model, which allows automated detection of ascending, descending and flat replication fork directionality segments revealing the zones of replication initiation, termination and unidirectional fork movement across the entire genome. These tools are essential for the accurate interpretation of human and yeast replication programs. The experiments and the data processing can be accomplished within six days. Besides revealing the genome replication program in fine detail, OK-seq has been instrumental in numerous studies unravelling mechanisms of genome stability, epigenome maintenance and genome evolution.

Prospectively defined patterns of APOBEC3A mutagenesis are prevalent in human cancers.

Mutational signatures defined by single base substitution (SBS) patterns in cancer have elucidated potential mutagenic processes that contribute to malignancy. Two prevalent mutational patterns in human cancers are attributed to the APOBEC3 cytidine deaminase enzymes. Among the seven human APOBEC3 proteins, APOBEC3A is a potent deaminase and proposed driver of cancer mutagenesis. In this study, we prospectively examine genome-wide aberrations by expressing human APOBEC3A in avian DT40 cells. From whole-genome sequencing, we detect hundreds to thousands of base substitutions per genome. The APOBEC3A signature includes widespread cytidine mutations and a unique insertion-deletion (indel) signature consisting largely of cytidine deletions. This multi-dimensional APOBEC3A signature is prevalent in human cancer genomes. Our data further reveal replication-associated mutations, the rate of stem-loop and clustered mutations, and deamination of methylated cytidines. This comprehensive signature of APOBEC3A mutagenesis is a tool for future studies and a potential biomarker for APOBEC3 activity in cancer.

Genome-wide and sister chromatid-resolved profiling of protein occupancy in replicated chromatin with ChOR-seq and SCAR-seq.

Elucidating the mechanisms underlying chromatin maintenance upon genome replication is critical for the understanding of how gene expression programs and cell identity are preserved across cell divisions. Here, we describe two recently developed techniques, chromatin occupancy after replication (ChOR)-seq and sister chromatids after replication (SCAR)-seq, that profile chromatin occupancy on newly replicated DNA in mammalian cells in 5 d of bench work. Both techniques share a common strategy that includes pulse labeling of newly synthesized DNA and chromatin immunoprecipitation (ChIP), followed by purification and high-throughput sequencing. Whereas ChOR-seq quantitatively profiles the post-replicative abundance of histone modifications and chromatin-associated proteins, SCAR-seq distinguishes chromatin occupancy between nascent sister chromatids. Together, these two complementary techniques have unraveled key mechanisms controlling the inheritance of modified histones during replication and revealed locus-specific dynamics of histone modifications across the cell cycle. Here, we provide the experimental protocols and bioinformatic pipelines for these methods.

DNA molecular combing-based replication fork directionality profiling.

The replication strategy of metazoan genomes is still unclear, mainly because definitive maps of replication origins are missing. High-throughput methods are based on population average and thus may exclusively identify efficient initiation sites, whereas inefficient origins go undetected. Single-molecule analyses of specific loci can detect both common and rare initiation events along the targeted regions. However, these usually concentrate on positioning individual events, which only gives an overview of the replication dynamics. Here, we computed the replication fork directionality (RFD) profiles of two large genes in different transcriptional states in chicken DT40 cells, namely untranscribed and transcribed DMD and CCSER1 expressed at WT levels or overexpressed, by aggregating hundreds of oriented replication tracks detected on individual DNA fibres stretched by molecular combing. These profiles reconstituted RFD domains composed of zones of initiation flanking a zone of termination originally observed in mammalian genomes and were highly consistent with independent population-averaging profiles generated by Okazaki fragment sequencing. Importantly, we demonstrate that inefficient origins do not appear as detectable RFD shifts, explaining why dispersed initiation has remained invisible to population-based assays. Our method can both generate quantitative profiles and identify discrete events, thereby constituting a comprehensive approach to study metazoan genome replication.

Staying true to yourself: mechanisms of DNA methylation maintenance in mammals.

DNA methylation is essential to development and cellular physiology in mammals. Faulty DNA methylation is frequently observed in human diseases like cancer and neurological disorders. Molecularly, this epigenetic mark is linked to other chromatin modifications and it regulates key genomic processes, including transcription and splicing. Each round of DNA replication generates two hemi-methylated copies of the genome. These must be converted back to symmetrically methylated DNA before the next S-phase, or the mark will fade away; therefore the maintenance of DNA methylation is essential. Mechanistically, the maintenance of this epigenetic modification takes place during and after DNA replication, and occurs within the very dynamic context of chromatin re-assembly. Here, we review recent discoveries and unresolved questions regarding the mechanisms, dynamics and fidelity of DNA methylation maintenance in mammals. We also discuss how it could be regulated in normal development and misregulated in disease.

Chromatin replication and epigenetic cell memory.

Propagation of the chromatin landscape across cell divisions is central to epigenetic cell memory. Mechanistic analysis of the interplay between DNA replication, the cell cycle, and the epigenome has provided insights into replication-coupled chromatin assembly and post-replicative chromatin maintenance. These breakthroughs are critical for defining how proliferation impacts the epigenome during cell identity changes in development and disease. Here we review these findings in the broader context of epigenetic inheritance across mitotic cell division.

Developmental and cancer-associated plasticity of DNA replication preferentially targets GC-poor, lowly expressed and late-replicating regions.

The spatiotemporal program of metazoan DNA replication is regulated during development and altered in cancers. We have generated novel OK-seq, Repli-seq and RNA-seq data to compare the DNA replication and gene expression programs of twelve cancer and non-cancer human cell types. Changes in replication fork directionality (RFD) determined by OK-seq are widespread but more frequent within GC-poor isochores and largely disconnected from transcription changes. Cancer cell RFD profiles cluster with non-cancer cells of similar developmental origin but not with different cancer types. Importantly, recurrent RFD changes are detected in specific tumour progression pathways. Using a model for establishment and early progression of chronic myeloid leukemia (CML), we identify 1027 replication initiation zones (IZs) that progressively change efficiency during long-term expression of the BCR-ABL1 oncogene, being twice more often downregulated than upregulated. Prolonged expression of BCR-ABL1 results in targeting of new IZs and accentuation of previous efficiency changes. Targeted IZs are predominantly located in GC-poor, late replicating gene deserts and frequently silenced in late CML. Prolonged expression of BCR-ABL1 results in massive deletion of GC-poor, late replicating DNA sequences enriched in origin silencing events. We conclude that BCR-ABL1 expression progressively affects replication and stability of GC-poor, late-replicating regions during CML progression.

MCM2 promotes symmetric inheritance of modified histones during DNA replication.

During genome replication, parental histones are recycled to newly replicated DNA with their posttranslational modifications (PTMs). Whether sister chromatids inherit modified histones evenly remains unknown. We measured histone PTM partition to sister chromatids in embryonic stem cells. We found that parental histones H3-H4 segregate to both daughter DNA strands with a weak leading-strand bias, skewing partition at topologically associating domain (TAD) borders and enhancers proximal to replication initiation zones. Segregation of parental histones to the leading strand increased markedly in cells with histone-binding mutations in MCM2, part of the replicative helicase, exacerbating histone PTM sister chromatid asymmetry. This work reveals how histones are inherited to sister chromatids and identifies a mechanism by which the replication machinery ensures symmetric cell division.

Accurate Recycling of Parental Histones Reproduces the Histone Modification Landscape during DNA Replication.

Chromatin organization is disrupted genome-wide during DNA replication. On newly synthesized DNA, nucleosomes are assembled from new naive histones and old modified histones. It remains unknown whether the landscape of histone post-translational modifications (PTMs) is faithfully copied during DNA replication or the epigenome is perturbed. Here we develop chromatin occupancy after replication (ChOR-seq) to determine histone PTM occupancy immediately after DNA replication and across the cell cycle. We show that H3K4me3, H3K36me3, H3K79me3, and H3K27me3 positional information is reproduced with high accuracy on newly synthesized DNA through histone recycling. Quantitative ChOR-seq reveals that de novo methylation to restore H3K4me3 and H3K27me3 levels occurs across the cell cycle with mark- and locus-specific kinetics. Collectively, this demonstrates that accurate parental histone recycling preserves positional information and allows PTM transmission to daughter cells while modification of new histones gives rise to complex epigenome fluctuations across the cell cycle that could underlie cell-to-cell heterogeneity.

Replication landscape of the human genome.

Despite intense investigation, human replication origins and termini remain elusive. Existing data have shown strong discrepancies. Here we sequenced highly purified Okazaki fragments from two cell types and, for the first time, quantitated replication fork directionality and delineated initiation and termination zones genome-wide. Replication initiates stochastically, primarily within non-transcribed, broad (up to 150 kb) zones that often abut transcribed genes, and terminates dispersively between them. Replication fork progression is significantly co-oriented with the transcription. Initiation and termination zones are frequently contiguous, sometimes separated by regions of unidirectional replication. Initiation zones are enriched in open chromatin and enhancer marks, even when not flanked by genes, and often border 'topologically associating domains' (TADs). Initiation zones are enriched in origin recognition complex (ORC)-binding sites and better align to origins previously mapped using bubble-trap than λ-exonuclease. This novel panorama of replication reveals how chromatin and transcription modulate the initiation process to create cell-type-specific replication programs.

Functional study of the Hap4-like genes suggests that the key regulators of carbon metabolism HAP4 and oxidative stress response YAP1 in yeast diverged from a common ancestor.

The transcriptional regulator HAP4, induced by respiratory substrates, is involved in the balance between fermentation and respiration in S. cerevisiae. We identified putative orthologues of the Hap4 protein in all ascomycetes, based only on a conserved sixteen amino acid-long motif. In addition to this motif, some of these proteins contain a DNA-binding motif of the bZIP type, while being nonetheless globally highly divergent. The genome of the yeast Hansenula polymorpha contains two HAP4-like genes encoding the protein HpHap4-A which, like ScHap4, is devoid of a bZIP motif, and HpHap4-B which contains it. This species has been chosen for a detailed examination of their respective properties. Based mostly on global gene expression studies performed in the S. cerevisiae HAP4 disruption mutant (ScΔhap4), we show here that HpHap4-A is functionally equivalent to ScHap4, whereas HpHap4-B is not. Moreover HpHAP4-B is able to complement the H2O2 hypersensitivity of the ScYap1 deletant, YAP1 being, in S. cerevisiae, the main regulator of oxidative stress. Finally, a transcriptomic analysis performed in the ScΔyap1 strain overexpressing HpHAP4-B shows that HpHap4-B acts both on oxidative stress response and carbohydrate metabolism in a manner different from both ScYap1 and ScHap4. Deletion of these two genes in their natural host, H. polymorpha, confirms that HpHAP4-A participates in the control of the fermentation/respiration balance, while HpHAP4-B is involved in oxidative stress since its deletion leads to hypersensitivity to H2O2. These data, placed in an evolutionary context, raise new questions concerning the evolution of the HAP4 transcriptional regulation function and suggest that Yap1 and Hap4 have diverged from a unique regulatory protein in the fungal ancestor.

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.