Spatiotemporal kinetics of CAF-1-dependent chromatin maturation ensures transcription fidelity during S-phase.

Proper maintenance of epigenetic information after replication is dependent on the rapid assembly and maturation of chromatin. Chromatin Assembly Complex 1 (CAF-1) is a conserved histone chaperone that deposits (H3-H4)2 tetramers as part of the replication-dependent chromatin assembly process. Loss of CAF-1 leads to a delay in chromatin maturation, albeit with minimal impact on steady-state chromatin structure. However, the mechanisms by which CAF-1 mediates the deposition of (H3-H4)2 tetramers and the phenotypic consequences of CAF-1-associated assembly defects are not well understood. We used nascent chromatin occupancy profiling to track the spatiotemporal kinetics of chromatin maturation in both wild-type (WT) and CAF-1 mutant yeast cells. Our results show that loss of CAF-1 leads to a heterogeneous rate of nucleosome assembly, with some nucleosomes maturing at near WT kinetics and others showing significantly slower maturation kinetics. The slow-to-mature nucleosomes are enriched in intergenic and poorly transcribed regions, suggesting that transcription-dependent assembly mechanisms can reset the slow-to-mature nucleosomes following replication. Nucleosomes with slow maturation kinetics are also associated with poly(dA:dT) sequences, which implies that CAF-1 deposits histones in a manner that counteracts resistance from the inflexible DNA sequence, promoting the formation of histone octamers as well as ordered nucleosome arrays. In addition, we show that the delay in chromatin maturation is accompanied by a transient and S-phase-specific loss of gene silencing and transcriptional regulation, revealing that the DNA replication program can directly shape the chromatin landscape and modulate gene expression through the process of chromatin maturation.

Spatiotemporal kinetics of CAF-1-dependent chromatin maturation ensures transcription fidelity during S-phase.

Proper maintenance of epigenetic information after replication is dependent on the rapid assembly and maturation of chromatin. Chromatin Assembly Complex 1 (CAF-1) is a conserved histone chaperone that deposits (H3-H4)2 tetramers as part of the replication-dependent chromatin assembly process. Loss of CAF-1 leads to a delay in chromatin maturation, albeit with minimal impact on steady-state chromatin structure. However, the mechanisms by which CAF-1 mediates the deposition of (H3-H4)2 tetramers and the phenotypic consequences of CAF-1-associated assembly defects are not well understood. We used nascent chromatin occupancy profiling to track the spatiotemporal kinetics of chromatin maturation in both wild-type (WT) and CAF-1 mutant yeast cells. Our results show that loss of CAF-1 leads to a heterogeneous rate of nucleosome assembly, with some nucleosomes maturing at near WT kinetics and others exhibiting significantly slower maturation kinetics. The slow-to-mature nucleosomes are enriched in intergenic and poorly transcribed regions, suggesting that transcription-dependent assembly mechanisms can reset the slow-to-mature nucleosomes following replication. Nucleosomes with slow maturation kinetics are also associated with poly(dA:dT) sequences, which implies that CAF-1 deposits histones in a manner that counteracts resistance from the inflexible DNA sequence, promoting the formation of histone octamers as well as ordered nucleosome arrays. In addition, we demonstrate that the delay in chromatin maturation is accompanied by a transient and S-phase specific loss of gene silencing and transcriptional regulation, revealing that the DNA replication program can directly shape the chromatin landscape and modulate gene expression through the process of chromatin maturation.

Disruption of origin chromatin structure by helicase activation in the absence of DNA replication.

Prior to initiation of DNA replication, the eukaryotic helicase, Mcm2-7, must be activated to unwind DNA at replication start sites in early S phase. To study helicase activation within origin chromatin, we constructed a conditional mutant of the polymerase α subunit Cdc17 (or Pol1) to prevent priming and block replication. Recovery of these cells at permissive conditions resulted in the generation of unreplicated gaps at origins, likely due to helicase activation prior to replication initiation. We used micrococcal nuclease (MNase)-based chromatin occupancy profiling under restrictive conditions to study chromatin dynamics associated with helicase activation. Helicase activation in the absence of DNA replication resulted in the disruption and disorganization of chromatin, which extends up to 1 kb from early, efficient replication origins. The CMG holohelicase complex also moves the same distance out from the origin, producing single-stranded DNA that activates the intra-S-phase checkpoint. Loss of the checkpoint did not regulate the progression and stalling of the CMG complex but rather resulted in the disruption of chromatin at both early and late origins. Finally, we found that the local sequence context regulates helicase progression in the absence of DNA replication, suggesting that the helicase is intrinsically less processive when uncoupled from replication.

Linking the dynamics of chromatin occupancy and transcription with predictive models.

Though the sequence of the genome within each eukaryotic cell is essentially fixed, it exists within a complex and changing chromatin state. This state is determined, in part, by the dynamic binding of proteins to the DNA. These proteins-including histones, transcription factors (TFs), and polymerases-interact with one another, the genome, and other molecules to allow the chromatin to adopt one of exceedingly many possible configurations. Understanding how changing chromatin configurations associate with transcription remains a fundamental research problem. We sought to characterize at high spatiotemporal resolution the dynamic interplay between transcription and chromatin in response to cadmium stress. Whereas gene regulatory responses to environmental stress in yeast have been studied, how the chromatin state changes and how those changes connect to gene regulation remain unexplored. By combining MNase-seq and RNA-seq data, we found chromatin signatures of transcriptional activation and repression involving both nucleosomal and TF-sized DNA-binding factors. Using these signatures, we identified associations between chromatin dynamics and transcriptional regulation, not only for known cadmium response genes, but across the entire genome, including antisense transcripts. Those associations allowed us to develop generalizable models that predict dynamic transcriptional responses on the basis of dynamic chromatin signatures.

Local nucleosome dynamics and eviction following a double-strand break are reversible by NHEJ-mediated repair in the absence of DNA replication.

We interrogated at nucleotide resolution the spatiotemporal order of chromatin changes that occur immediately following a site-specific double-strand break (DSB) upstream of the PHO5 locus and its subsequent repair by nonhomologous end joining (NHEJ). We observed the immediate eviction of a nucleosome flanking the break and the repositioning of adjacent nucleosomes away from the break. These early chromatin events were independent of the end-processing Mre11-Rad50-Xrs2 (MRX) complex and preceded the MRX-dependent broad eviction of histones and DNA end-resectioning that extends up to ∼8 kb away from the break. We also examined the temporal dynamics of NHEJ-mediated repair in a G1-arrested population. Concomitant with DSB repair by NHEJ, we observed the redeposition and precise repositioning of nucleosomes at their originally occupied positions. This re-establishment of the prelesion chromatin landscape suggests that a DNA replication-independent mechanism exists to preserve epigenome organization following DSB repair.

Nascent chromatin occupancy profiling reveals locus- and factor-specific chromatin maturation dynamics behind the DNA replication fork.

Proper regulation and maintenance of the epigenome is necessary to preserve genome function. However, in every cell division, the epigenetic state is disassembled and then reassembled in the wake of the DNA replication fork. Chromatin restoration on nascent DNA is a complex and regulated process that includes nucleosome assembly and remodeling, deposition of histone variants, and the re-establishment of transcription factor binding. To study the genome-wide dynamics of chromatin restoration behind the DNA replication fork, we developed nascent chromatin occupancy profiles (NCOPs) to comprehensively profile nascent and mature chromatin at nucleotide resolution. Although nascent chromatin is inherently less organized than mature chromatin, we identified locus-specific differences in the kinetics of chromatin maturation that were predicted by the epigenetic landscape, including the histone variant H2AZ, which marked loci with rapid maturation kinetics. The chromatin maturation at origins of DNA replication was dependent on whether the origin underwent initiation or was passively replicated from distal-originating replication forks, suggesting distinct chromatin assembly mechanisms surrounding activated and disassembled prereplicative complexes. Finally, we identified sites that were only occupied transiently by DNA-binding factors following passage of the replication fork, which may provide a mechanism for perturbations of the DNA replication program to shape the regulatory landscape of the genome.

Mcm2-7 Is an Active Player in the DNA Replication Checkpoint Signaling Cascade via Proposed Modulation of Its DNA Gate.

The DNA replication checkpoint (DRC) monitors and responds to stalled replication forks to prevent genomic instability. How core replication factors integrate into this phosphorylation cascade is incompletely understood. Here, through analysis of a unique mcm allele targeting a specific ATPase active site (mcm2DENQ), we show that the Mcm2-7 replicative helicase has a novel DRC function as part of the signal transduction cascade. This allele exhibits normal downstream mediator (Mrc1) phosphorylation, implying DRC sensor kinase activation. However, the mutant also exhibits defective effector kinase (Rad53) activation and classic DRC phenotypes. Our previous in vitro analysis showed that the mcm2DENQ mutation prevents a specific conformational change in the Mcm2-7 hexamer. We infer that this conformational change is required for its DRC role and propose that it allosterically facilitates Rad53 activation to ensure a replication-specific checkpoint response.

Dynamic loading and redistribution of the Mcm2-7 helicase complex through the cell cycle.

Eukaryotic replication origins are defined by the ORC-dependent loading of the Mcm2-7 helicase complex onto chromatin in G1. Paradoxically, there is a vast excess of Mcm2-7 relative to ORC assembled onto chromatin in G1. These excess Mcm2-7 complexes exhibit little co-localization with ORC or replication foci and can function as dormant origins. We dissected the mechanisms regulating the assembly and distribution of the Mcm2-7 complex in the Drosophila genome. We found that in the absence of cyclin E/Cdk2 activity, there was a 10-fold decrease in chromatin-associated Mcm2-7 relative to the levels found at the G1/S transition. The minimal amounts of Mcm2-7 loaded in the absence of cyclin E/Cdk2 activity were strictly localized to ORC binding sites. In contrast, cyclin E/Cdk2 activity was required for maximal loading of Mcm2-7 and a dramatic genome-wide reorganization of the distribution of Mcm2-7 that is shaped by active transcription. Thus, increasing cyclin E/Cdk2 activity over the course of G1 is not only critical for Mcm2-7 loading, but also for the distribution of the Mcm2-7 helicase prior to S-phase entry.

Genome-wide chromatin footprinting reveals changes in replication origin architecture induced by pre-RC assembly.

Start sites of DNA replication are marked by the origin recognition complex (ORC), which coordinates Mcm2-7 helicase loading to form the prereplicative complex (pre-RC). Although pre-RC assembly is well characterized in vitro, the process is poorly understood within the local chromatin environment surrounding replication origins. To reveal how the chromatin architecture modulates origin selection and activation, we "footprinted" nucleosomes, transcription factors, and replication proteins at multiple points during the Saccharomyces cerevisiae cell cycle. Our nucleotide-resolution protein occupancy profiles resolved a precise ORC-dependent footprint at 269 origins in G2. A separate class of inefficient origins exhibited protein occupancy only in G1, suggesting that stable ORC chromatin association in G2 is a determinant of origin efficiency. G1 nucleosome remodeling concomitant with pre-RC assembly expanded the origin nucleosome-free region and enhanced activation efficiency. Finally, the local chromatin environment restricts the loading of the Mcm2-7 double hexamer either upstream of or downstream from the ARS consensus sequence (ACS).

Heterogeneous polymerase fidelity and mismatch repair bias genome variation and composition.

Mutational heterogeneity must be taken into account when reconstructing evolutionary histories, calibrating molecular clocks, and predicting links between genes and disease. Selective pressures and various DNA transactions have been invoked to explain the heterogeneous distribution of genetic variation between species, within populations, and in tissue-specific tumors. To examine relationships between such heterogeneity and variations in leading- and lagging-strand replication fidelity and mismatch repair, we accumulated 40,000 spontaneous mutations in eight diploid yeast strains in the absence of selective pressure. We found that replicase error rates vary by fork direction, coding state, nucleosome proximity, and sequence context. Further, error rates and DNA mismatch repair efficiency both vary by mismatch type, responsible polymerase, replication time, and replication origin proximity. Mutation patterns implicate replication infidelity as one driver of variation in somatic and germline evolution, suggest mechanisms of mutual modulation of genome stability and composition, and predict future observations in specific cancers.

Genome-wide localization of replication factors.

Chromatin Immunoprecipitation (ChIP) is a powerful tool for the identification and characterization of protein-DNA interactions in vivo. ChIP has been utilized to study diverse nuclear processes such as transcription regulation, chromatin modification, DNA recombination and DNA replication at specific loci and, more recently, across the entire genome. Advances in genomic approaches, and whole genome sequencing in particular, have made it possible and affordable to comprehensively identify specific protein binding sites throughout the genomes of higher eukaryotes. The dynamic nature of the DNA replication program and the transient occupancy of many replication factors throughout the cell cycle present additional challenges that may not pertain to the mapping of site specific transcription factors. Here we discuss the specific considerations that need to be addressed in the application of ChIP to the genome-wide location analysis of protein factors involved in DNA replication.

Chromatin signatures of the Drosophila replication program.

DNA replication initiates from thousands of start sites throughout the Drosophila genome and must be coordinated with other ongoing nuclear processes such as transcription to ensure genetic and epigenetic inheritance. Considerable progress has been made toward understanding how chromatin modifications regulate the transcription program; in contrast, we know relatively little about the role of the chromatin landscape in defining how start sites of DNA replication are selected and regulated. Here, we describe the Drosophila replication program in the context of the chromatin and transcription landscape for multiple cell lines using data generated by the modENCODE consortium. We find that while the cell lines exhibit similar replication programs, there are numerous cell line-specific differences that correlate with changes in the chromatin architecture. We identify chromatin features that are associated with replication timing, early origin usage, and ORC binding. Primary sequence, activating chromatin marks, and DNA-binding proteins (including chromatin remodelers) contribute in an additive manner to specify ORC-binding sites. We also generate accurate and predictive models from the chromatin data to describe origin usage and strength between cell lines. Multiple activating chromatin modifications contribute to the function and relative strength of replication origins, suggesting that the chromatin environment does not regulate origins of replication as a simple binary switch, but rather acts as a tunable rheostat to regulate replication initiation events.

Identification of functional elements and regulatory circuits by Drosophila modENCODE.

To gain insight into how genomic information is translated into cellular and developmental programs, the Drosophila model organism Encyclopedia of DNA Elements (modENCODE) project is comprehensively mapping transcripts, histone modifications, chromosomal proteins, transcription factors, replication proteins and intermediates, and nucleosome properties across a developmental time course and in multiple cell lines. We have generated more than 700 data sets and discovered protein-coding, noncoding, RNA regulatory, replication, and chromatin elements, more than tripling the annotated portion of the Drosophila genome. Correlated activity patterns of these elements reveal a functional regulatory network, which predicts putative new functions for genes, reveals stage- and tissue-specific regulators, and enables gene-expression prediction. Our results provide a foundation for directed experimental and computational studies in Drosophila and related species and also a model for systematic data integration toward comprehensive genomic and functional annotation.

Drosophila ORC localizes to open chromatin and marks sites of cohesin complex loading.

The origin recognition complex (ORC) is an essential DNA replication initiation factor conserved in all eukaryotes. In Saccharomyces cerevisiae, ORC binds to specific DNA elements; however, in higher eukaryotes, ORC exhibits little sequence specificity in vitro or in vivo. We investigated the genome-wide distribution of ORC in Drosophila and found that ORC localizes to specific chromosomal locations in the absence of any discernible simple motif. Although no clear sequence motif emerged, we were able to use machine learning approaches to accurately discriminate between ORC-associated sequences and ORC-free sequences based solely on primary sequence. The complex sequence features that define ORC binding sites are highly correlated with nucleosome positioning signals and likely represent a preferred nucleosomal landscape for ORC association. Open chromatin appears to be the underlying feature that is deterministic for ORC binding. ORC-associated sequences are enriched for the histone variant, H3.3, often at transcription start sites, and depleted for bulk nucleosomes. The density of ORC binding along the chromosome is reflected in the time at which a sequence replicates, with early replicating sequences having a high density of ORC binding. Finally, we found a high concordance between sites of ORC binding and cohesin loading, suggesting that, in addition to DNA replication, ORC may be required for the loading of cohesin on DNA in Drosophila.