Next-Generation Sequencing Enables Spatiotemporal Resolution of Human Centromere Replication Timing

Centromeres serve a critical function in preserving genome integrity across sequential cell divisions, by mediating symmetric chromosome segregation. The repetitive, heterochromatic nature of centromeres is thought to be inhibitory to DNA replication, but has also led to their underrepresentation in human reference genome assemblies. Consequently, centromeres have been excluded from genomic replication timing analyses, leaving their time of replication unresolved. However, the most recent human reference genome, hg38, included models of centromere sequences. To establish the experimental requirements for achieving replication timing profiles for centromeres, we sequenced G1- and S-phase cells from five human cell lines, and aligned the sequence reads to hg38. We were able to infer DNA replication timing profiles for the centromeres in each of the five cell lines, which showed that centromere replication occurs in mid-to-late S phase. Furthermore, we found that replication timing was more variable between cell lines in the centromere regions than expected, given the distribution of variation in replication timing genome-wide. These results suggest the potential of these, and future, sequence models to enable high-resolution studies of replication in centromeres and other heterochromatic regions.

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High-throughput analysis of DNA replication in single human cells reveals constrained variability in the location and timing of replication initiation

10.1101/2021.05.14.443897 ◽

2021 ◽

Author(s):

Dashiell J Massey ◽

Amnon Koren

Keyword(s):

Dna Replication ◽

Replication Origin ◽

Replication Timing ◽

S Phase ◽

Human Cells ◽

Replication Initiation ◽

High Throughput Analysis ◽

Timing Variability ◽

Fixed Set ◽

Fixed Order

DNA replication occurs throughout the S phase of the cell cycle, initiating from replication origin loci that fire at different times. Debate remains about whether origins are a fixed set of loci used across all cells or a loose agglomeration of potential origins used stochastically in individual cells, and about how consistent their firing time during S phase is across cells. Here, we develop an approach for profiling DNA replication in single human cells and apply it to 2,305 replicating cells spanning the entire S phase. The resolution and scale of the data enabled us to specifically analyze initiation sites and show that these sites have confined locations that are consistently used among individual cells. Further, we find that initiation sites are activated in a similar, albeit not fixed, order across cells. Taken together, our results suggest that replication timing variability is constrained both spatially and temporally, and that the degree of variation is consistent across human cell lines.

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Genomic Analysis of the DNA Replication Timing Program during Mitotic S Phase in Maize (Zea mays) Root Tips

The Plant Cell ◽

10.1105/tpc.17.00037 ◽

2017 ◽

Vol 29 (9) ◽

pp. 2126-2149 ◽

Cited By ~ 11

Author(s):

Emily E. Wear ◽

Jawon Song ◽

Gregory J. Zynda ◽

Chantal LeBlanc ◽

Tae-Jin Lee ◽

...

Keyword(s):

Zea Mays ◽

Dna Replication ◽

Genomic Analysis ◽

Replication Timing ◽

S Phase ◽

Root Tips ◽

Dna Replication Timing

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Liftoff: accurate mapping of gene annotations

Bioinformatics ◽

10.1093/bioinformatics/btaa1016 ◽

2020 ◽

Author(s):

Alaina Shumate ◽

Steven L Salzberg

Keyword(s):

Reference Genome ◽

Supplementary Information ◽

Closely Related Species ◽

Protein Coding ◽

Human Reference Genome ◽

Sequence Identity ◽

Gene Annotations ◽

Genome Assemblies ◽

Average Sequence Identity ◽

High Quality Genome

Abstract Motivation Improvements in DNA sequencing technology and computational methods have led to a substantial increase in the creation of high-quality genome assemblies of many species. To understand the biology of these genomes, annotation of gene features and other functional elements is essential; however for most species, only the reference genome is well-annotated. Results One strategy to annotate new or improved genome assemblies is to map or ‘lift over’ the genes from a previously-annotated reference genome. Here we describe Liftoff, a new genome annotation lift-over tool capable of mapping genes between two assemblies of the same or closely-related species. Liftoff aligns genes from a reference genome to a target genome and finds the mapping that maximizes sequence identity while preserving the structure of each exon, transcript, and gene. We show that Liftoff can accurately map 99.9% of genes between two versions of the human reference genome with an average sequence identity >99.9%. We also show that Liftoff can map genes across species by successfully lifting over 98.3% of human protein-coding genes to a chimpanzee genome assembly with 98.2% sequence identity. Availability and Implementation Liftoff can be installed via bioconda and PyPI. Additionally, the source code for Liftoff is available at https://github.com/agshumate/Liftoff Supplementary information Supplementary data are available at Bioinformatics online.

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Regulation of Epstein-Barr Virus Origin of Plasmid Replication (OriP) by the S-Phase Checkpoint Kinase Chk2

Journal of Virology ◽

10.1128/jvi.01300-09 ◽

2010 ◽

Vol 84 (10) ◽

pp. 4979-4987 ◽

Cited By ~ 11

Author(s):

Jing Zhou ◽

Zhong Deng ◽

Julie Norseen ◽

Paul M. Lieberman

Keyword(s):

Dna Replication ◽

Epstein Barr Virus ◽

Replication Timing ◽

S Phase ◽

Plasmid Replication ◽

Acceptor Site ◽

Plasmid Maintenance ◽

Barr Virus ◽

Amino Terminal ◽

Epstein Barr

ABSTRACT The Epstein-Barr virus (EBV) origin of plasmid replication (OriP) is required for episome stability during latent infection. Telomere repeat factor 2 (TRF2) binds directly to OriP and facilitates DNA replication and plasmid maintenance. Recent studies have found that TRF2 interacts with the DNA damage checkpoint protein Chk2. We show here that Chk2 plays an important role in regulating OriP plasmid stability, chromatin modifications, and replication timing. The depletion of Chk2 by small interfering RNA (siRNA) leads to a reduction in DNA replication efficiency and a loss of OriP-dependent plasmid maintenance. This corresponds to a change in OriP replication timing and an increase in constitutive histone H3 acetylation. We show that Chk2 interacts with TRF2 in the early G1/S phase of the cell cycle. We also show that Chk2 can phosphorylate TRF2 in vitro at a consensus acceptor site in the amino-terminal basic domain of TRF2. TRF2 mutants with a serine-to-aspartic acid phosphomimetic substitution mutation were reduced in their ability to recruit the origin recognition complex (ORC) and stimulate OriP replication. We suggest that the Chk2 phosphorylation of TRF2 is important for coordinating ORC binding with chromatin remodeling during the early S phase and that a failure to execute these events leads to replication defects and plasmid instability.

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Deregulation of cyclin E in human cells interferes with prereplication complex assembly

The Journal of Cell Biology ◽

10.1083/jcb.200404092 ◽

2004 ◽

Vol 165 (6) ◽

pp. 789-800 ◽

Cited By ~ 190

Author(s):

Susanna Ekholm-Reed ◽

Juan Méndez ◽

Donato Tedesco ◽

Anders Zetterberg ◽

Bruce Stillman ◽

...

Keyword(s):

Dna Replication ◽

Cell Lines ◽

Broad Spectrum ◽

Cyclin E ◽

Chromosome Instability ◽

S Phase ◽

Potential Mechanism ◽

Minichromosome Maintenance ◽

Origins Of Replication ◽

Initiation Of Replication

Deregulation of cyclin E expression has been associated with a broad spectrum of human malignancies. Analysis of DNA replication in cells constitutively expressing cyclin E at levels similar to those observed in a subset of tumor-derived cell lines indicates that initiation of replication and possibly fork movement are severely impaired. Such cells show a specific defect in loading of initiator proteins Mcm4, Mcm7, and to a lesser degree, Mcm2 onto chromatin during telophase and early G1 when Mcm2–7 are normally recruited to license origins of replication. Because minichromosome maintenance complex proteins are thought to function as a heterohexamer, loading of Mcm2-, Mcm4-, and Mcm7-depleted complexes is likely to underlie the S phase defects observed in cyclin E–deregulated cells, consistent with a role for minichromosome maintenance complex proteins in initiation of replication and fork movement. Cyclin E–mediated impairment of DNA replication provides a potential mechanism for chromosome instability observed as a consequence of cyclin E deregulation.

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Replication timing analysis in polyploid cells reveals Rif1 uses multiple mechanisms to promote underreplication in Drosophila

Genetics ◽

10.1093/genetics/iyab147 ◽

2021 ◽

Author(s):

Souradip Das ◽

Madison Caballero ◽

Tatyana Kolesnikova ◽

Igor Zhimulev ◽

Amnon Koren ◽

...

Keyword(s):

Dna Replication ◽

Copy Number ◽

Genome Stability ◽

Timing Analysis ◽

Replication Timing ◽

Critical Function ◽

Replication Fork Progression ◽

Polyploid Cells ◽

Genomic Regions ◽

Insight Into

Abstract Regulation of DNA replication and copy number is necessary to promote genome stability and maintain cell and tissue function. DNA replication is regulated temporally in a process known as replication timing (RT). Rap1-interacting factor 1 (Rif1) is a key regulator of RT and has a critical function in copy number control in polyploid cells. Previously, we demonstrated that Rif1 functions with SUUR to inhibit replication fork progression and promote underreplication (UR) of specific genomic regions. How Rif1-dependent control of RT factors into its ability to promote UR is unknown. By applying a computational approach to measure RT in Drosophila polyploid cells, we show that SUUR and Rif1 have differential roles in controlling UR and RT. Our findings reveal that Rif1 acts to promote late replication, which is necessary for SUUR-dependent underreplication. Our work provides new insight into the process of UR and its links to RT.

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Cohesin-Mediated Genome Architecture Does Not Define DNA Replication Timing Domains

Genes ◽

10.3390/genes10030196 ◽

2019 ◽

Vol 10 (3) ◽

pp. 196 ◽

Cited By ~ 6

Author(s):

Phoebe Oldach ◽

Conrad A. Nieduszynski

Keyword(s):

Dna Replication ◽

Genome Organization ◽

Mammalian Cells ◽

Replication Timing ◽

S Phase ◽

Genome Architecture ◽

3D Genome ◽

Synchronized Cells ◽

Dna Replication Timing ◽

Phase Entry

3D genome organization is strongly predictive of DNA replication timing in mammalian cells. This work tested the extent to which loop-based genome architecture acts as a regulatory unit of replication timing by using an auxin-inducible system for acute cohesin ablation. Cohesin ablation in a population of cells in asynchronous culture was shown not to disrupt patterns of replication timing as assayed by replication sequencing (RepliSeq) or BrdU-focus microscopy. Furthermore, cohesin ablation prior to S phase entry in synchronized cells was similarly shown to not impact replication timing patterns. These results suggest that cohesin-mediated genome architecture is not required for the execution of replication timing patterns in S phase, nor for the establishment of replication timing domains in G1.

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Identification of regulatory pathways across different cell lines (NHBE and PBMC) in COVID-19 patients using transcriptome analysis v1

10.17504/protocols.io.bx6aprae ◽

2021 ◽

Author(s):

Lavanya not provided C ◽

Vidya Niranjan ◽

Aajnaa not provided Upadhyaya ◽

Arpita not provided Guha Neogi

Keyword(s):

Gene Expression ◽

Cell Line ◽

Cell Lines ◽

Expression Analysis ◽

Pathway Analysis ◽

Gene Expression Analysis ◽

Reference Genome ◽

Host Cells ◽

Human Reference Genome ◽

Regulatory Pathways

The Sars-CoV-2 virus is a previously uncharacterized coronavirus and causative agent of the COVID-19 pandemic. Gene expression analysis followed by pathway analysis helps researchers to find possible key targets present in biological pathways of host cells that are targeted by the SARS-CoV-2 virus. This review considers the peripheral blood mononuclear cell line (PBMC) and the normal human bronchial epithelial (NHBE) cell line, both of which support SARS-CoV-2 viral replication. Pathway analysis between the healthy and patient samples of the respective cell lines shall provide useful insights on the COVID-19 disease. Initially, the datasets from the respective cell lines were collected from the NCBI databank. These datasets underwent further analysis and were mapped and aligned to the human reference genome. This outputs the file in the BAM format. The BAM files along with the human reference genome in the GFF format were uploaded to an open-source software called OmicsBox 2.0 for differential gene expression analysis. This resulted in the generation of a table containing the differentially expressed genes which were upregulated and downregulated. These gene lists were uploaded to various pathway analyzers that map the significant genes to the most significant pathways. In this project, KOBAS 3.0 and Enrichr were used for pathway analysis. The pathways obtained from the above-mentioned pathway analyzers were further narrowed down by manual comparison. It was observed that many pathways were similar between the NHBE and PBMC cell lines. However, they were also different in terms of their overall nature. In this project, many patterns were seen through the pathways obtained, however, further optimization and functionality studies must be performed in order to establish conclusive results on the scope of the COVID-19 disease. Expanding research on the scope of the disease by going back to the basics will generate new and valuable information about the virus. This knowledge will help us combat the disease in a better and appropriate manner.

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

The Journal of Cell Biology ◽

10.1083/jcb.200103001 ◽

2001 ◽

Vol 154 (4) ◽

pp. 683-690 ◽

Cited By ~ 54

Author(s):

Beth Sullivan ◽

Gary Karpen

Keyword(s):

Cell Lines ◽

Replication Timing ◽

S Phase ◽

Drosophila Cell ◽

Centromeric Chromatin ◽

Nucleotide Incorporation

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.

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Precise switching of DNA replication timing in the GC content transition area in the human major histocompatibility complex.

Molecular and Cellular Biology ◽

10.1128/mcb.17.7.4043 ◽

1997 ◽

Vol 17 (7) ◽

pp. 4043-4050 ◽

Cited By ~ 62

Author(s):

T Tenzen ◽

T Yamagata ◽

T Fukagawa ◽

K Sugaya ◽

A Ando ◽

...

Keyword(s):

Major Histocompatibility Complex ◽

Dna Replication ◽

Replication Timing ◽

S Phase ◽

Chromosome Band ◽

Switch Region ◽

Switch Point ◽

Junction Area ◽

Histocompatibility Complex ◽

Dna Replication Timing

The human genome is composed of long-range G+C% (GC%) mosaic structures thought to be related to chromosome bands. We previously reported a boundary of megabase-sized GC% mosaic domains at the junction area between major histocompatibility complex (MHC) classes II and III, proposing it as a possible chromosome band boundary. DNA replication timing during the S phase is known to be correlated cytogenetically with chromosome band zones, and thus the band boundaries have been predicted to contain a switch point for DNA replication timing. In this study, to identify to the nucleotide sequence level the replication switch point during the S phase, we determined the precise DNA replication timing for MHC classes II and III, focusing on the junction area. To do this, we used PCR-based quantitation of nascent DNA obtained from synchronized human myeloid leukemia HL60 cells. The replication timing changed precisely in the boundary region with a 2-h difference between the two sides, supporting the prediction that this region may be a chromosome band boundary. We supposed that replication fork movement terminates (pauses) or significantly slows in the switch region, which contains dense Alu clusters; polypurine/polypyrimidine tracts; di-, tri-, or tetranucleotide repeats; and medium-reiteration-frequency sequences. Because the nascent DNA in the switch region was recovered at low efficiency, we investigated whether this region is associated with the nuclear scaffold and found three scaffold-associated regions in and around the switch region.

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