Comprehensive analysis of DNA replication timing in genetic diseases and gene knockouts identifies MCM10 as a novel regulator of the replication program

AbstractCellular proliferation depends on the accurate and timely replication of the genome. Several genetic diseases are caused by mutations in key DNA replication genes; however, it remains unclear whether these genes influence the normal program of DNA replication timing. Similarly, the factors that regulate DNA replication dynamics are poorly understood. To systematically identify trans-acting modulators of replication timing, we profiled replication in 184 cell lines from three cell types, encompassing 60 different gene knockouts or genetic diseases. Through a rigorous approach that considers the background variability of replication timing, we concluded that most samples displayed normal replication timing. However, mutations in two genes showed consistently abnormal replication timing. The first gene was RIF1, a known modulator of replication timing. The second was MCM10, a highly conserved member of the pre-replication complex. MCM10 mutant cells demonstrated replication timing variability comprising 46% of the genome and at different locations than RIF1 knockouts. Replication timing alterations in MCM10-mutant cells was predominantly comprised of replication initiation defects. Taken together, this study demonstrates the remarkable robustness of the human replication timing program and reveals MCM10 as a novel modulator of DNA replication timing.

Download Full-text

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.

Download Full-text

Fused in sarcoma regulates DNA replication timing and progression

10.1101/2020.04.22.055343 ◽

2020 ◽

Author(s):

Weiyan Jia ◽

Mark A. Scalf ◽

Peter Tonzi ◽

Robert J. Millikin ◽

Sang Hwa Kim ◽

...

Keyword(s):

Dna Replication ◽

Transcriptional Activation ◽

Rna Binding ◽

Soft Tissue Sarcomas ◽

Replication Timing ◽

Strand Break ◽

Replication Initiation ◽

Proliferative Potential ◽

Fused In Sarcoma ◽

Dna Replication Timing

AbstractFused in sarcoma (FUS) encodes a low complexity RNA-binding protein with diverse roles in transcriptional activation and RNA processing. While oncogenic fusions of FUS and transcription factor DNA-binding domains are associated with soft tissue sarcomas, dominant mutations in FUS cause amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). FUS has also been implicated in DNA double-strand break repair (DSBR) and genome maintenance. However, the underlying mechanisms are unknown. Here we employed quantitative proteomics, transcriptomics, and DNA copy number analysis (Sort-Seq), in conjunction with FUS-/- cells to ascertain roles of FUS in genome protection. FUS-/- cells exhibited alterations in the recruitment and retention of DSBR factors BRCA1 and 53BP1 but were not overtly sensitive to genotoxins. By contrast, FUS-deficient cells had reduced proliferative potential that correlated with reduced replication fork speed, diminished loading of pre-replication complexes, and attenuated expression of S-phase associated genes. FUS interacted with lagging strand DNA synthesis factors and other replisome components, but did not translocate with active replication forks. Using a Sort-Seq workflow, we show that FUS contributes to genome-wide control of DNA replication timing and is essential for the early replication of transcriptionally active DNA. These findings illuminate new roles for FUS in DNA replication initiation and timing that may contribute to genome instability and functional defects in cells harboring disease-associated FUS fusions.

Download Full-text

The Genetic Architecture of DNA Replication Timing in Human Pluripotent Stem Cells

10.1101/2020.05.08.085324 ◽

2020 ◽

Cited By ~ 1

Author(s):

Qiliang Ding ◽

Matthew M. Edwards ◽

Michelle L. Hulke ◽

Alexa N. Bracci ◽

Ya Hu ◽

...

Keyword(s):

Dna Replication ◽

Dna Sequences ◽

Timing Analysis ◽

Replication Timing ◽

Histone Code ◽

Replication Initiation ◽

Histone Hyperacetylation ◽

Stem Cell Lines ◽

Early Replication ◽

Dna Replication Timing

AbstractDNA replication follows a strict spatiotemporal program that intersects with chromatin structure and gene regulation. However, the genetic basis of the mammalian DNA replication timing program is poorly understood1–3. To systematically identify genetic regulators of DNA replication timing, we exploited inter-individual variation in 457 human pluripotent stem cell lines from 349 individuals. We show that the human genome’s replication program is broadly encoded in DNA and identify 1,617 cis-acting replication timing quantitative trait loci (rtQTLs4) – base-pair-resolution sequence determinants of replication initiation. rtQTLs function individually, or in combinations of proximal and distal regulators, to affect replication timing. Analysis of rtQTL locations reveals a histone code for replication initiation, composed of bivalent histone H3 trimethylation marks on a background of histone hyperacetylation. The H3 trimethylation marks are individually repressive yet synergize to promote early replication. We further identify novel positive and negative regulators of DNA replication timing, the former comprised of pluripotency-related transcription factors while the latter involve boundary elements. Human replication timing is controlled by a multi-layered mechanism that operates on target DNA sequences, is composed of dozens of effectors working combinatorially, and follows principles analogous to transcription regulation: a histone code, activators and repressors, and a promoter-enhancer logic.

Download Full-text

The genetic architecture of DNA replication timing in human pluripotent stem cells

Nature Communications ◽

10.1038/s41467-021-27115-9 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Qiliang Ding ◽

Matthew M. Edwards ◽

Ning Wang ◽

Xiang Zhu ◽

Alexa N. Bracci ◽

...

Keyword(s):

Stem Cells ◽

Dna Replication ◽

Pluripotent Stem Cells ◽

Genetic Basis ◽

Replication Timing ◽

Human Pluripotent Stem Cells ◽

Replication Initiation ◽

Histone Hyperacetylation ◽

Early Replication ◽

Dna Replication Timing

AbstractDNA replication follows a strict spatiotemporal program that intersects with chromatin structure but has a poorly understood genetic basis. To systematically identify genetic regulators of replication timing, we exploited inter-individual variation in human pluripotent stem cells from 349 individuals. We show that the human genome’s replication program is broadly encoded in DNA and identify 1,617 cis-acting replication timing quantitative trait loci (rtQTLs) – sequence determinants of replication initiation. rtQTLs function individually, or in combinations of proximal and distal regulators, and are enriched at sites of histone H3 trimethylation of lysines 4, 9, and 36 together with histone hyperacetylation. H3 trimethylation marks are individually repressive yet synergistically associate with early replication. We identify pluripotency-related transcription factors and boundary elements as positive and negative regulators of replication timing, respectively. Taken together, human replication timing is controlled by a multi-layered mechanism with dozens of effectors working combinatorially and following principles analogous to transcription regulation.

Download Full-text

Faculty Opinions recommendation of Conserved forkhead dimerization motif controls DNA replication timing and spatial organization of chromosomes in S. cerevisiae.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.727380810.793535287 ◽

2017 ◽

Author(s):

Catherine Fox

Keyword(s):

Dna Replication ◽

Spatial Organization ◽

Replication Timing ◽

Dna Replication Timing

Download Full-text

Faculty Opinions recommendation of Stability of patient-specific features of altered DNA replication timing in xenografts of primary human acute lymphoblastic leukemia.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.727545410.793549651 ◽

2018 ◽

Author(s):

Domenico Maiorano

Keyword(s):

Acute Lymphoblastic Leukemia ◽

Dna Replication ◽

Lymphoblastic Leukemia ◽

Replication Timing ◽

Patient Specific ◽

Dna Replication Timing ◽

Human Acute Lymphoblastic Leukemia

Download Full-text

TIGER: inferring DNA replication timing from whole-genome sequence data

Bioinformatics ◽

10.1093/bioinformatics/btab166 ◽

2021 ◽

Cited By ~ 1

Author(s):

Amnon Koren ◽

Dashiell J Massey ◽

Alexa N Bracci

Keyword(s):

Dna Replication ◽

Genome Sequence ◽

Genomic Dna ◽

Sequence Data ◽

Replication Timing ◽

Whole Genome Sequence ◽

Supplementary Information ◽

Whole Genome ◽

Genome Sequence Data ◽

Dna Replication Timing

Abstract Motivation Genomic DNA replicates according to a reproducible spatiotemporal program, with some loci replicating early in S phase while others replicate late. Despite being a central cellular process, DNA replication timing studies have been limited in scale due to technical challenges. Results We present TIGER (Timing Inferred from Genome Replication), a computational approach for extracting DNA replication timing information from whole genome sequence data obtained from proliferating cell samples. The presence of replicating cells in a biological specimen leads to non-uniform representation of genomic DNA that depends on the timing of replication of different genomic loci. Replication dynamics can hence be observed in genome sequence data by analyzing DNA copy number along chromosomes while accounting for other sources of sequence coverage variation. TIGER is applicable to any species with a contiguous genome assembly and rivals the quality of experimental measurements of DNA replication timing. It provides a straightforward approach for measuring replication timing and can readily be applied at scale. Availability and Implementation TIGER is available at https://github.com/TheKorenLab/TIGER. Supplementary information Supplementary data are available at Bioinformatics online

Download Full-text

Asymmetrical deposition and modification of histone H3 variants are essential for zygote development

Life Science Alliance ◽

10.26508/lsa.202101102 ◽

2021 ◽

Vol 4 (8) ◽

pp. e202101102

Author(s):

Machika Kawamura ◽

Satoshi Funaya ◽

Kenta Sugie ◽

Masataka G Suzuki ◽

Fugaku Aoki

Keyword(s):

Dna Replication ◽

Mrna Expression ◽

Detrimental Effect ◽

Histone H3 ◽

Replication Timing ◽

Male And Female ◽

Zygote Development ◽

The One ◽

Dna Replication Timing ◽

Incorporation Efficiency

The pericentromeric heterochromatin of one-cell embryos forms a unique, ring-like structure around the nucleolar precursor body, which is absent in somatic cells. Here, we found that the histone H3 variants H3.1 and/or H3.2 (H3.1/H3.2) were localized asymmetrically between the male and female perinucleolar regions of the one-cell embryos; moreover, asymmetrical histone localization influenced DNA replication timing. The nuclear deposition of H3.1/3.2 in one-cell embryos was low relative to other preimplantation stages because of reduced H3.1/3.2 mRNA expression and incorporation efficiency. The forced incorporation of H3.1/3.2 into the pronuclei of one-cell embryos triggered a delay in DNA replication, leading to developmental failure. Methylation of lysine residue 27 (H3K27me3) of the deposited H3.1/3.2 in the paternal perinucleolar region caused this delay in DNA replication. These results suggest that reduced H3.1/3.2 in the paternal perinucleolar region is essential for controlled DNA replication and preimplantation development. The nuclear deposition of H3.1/3.2 is presumably maintained at a low level to avoid the detrimental effect of K27me3 methylation on DNA replication in the paternal perinucleolar region.

Download Full-text