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Transcription shapes DNA replication initiation and termination in human cells

Nature Structural & Molecular Biologyvolume 26pages6777 (2019) | Download Citation

Abstract

Although DNA replication is a fundamental aspect of biology, it is not known what determines where DNA replication starts and stops in the human genome. We directly identified and quantitatively compared sites of replication initiation and termination in untransformed human cells. We found that replication preferentially initiates at the transcription start site of genes occupied by high levels of RNA polymerase II, and terminates at their polyadenylation sites, thereby ensuring global co-directionality of transcription and replication, particularly at gene 5′ ends. During replication stress, replication initiation is stimulated downstream of genes and termination is redistributed to gene bodies; this globally reorients replication relative to transcription around gene 3′ ends. These data suggest that replication initiation and termination are coupled to transcription in human cells, and propose a model for the impact of replication stress on genome integrity.

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Data availability

Data, including raw sequencing reads and tables used to generate source data for graphs in Figs. 16, are publicly available under accession number GSE114017. Custom scripts are available upon request from the corresponding authors.

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Acknowledgements

We thank the NYU Genome Technology Center for assistance with TapeStation and sequencing. We thank D. Remus, I. Whitehouse, E. Mazzoni, and H. Klein for helpful discussions, and G Sanchez for sharing RPE-1 enhancer data. Y.-H.C. was funded in part by the Molecular Oncology and Immunology NCI training program through NYU School of Medicine (no. 5T32CA009161-40). Work in T.T.H.'s laboratory is supported by grants from the NIH (ES025166), V Foundation BRCA Research and Basser Innovation Award. Work in D.J.S.'s laboratory is supported by grants from the NIH (nos. GM127336, GM114340) and the Searle Scholars Program.

Author information

Author notes

    • Yu-Hung Chen

    Present address: Miroculus, Inc., San Francisco, CA, USA

  1. These authors contributed equally: Yu-Hung Chen, Sarah Keegan, Malik Kahli.

Affiliations

  1. Department of Biochemistry and Molecular Pharmacology, New York University School of Medicine, New York, NY, USA

    • Yu-Hung Chen
    • , Sarah Keegan
    • , Peter Tonzi
    • , David Fenyö
    •  & Tony T. Huang
  2. Institute for Systems Genetics, New York University School of Medicine, New York, NY, USA

    • Sarah Keegan
    •  & David Fenyö
  3. Department of Biology, New York University, New York, NY, USA

    • Malik Kahli
    •  & Duncan J. Smith

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Contributions

Y.-H. C., M.K., and P.T. performed the experiments, S.K. and D.J.S. analyzed the data and D.F., T.T.H., and D.J.S. conceived and supervised the study. All of the authors interpreted the data. D.J.S. wrote the manuscript with input from all authors.

Competing interests

The authors declare no competing interests.

Corresponding authors

Correspondence to David Fenyö or Tony T. Huang or Duncan J. Smith.

Integrated supplementary information

  1. Supplementary Fig 1 Reproducibility of TSS data across replicate datasets.

    a, Percentage of Okazaki fragments (OFs) mapping to the Crick strand across a region ±50 kb from random genomic loci. b, Percentage of OFs mapping to the Crick strand across a ±50-kb window around the TSS of Watson (W) or Crick (C) genes. Data were analyzed as in Fig. 1e for two replicate datasets. c, Percentage of replication forks moving left to right around TSS binned by transcriptional volume (FPKM from 19 × gene length). Data were analyzed as in Fig. 2g, for two replicate datasets. d, Percentage of replication forks moving left to right around TSS binned by transcriptional volume, for cells treated with siRNAs against FANCD2 (green), FANCI (blue), or mock-treated (black), grown in 0.2 mM hydroxyurea for 4 h before OF collection. Data were analyzed as in Fig. 4d, using the second replicate datasets for each knockdown condition.

  2. Supplementary Fig 2 The effect of gene length on TSS-proximal origin firing efficiency is not solely a result of passive replication.

    a, Percentage of replication forks moving left to right around the TSS of actively transcribed genes (FPKM > median), where the TSS of the most proximal upstream gene is under (black) or over (green) 50 kb from the TSS being analyzed. b, Percentage of replication forks moving left to right around the TSS of actively transcribed genes (FPKM > median), where the TTS of the most proximal upstream gene is under (black) or over (red) 50 kb from the TSS being analyzed. c, Percentage of replication forks moving left to right around the TSS of actively transcribed genes (FPKM > median), where the TSS of the most proximal downstream gene is under (black) or over (green) 50 kb from the TSS being analyzed. d, Percentage of replication forks moving left to right around the TSS of actively transcribed genes (FPKM > median), where the most proximal downstream TTS is under (black) or over (red) 50 kb from the TSS being analyzed. Note that this TSS–TTS distance is equivalent to the length of the gene.

  3. Supplementary Fig 3 Replication initiation is most efficient at high-volume TSS in HeLa and GM06990 cells.

    Data in af were analyzed using Ok-seq data from HeLa cells (7) and HeLa RNA-seq data. a, Percentage of HeLa OFs mapping to the Crick strand (indicating rightward-moving replication forks) across a ±50-kb window around annotated TSS. Data were analyzed as in Fig. 1d. b, Percentage of OFs mapping to the Crick strand across a ±50-kb window around the TSS of Watson (W) or Crick (C) genes. Data were analyzed as in Fig. 1e. c, Percentage of replication forks moving from left to right around the TSS of all genes, oriented such that transcription runs from left to right. Data were analyzed as in Fig. 1f. d, Replication initiation frequency, calculated as the first derivative of Okazaki fragment strand bias as a function of position, across a ±50-kb window around the TSS. Data were analyzed as in Fig. 1g. e, Percentage of replication forks moving left to right around TSS binned by RNA-seq read depth quartile. Data were analyzed as in Fig. 2a. f, Percentage of replication forks moving left to right around TSS binned by gene length. Data were analyzed as in Fig. 2e. gl, Data were analyzed as in af, using Ok-seq data from GM06990 cells (7) and GM06990 RNA-seq data.

  4. Supplementary Fig 4 Comparison of OF strand bias around enhancers and equivalently selected random sites at various distances from TSS.

    Percentage of OFs mapping to the Crick strand around enhancer midpoints, for enhancers or random sites within the indicated distance of annotated TSS. Enhancers are binned according to transcription level (above or below median). Data were analyzed as in Fig. 3c.

  5. Supplementary Fig 5 The effect of FANCI knockdown is not related to gene length or increased replication termination.

    Percentage of replication forks moving left to right around TSS binned by transcriptional volume (FPKM from Harenza et al. × gene length) for cells treated with siRNAs against FANCI (blue) or mock-treated (black), grown in 0.2 mM hydroxyurea for 4 h before OF collection. The TSS is denoted by a gray dotted line: lower and upper bounds for gene length in each quartile are denoted by dotted and dashed black lines, respectively.

  6. Supplementary Fig 6 Reproducibility of TTS data across replicate datasets.

    a, Percentage of replication forks moving left to right around transcription termination sites (TTS) binned by RNA-seq read depth quartile. Data were analyzed as in Fig. 5a, for two replicate datasets. b, Percentage of replication forks moving left to right around (TTS) binned by RNA-seq read depth quartile, from cells grown in 0.2 mM HU for 4 h before OF collection. Data were analyzed as in Fig. 6a, for two replicate datasets.

  7. Supplementary Fig 7 Transcription-dependent, R-loop-independent replication termination at TTS in HeLa and GM06990 cells.

    Data in ad were analyzed using Ok-seq data from HeLa cells (7) and HeLa RNA-seq data. DRIP-seq data are from HeLa cells (14). a, Percentage of replication forks moving left to right around transcription termination sites (TTS) binned by RNA-seq read depth quartile. Data were analyzed as in Fig. 5a. b, Replication initiation frequency, calculated as the first derivative of Okazaki fragment strand bias, around TTS binned by RNA-seq read density in the gene body. Data were analyzed as in Fig. 5c. c, Percentage of replication forks moving left to right around TTS of actively transcribed (FPKM > median) high-DRIP versus low-DRIP genes. Data were analyzed as in Fig. 5g. d, Replication initiation frequency, calculated as the first derivative of Okazaki fragment strand bias, around TTS of actively transcribed (FPKM > median) high-DRIP versus low-DRIP genes. Data were analyzed as in Fig. 5h. eh, Data were analyzed as in ad using Ok-seq data from GM06990 cells (7) and GM06990 RNA-seq data.

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https://doi.org/10.1038/s41594-018-0171-0