Human ORC/MCM density is low in active genes and correlates with replication time but does not delimit initiation zones
Abstract
Eukaryotic DNA replication initiates during S phase from origins that have been licensed in the preceding G1 phase. Here, we compare ChIP-seq profiles of the licensing factors Orc2, Orc3, Mcm3, and Mcm7 with gene expression, replication timing and fork directionality profiles obtained by RNA-seq, Repli-seq and OK-seq. ORC and MCM are significantly and homogeneously depleted from transcribed genes, enriched at gene promoters, and more abundant in early- than in late-replicating domains. Surprisingly, after controlling these variables, no difference in ORC/MCM density is detected between initiation zones, termination zones, unidirectionally replicating and randomly replicating regions. Therefore, ORC/MCM density correlates with replication timing but does not solely regulate the probability of replication initiation. Interestingly, H4K20me3, a histone modification proposed to facilitate late origin licensing, was enriched in late replicating initiation zones and gene deserts of stochastic replication fork direction. We discuss potential mechanisms specifying when and where replication initiates in human cells.
Data availability
Sequencing data have been deposited ad the ENA European Nucleotide Archive and NCBI Gene Expression Omnibus as indicatedChIP-Seq: PRJEB32855, RNA-seq Raji: PRJEB31867 OK-seq Raji: PRJEB25180, Repli-seq Raji: GSE102522, OK-seq mESC: SRR,7535256, OK-seq mouse B-cells: GSE116319All data generated or analysed during this study are included in the manuscript and supporting files. Source data files have been provided for Figures 1-6; Fig. 1-Fig.1-Suppl. 3; Fig2-Fig2 Suppl. 1,2; Fig3-Fig3-Suppl. 1; Fig. 4-Fig.4 Suppl. 1,2; Fig5-Fig.5-Suppl. 1; Fig7-Fig.7-Suppl. 1
-
Human ORC/MCM density is low in active genes and correlates with replication time but does not delimit initiation zonesENA European Nucleotide Archive, PRJEB32855.
-
RNA-seq in Raji cells with inducible BZLF1 prior to and after induction of EBV's lytic cycle by doxycyclineENA European Nucleotide Archive, PRJEB31867.
-
MCM2 promotes symmetric inheritance of modified histones during DNA replicationENA European Nucleotide Archive, SRR7535256.
-
OK-seq profile from cycling (S) phase untreated B cellsNCBI Gene Expression Omnibus, GSE116319.
Article and author information
Author details
Funding
Helmholtz Zentrum Muenchen
- Nina Kirstein
- Alexander Buschle
- Wolfgang Hammerschmidt
- Aloys Schepers
National Cancer Institute (CA70723)
- Wolfgang Hammerschmidt
Deutsche Forschungsgemeinschaft (SFB 1064 TP05; SFB1064/TP A13,SFB-TR36/TP A04)
- Wolfgang Hammerschmidt
- Aloys Schepers
Agence Nationale de la Recherche (ANR-15-CE12-0011,ANR-18-CE45-0002,ANR-19-CE12-0028,ANR-10-IDEX-0001-02)
- Olivier Hyrien
- Benjamin Audit
Fondation pour la Recherche Médicale (FRM DEI201512344404)
- Olivier Hyrien
- Benjamin Audit
Canceropole Ile-de-France (PL-BIO16-302)
- Olivier Hyrien
- Benjamin Audit
INCa
- Olivier Hyrien
Ligue Nationale Contre le Cancer (RS19/75-75)
- Olivier Hyrien
Association pour la Recherche sur le Cancer (PJA 20171206387)
- Olivier Hyrien
Deutsche Krebshilfe (70112875)
- Wolfgang Hammerschmidt
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Copyright
© 2021, Kirstein et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
Metrics
-
- 2,234
- views
-
- 358
- downloads
-
- 27
- citations
Views, downloads and citations are aggregated across all versions of this paper published by eLife.
Download links
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Further reading
-
- Chromosomes and Gene Expression
- Genetics and Genomics
The preservation of genome integrity during sperm and egg development is vital for reproductive success. During meiosis, the tumor suppressor BRCA1/BRC-1 and structural maintenance of chromosomes 5/6 (SMC-5/6) complex genetically interact to promote high fidelity DNA double strand break (DSB) repair, but the specific DSB repair outcomes these proteins regulate remain unknown. Using genetic and cytological methods to monitor resolution of DSBs with different repair partners in Caenorhabditis elegans, we demonstrate that both BRC-1 and SMC-5 repress intersister crossover recombination events. Sequencing analysis of conversion tracts from homolog-independent DSB repair events further indicates that BRC-1 regulates intersister/intrachromatid noncrossover conversion tract length. Moreover, we find that BRC-1 specifically inhibits error prone repair of DSBs induced at mid-pachytene. Finally, we reveal functional interactions of BRC-1 and SMC-5/6 in regulating repair pathway engagement: BRC-1 is required for localization of recombinase proteins to DSBs in smc-5 mutants and enhances DSB repair defects in smc-5 mutants by repressing theta-mediated end joining (TMEJ). These results are consistent with a model in which some functions of BRC-1 act upstream of SMC-5/6 to promote recombination and inhibit error-prone DSB repair, while SMC-5/6 acts downstream of BRC-1 to regulate the formation or resolution of recombination intermediates. Taken together, our study illuminates the coordinate interplay of BRC-1 and SMC-5/6 to regulate DSB repair outcomes in the germline.
-
- Chromosomes and Gene Expression
Two different models have been proposed to explain how the endpoints of chromatin looped domains (‘TADs’) in eukaryotic chromosomes are determined. In the first, a cohesin complex extrudes a loop until it encounters a boundary element roadblock, generating a stem-loop. In this model, boundaries are functionally autonomous: they have an intrinsic ability to halt the movement of incoming cohesin complexes that is independent of the properties of neighboring boundaries. In the second, loops are generated by boundary:boundary pairing. In this model, boundaries are functionally non-autonomous, and their ability to form a loop depends upon how well they match with their neighbors. Moreover, unlike the loop-extrusion model, pairing interactions can generate both stem-loops and circle-loops. We have used a combination of MicroC to analyze how TADs are organized, and experimental manipulations of the even skipped TAD boundary, homie, to test the predictions of the ‘loop-extrusion’ and the ‘boundary-pairing’ models. Our findings are incompatible with the loop-extrusion model, and instead suggest that the endpoints of TADs in flies are determined by a mechanism in which boundary elements physically pair with their partners, either head-to-head or head-to-tail, with varying degrees of specificity. Although our experiments do not address how partners find each other, the mechanism is unlikely to require loop extrusion.