Clustering of strong replicators associated with active promoters is sufficient to establish an early‐replicating domain
2020; Springer Nature; Volume: 39; Issue: 21 Linguagem: Inglês
10.15252/embj.201899520
ISSN1460-2075
AutoresCaroline Brossas, Anne-Laure Valton, Sergey V. Venev, Sabarinadh Chilaka, Antonin Counillon, Marc Laurent, Coralie Goncalves, Bénédicte Duriez, Franck Picard, Job Dekker, Marie‐Noëlle Prioleau,
Tópico(s)Gene expression and cancer classification
ResumoArticle16 September 2020Open Access Transparent process Clustering of strong replicators associated with active promoters is sufficient to establish an early-replicating domain Caroline Brossas CNRS, Institut Jacques Monod, Equipe Labellisée Association Pour la Recherche sur le Cancer, Université de Paris, Paris, France Search for more papers by this author Anne-Laure Valton Department of Biochemistry and Molecular Pharmacology, Program in Systems Biology, University of Massachusetts Medical School, Worcester, MA, USA Howard Hughes Medical Institute, Chevy Chase, MD, USA Search for more papers by this author Sergey V Venev Department of Biochemistry and Molecular Pharmacology, Program in Systems Biology, University of Massachusetts Medical School, Worcester, MA, USA Search for more papers by this author Sabarinadh Chilaka Centre for Immunology, University of Glasgow, Glasgow, UK Search for more papers by this author Antonin Counillon CNRS, Institut Jacques Monod, Equipe Labellisée Association Pour la Recherche sur le Cancer, Université de Paris, Paris, France Search for more papers by this author Marc Laurent CNRS, Institut Jacques Monod, Equipe Labellisée Association Pour la Recherche sur le Cancer, Université de Paris, Paris, France Search for more papers by this author Coralie Goncalves CNRS, Institut Jacques Monod, Equipe Labellisée Association Pour la Recherche sur le Cancer, Université de Paris, Paris, France Search for more papers by this author Bénédicte Duriez CNRS, Institut Jacques Monod, Equipe Labellisée Association Pour la Recherche sur le Cancer, Université de Paris, Paris, France Search for more papers by this author Franck Picard Laboratoire de Biométrie et Biologie Evolutive UMR 5558, CNRS, Université de Lyon, Université Lyon 1, Villeurbanne, France Search for more papers by this author Job Dekker Department of Biochemistry and Molecular Pharmacology, Program in Systems Biology, University of Massachusetts Medical School, Worcester, MA, USA Howard Hughes Medical Institute, Chevy Chase, MD, USA Search for more papers by this author Marie-Noëlle Prioleau Corresponding Author [email protected] orcid.org/0000-0003-2585-4005 CNRS, Institut Jacques Monod, Equipe Labellisée Association Pour la Recherche sur le Cancer, Université de Paris, Paris, France Search for more papers by this author Caroline Brossas CNRS, Institut Jacques Monod, Equipe Labellisée Association Pour la Recherche sur le Cancer, Université de Paris, Paris, France Search for more papers by this author Anne-Laure Valton Department of Biochemistry and Molecular Pharmacology, Program in Systems Biology, University of Massachusetts Medical School, Worcester, MA, USA Howard Hughes Medical Institute, Chevy Chase, MD, USA Search for more papers by this author Sergey V Venev Department of Biochemistry and Molecular Pharmacology, Program in Systems Biology, University of Massachusetts Medical School, Worcester, MA, USA Search for more papers by this author Sabarinadh Chilaka Centre for Immunology, University of Glasgow, Glasgow, UK Search for more papers by this author Antonin Counillon CNRS, Institut Jacques Monod, Equipe Labellisée Association Pour la Recherche sur le Cancer, Université de Paris, Paris, France Search for more papers by this author Marc Laurent CNRS, Institut Jacques Monod, Equipe Labellisée Association Pour la Recherche sur le Cancer, Université de Paris, Paris, France Search for more papers by this author Coralie Goncalves CNRS, Institut Jacques Monod, Equipe Labellisée Association Pour la Recherche sur le Cancer, Université de Paris, Paris, France Search for more papers by this author Bénédicte Duriez CNRS, Institut Jacques Monod, Equipe Labellisée Association Pour la Recherche sur le Cancer, Université de Paris, Paris, France Search for more papers by this author Franck Picard Laboratoire de Biométrie et Biologie Evolutive UMR 5558, CNRS, Université de Lyon, Université Lyon 1, Villeurbanne, France Search for more papers by this author Job Dekker Department of Biochemistry and Molecular Pharmacology, Program in Systems Biology, University of Massachusetts Medical School, Worcester, MA, USA Howard Hughes Medical Institute, Chevy Chase, MD, USA Search for more papers by this author Marie-Noëlle Prioleau Corresponding Author [email protected] orcid.org/0000-0003-2585-4005 CNRS, Institut Jacques Monod, Equipe Labellisée Association Pour la Recherche sur le Cancer, Université de Paris, Paris, France Search for more papers by this author Author Information Caroline Brossas1, Anne-Laure Valton2,3, Sergey V Venev2, Sabarinadh Chilaka4, Antonin Counillon1, Marc Laurent1, Coralie Goncalves1, Bénédicte Duriez1, Franck Picard5, Job Dekker2,3 and Marie-Noëlle Prioleau *,1 1CNRS, Institut Jacques Monod, Equipe Labellisée Association Pour la Recherche sur le Cancer, Université de Paris, Paris, France 2Department of Biochemistry and Molecular Pharmacology, Program in Systems Biology, University of Massachusetts Medical School, Worcester, MA, USA 3Howard Hughes Medical Institute, Chevy Chase, MD, USA 4Centre for Immunology, University of Glasgow, Glasgow, UK 5Laboratoire de Biométrie et Biologie Evolutive UMR 5558, CNRS, Université de Lyon, Université Lyon 1, Villeurbanne, France *Corresponding author. Tel: +33 (0) 157278102; E-mail: [email protected] EMBO J (2020)39:e99520https://doi.org/10.15252/embj.201899520 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Vertebrate genomes replicate according to a precise temporal program strongly correlated with their organization into A/B compartments. Until now, the molecular mechanisms underlying the establishment of early-replicating domains remain largely unknown. We defined two minimal cis-element modules containing a strong replication origin and chromatin modifier binding sites capable of shifting a targeted mid-late-replicating region for earlier replication. The two origins overlap with a constitutive or a silent tissue-specific promoter. When inserted side-by-side, these modules advance replication timing over a 250 kb region through the cooperation with one endogenous origin located 30 kb away. Moreover, when inserted at two chromosomal sites separated by 30 kb, these two modules come into close physical proximity and form an early-replicating domain establishing more contacts with active A compartments. The synergy depends on the presence of the active promoter/origin. Our results show that clustering of strong origins located at active promoters can establish early-replicating domains. Synopsis How vertebrate genomes organize a precise replication timing program that is strongly correlated with their organization into so-called A/B compartments has remained unclear. Here, clustering of strong origins located at active promoters is shown to be able to establish early-replicating domains. Two replicators carrying timing information can act in cooperation if located in close proximity. Strong autonomous replicons overlapping an active promoter can act in synergy on replication timing even when separated by 30 kb. Formation of an early-replicating domain results locally in formation of a spatial connection between advanced replicons, and in an increase of contacts to active A compartments. Replication timing of late-replicating domains can be advanced locally by a strong autonomous replicon. Introduction A precise, cell type-specific temporal program governs the duplication of vertebrate genomes (Ryba et al, 2010). The genome-wide mapping of replication origins with different methods has revealed a high density of efficient site-specific origins in early-replicated domains, whereas late regions are usually origin-poor (Prioleau & MacAlpine, 2016). Replication timing (RT) domains are correlated with the organization of chromosomes into two main types of compartments: compartment A, which is accessible and replicated early, and compartment B, which is more condensed and replicated late (Ryba et al, 2010). These compartments, which display greater interaction within themselves rather than across them, were initially defined by the Hi-C method, at a resolution of 1 megabase (Lieberman-Aiden et al, 2009). The only major player in the RT program for which the mode of action has been elucidated is Rif1 (Cornacchia et al, 2012; Knott et al, 2012; Yamazaki et al, 2012). Rif1 has a global repressive effect on genome-wide DNA replication. This effect is mediated by the recruitment of protein phosphatase 1, which opposes the Dbf4-dependent kinase (DDK) activity required for origin firing (Hiraga et al, 2014). Consistent with this direct role, ChIP analyses of Rif1 throughout the mouse genome revealed an overlap between Rif1 associated domains and late replication (Foti et al, 2016). However, only regions associated with Rif1 but not with the nuclear lamina switch to early replication in a context of Rif1 depletion. This finding suggests that other major players, such as the nuclear lamina, are also involved in controlling late replication (Duriez et al, 2019). One key question that remains concerns whether some late-replicating domains are so robustly constrained by their nuclear compartmentalization that the targeting of a very efficient origin associated with early timing control elements could not locally advance the timing of their replication. The underlying question is whether a late domain is defined by the deficiency of an early-firing signal together with an accumulation of signals imposing the late-firing of many potential initiation sites. A related question is whether early-replicated domains are defined solely by the absence of a strong negative signal, such as association with the nuclear lamina and/or Rif1. Alternatively, early constant timing regions (CTRs) may result fortuitously from the more or less synchronous firing of a cluster of replicons, each with its own individual local early timing control elements. This hypothesis is supported by the high density of efficient origins mostly associated with transcription start sites (TSS) and thus proximal to sites associated with open chromatin marks in early-replicated domains (Picard et al, 2014). In agreement with this model, a recent study showed that stem cell-specific early-replicating domains in mouse are controlled by stem cell-specific cis-elements located in promoter and enhancer regions (Sima et al, 2019). Moreover, several elements spanning altogether 30 kb had to be deleted so that the early domain switched from early to late, suggesting redundancy between these elements along early domains and their potential capacity to act remotely. Here we put forward the central role of constitutive promoters in the formation of constitutive early-replicating domains and strongly suggests that spatial connections between strong initiation sites play a key role in this fundamental process. Results Cooperation between two minimal autonomous replicons impacts on RT at a large scale The current study is based on the method we developed to quantify the magnitude of the RT shift induced by the insertion of an ectopic DNA sequence into a specific mid-late-replicating locus (chr1:72,565,520 bp, galGal5) (Hassan-Zadeh et al, 2012 and Appendix Fig S1). Here we confirmed our previous data on a much larger number of cell lines modified in the same targeted region, allowing a statistical quantification of RT shifts (Fig 1 and Appendix Table S1). The tissue-specific βA-globin promoter containing a strongly active replication origin flanked by 2XFIV [Footprint IV of cHS4 insulator element is a binding site for the Upstream Stimulatory Factor (USF) (West et al, 2004)] significantly advanced RT (Fig 1 (ii), P-value = 4.57E-05) and to the same extent as the active β-actin constitutive promoter containing an active replication origin (Figs EV1A and 1 (iii), P-value = 3.38E-03). Finally, the combination of these two minimal modules (βA-globin+β-actin constructs) imposed a stronger shift to earlier replication at the inserted locus than the presence of a single minimal module alone (Fig 1 (iv), P-value = 3.25E-03 between (ii) and (iv); P-value = 2.66E-03 between (iii) and (iv) and Fig EV1 and Hassan-Zadeh et al, 2012). These endogenous origins/promoters are naturally found in early-replicating domains in DT40 cells (Appendix Fig S2) and therefore constitute excellent models to understand how early domains might be established. Insertion of this large construct on the two homologous chromosomes (2 × (βA-globin + β-actin) cell line) allowed us to observe a 250 kb region displaying an advance RT compared to a WT cell line (Fig 2A and Appendix Fig S3). A zoom in centered on the site of insertion confirmed that this site is naturally a termination zone (TZ) flanked by two initiation zones (IZ) (Fig 2B). RT profiles obtained from S1 to S4 fractions showed that the IZ about 30 kb upstream of the site of insertion (IZ.1) is activated in S3 in WT cells, whereas the IZ located 60 kb downstream (IZ.2) is activated already in S2. IZ.1 and IZ.2 correspond to strong initiation sites detected by the short nascent strand (SNS) assay (Massip et al, 2019). Profiles observed at the same region in a cell line modified on both chromosomes (2 × (βA-globin + β-actin) cell line) showed firing of the IZ.1 in S1, whereas the IZ.2 is unchanged compared to the WT cell line. Moreover, in this line, IZ.1 is then extended in S2 on the 3′ direction toward the insertion site, revealing the activation of origins brought by the inserted construct. These changes led to a 30 kb shift of the TZ downstream of the site of insertion. Overall, this result suggested that the large βA-globin + β-actin construct impacted strongly on the RT profile of its surrounding regions in two different ways: It advanced significantly the RT of a 30 kb upstream strong endogenous origin from S3 to S1 and induced strong initiation in S2 due to efficient firing of replication origins present inside the construct. Figure 1. Quantitative analysis of RT shifts reveals cooperation between two combinations of cis-regulatory elementsDistribution of RT shifts calculated with the –ΔL + ΔE method described in Appendix Fig S1. Different transgenes in a mid-late-replicating region are compared: the IL2R reporter gene under the control of the βA-globin promoter (βA pro) containing an inactive (i, N = 10) or an active origin (ii, N = 8) flanked by two copies of FIV (2xFIV), the blasticidin resistance gene (BsR) under the control of the β-actin promoter (β-act pro) (iii, N = 7), or a combination of these two transgenes (iv, N = 8). Blue and black triangles represent reactive loxP sites and recombined inactive loxP sites, respectively. Rectangle edges correspond to the 0.25 and 0.75 quartiles, the thick black lines represent the median, the white triangles represent the mean, and the whiskers extend to the smallest and largest –ΔL + ΔE values. Statistical analysis was performed with Wilcoxon nonparametric two-tailed tests (ns, not significant; **P < 0.01; ***P < 0.001). Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Additional RT assays for transgenes shifting the timing of replication A, B. RT profiles of each chromosomal allele are determined after targeted transgene integration using the allele-specific analysis of RT method by real-time PCR quantification described in Appendix Fig S1. Differences in –ΔL + ΔE values calculated at the target site following transgene integration are indicated. Blue triangles represent reactive loxP sites. Error bars correspond to the standard deviation for qPCR duplicates. Analysis of six β-actin clonal cell lines (A) or two βA-globin + β-actin clonal cell lines (B) described in Fig 1 is reported. Black vertical bars represent insertion sites. Download figure Download PowerPoint Figure 2. One autonomous replicon inserted in the mid-late region perturbs the RT over a 250 kb region through cooperation with an endogenous strong origin UCSC genome browser visualization of the mid-late insertion site of chromosome 1 (genomic positions: chr1:71,000,000–74,100,000 bp, 3.1 Mb; galGal5). RT-weighted average (WA) values for the wt and the βA-globin + β-actin cell lines are shown. Early-replicated regions (E) are represented in orange and late-replicating regions (L) in blue. Annotated genes (Ref Seq genes) are represented below. The mid-late insertion site is indicated with a red arrow and dotted line. UCSC genome browser visualization of the mid-late insertion site of chromosome 1 (genomic positions: chr1:72,450,000–72,650,000 bp; 200 kb; galGal5). Tracks of nascent strands (NS) enrichments in the four S-phase fractions were represented separately (S1–S4) for the wt and the 2 × (βA-globin + β-actin) cell lines. NS-enriched and depleted regions for each fraction are represented in purple and blue, respectively. Single reads form SNS aligned and track of replication origins (Ori peaks) determined in (Massip et al, 2019) are reported in between. The three mid-late insertion sites 1, 2, and 3 are indicated with red arrows. Annotated genes and CpG Islands are shown below. Initiation zones (IZ) and termination zones (TZ) are reported. Download figure Download PowerPoint Two advanced replicons separated by 30 kb synergize to form a synthetic early-replicating region that interacts more with A compartments Our observation suggested that the large βA-globin + β-actin construct might cooperate with endogenous replication origins located about 30 kb upstream. To further test this hypothesis, we inserted two large autonomous replicons 30 kb apart at sites 1 and 3 (insertion site 1; chr1:72,565,520 bp and insertion site 3; chr1: 72,536,061 bp, galGal5, Fig 2B) and tested their impact on RT. The autonomous replicon at site 3 was similar to the one at site 1 except that the gene used conferred puromycin-N-acetyltransferase resistance (PuroR, insertion site 3, Fig 3A). We assessed the impact of RT changes in the middle of our modified region, by introducing a reporter construct in a central position (Insertion site 2; chr1: 72,548,590 bp, galGal5, Fig 2B) between the two autonomous replicons (1 + 2 + 3) or on the other chromosome (1 + 3) (Fig 3A). The reporter construct consisted of the erythroid-specific βA-globin origin/promoter linked to the green fluorescent protein (GFP) reporter gene and a 1.6 kb fragment of human chromosome 7 (h.K7; chr7:26,873,165–26,874,805 bp, hg38) containing no replication origin (Fig 3A). We found that the GFP reporter construct had no impact on RT when inserted alone at site 2 (−ΔL + ΔE = −5.1%, Fig EV2A). We then showed that the βA-globin + β-actin construct inserted at site 1 had similar effects on RT when associated with GFP reporter construct insertion at site 2 on the same or the other chromosome (compare Figs EV1B with EV2B and C and Hassan-Zadeh et al, 2012). These results confirm the neutral impact on RT of inserting the GFP reporter construct at site 2. We then investigated whether the insertion of a single autonomous replicon at site 3 was able to advance RT as efficiently as insertion at site 1. We confirmed that the βA-globin + β-actin construct induced a substantial shift in RT when inserted at this new genomic position (−ΔL + ΔE = +26.8% for clone 1 and –ΔL + ΔE = +22% for clone 2, Fig EV2D). This shift was similar to the one observed in βA-globin + β-actin clones after insertion at site 1 (Fig 3). As previously shown, the insertion of the GFP reporter construct at site 2 had no impact on RT when associated with a single autonomous replicon at site 3 on either the same or the other chromosome (Fig EV2, compare –ΔL + ΔE values between D and E). Insertion of two autonomous replicons at sites 1 and 3, with (1 + 2 + 3) or without (1 + 3) the central GFP reporter construct, resulted in larger shift in RT over the whole of the genomic region targeted (Fig EV3A and B, −ΔL + ΔE = +63.3 and +70.3% for 1 + 2 + 3 insertions and –ΔL + ΔE = +65.2, +84.2, +57.3%, for 1 + 3 insertions). A quantitative analysis revealed that the differences in RT between cell clones with one and two autonomous replicons were significant (P-value = 0.01554 between (i) and (ii) Fig 3A). Overall, these results strongly suggest that two similar independent advanced replicons, 30 kb apart, can synergize to control the extent of the RT shift. Figure 3. Two advanced replicons separated by 30 kb cooperate to form a synthetic early-replicated region that is more associated with A compartments Distribution of –ΔL + ΔE values for clonal cell lines containing one advanced replicon (βA-globin + β-actin) inserted at site 1 (i, N = 8) or 3 (iii, N = 3) or at both sites on the same chromosome with one GFP reporter construct composed of the GFP reporter gene under the control of the βA-globin promoter/origin linked to a 1.6 kb fragment of human chromosome 7 (h.K7) inserted at site 2, either on the same chromosome as 1 and 3 (1 + 2 + 3), or on the other chromosome (1 + 3) (ii, N = 5). Blue and black triangles represent reactive loxP sites and recombined inactive loxP sites, respectively. Black vertical bars represent insertion sites. Rectangle edges correspond to the 0.25 and 0.75 quartiles, the thick black lines represent the median, the white triangles represent the mean and the whiskers extend to the smallest and largest –ΔL + ΔE values. Statistical analysis was performed with Wilcoxon nonparametric two-tailed tests (**P < 0.01). Schematic representation of alleles 1 + 3 and 2. Blue and black triangles represent reactive loxP sites and recombined inactive loxP sites, respectively. Black vertical bars represent insertion sites. Interaction profiles of allele 1 + 3/2 with chr1 for combined Hi-C libraries: distance-corrected interactions, i.e., observed/expected (OE), binned at 500 kb with corresponding compartment (EV1) tracks. Red arrows represent the loci that show preferred interactions with allele 1 + 3 and that coincide with A compartment. OE interactions of allele 1 + 3 (orange rectangles) and allele 2 (blue rectangles) averaged over A and B compartments for p and q arms at 100 kb bin size. A random compartment status permutation test was made, and then, we calculated the one-sided P-value for the observed value of "difference allele 1 + 3/2 A − B". Values corresponding to OE interactions of allele 1 + 3 and allele 2 averaged over shuffled A (red circles, N = 1,000) and B compartments (blue circles, N = 1,000) for p and q arms. ns, not significant; **P < 0.01; ***P < 0.001. Scatterplot of density of interactions between allele 1 + 3 and allele 2 with chromosomes larger than 5,000,000 bp. Download figure Download PowerPoint Click here to expand this figure. Figure EV2. One advanced replicon inserted at site 1 or at site 3 directs a shift to earlier replication independently of the presence of a GFP reporter construct inserted at site 2RT profiles of each chromosomal allele are determined after targeted transgene integration using the allele-specific analysis of RT method by real-time PCR quantification described in Appendix Fig S1. Differences in –ΔL + ΔE values calculated at the target site following transgene integration are indicated. A. Analysis of one clonal cell line containing one GFP reporter construct composed of the GFP reporter gene under the control of the βA-globin promoter (βA pro) and linked to a 1.6 kb fragment of human chromosome 7 (h.K7) inserted at site 2. B, C. Analysis of clonal cell lines containing one βA-globin + β-actin construct described in Fig 1 inserted at site 1 and one GFP reporter construct inserted at site 2 on the same chromosome (B) or on the other chromosome (C). D, E. Analysis of clonal cell lines containing one βA-globin+β-actin construct inserted at site 3 and one GFP reporter construct inserted at site 2 on the same chromosome (D) or on the other chromosome (E). Data information: Blue and black triangles represent reactive loxP sites and recombined inactive loxP sites, respectively. Black vertical bars represent insertion sites. Error bars correspond to the standard deviation for qPCR duplicates. Download figure Download PowerPoint Click here to expand this figure. Figure EV3. Two βA-globin + β-actin constructs as well as two β-actin constructs inserted at sites 1 and 3 form an early-replicated domain A–C. RT profiles of each chromosomal allele are determined after targeted transgene integration using the allele-specific analysis of RT method by real-time PCR quantification described in Appendix Fig S1. Differences in −ΔL + ΔE values calculated at the target site following transgene integration are indicated. Error bars correspond to the standard deviation for qPCR duplicates. (A, B) Analysis of two 1 + 2 + 3 (A) and three 1 + 3 (B) clonal cell lines described in Fig 3A. (C) Analysis of five clonal cell lines containing two β-actin constructs inserted at sites 1 and 3 on the same chromosome described in Fig 3B. Blue and black triangles represent reactive loxP sites and recombined inactive loxP sites, respectively. Black vertical bars represent insertion sites. Error bars correspond to the standard deviation for qPCR duplicates. Download figure Download PowerPoint Genome-scale studies have shown that RT is strongly correlated with A/B compartments in human cells, suggesting spatial proximity of regions of similar RT within the nucleus (Ryba et al, 2010). We tested whether the large change in RT of the modified chromosome is accompanied by a change in the way the inserted origins interact with A and B compartments. This system allows us to assess how the early-replicating allele (allele 1 + 3) interacts with A and B compartments compared to the allele with the endogenous RT (allele 2) (Fig 3B). We conducted Hi-C to assess compartment status genome wide and to measure long-range chromatin interactions for allele 1 + 3 and allele 2 at the same time. We first examined interactions between inserted sequences at alleles 1 + 3 and 2 and chromosome 1 (chr1). The profile of interactions of allele 1 + 3 with chr1 and allele 2 with chr1 demonstrates alternating patterns in the p-arm of chr1, whereas in the q-arm the two profiles show more overlap (Fig 3C, and Appendix Fig S4A and D). Most of the loci that show preferred interactions with allele 1 + 3 coincide with loci located within A compartment domains (Fig 3C, and Appendix Fig S4A and D). To further quantify the observed preference, we calculated average distance-corrected interactions between allele 1 + 3/allele 2 and regions of chr1 according to their compartment status (Fig 3D and Appendix Fig S4B and E). Averaged interactions confirm that allele 1 + 3 interacts more with A compartment than with B compartment both for p and q arms of chr1, unlike allele 2 which shows smaller preference for A compartment in the q-arm, and no association with either A or B compartment in the p-arm. Our observations regarding allele 2 are in line with the WT compartment status of the mid-late insertion site, as shown by an eigenvector 1 (EV1) value near zero (see also Fig 7A below). We assessed the significance of the interaction preferences by re-calculating the average interactions of allele 1 + 3/allele 2 and regions of chr1 with randomly shuffled compartment status (EV1) (Fig 3D, Appendix Fig S4B and E) (see Materials and Methods for details). We further looked if allele 1 + 3 and allele 2 demonstrated any preferential interactions with other chromosomes in the genome, by comparing their density of interactions (averaged interactions normalized by chromosome length, see Materials and Methods for details). The number of interactions with chr9 is disproportionately higher for allele 1 + 3 than for allele 2 compared to other chromosomes (Fig 3E, Appendix Fig S4C and F) showing that allele 1 + 3 interacts more with chr9. Our results show that allele 1 + 3 interacts more with specific A compartments located on chr1 and globally more with chr9. These specific interactions coincide with several strong replication origins and high gene density for chr9; however, precise mechanistic understanding will require further investigation. The constitutive β-actin promoter/origin is required for the synergic action of the two replicons separated by 30 kb We have already shown that the large autonomous replicon used in this study is composed of two strong origins associated either with an active (β-actin) or an inactive (βA-globin) promoter (Hassan-Zadeh et al, 2012 and Appendix Fig S2 and Fig EV4A). MNase (micrococcal nuclease) titration analyses confirmed a differential chromosomal accessibility of the two minimal modules inserted separately at site 1 (Fig EV4B and C, and Appendix Fig S5). However, both minimal modules individually can induce a significant RT shift (Fig 1). To understand in more details the mechanisms involved in the synergy observed in the large βA-globin + β-actin construct, we tested for each minimal construct the effect of a double insertion at sites 1 and 3 on the same chromosome. We observed that similarly to the large autonomous replicon, the β-actin promoter induced a stronger shift in RT when inserted at 1 + 3 positions (Figs 4A and EV3C). By contrast, two inactive βA-globin promoters did not produce any advanced RT when inserted at 1 + 3 sites (Figs 4B and EV5). We observed for a subset of clones even a decrease in the intensity of the RT after the second insertion, a reproducible obse
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