15 research outputs found
The replication timing shift affects only the replicon located inside the transgene.
<p>Replication timing profiles surrounding DT40 chromosomal alleles following transgene integration into both alleles. (A) Schematic representation of the transgenic locus containing the <i>IL-2R</i> transgene flanked by two copies of the FIV USF binding site linked to the <i>β-actin</i> blasticidin resistance transgene on one allele and puromycin resistance transgene on the other. Primer pairs used are located at the top of the figure, and their positions relative to the site of insertion are indicated. The global replication forks' directions deduced from panel B are shown. (B) Analysis of replication timing at the integration site and over a ∼300 kb region surrounding the transgene integration site in two clonal cell lines. The differences in late or early replication (ΔL and ΔE) at flanking regions compared with the integration site (With) are shown.</p
A strong origin linked to a transcriptionally active transgene has a minimal effect on replication timing.
<p>(A and B) Replication timing profiles of chromosomal alleles following targeted integration of transgenes into a late replicating locus of DT40 cells described in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001277#pbio-1001277-g002" target="_blank">Figure 2</a>. (A) Analysis of a transgenic line containing a blasticidin resistance gene (grey rectangle) under the control of the <i>β-actin</i> promoter (white rectangle) targeted into the site of integration (red vertical bar). The modified (Mut) and wild type (Wt) alleles are represented together with the location of amplicons used for timing analysis. (B) Analysis of a transgenic line containing the same transgene linked to the <i>β<sup>A</sup>-globin</i> promoter/replicator. Only the modified allele is shown with the position of amplicons used in QPCR analyses. (A and B) Cells were BrdU pulse labeled, sorted into four S-phase fractions and nascent strands quantified by real-time PCR (S1 in red, S2 in yellow, S3 in green, and S4 in blue). Three different PCR primer sets were used to measure the replication timing at the site of integration on either the transgenic allele (With), the endogenous allele that lacks transgene integration (Without), or both alleles (Both). The endogenous <i>β-globin</i> locus was analyzed as an early replicated control. The differences in late or early replication (ΔL and ΔE) at the target site following transgene integration are shown. (C) Quantification of SNS enrichment over the <i>β<sup>A</sup>-globin</i>/<i>β-actin</i> transgene. Three transgene-specific primer sets were used (1–3, indicated in B); primer set 4 is located 5 kb from the integration site. SNS enrichments are relative to those of the endogenous <i>ρ-globin</i> positive control origin (P). A region located 21 kb upstream of the endogenous HS4 insulator that is devoid of origin activity was analyzed as a negative control (N). (D) ChIP analysis of H3K9acK14ac and H4 density along the <i>β<sup>A</sup>-globin/β-actin</i> transgene. Histone H3 acetylation levels are relative the <i>β-actin</i> promoter (region 2) and H4 density is relative to distal region 4.</p
Analysis of replication timing changes after the insertion of <i>cis</i>-regulatory elements.
<p>(A) Diagram showing the modified allele after insertion of a transgene by homologous recombination and below the wild type allele. Primer pairs used for replication timing analyses are shown and can detect replication timing of the modified allele (With), the wild type allele (Without), or both alleles (Both). (B) After insertion by homologous recombination, selected clones are pulse labeled with BrdU and cell sorted into four fractions from early to late S-phase. BrdU labeled nascent strand DNA is immunoprecipitated and quantified by real-time PCR. The nascent strands produced at the site of integration on the allele containing the transgene (With), the wild type allele (Without), or both alleles (Both) were quantified. Analyses of both alleles should display an average picture of the profiles obtained on each allele, thus providing a validation of the PCR quantification. For each clone, we calculate differences in the timing of replication between the unmodified (Without) and targeted (With) alleles, where ΔL and ΔE describe the differences in late and early S-phase, respectively.</p
An origin flanked by HS4 insulator elements induces an important early replication shift.
<p>Replication timing profiles of DT40 chromosomal alleles following targeted integration of insulator containing transgenes. (A) Analysis of a transgenic line containing an <i>IL-2R</i> transgene flanked by two copies of the 275 bp HS4 insulator linked to the <i>β-actin</i> transgene. The <i>IL-2R</i> cDNA (grey rectangle) and the SV40 polyA signal were linked to the <i>β<sup>A</sup>-globin</i> promoter (white rectangle) and the <i>β/ε</i> enhancer (grey oval). Flow cytometric analysis of <i>IL-2R</i> expression is shown below for the two transgenic lines (clones 1 and 2). The percentage of cells with fluorescence levels higher than control cells (R2) are indicated. (B) Analysis of a transgenic line containing two copies of the 275 bp HS4 insulator upstream of the <i>β<sup>A</sup>-globin</i>/<i>β-actin</i> transgene described in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001277#pbio-1001277-g004" target="_blank">Figure 4B</a>.</p
Mutation of USF elements abolishes replication timing control.
<p>Replication timing profiles of DT40 chromosomal alleles following targeted integration of insulator containing transgenes. (A) Analysis of two transgenic lines containing the <i>IL-2R</i> transgene flanked by two copies of FIV as described in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001277#pbio-1001277-g006" target="_blank">Figure 6B</a>, except that the FIV sites carry E-box mutations known to disrupt USF binding. The sequences of wild type FIV (FIV WT) and mutated FIV (FIV mut) are shown; bases footprinted by USF binding are underlined and the degenerate E-box motif is boxed. (B) Analysis of two transgenic lines containing the <i>IL-2R</i> transgene flanked on one side by four copies of FIV. The differences in late or early replication (ΔL and ΔE) at the target site following transgene integration are shown.</p
Role of <i>cis</i>-regulatory element in controlling replication timing.
<p>(A) In wt DT40 cells, the selected site of insertion is located in a mid-late replicating region devoid of strong replication origin. One hypothesis is that this locus is naturally located more toward the nuclear envelope (NE). (B) Insertion of a strong replicator linked to two copies of the HS4 insulator or four binding sites for USF (FIV) does not induce a replication timing shift. The replication timing of the transgenic replicator follows that of its chromosomal environment. (C) Flanking the strong replicator with two copies of FIV induces a significant replication timing shift and recruitment of histone modifying enzymes. This could be accompanied by the formation of a small loop that relocates the replicator more toward the interior of the nucleus. The increased accessibility to S-phase kinases could be either due to local decondensation of the chromatin structure and/or to the nuclear repositioning of the modified chromosomal region. (D) The addition of a strong promoter nearby increases the replication timing shift and leads to the formation of an independent mid-early replicon.</p
Mapping of replication origins around the selected site of insertion in DT40 cells.
<p>(A) UCSC genome browser visualization (May 2006 build) of unique short nascent strand sequence tags aligned to a 300 kb mid-late replicated region of Chromosome 1. Four peaks were identified among which two were validated by qPCR. Tracks showing GC percentage, annotated genes, and CpG islands are below. (B) Precise quantification of replication timing profiles near the site of insertion and along flanking regions. The position 0 corresponds to the site of insertion. The early control is located inside the <i>β-globin</i> locus. Cells were BrdU pulse labeled, sorted into four S-phase fractions, and nascent strands were quantified by real-time PCR (S1 in red, S2 in yellow, S3 in green, and S4 in blue).</p
The USF binding site from the HS4 insulator is sufficient to direct early replication.
<p>(A and B) Replication timing profiles of chromosomal alleles following targeted transgene integration. The differences in late or early replication (ΔL and ΔE) at the target site following transgene integration are shown. (A) Analyses of two clonal cell lines containing the <i>IL-2R</i> transgene flanked by two copies of the FIV USF binding site linked to the <i>β-actin</i> transgene. (B) Analyses of two clonal cell lines containing the same construct after recombinase-mediated excision of the <i>β-actin</i> transgene. Flow cytometric analysis of <i>IL-2R</i> expression is shown below for the two transgenic lines (clones 1 and 2).</p
Integration of a transgenic <i>β<sup>A</sup></i> replicator flanked by insulators induces early replication timing at two other chromosomal loci.
<p>Replication timing profiles following transgene integration into erythroid 6C2 cells. (A) Schematic representation of the <i>IL-2R</i> transgene drawn to scale. The construct is identical to the one described in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001277#pbio-1001277-g005" target="_blank">Figure 5A</a>, except that HS4 carries deletions of the CTCF (ΔFII) or VEZF1 (ΔFIII) binding sites. This construct is linked to the hygromicin antibiotic resistance gene under the control of the HSV-thymidine kinase promoter. (B) The mapped sites of integration (red dots) for the transgenes flanked by ΔFII or ΔFIII insulators are shown. UCSC genome browser views (May 2006 build) of 100 kb regions with the chromosomal position, GC percentage, annotated genes, and CpG islands shown. (C) Replication timing profiles of chromosomal alleles at the mapped sites of transgene integration. Late and early replicated controls correspond to the endogenous <i>amylase</i> α 2A and <i>β-globin</i> loci, respectively. The differences in late or early replication (ΔL and ΔE) at flanking regions compared with the integration site are shown.</p
Analysis of induction in cadmium chloride-treated cells transfected with TFBS-UR plasmids.
<p>HEK293 cells transfected with a plasmid pool, that included the plasmids listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050521#pone.0050521.s003" target="_blank">Table S2</a> and pRL-SV40 and were subsequently treated with cadmium. (A) Microarray-based detection of TF derived activation of UR expression. (B) qPCR-based detection of TF-derived activation of UR expression. Values are presented as log2 treatments of the fold induction of the TFBS-directed UR expression after treatment with the inducer of interest. The grey bar represents treatment-independent changes in the system. TFBS marked with * represent treatment-dependent effects on the TF library. Numerical data is presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050521#pone.0050521.s004" target="_blank">Table S3</a>. A statistical analysis of the qPCR assay data is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050521#pone-0050521-g003" target="_blank">Figure 3</a>.</p