Dissection of the ß-globin replicator using a plasmid-stability assay

<p><b>(1)</b>. The ß-globin locus and the positions of two reported replicators, Rep-P and Rep-I [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077350#B20" target="_blank">20</a>], are drawn in A. We inserted wild type Rep-P as well as fragments bearing four deletions or an AG1 point mutation (orange bars in C) into the <i>Not</i>I site of the vector plasmid pTV-MCS (B). In panel C, RepP-1 and RepP-2 regions are indicated as gray rectangles. The plasmids were transfected into COLO 320DM cells, and polyclonal transfectants were selected in blasticidine for about 1 month. Metaphase spreads from these cells were analyzed by FISH using a pTV-MCS plasmid-derived probe. The frequencies of cells bearing HSRs of plasmid repeats were scored in both metaphase and the interphase cells, and the data are plotted in panel E.</p

Requirement of MAR for HSR generation.

<p>A. To examine the effect of the AR1 MAR sequence of the vector plasmid, we removed it and constructed three new plasmids, pTV-Rep-P∆AR1, pTV-MCS∆AR1, and pG5∆AR1. We transfected plasmids with or without the AR1 MAR into COLO 320DM cells, selected transfectants, and analyzed them by FISH. Frequency of HSR is plotted in the graph. B–D. The IR sequences from ß-globin Rep-P (B), c-myc (C), and <i>DHFR</i> (D) were analyzed using MAR-Wiz program, and the results (blue graph area) are shown. In each graph, regions with HSR-generation activity revealed by our previous study (<i>DHFR</i> and c-myc; [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077350#B18" target="_blank">18</a>]) and this study (ß-globin) are shown as gray rectangle(s). AT-rich and AG-rich tracts are also indicated.</p

Amplification of the IR/MAR plasmid in transfected cells.

<p>After transfection, a conventional plasmid without an IR/MAR is integrated into the chromosome arm at low copy number in a stable transfectant. Such a sequence is barely detectable by FISH (noted as “0”). By contrast, an IR/MAR-bearing plasmid is amplified at an extrachromosomal location as a large circular molecule of tandem plasmid repeats (step 1; [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077350#B2" target="_blank">2</a>]). If such a molecule suffers from a DNA break (step 2a), it is eliminated from cells (step 2c; [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077350#B5" target="_blank">5</a>]) or integrated to a chromosome arm, generating a small HSR that is visible by FISH as paired dots or a line (step 2b). There, the plasmid repeat is elongated to generate a large HSR (step 3; [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077350#B13" target="_blank">13</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077350#B14" target="_blank">14</a>]). These three steps occur spontaneously in human COLO 320DM cells, whereas step 3 does not occur spontaneously in CHO DG44 cells [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077350#B11" target="_blank">11</a>].</p

Dissection of ß-globin replicator using a plasmid stability assay

<p><b>(3)</b>. The G5 fragment, which exhibited the highest HSR-generation activity in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077350#pone-0077350-g003" target="_blank">Figure 3</a>, was further dissected. The positions of AT-rich, AG-rich, palindrome, topo II binding-site, and CAAT-box regions are indicated. “MAR?” indicates the position of a MAR-like element predicted by the MAR-Wiz program. The regions indicated by orange bars in panel A were subjected to the plasmid-stability assay as in Figure 2. In this case, we examined HSR generation in both COLO 320DM cells and CHO-DG44 cells, in which gene amplification progresses differently, as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077350#pone-0077350-g001" target="_blank">Figure 1</a>. The results from both cell lines are shown in panels B and C. The data suggest that the gray rectangular regions drawn in the left panel have HSR-generation activity.</p

Detection of the amplified plasmid sequence by FISH.

<p>Representative images obtained in this study are shown. Plasmid pTV-Rep-P bearing full-length Rep-P sequence was transfected to COLO 320DM cells (A to C) or CHO-DG44 cells (D). Metaphase chromosome spreads were prepared from the stable transfectants, and the plasmid sequence was detected by FISH. Long HSR appeared in the metaphase cells (A and B) or a interphase cell (A). The DMs with plasmid sequence appear in the metaphase cells in panel B and C as well as an interphase cell in panel C. In CHO-DG44 cells, the same plasmid did not generate long HSR, instead it remained as the short HSRs that were arrowed (D), as explained in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077350#pone-0077350-g001" target="_blank">Figure 1</a>.</p

Dissection of ß-globin replicator using a plasmid-stability assay (2).

<p>The regions indicated by the orange bars in the left panel were subjected to the plasmid-stability assay in COLO 320DM cells, as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077350#pone-0077350-g002" target="_blank">Figure 2</a>. The results are shown in the right panel. The data suggest that the gray rectangular region drawn in the left panel has HSR-generation activity.</p

A model from this study.

<p>The replication initiation at the chromosomal context, the gene amplification from the endogenous chromosomal sequence and the gene amplification from the plasmid sequence require the same MAR-like element and the same AT-rich element, whereas the former one and the latter two events require similar but different AG-rich elements. The MAR elements is also required for these three events, and it is included in the plasmid (AR1) or it may be located at the chromosomal flanking region.</p

Epigenetic Repeat-Induced Gene Silencing in the Chromosomal and Extrachromosomal Contexts in Human Cells

<div><p>A plasmid bearing both a replication initiation region and a matrix attachment region is spontaneously amplified in transfected mammalian cells and generates plasmid repeats in the extrachromosomal double minutes (DMs) or the chromosomal homogeneously staining region (HSR). Generally, the repeat sequences are subject to repeat-induced gene silencing, the mechanism of which remains to be elucidated. Previous research showed that gene expression from the same plasmid repeat was higher from repeats located at DMs than at the HSR, which may reflect the extrachromosomal environment of the DMs. In the current study, plasmid repeats in both DMs and HSR were associated with repressive histone modifications (H3K9me3, H3K9me2), and the levels of repressive chromatin markers were higher in HSR than in DMs. Inactive chromatin is known to spread to neighboring regions in chromosome arm. Here, we found that such spreading also occurs in extrachromosomal DMs. Higher levels of active histone modifications (H3K9Ac, H3K4me3, and H3K79me2) were detected at plasmid repeats in DMs than in HSR. The level of DNA CpG methylation was generally low in both DMs and HSR; however, there were some hypermethylated copies within the population of repeated sequences, and the frequency of such copies was higher in DMs than in HSR. Together, these data suggest a “DNA methylation-core and chromatin-spread” model for repeat-induced gene silencing. The unique histone modifications at the extrachromosomal context are discussed with regard to the model.</p></div

A “DNA methylation-core and chromatin-spread” model of RIGS.

<p>Repeated copies of plasmid sequences are shown with gray arrows. Restricted copies adjacent to the genomic sequence (bold navy line) at either end of the full repeat sequence (A) or internal repeat copies (B) are methylated (m) at CpG dinucleotides. Histone (circles) is then modified to the repressed state (red) at the DNA methylation site, and chromatin repression spreads in both directions along the repeats.</p

Repressive histone modifications at plasmid repeats in DMs and HSR.

<p>(A, B) Metaphase chromosome spreads from COLO 320 clone 12 (DM) and clone 22 (HSR) were hybridized with a probe prepared from plasmid pSFVdhfr. The hybridized probe was detected as green fluorescence, and DNA counterstained by propidium iodide (PI) is shown in red. The hybridized plasmid probe appears at the multiple DMs (A; arrowheads) and at the chromosomal HSR (B; arrow). (C) The structure of the pSFVdhfr plasmid is depicted, with the positions of regions used for real-time PCR analysis shown as red bars. (D, E) Chromatin from clone 12 (DM; green bars) and clone 22 (HSR; orange bars) was precipitated using antibody specific to H3K9me2 or H3K9me3. Sequences along the plasmid were quantitated in immunoprecipitated DNA using real-time PCR. The housekeeping gene <i>GAPDH</i> was used as a control. Results (mean +/- SD; n = 3) are shown as a percentage of input chromatin. The statistical significance of the difference between clone 12 (DM) and clone 22 (HSR) is analyzed by student’s t-test. ***; p<0.001, **; p<0.01, *; p<0.1.</p