The matrix attachment region in the Chinese hamster dihydrofolate reductase origin of replication may be required for local chromatid separation

Abstract

Centered in the Chinese hamster dihydrofolate reductase origin of replication is a prominent nuclear matrix attachment region (MAR). Indirect lines of evidence suggested that this MAR might be required for origin activation in early S phase. To test this possibility, we have deleted the MAR from a Chinese hamster ovary variant harboring a single copy of the dihydrofolate reductase locus. However, 2D gel replicon mapping shows that removal of the MAR has no significant effect either on the frequency or timing of initiation in this locus. Rather, fluorescence in situ hybridization studies on cells swollen under either neutral or alkaline conditions show that deletion of the MAR interferes with local separation of daughter chromatids. This surprising result provides direct genetic evidence that at least a subset of MARs performs an important biological function, possibly related to chromatid cohesion and separation.

Figure 1

The DHFR locus and the ROKO approach. (A) The 120-kb region encompassing the DHFR and 2BE2121 (53, 54) genes in the WT hemizygote, UA21, showing the position of three preferred replication initiation sites (ori-β, ori-β′, and ori-γ; refs. , , and 21) and the intergenic MAR (black square; ref. 22). Positions of relevant hybridization probes and an EcoRI map of the region are shown above and below the map, respectively. (B) The DHFR-deficient variant, DR-8A7, with the diagnostic deletion junction EcoRI fragment shown below. (C) The BAC donor used to restore the DHFR gene and knock out the downstream sequence of interest, showing the XhoI fragment into which each deletion was engineered (in the example shown, a 7-kb region encompassing the MAR; see text). (D and E) Recombination near sites a and b (C) leads to a restored WT derivative (D), whereas recombination near sites a and c leads to restoration of the gene and simultaneous deletion of the downstream target (E). (F) Detailed maps showing positions of deletions (see text) and sizes of resulting diagnostic EcoRI fragments. X, XhoI; R, EcoRI; P, PstI; B, BstEII. (G) Southern analysis of EcoRI digests of each cell line, hybridized with a mixture of probes 100, 35, and 71 (see A and F). Size markers are a mixture of a 1-kb ladder and high molecular weight standard (Invitrogen).

Figure 2

Defining the minimal MAR-binding sequence. (A) Matrix-halo structures were prepared from CHO and CHOC400 cells as described in Materials and Methods, and loop DNA was removed with a combination of PvuII and HinfI. Equal weights of matrix and loop DNA fractions were separated on an agarose gel and blotted to a nylon membrane. The transfer was then hybridized with a combination of 450- and 379-bp PvuII/HinfI subfragments. The marker is an end-labeled 123-bp ladder (BRL). (B) Matrix/halo structures were prepared from CHOC400 cells and all attached DNA was completely removed with DNaseI. Isolated DNA-free matrices were then incubated with end-labeled fragments from the 3.4-kb PvuII MAR-containing fragment in the presence and absence of cold competitor DNA. The bound radioactive DNA was analyzed by Southern blotting. (C) HinfI subfragments of the 3.4-kb PvuII MAR-binding fragment (22), which were tested for association with the matrix in the in vivo assay illustrated in A (additional probings not shown). (D) Primary sequence of the 378-bp PvuII/HinfI MAR-binding fragment. Relevant restriction enzyme sites are shown above the sequence, and the 78-bp AT-rich deletion in the AT-MARKO cell line is shaded. At position 144, a C was inserted in the donor BAC that gave rise to the AT-MARKO cell line to create an SpeI site for diagnosis of recombinants. The sequence shown is from PCR products obtained from DR8A-7, as well as a subclone from a CHOC400 cosmid library. Sequence was also obtained from the AT-MARKO cell line to confirm deletion of the shaded region.

Figure 3

The MARKO deletion variants appear to be very late replicating by the criterion of the FISH-based replication timing assay. (A) Principle of the FISH-based replication timing assay (37). Cells are swollen spread on microscope slides (34, 40), and hybridized with a digoxygenin-labeled cosmid specific for the intergenic region (KD504) and with a biotin-labeled cosmid specific for the control, early-replicating, rhodopsin origin. The respective signals are detected with fluorescein-labeled antidigoxygenin or Texas red-labeled antiavidin, as outlined in Materials and Methods (40). The number of dots of each color is recorded for at least 100 interphase nuclei from each cell line. Boxes B–F represent cells in different stages of the S-period or G₂. Box G defines a replication index, which is calculated by dividing the number of cells that have replicated the DHFR locus (as in boxes D–F) by the number that has doubled the rhodopsin control locus (as in boxes B, C, E, and F). (B) Cells were sampled from synchronized cultures at the indicated times and swollen under neutral conditions. Resulting replication indices for each cell line are plotted as a function of time after release from the G₁/S mimosine block. (C) Synchronized AT-MARKO and DR8-A7 cell lines were harvested at the indicated times, divided into two, and prepared for the FISH assay under either neutral or alkaline conditions. After hybridization with the mixed DHFR/rhodopsin probe, replication indices were determined. (D) Unsynchronized cultures of each of the indicated cell lines were divided into two and prepared for FISH under either neutral or alkaline swelling conditions. Replication indices were determined as described above.

Figure 4

2D gel analysis of the MARKO variants suggests that deletion of the MAR has no effect on either the efficiency or timing of initiation in the DHFR locus. Replication intermediates were purified from aliquots of the same synchronized cell populations analyzed by the FISH-based assay in Fig. 3B. After digestion with EcoRI, intermediates were separated on a neutral/neutral 2D gel, transferred to Hybond N⁺, and hybridized with a combination of probes 12 and 38, which are specific for a fragment containing ori-β. Each cell line was additionally analyzed with a probe specific for the early-replicating rhodopsin standard (shown here for the DR8-A7 variant only). The principle of the method is outlined in A Right. Replication intermediates are separated in the first dimension according to molecular mass, which for any fragment will vary from 1n (unreplicated) to just less than 2n. The first dimension lane is excised, turned through 90°, and separated in the second dimension according to both mass and shape (32). Linear nonreplicating fragments trace a diagonal (curve a). If a fragment is replicated passively by a fork originating from a site outside of the fragment, it will display a single fork arc (curve b). However, if the fragment contains a centered initiation site, it will display an arching bubble arc that extends from the 1n to the 2n positions (curve c).