Co-localization of centromere activity, proteins and topoisomerase II within a subdomain of the major human X alpha-satellite array

Abstract

Dissection of human centromeres is difficult because of the lack of landmarks within highly repeated DNA. We have systematically manipulated a single human X centromere generating a large series of deletion derivatives, which have been examined at four levels: linear DNA structure; the distribution of constitutive centromere proteins; topoisomerase IIalpha cleavage activity; and mitotic stability. We have determined that the human X major alpha-satellite locus, DXZ1, is asymmetrically organized with an active subdomain anchored approximately 150 kb in from the Xp-edge. We demonstrate a major site of topoisomerase II cleavage within this domain that can shift if juxtaposed with a telomere, suggesting that this enzyme recognizes an epigenetic determinant within the DXZ1 chromatin. The observation that the only part of the DXZ1 locus shared by all deletion derivatives is a highly restricted region of <50 kb, which coincides with the topo isomerase II cleavage site, together with the high levels of cleavage detected, identify topoisomerase II as a major player in centromere biology.

Fig. 1. Immuno-FISH showing CENP-A and CENP-C relative to the human X α-satellite array DXZ1. (A) Examples of indirect immunofluorescence to detect CENP-A (green) in combination with FISH to detect DXZ1 (red) on mechanically stretched human X chromosomes present in various cell lines. DNA was counterstained using DAPI (blue). The images show partial chromosome complements and in some instances encompass chromosomes from more than one nucleus and/or show sister chromatids with varying degrees of separation. (B) A tabulated summary of the observed distribution. The arm distribution was based on chromosome morphology. Where displacement was apparent but the morphology too distorted for assignment the signals were counted as ‘displaced, but unknown’. Chi-square analysis of this data indicates a highly significant p-arm bias in the CENP distribution (1 df, probability of a random distribution = <0.0001).

Fig. 2. Strategy for dissecting the human X centromere. (A) The DXZ1 array (grey box) on the IKNFA3 minichromosome appears contiguous with that on the X from which it was derived except for the removal of 700 kb from the Xq-side (Farr et al., 1992; Bayne et al., 1994; Mills et al., 1999). The Xp pericentromeric region is rich in monomeric α-satellite DNA lacking higher order repeat organization (thick black line) (Schueler et al., 2001) and has embedded in it a 40 kb γ-satellite array (white box) (Lee et al., 2000). Experimentally seeded telomeres are represented as open arrows with the selectable marker indicated. To generate smaller derivatives a telomere-seeding construct was designed to target the DXZ1 array (pRSTBloxα). Two versions, differing only in the orientation of the 2 kb block of DXZ1 relative to the ds(TTAGGG)_n, were generated (α7 and α9) and independently transfected into a DT40 cell line carrying the IKNFA3 chromosome. Blasticidin-resistant transfectants were screened for new minichromosomes, either by PFGE or for loss of terminal markers. (B) The IKNFA3 minichromosome and examples of derivatives resolved as intact HMW DNA by PFGE, Southern blotted and detected using DXZ1, RS416 (specific for the targeting construct) and γ-satellite DNA (gamma-X). Co-localization of new DXZ1 hybridizing linear DNA molecules with the introduced construct is consistent with targeted breakage events (indicated by an arrowhead for cell lines α7: 6, 23, 28, 127, 135, 182, 238, 266, 287, 347, 367, 376, 21G11, 21F4 and α9 8G6). The minichromosome in cell line α7 148 appears structurally unstable. Cell line α7 21G11 was confirmed as having two minichromosomes per cell. Cell line α9 11H12 contains a ∼1.8 Mb DXZ1 molecule (indicated by an asterisk), but has the construct randomly integrated into the DT40 genome. The other cell lines shown are not targeted.

Fig. 3. Molecular characterization of minichromosomes by restriction enzyme analysis. (A) Examples of SpeI PFGE analysis. DXZ1-hybridizing fragments that cohybridize with the RS416 DNA probe are indicated. PFGE conditions were 20–60 s pulse at 200 V for 24 h. Sizes of the DXZ1-hybridizing SpeI fragments present in the IKNFA3 DXZ1 array are indicated. (B) The linear DNA structure of IKNFA3 and various DXZ1 truncation derivatives. The structures of all new minichromosomes consistent with a straightforward targeted breakage event is presented. Each derivative is shown as a black line which directly reflects the region of the starting IKNFA3 molecule that is retained. In all cases there has been loss of varying amounts of the DXZ1 array together with replacement of the Xq(hyg^r)-tagged telomere with a bs^r-tagged telomere. Mitotic loss rates per generation (R) and the range in copy number per DT40 cell for various minichromosomes are given.

Fig. 4. Xp DNA sequences are dispensible for mitotic chromosome function. (A) Schematic of the two-step targeting strategy used to remove the remaining 650 kb of Xp DNA from α9 2F6 (2.4 Mb). A de novo telomere tagged by a CMV HyTK fusion gene was targeted into the region immediately distal to the γ-satellite array using pHyNOTγ (5/246 transfectants). One correctly targeted cell line 2F6γ16–119, was retargeted using pRSTH3/FRT/Xα9. G418- and ganciclovir-resistant clones (170) were PCR screened for loss of the HyTK locus and γ-satellite DNA. (B) Examples of Xp-truncation derivatives of α9 2F6 resolved as intact HMW DNA by PFGE and Southern blotted for DXZ1.

Fig. 5. Molecular characterization of minichromosomes in the Xp-truncation series. (A) Examples of SpeI PFGE analysis, Southern blotting and hybridization with DXZ1 and RS416. Novel DXZ1-hybridizing fragments that co-hybridize with the RS416 DNA probe (present on the DXZ1-targeting constructs) are indicated. The 180 kb DXZ1 fragment that co-hybridizes with RS416 and is present in all clones originates from the construct associated with the Xq(bs^r)-tagged telomere of α9 2F6 (see also Figure 3A). Sizes of the DXZ1-hybridizing SpeI fragments present in α9 2F6/2F6γ16–119 are indicated. In clone 6B8 a rearrangement appears to have accompanied the targeted breakage event resulting in a novel SpeI fragment slightly greater than the starting 670 kb. PFGE conditions were 30–70 s pulse at 200 V for 24 h. (B) The molecular structure of the ganciclovir- resistant 2F6γ16–119 derivatives consistent with a straightforward targeted breakage event is presented (n = 25). Mitotic loss rates per generation (R) and minichromosome copy number per DT40 cell for selected minichromosomes are shown.

Fig. 6. Localization of CENP-H and -C to the Xp-side of the DXZ1 array using immuno-FISH. (A and A′) Chromatin fibres from 1aH2, a DT40 line carrying the IKNFA3 minichromosome and producing a CENPH–GFP fusion protein, were generated. The centromerically located fusion protein was detected (on all chromosomes in the cell) using anti-GFP (green). This was followed by dual FISH for the human X centromere-based minichromosome using DXZ1 (red) and BAC 342O19 (which spans the γ-satellite array DNA) (white). (B and B′) Chromatin fibres from the DT40 cell line IKNFA3. CENP-C has been detected using antibodies to chicken CENP-C (green) followed by dual FISH for DXZ1 (red) and BAC342O19 (white).

Fig. 7. Mapping topo II activity within the DXZ1 array. (A) DT40 cells carrying the α9 2F6 minichromosome were incubated with etoposide in vivo for 30 min and embedded in agarose. HMW DNA was cut with KpnI, PvuII or BglI, resolved by PFGE and Southern blotted with DXZ1. Etoposide-specific DXZ1-hybridizing fragments are indicated. PFGE conditions: 20–60 s pulse, 200 V, 24 h. (B) Uncut HMW DNA from various 50 µM etoposide-treated (+) or DMSO only (–) minichromosome-carrying DT40 lines from the Xq-truncation series was resolved by PFGE. The Southern blot filter was hybridized sequentially for X γ-satellite (gamma-X) and DXZ1. Etoposide-specific hybridizing fragments are indicated. (The reason for the slight mobility shift of the intact minichromosomes following etoposide exposure is unknown, but may be due to an effect of the apoptotic genome-wide chromosome fragmentation on HMW DNA migration conditions.) (C) Schematic showing localization of the topo II cleavage site on IKNFA3 and various minichromosomes from the Xq-truncation series. Also indicated are the positions of BglI, PvuII and KpnI sites within and flanking the DXZ1 array (grey box). (D) The IKNFA3 and α7 3C3 DT40 cell lines treated with various topo II inhibitors: AMSA (amsacrine), Ellipt (ellipticine), Etop (etoposide) and Gen (genistein), or with DMSO only. HMW DNA was digested with BglI or KpnI, resolved by PFGE and Southern blotted with DXZ1. PFGE conditions: 40–80 s pulse, 200 V, 24 h. The 520 and 150 kb inhibitor-specific cleavage products are indicated. (E) Preservation of the position of topo II cleavage on the IKNFA3 minichromosome in human and chicken. FA3HT4.2 (minichromosome in HT1080), HT1080 and IKNFA3 (minichromosome in DT40) were treated with etoposide for 60 min before preparation for PFGE. KpnI-cut HMW DNA was Southern blotted with DXZ1. PFGE conditions: 20–60 s pulse, 200 V, 24 h. Etoposide-specific DXZ1-hybridizing KpnI fragments are indicated. (F and F′) Analysis of etoposide-sensitive topo II cleavage in the α7 6 minichromosome using KpnI-cut and uncut HMW DNA. Samples are 50 µM etoposide-treated (+) or DMSO only (–). PFGE conditions for (F): 20–60 s pulse, 200 V, 24 h and for (F′): 10–30 s pulse, 200 V, 18 h in 1.5% agarose. Etoposide-specific DXZ1-hybridizing fragments are indicated. (G) Analysis of topo II cleavage in various 2F6α9 clones from the Xp- truncation series. HMW DNA from 10 µM etoposide-treated (+) and untreated (–) cells was digested with BglI, resolved by PFGE and Southern blotted. Etoposide-specific DXZ1-hybridizing fragments are indicated. PFGE conditions were 40–80 s pulse at 200 V for 24 h. (H) Analysis of topo II cleavage in various 2F6α9 clones from the Xp-truncation series. Uncut HMW DNA from etoposide-treated (+) and untreated (–) cells was resolved by PFGE and the Southern blotted for DXZ1. Etoposide-specific hybridizing fragments are indicated. (I) Schematic showing localization of topo II cleavage. The position of BglI sites relative to the DXZ1 array (grey box) is indicated.