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First published online December 9, 2005
doi: 10.1242/10.1242/jcs.02702

Journal of Cell Science 118, 5885-5898 (2005)
Published by The Company of Biologists 2005
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Assembly of additional heterochromatin distinct from centromere-kinetochore chromatin is required for de novo formation of human artificial chromosome

Hiroshi Nakashima1,2,3, Megumi Nakano1,*, Ryoko Ohnishi1, Yasushi Hiraoka4, Yasufumi Kaneda2, Akio Sugino1,3 and Hiroshi Masumoto1,*,{ddagger}

1 Division of Biological Science, Graduate School of Science, Nagoya University, Chikusa-ku, Nagoya 464-8602, Japan
2 Division of Gene Therapy Science, Osaka University Graduate School of Medicine, 2-2 Yamada-oka, Suita, Osaka 565-0871, Japan
3 Laboratories for Biomolecular Networks, Graduate School of Frontier Biosciences, Osaka University, 1-3 Yamada-oka, Suita, Osaka 565-0871, Japan
4 Kansai Advanced Research Center, National Institute of Information and Communications Technology, 588-2 Iwaoka, Iwaoka-cho, Nishi-ku, Kobe 651-2492, Japan



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  		Fig. 1. Structures of HAC vector constructs and transcriptional activities. (A) 7C5-basic BAC DNA contained an alphoid repeat (~70 kb) sequence, a mammalian selectable maker gene (SV40-bsr), human inverted telomeres (each ~1.1 kb), a loxP site, and a chloramphenicol resistance (CmR) and ampicillin resistance (AmpR) gene. Insertion vectors contained a kanamycin resistance (KanR) gene, sequences of interest (SOI) and a loxP site, from which the replication origin had been removed (see Materials and Methods). (B) HAC vector constructs were linearized with I-SceI endonuclease. Each HAC vector had an insertion of different DNA at the loxP site on the left arm of 7C5-basic BAC: 7C5-SV BAC lacks the promoter of ßgeo gene. 7C5-SV/CMV BAC contains the ßgeo gene with CMV promoter. 7C5-INS BAC possesses the chicken ß-globin insulator sequences (cHS4) flanking both sides of the CMV-ßgeo cassette. 7C5-SV/CMVrev BAC is identical to 7C5-SV/CMV BAC but contains the CMV-ßgeo cassette in reversed orientation. (C) ß-Galactosidase activity of HT1080 cells was analyzed 24 hours after transfection of either SV-ßgeo or CMV-ßgeo plasmid DNA, or after trasfection of pBluescript II as a control. Luciferase activity of co-transfected luciferase expression vector (pRL-CMV, wako) was used for normalization. Data are the averages of three independent experiments. Error bar shows the s.e.m. (D) RT-PCR was performed 24 hours after transfection of each HAC vector DNA (7C5-SV, 7C5-SV/CMV or 7C5-INS BAC). PCR was carried out with 25 or 30 cycles against reverse transcribed cDNAs with specific primers (from top: ß-actin, bsr, bsr-pA, right junction, left junction, kanR, ßgeo-pA, ßgeo1). Control reactions were performed against mock-transcribed cDNAs without reverse transcriptase (–RT).

 


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  		Fig. 2. Cytological analyses of centromere-kinetochore proteins assembled on HAC and integration sites. (A,B) Metaphase chromosomes from different cell lines (S026 in A, and K031 in B and C) were analyzed by (a) FISH with an {alpha}21-I probe (green) and a BAC probe (red), or by (b-j and k-l) FISH with a BAC probe (red) in combination with immunofluorescence, using antibodies against CENPs (green). Chromosomes were counterstained with DAPI (blue). Arrows indicate the HAC or integration sites of the alphoid BAC. Arrowheads indicate the canonical centromeres on the host chromosomes. The typical variegated assemblies of CENPs at the integration site in BS-resistant K031 cells are shown in panel B as presence (assembly+) or absence (assembly–) of each CENP signal. (C) Small and large reformed minichromosomes (k and l, respectively) contain BAC integration signals (red) that overlap with CENP-A signals (green) in K031 cells under BS and G418 double-selective conditions. Bars, 10 µm.

 


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  		Fig. 3. Levels of transcription and acetylated-histone H3 on HAC or integrated sites. (A) RT-PCR analysis of the transformed cell lines (S026, S013 and K031) was performed as described in Fig. 1D. The schematic map of 7C5-SV/CMV BAC indicates PCR probe sites. The primers used are, from top: ß-actin, bsr, bsr-pA, right junction, left junction, kanR, ßgeo-pA, ßgeo1. (B) ChIP and real-time PCR analysis of alphoid BAC using antibody against acetylated histone H3 and normal IgG (as a control). Recovery rate of immunoprecipitated DNA against the input DNA is shown in the histogram, error bars give the s.e.m. (n=3). As controls, an endogenous promoter region of the CENP-B gene (PCENP-B) and the pericentromeric satellite 2 repeat loci (sat2) were used.

 


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  		Fig. 4. Distribution of CENP-A and heterochromatin-assemblies on HAC and integrated sites analyzed by ChIP. (A) Positions of PCR probes on alphoid BAC DNA are indicated (left arm: L1, L2, L3 and L4, right arm: R1, R2). ChIP and real-time PCR analysis of the transformed cell lines (S026, K031 and original HT1080) were performed with antibodies against CENP-A and triMet H3-K9, and normal IgG. Recovery rate of immunoprecipitated DNA against the input DNA is shown as histogram with s.e.m. (n=2). (B) ChIP analysis with anti-GFP antibody against YFP-HP1{alpha} (blue) or YFP (white) was performed. Relative enrichment was calculated by dividing the immunoprecipitated fraction of each probe by the one of the endogenous promoter of the CENP-B gene (PCENP-B), and is shown in the histogram. Error bars give the s.e.m. (n=2).

 


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  		Fig. 5. HP1{alpha} and aurora B kinase protein assemblies on HAC and extra-chromosomes, not at integrated sites in mitosis. (A-D) Metaphase chromosomes were immunofluorescent-stained with (A-D) specific antibodies and (B-D) by using a combination of FISH with the BAC probe. The localization of HP1{alpha} (red), compared with kinetochore protein CENP-C (green) in metaphase HT1080 cell is indicated in A. The presence or absence of HP1{alpha} (green in c-f, h, i) or aurora B kinase (green in g-j) on the BAC (red) locus was analyzed in (B) de novo HACs (S026), (C) the integration site (K031) or (D) the reformed minichromosome (K031) in metaphase-arrested cells. Chromosomes were counterstained with DAPI (blue or gray). Arrows indicate the HACs or the integration sites of the alphoid BAC. Arrowheads indicate canonical centromeres on the host chromosomes. Bars, 10 µm.

 


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  		Fig. 6. Models of chromatin assembly and structural organization on input alphoid BACs. Chromatin assemblies and transcripts on one unit of multimerized alphoid BAC DNA (7C5-SV BAC in A, 7C5-SV/CMV BAC in B–D) are shown as hypothetical models. Chromatin states supported by RT-PCR and ChIP analyses are indicated by colored lines. Arrows below the vector maps show transcription level (width) and length. Even though the ChIP analysis represents the sum of chromatin structures formed on multimerized alphoid BAC units, our ChIP data show a tendency for distinct chromatin structures to correspond to the sequence structures of the alphoid BAC constructs, implying that the HAC is maintained as punctuated blocks of chromatin structures. (A) Open chromatin or euchromatin (green) at the transcriptional gene on the right arm enhances the assembly of centromere chromatin (red) on the inserted alphoid DNA. In addition, when heterochromatin assembly (blue) occurs at the left arm and at a part of alphoid repeats (Fig. 4), functional HAC is generated and stably maintained. (B) If the heterochromatin-formation domain on the left arm is replaced with euchromatin by inserting a transcriptional gene unit, centromere-kinetochore components still can assemble on the alphoid array, but cohesion and inner centromere functions are absent. As a result, the unstable structure of the extra-chromosome is lost or integrated into a host chromosome. (C) Otherwise, at the integration site of a host chromosome, a silencing effect may be induced and/or heterochromatin spreads as indicated by triMet H3-K9 (Fig. 4A) into both non-selective marker gene and the insert alphoid DNA; consequently, centromere assembly on the alphoid DNA would be inactivated. (D) On the ectopic integration sites of multiple alphoid BAC DNAs, however, a chromatin opening also occurs stochastically in a part of the multiple array as indicated with the variegate assembly of the CENPs at the sites. If the open chromatin is selected again by the double selection as described in B, the chromatin opening and the functional assembly of kinetochore components on the ectopic alphoid array are accelerated and causes the reformation of minichromosomes accompanied by chromosome breakage events and with a part of the host chromosome fragment as a donor of heterochromatin-cohesion.

 

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© The Company of Biologists Ltd 2005