Inactivation of a Human Kinetochore by Specific Targeting of Chromatin Modifiers

Summary We have used a human artificial chromosome (HAC) to manipulate the epigenetic state of chromatin within an active kinetochore. The HAC has a dimeric α-satellite repeat containing one natural monomer with a CENP-B binding site, and one completely artificial synthetic monomer with the CENP-B box replaced by a tetracycline operator (tetO). This HAC exhibits normal kinetochore protein composition and mitotic stability. Targeting of several tet-repressor (tetR) fusions into the centromere had no effect on kinetochore function. However, altering the chromatin state to a more open configuration with the tTA transcriptional activator or to a more closed state with the tTS transcription silencer caused missegregation and loss of the HAC. tTS binding caused the loss of CENP-A, CENP-B, CENP-C, and H3K4me2 from the centromere accompanied by an accumulation of histone H3K9me3. Our results reveal that a dynamic balance between centromeric chromatin and heterochromatin is essential for vertebrate kinetochore activity.


BAC Transfection
The alphoid tetO BAC DNAs were purified using a Qiagen large construction kit (QIAGEN). Using 4.5 μl of Lipofectamine Reagent (Invitrogen Corporation), 0.4 μg of purified BAC DNA was transfected into HT1080 cells. Bs-resistant cell lines were selected with 4 μg/ml blasticidin S hydrochloride (MP Biomedicals, Inc.) and analyzed by FISH. To obtain homogeneous populations of sub-lines containing HACs, single colonies were picked up from the original alphoid tetO HAC clones AB 2.2.18 and AB 2.5.4. Throughout the sub-cloning process, cells were cultured in non-selective medium. Loss rates (R) of HACs per generation were calculated using the following formula: N 37 = N 0 x (1-R) 37 . N 37 is the average number of HACs per cell from 20 observed metaphase cells at day 37 and N 0 =1 in this case because of the sub-cloning.

De novo HAC Formation Analyses by Fluorescence In Situ Hybridization (FISH)
Standard FISH techniques were carried out for the alphoid tetO BACtransformed cell lines as previously described (Masumoto et al., 1989). The probes used were PCR products of p3.5α for the alphoid tetO dimer and RSA/SAT43 for the BAC vector DNA. Alphoid tetO dimer template was amplified by PCR using TaKaRa LA Taq (Takara Bio Inc.) with M13 universal and reverse primers. Alphoid DNA hooks were eliminated from the BAC vector by restriction enzyme treatments and the fragment containing the YAC and BAC cassettes was purified from the gel. PCR amplified alphoid tetO and the BAC vector DNA fragment were labeled using a nick-translation kit with digoxigenin-11dUTP or biotin16-dUTP (Roche Diagnostics). Images were captured using a cooled-charge-coupled device (CCD) camera (Cool SNAP HQ, Photometrics) and analyzed by IPLab software (Signal Analytics).

Construction of tetR-Fusion Protein Expression Vectors
The tetR coding sequence of E. coli Tn10 was cloned with and without the stop codon into pZeoSV(-) (Invitrogen) using EcoRI and BamHI.
TetR-fusion protein genes and the neomycin resistance gene were cloned into a retrovirus vector bearing an internal ribosome entry sequence (IRES). Virus-infected cells were maintained in medium containing neomycin and/or 1 μg/ml of doxycycline.

Microscopy and Image Analysis
Cells were transfected, fixed for 10 min with 4% PFA and mounted with VectaShied for microscopy, which was performed with a DeltaVision (Applied Precision, Issequah, WA) inverted microscope. For analysis of the intensity of various FPs on the HAC, Z-stacks were acquired using the same Z-spacing and exposure without deconvolution. We then defined a cylindrical region of interest through the stacks using the image analysis tool of Softworx and summed the intensity within this region for each image plane. The total intensity for image planes containing the HAC is shown in Fig. 4B. This was normalized for background by division by the summed intensity above and below the HAC within the cylindrical ROI.

ImmunoFISH and Cytological Preparations
ImmunoFISH experiments were performed on chromosome spreads. Cells from alphoid tetO HAC cell line AB2.2.18 were incubated for 4 hours in 0.1 μg/ml colcemid and after mitotic shake-off, mitotic cells were resuspended in were fixed for 15 min with cold (-20 °C) MeOH. Mitotic spreads were transferred by dropping onto a clean glass slide, dried and incubated in PBST (1x PBS + 0.05% Tween 20) for 5 min. After pre-block with 1% BSA in PBST for 30 min at 37 °C, incubation with primary antibody was done o/n at 4 °C and with secondary for 30 min at 37 °C. After the second incubation, cells were fixed again with 4% PFA for 8 min, washed twice with 2x SSC buffer for 5 min and EtOH dehydrated. Another wash was done with 2x SSC for 45 min at increasing temperature from 25 to 70 °C, followed by EtOH dehydration.
DNA was denaturated with 0.1 M NaOH for 10 min, the slides were washed 3 times with 2x SSC and dehydrated. BAC-probe (obtained using Bionick Labelling kit from Invitrogen) was denaturated and applied to the slides, which were incubated for 2 min on a thermoblock at 75 °C. Hybridization was o/n at 39 °C in a humidified chamber. The following day slides were washed 3 times for 5 min with 2x SSC at 45 °C and again for 5 min at room temperature.

Chromatin Immunoprecipitation (ChIP) and Real-Time PCR
ChIP with CENP-B antibody (2D8D8 and 5E6C1) was carried out according to a previously described method (Nakano et al., 2003). ChIP with antibody against EYFP (anti-Green Fluorescent Protein, Roche) was done using a modified method. Cultured cells were cross-linked in 1.0% formaldehyde.

Indirect Immunofluorescence
Indirect immunofluorescence was carried out as previously described. Cells expressing EYFP-TetR or EYFP-tTS were cultured on poly-D-Lysine-coated coverslips, fixed in 1% formaldehyde for 10 min, treated with methanol for 5 min and dried. The coverslips were then treated with 0.5% Triton X-100 and 0.1 M glycine for 5 min each. Antibodies used were anti-CENP-A (mAN1), anti CENP-C (Ra1), anti-GFP monoclonal antibody (Invitrogen), and/or anti-GFP polyclonal antibody (Medical & Biological Laboratories co., ltd, Japan). Images were captured using a Zeiss microscope (Axiophot) equipped with a cooled-charge-coupled device (CCD) camera (Cool SNAP HQ, Photometrics) and analyzed by IPLab software (Signal Analytics).

Quantification of Transcripts Derived from alphoid tetO HAC
Real-time RT-PCR was carried out using the iScript One-Step RT-PCR Kit with SYBR Green (Bio-Rad) according to the manufacturer's protocol, using total RNA prepared with the SV Total RNA Isolation system (Promega).

Methylated DNA Immunoprecipitation (MeDIP)
MeDIP with anti 5-meC antibody (Diagenode) was carried out using a modified method. Purified genomic DNA was sheared using a Bioruptor sonicator (Cosmo Bio) to an average DNA size of 0.5 kb and denatured at 94˚C for 10 minutes. The methylated DNA was immunoprecipitated in IP buffer (20 mM Tris, 150 mM NaCl, 1 mM EDTA, 20% glycerol, 1.5 μM aprotinin, 10 μM leupeptin, 1 mM DTT, 0.1% NP-40). Protein A agarose blocked with salmon sperm DNA (Upstate) was added, and the antibody-DNA complex was recovered by centrifugation. The recovery ratio of the immunoprecipitated DNA from input DNA was measured by real-time PCR using the following primer sets: 11-10R and mCbox-4 for 11-mer of chromosome 21 alphoid DNA (alphoid chr. 21 ), tet-1 and tet-3 for the alphoid tetO .
bsr-F and bsr-R for the marker gene (bsr) of alphoid tetO HAC.

Supplemental References
Agata, Y., Matsuda, E., and Shimizu, A. (1999). Two novel Kruppel-   containing 10 repeats of alphoid tetO dimer) were treated with StuI whose restriction site appears once per alphoid tetO dimer. Alphoid tetO BAC was simultaneously treated with StuI and SalI appears at alphoid tetO dimer cloning site, and was compared with empty BAC vector.

Immunoprecipitation (MeDIP) with Anti 5-Methyl Cytidine Antibody
Genomic DNA was purified from alphoid tetO HAC cells (AB2.2.18.21) expressing tetR or tTS cultured in doxycycline free medium for 3, 7, 10 and 14 days. The purified DNA was immunoprecipitated with antibody against 5-methylated Cytidine and quantitated by real-time PCR. As controls, the genomic DNA of the original cell (AB2.2.18.21) and purified alphoid tetO dimer plasmid DNA (p3.5α) were treated with or without Sss I CpG methylase in vitro and were analyzed by MeDIP assay. The bars show the relative enrichment of alphoid chr. 21 (white), alphoid tetO (black) and the bsr gene (gray). The level of the CpG methylated alphoid tetO HAC DNA did not increase drastically during 14 days of culture even after tTS binding.