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Originally published In Press as doi:10.1074/jbc.M401051200 on June 25, 2004

J. Biol. Chem., Vol. 279, Issue 36, 37631-37639, September 3, 2004
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Effects of Scaffold/Matrix Alteration on Centromeric Function and Gene Expression*

Huseyin Sumer{ddagger}§, Richard Saffery{ddagger}, Nicholas Wong{ddagger}, Jeffrey M. Craig{ddagger}, and K. H. Andy Choo{ddagger}

From the {ddagger}Murdoch Childrens Research Institute, Department of Pediatrics, Royal Children's Hospital, Flemington Road, Melbourne 3052, Australia

Received for publication, January 30, 2004 , and in revised form, June 1, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
We have previously described a 3.5-Mb domain of enhance scaffold/matrix attachment region (S/MAR) at a human neocentromere, and normal expression of underlying genes within this region. We also reported that partial inhibition of histone deacetylation using 33 nMtrichostatin A (TSA) resulted in a shift in the position of the CENP-A-binding domain within the neocentromere, with no noticeable effects on mitotic segregation function. In this study, 33 nM TSA caused a reduction in the size of the enhanced S/MAR domain of one-half to 1.7 Mb. Treatment with a DNA-intercalating drug distamycin A (DST) at 75 µg/ml resulted in a size reduction of the enhanced S/MAR domain at the neocentromere of two-thirds to 1.2 Mb, and that of the CENP-A-binding domain of 40%, from 330 to 196 kb, with no significant shift in the position of the latter domain. Other DST effects include mitotic chromosomal missegregation, reduction in the levels of Topo II{alpha}, CENP-A, CENP-C, and HP1{alpha}, and an increase in mitotic checkpoint protein BubR1. TSA or DST treatment similarly resulted in a significant reduction, by ~20 and 50%, respectively, in the size of the enhanced S/MAR domain at the {alpha}-satellite DNA of a native chromosome 10 centromere. Transcriptional competence within the neocentromere is overall not noticeably altered by either TSA or DST treatment, as is evident from the absence of any significant increase or decrease in the expression levels of 47 underlying genes tested. These results suggest that a substantial contraction of the S/MAR domain may not be deleterious to centromere function, that disruption of the S/MAR domain directly affects the binding properties of a host of scaffold/matrix and centromeric/pericentric proteins, and that the overall competence and regulation of transcription at the neocentromeric chromatin is similar to those found at the corresponding normal genomic sites.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The centromere of the eukaryotes is essential for the proper segregation of replicated sister chromatids during cell division. It appears as the primary constriction on condensed chromosomes and is the point of microtubule spindle attachment. Most eukaryotic centromeres are comprised of tandemly repetitive satellite DNA (1–3); however a different class of centromere (termed neocentromeres) lacking these repeats has been described (4). Neocentromeres arise at previously non-centromeric loci in the genome and form active kinetochores that are functionally equivalent to the satellite DNA-based centromeres, both in terms of mitotic stability and association with centromere proteins (5–7). The lack of any repetitive satellite DNA at neocentromeres supported the proposal of an epigenetic model for centromere chromatin formation and maintenance (8, 9).

The eukaryotic genome is organized into higher order chromatin domains consisting of euchromatin and heterochromatin. One of the proposed epigenetic markers for maintaining centromeric heterochromatin is histone acetylation (9, 10). The hypoacetylation of the terminal tails of core histones is essential for the correct loading of heterochromatin proteins (11, 12). In fission yeast, the formation of a heterochromatic structure is essential for full centromere function (13). The induction of histone hyperacetylation using the deacetylase inhibitor trichostatin A (TSA)1 causes chromosome loss and the disruption of the fission yeast heterochromatin protein Swi6 association from pericentric regions, interfering with the transcriptional repression in centromeric heterochromatin resulting in the expression of reporter genes (14). Similarly, in human cells, prolonged treatment with TSA disrupts Swi6 homologue HP1 localization and causes centromeric defects resulting in chromosomal missegregation (11).

Chromatin in the eukaryotic genome has also been shown to be organized into loops that attach to a protein-rich chromosomal scaffold/matrix mediated by AT-rich DNA sequences termed scaffold/matrix attachment regions (S/MARs) (15–17). Centromeric {alpha}-satellite DNA is packaged more tightly than other genomic DNA, with a higher frequency of S/MARs (18–20). Centromeric scaffold/matrix attachment has been shown to be sequence-independent, with the domain of increased S/MARs being mapped and shown to fully encompass the centromere-specific histone H3-related kinetochore protein domain of CENP-A on two human neocentromeres (21, 22). Furthermore, scaffold/matrix attachment of telomere sequences has also been shown to be epigenetic (23).

Chromosome condensation by scaffold/matrix attachment can be inhibited by the minor groove DNA-binding drug distamycin A (DST). In Drosophila, DST inhibits centromeric condensation at metaphase (24), preventing the activity of topoisomerase II, the major component of the chromosome scaffold/matrix (25). In mouse cells, DST reduces condensation of centromeric heterochromatin (26), by competing with centromeric repeat elements for binding sites of chromosomal proteins (27, 28). In human cells, DST has also been shown to inhibit the condensation of heterochromatic and centromeric regions on metaphase chromosomes (29, 30). Furthermore, the drug has been shown to inhibit S/MAR elements within genes, altering their expression (31, 32).

We have begun a systematic dissection of the chromatin structure and the effects chromatin-modification drugs on the previously reported 10q25 neocentromere of the human marker chromosome mardel (10) (6, 33, 34). This neocentromere has been extensively characterized, and the localization of several important centromeric chromatin domains have been mapped along its DNA base, including a 330-kb CENP-A-binding domain and a 1-Mb region of delayed replication timing (35). More recently, the distribution of the various domains corresponding to enriched S/MARs, centromere protein CENP-H, and heterochromatin protein HP1, as well as the expression status of endogenous gene loci within and flanking the 10q25 neocentromere, have been determined (21). In addition, we have described the effects of TSA-induced histone hyperacetylation that resulted in a unilateral shift (by ~320 kb) in the position of the CENP-A-associated domain and an apparent expansion in the size of this domain to ~480 kb (7). Here, we further investigate the effects of TSA-induced histone hyper-acetylation and DST-induced disruption of scaffold/matrix attachment on the organization of the chromosomal scaffold/matrix domain on the functional integrity and underlying gene expression properties of the neocentromere.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture and Drug Treatment—CHO-human somatic cell hybrids containing the mardel (10) chromosome (M10) and normal chromosome 10 (N10) were cultured as previously described (21). Cells were incubated with 33 nm trichostatin A (Calbiochem) for 17 h or 75 µg/ml distamycin A (SERVA) for 72 h before harvesting for analysis.

Genomic BAC Array Production—BAC clones were obtained from the human genomic library RPCI-11 (36), and DNA was isolated using standard techniques. 100 ng of BAC DNA was immobilized onto Hybond N+ nylon membranes in a dot blot format (minifold SRC-96, Schleicher and Schuell, Dassel, Germany).

Nuclei and S/MAR DNA Isolation—S/MAR array analysis was performed as previously described (21, 22). 2 x 108 cells were harvested and collected by centrifugation and the pellets washed in phosphate-buffered saline for 5 min at 500 x g. The pellet was resuspended and washed three times for 5 min at 500 x g in isolation buffer containing 3.75 mM Tris-HCl, 0.05 mM spermine, 0.125 mM spermidine, 1% (v/v) thiodyglycol (Fluka, Buchs, Switzerland), 20 mM KCl, 0.1 mM PMSF, 0.5 mM EDTA/KOH, pH 7.4, and 10 kIU/ml aprotinin. The pellet of washed cells was resuspended in 12 ml of ice-cold isolation buffer containing 0.1% digitonin and 100 kIU/ml aprotinin, and broken up in a Dounce-type tissue homogenizer with 12 strokes of a B (loose) pestle. The nuclei were collected by three washes in isolation buffer containing 0.1% digitonin and 10 kIU/ml aprotinin at 900 x g, 10 min at 4 °C. The washed pellet was resuspended in 5 ml of isolation buffer containing 0.1% digitonin, 100 kIU/ml aprotinin, and without EDTA/KOH. Nuclei were then filtered through a 40-micron filter (BD Biosciences) using gravity to remove nuclei clumps.

1 x 106 nuclei in 100 µl of isolation buffer with 0.1% digitonin, 100 kIU/ml aprotinin, and without EDTA/KOH were stabilized at 37 °C for 20 min. The nuclei were then diluted with 1 ml of LIS buffer consisting of 5 mM Hepes/NaOH, 0.25 mM spermidine, 2 mM EDTA/KOH, 2 mM KCl, and 50 mM 3,5-diiodasalicylic acid, lithium salt (LIS) (SERVA), and left to extract for 10 min at 4 °C. The extracted nuclei were centrifuged at 2,400 x g for 20 min at 4 °C. The pellet was then washed four times with 8 ml of digestion buffer. Digestion buffer consisted of 20 mM Tris-HCl, 0.05 mM spermine, 0.125 mM spermidine, 20 mM KCl, 0.1 mM PMSF, 0.1% digitonin, 50 mM NaCl, 5 mM MgCl2 and 100 kIU/ml aprotinin. Restriction enzymes (EcoRI, EcoRV, and BamHI) were then added at 1,000 units/ml and incubated at 37 °C for 5 h. The nuclear scaffold-attached DNA was pelleted from the digested loop DNA by centrifugation at 2,400 x g for 10 min at 4 °C. Each fraction was treated with RNase for 30 min and proteinase K for 4 h and DNA extracted using standard phenol:chloroform extraction.

S/MAR Array Analysis—Identical genomic array blots were preannealed with 5 µg of salmon sperm DNA and probed with 1 µg of scaffold-attached or loop DNA from the mardel (10) and control cell lines, radioactivity labeled by random priming according to the manufacturer's instructions (Roche Applied Science), and pre-annealed with 5 µg of human Cot-1 DNA. Hybridization and washes were performed at high stringency (0.1x SSC, 0.1% SDS, 65 °C). All blots were exposed to a Kodak PhosphorImager screen and were analyzed using the PhosphorImager system (Storm 860 Gel and Blot Imaging System, Molecular Dynamics) connected to an IBM-compatible computer controlled by Image QuaNT version 4.2 software (Molecular Dynamics). The signal obtained from each scaffold-attached DNA spot on the dot blot BAC array was compared with that on a duplicate blot hybridized with the loop DNA. Scaffold/matrix attachment for each BAC for both test and control cell lines was calculated as the percentage difference between the scaffold/matrix-attached/unattached signal ratio of mardel (10)-containing cell line and normal chromosome 10-containing cell lines (i.e.{[normal – marker]/normal} x 100). The data were represented graphically by plotting the average and standard deviation of the mean on the midpoint for each BAC/PAC on the array. A two-tailed Student's t test was performed on the results for each BAC/PAC to establish statistical significance.

Southern Blot Analysis—Five micrograms of DNA corresponding to total, pellet (S/MAR), and supernatant (loop) fractions were isolated from control and treated cells, electrophoresed on a 1% agarose gel, and transferred to Hybond-N+ nylon membranes (Amersham Biosciences). Nylon membranes were prehybridized at 65 °C for 4 h. Probes were radioactively labeled by the Random Primed DNA labeling kit (Roche Applied Science), and hybridization was performed at 65 °C for 18 h. Filters were washed at 65 °C twice in 1x SSC, 0.1% SDS and twice in 0.1x SSC, 0.1% SDS. Blots were exposed to a PhosphorImager screen and analyzed using the PhosphorImager system. Band intensities were quantified using Image QuaNT version 4.2 software. S/MAR strength values were calculated as described by Strissel et al. (20), relative strength (R) is the band intensity of the pellet S/MAR fraction divided by the sum of intensities of the pellet (S/MAR) and supernatant (loop) fraction (i.e. [{S/MAR}/{S/MAR + loop}] x 100).

Chromatin Immunoprecipitation (ChIP) and Array Analysis—ChIP array analysis using affinity-purified CENP-A was performed as previously described (35, 37). Exponentially growing cells were harvested and resuspended in TBS (0.01 M Tris-HCl, pH 7.5, 3 mM CaCl2, 2 mM MgCl2 with 0.1 mM PMSF and proteinase inhibitors (Complete, protein-ase inhibitor mixture tablet, Roche Applied Science) with 0.25% Tween 20 and incubated at 4 °C on a roller stirrer for 2 h. The suspension was homogenized to extract the nuclei using 30 strokes of the "Tight" pestle on a Dounce homogenizer (Wheaton). The nuclei were then pelleted by centrifugation at 1,500 rpm for 10 min at 4 °C (Sorvall RT-7). Nuclei were resuspended in 25% sucrose in TBS and carefully layered onto a 50% sucrose in TBS layer and centrifuged at 2,500 rpm at 4 °C to separate cytoplasmic debris through the discontinuous sucrose gradient. The nuclei pellet was resuspended in Digestion buffer (0.32 M sucrose, 50 mM Tris-HCl, pH 7.5, 4 mM CaCl2, 1 mM CaCl2 with 0.1 mM PMSF) to 500 ng/µl (obtained by measuring A260nm). Aliquots of nuclei were subjected to micrococcal nuclease digestion at a concentration of 80 units/mg DNA at 37 °C for 10 min. The reaction was stopped by chilling on ice and adding a 1:100 volume of 0.5 M EDTA. The preparation was centrifuged at 13,000 rpm for 5 min at 4 °C (Biofuge fresco, Heraeus). The supernatant containing mainly mononucleosomes was separated and kept on ice. The pellet fraction was further processed by incubation with lysis buffer (1 mM Tris-HCl, pH 7.5, 0.2 mM EDTA, 0.2 mM PMSF, and a proteinase inhibitor tablet in a 50-ml volume) on ice for 1 h. The sample was vortexed, and the supernatant containing oligonucleosomes was obtained after centrifugation at 13,000 rpm for 5 min at 4 °C. The two supernatants were pooled and precleared by incubation with an equal volume of protein A-Sepharose (Amersham Biosciences) at 4 °C for at least 4 h. The samples were then centrifuged at 1,500 rpm for 5 min at 4 °C to isolate the supernatant. The supernatant was stored at –20 °Cor used immediately for immunoprecipitation.

An equal volume of oligonucleosomes was mixed with incubation buffer (50 mM NaCl, 20 mM Tris-HCl, pH 7.5, 5 mM EDTA, 0.1 mM PMSF, and a proteinase inhibitor tablet per 50 mls). Affinity-purified CENP-A antibody was added at a 1:500 dilution and incubated on a rolling stirrer at 4 °C overnight. The immune complex was then captured by incubation at 4 °C with 12.5% protein A-Sepharose that had been washed in incubation buffer for 2 h. After incubation the protein A-Sepharose was washed stepwise in buffer A (50 mM Tris-HCl, pH 7.5, 10 mM EDTA) containing 50, 100, and 150 mM NaCl. The immune complex was then eluted twice with 1% SDS. The "bound" DNA was extracted from the eluted fraction as described above.

Duplicate BAC arrays were probed with 1 µg of radioactivity labeled "input" or "bound" DNA from the patient and control cell lines. The signal obtained from each bound DNA spot on the dot blot BAC array was compared with that on a duplicate blot hybridized with the input DNA. First, normalization was performed with each value by dividing the signal from an outlying BAC bA313D6, and the ratio for each spot of "bound" compared with "input" obtained. The ratio of enhancement of the bound/input (R) of the patient cell line was compared with a control cell line, i.e.{[Test (R) – control (R)]/control (R)} x 100. The data were represented graphically by plotting the average and standard error of the mean on the midpoint for each BAC on the array. A two-tailed Student's t test was performed on the results for each BAC to establish statistical significance.

Quantitative RT-PCR Analysis—Quantitative RT-PCR analysis was performed as described (21). Primers for PCR amplification were designed using primer express software (ABI). All primer pairs were designed so that at least one of the pair spanned a genomic exon/intron boundary to avoid the amplification of contaminating genomic DNA or total RNA, and no amplification was detected from human genomic DNA or total RNA prior to reverse transcription as described in Saffery et al. (21). Quantitative RT-PCR was carried out using SYBR green technology with the Applied Biosystems SYBR green master mix, and reactions were performed on an ABI 7700 Sequence Detection System.

{Delta}CT analysis was used to calculate the relative amount of expression of individual genes in relation to an 18 S rRNA control amplicon (Ambion Inc.). {Delta}CT for each dilution was then calculated, and if efficiencies of amplifications were comparable, this value did not change significantly with each dilution. For TAQman-based quantitative RTPCR, Assay On Demand preoptimized primer and probe mix were employed with TAQman master mix and TAQman 18 S rRNA control reagents (Applied Biosystems).

Mitotic Block and Release—Fibroblasts cells were grown on Super-Frost plus slides (Menzel-Glaser, Braunschweig, Germany) in Quadriperm (Vivascience, Selangor Darul Ehsan, Malaysia) slide chambers. Once cells had adhered, nocodazole to a final concentration of 100 nM was added and incubated overnight to block cells in mitosis. Cells were released from mitosis by removing media, washing twice in phosphate-buffered saline, and adding fresh medium containing 10 µM cytochalasin B for 1 h to block cytokinesis.

Immunofluorescence—Cells incubated in colcemid for 1 h were spun onto microscope slides using a cytospin centrifuge (Shandon) at 1000 rpm for 10 min. Slides were permeabilized in KCM (120 mM KCl, 20 mM NaCl, 10 mM Tris-HCl, 0.5 mM NaEDTA, 0.1% (v/v) Triton X-100) for 10 min. Primary antibodies were diluted in KCM and incubated on slides for 1 h at 37 °C. Slides were washed three times in KCM, and fluorescent-conjugated secondary antibodies diluted in KCM (according to the supplier's instructions) were incubated on slides for 1 h at 37 °C. Antibodies were then washed off the slides in KCM, fixed in 10% formalin, and then mounted in DAPI (2 µg/ml) in Vectashield antifade mountant (Vector Laboratories). Antibodies used in this study include polyclonal anti-human BubR1 (38), anti-mouse CENP-A (39), CENP-C (40), CREST6 (33), HP1{alpha} (41), Topo II{alpha} (TopoGEN), and {beta}-tubulin (Sigma-Aldrich).

Fluorescent in Situ Hybridization (FISH) Analysis—Cells incubated in colcemid for 1 h were dropped onto microscope slides in 3:1 methanol: acetic acid. Slides were dehydrated by sequential emersion in 75, 95, and 100% ethanol for 5 min, and air-dried at room temperature. Slides were then immersed in 70% formamide in SSC (83 °C) for 4 min followed by sequential emersion in 75, 95, and 100% ice-cold ethanol. Slides were then air-dried again at room temperature. 200 ng of BAC (bA153G5) probe labeled by nick translation with biotin-16-dUTP and pre-annealed with Cot-1 DNA (Roche Applied Science) were hybridized to slides at high stringency (50% formamide at 37 °C) overnight. Slides were then washed three times in 0.1x SSC at 60 °C. Hybridization was detected by dual-layer detection using avidin-fluorescein isothiocyanate (FITC), followed by FITC-conjugated goat anti-avidin. Chromosomes were counterstained using DAPI in Vectashield.

Slides were examined on a Zeiss Axiioplan II fluorescence microscope with a 100x oil objective and the appropriate filters. Images were captured with a cooled charge-coupled device video camera (SenSys 2), connected to a PowerMac G4 computer controlled by IP Lab version 2.5.5 software (Scanalytics). Quantification of fluorescence signal was performed as described essentially as described in Ref. 42. The best in-focus image of centromeres was determined visually, and a region corresponding to the centromere was measured for the mean fluorescence intensity minus background fluorescence using IP Lab software. The average of antibody fluorescence was calculated for at least 100 centromeres, and results compared using a Student's t test.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
TSA Contracts the Domain of Enhanced S/MAR—We have investigated the effects of TSA-induced histone hyperacetylation on scaffold/matrix attachment and gene expression at the mardel (10) neocentromere using a moderate dose of 33 nM TSA. A previous study has shown that although measurable changes to histone acetylation levels and centromeric domain organization were observed, this drug concentration caused no deleterious effects on chromosomal segregation functions (7). Here, we examined the effects of this treatment on S/MAR at the neocentromere.

S/MAR analysis involved the differential isolation of scaffold/matrix attached and non-attached DNA from cell lines containing the neocentromeric mardel (10) chromosome (M10) or normal chromosome 10 (N10), and comparing the hybridization profiles of these DNA fractions across a genomic array (see "Experimental Procedures"). Using this technique, we have previously identified an ~3.5-Mb region of enhanced S/MARs at the 10q25 neocentromere, spanning BACs bA190F19 to bA501J20 (21) (Fig. 1). In the present study, incubation with 33 nM TSA for 17 h resulted in a significant reduction in the overall size of the S/MAR-enriched domain at the neocentro-mere by approximately one-half, from ~3.5 Mb to ~1.7 Mb (Fig. 1A). The reduced region of enriched S/MARs at the neocentromere continued to fully encompass the CENP-A-binding domain following TSA treatment (Fig. 1B).



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  		FIG. 1.
Effects of TSA-induced hyperacetylation on neocentromeric and centromeric scaffold/matrix attachment and gene expression. A, scaffold/matrix attachment of the 10q25 neocentromere. The x-axis represents the position along the 10q25 BAC array across the neocentromeric region. Data were collected using a previously reported S/MAR array analysis (22). The y-axis represents the difference in S/MAR between the mardel (10) and control normal chromosome 10 hybrid cell lines. Each data point represents the mean and standard deviation of the mean from six independent experiments and is plotted as the midpoint of the corresponding BAC in the contig shown at the top of the graph. The linear expanse of the previously defined enriched S/MAR domain is indicated at the bottom of the graph with hatched lines while that of the domain after TSA treatment is indicated by gray shading. Statistical analysis was performed using a Student's t test, where asterisks indicate data points that are significant (p < 0.05). B, positions of genes within the 10q25 region (21), where the active and inactive genes are indicated by closed and open arrows, respectively. The positions of the previously reported CENP-A-binding domains before and after TSA treatment are shown by hatched and gray boxes, respectively (7). C, Southern blot scaffold/matrix attachment analysis of native human chromosome 10 centromere. Hybridization of chromosome 10-specific probe PZ10–1.3 on EcoRI-digested total genomic, S/MAR-, and loop DNA fractions before and after TSA treatment are shown. The position of the 342-bp 2-mer repeat unit of genomic PZ10–1.3 is indicated.

 
To investigate whether TSA-induced hyperacetylation resulted in loss of S/MAR in native human centromeres, we analyzed S/MARs at the centromere of human chromosome 10 using Southern blotting as previously described for other human centromeres (20). In this method, equal amounts of S/MAR and loop DNA were hybridized with a chromosome 10-specific {alpha}-satellite probe and the percentage scaffold attachment determined. In untreated cell the 342-bp 2-mer {alpha}-satellite repeat unit on the chromosome 10 centromere was strongly scaffold/matrix-attached, as shown by a relative strength of 83% in the S/MAR lane in Fig. 1C. After TSA-treatment, the relative strength of the chromosome 10 {alpha}-satellite repeat was reduced to 67%, with a concomitant increase in the hybridization signal intensity of the loop fraction (Fig. 1C). These results corresponded to an approximately overall 20% reduction of scaffold/matrix attachment of the chromosome 10 centromere.

Transcription Is Not Differentially Regulated at the Neocentromere by TSA—A host of endogenous genes residing at the 10q25 neocentromere were previously shown to be actively transcribed while others were transcriptionally inactive, although, importantly, no significant difference in transcriptional competence was observed at the neocentromere compared with the corresponding normal genomic region (21). Here we investigated the effects of 33 nM TSA treatment on the transcriptional activities of 47 different genes (Fig. 1B) in the M10 and N10 cell lines, and compared them to untreated cells. The results indicated that TSA-induced hyperacetylation did not result in the activation of any of the 36 previously inactive genes. Of the 11 genes that were previously shown to be expressed, no significant change in expression level was observed following TSA treatment, except for the gene HCG39837 (KIAA0534) spanning the CENP-A-binding domain, which was found to be more highly expressed in both the M10 and N10 cell lines (Fig. 1B and Table I). These results therefore indicated the absence of any overall dramatic effect of moderate hyper-acetylation on transcription and that transcription within the neocentromere is not significantly regulated differently from that of its corresponding normal genomic site.


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  		TABLE I
Gene expression analysis

Gene letter refers to those shown in Fig. 1B. Celera Gene ID and corresponding gene name (where applicable) are shown. Domain refers to the location of the gene in relation to chromatin domains identified in Saffery et al. (21). The relative expression levels of genes are shown for the somatic hybrid cell lines M10 and N10 containing the neocentromeric mardel (10) and normal chromosome 10, respectively, comparing each cell line before and after TSA and DST treatment (i.e. expression value with drug treatment divided by value without treatment) (21). Data points are the mean of at least four different experiments. Statistical significance was determined using {Delta}CT values in a Student's t test.

 
DST Causes Mitotic Chromosome Segregation Defects—In initial experiments, we tested the viability of the somatic hybrid cell lines grown in 10, 25, 50, 75, and 100 µg/ml of DST. We determined that 75 µg/ml of the drug was the threshold concentration at which cell division was not severely affected after 72 h of culture (data not shown). To further assess the effects of this drug concentration on mitotic phenotype, DST-treated and untreated control cells were incubated in 100 nM nocodazole for 12–16 h, released from the metaphase block by media change, and further incubated in 10 µM cytochalasin B for 1 h to accumulate cells in anaphase and telophase. The results indicated a noticeable proportion of the DST-treated cells showing lagging chromosomes, chromosome bridges, and micronuclei (Fig. 2A). Statistical analysis of 400 cells revealed significantly more anaphase/telophase abnormalities in the DST-treated cells (39%) compared with the control cells (6%) (p < 0.05) (Fig. 2B). Immunofluorescence revealed that the lagging chromosomes were binding centromere antigens (Fig. 2C). Comparison of the mitotic loss rate between mardel (10) and normal chromosome 10 in DST-treated cells indicated retention of these chromosomes in 82 and 84% of cells, respectively, suggesting that DST reduced the mitotic stabilities of the mardel (10) and normal chromosome 10 to the same extent.



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  		FIG. 2.
Effects of DST (75 µg/ml) on centromere function. A, mitotic segregation defects following treatments with nocodazole and cytochalasin B. Panels i and ii, chromatin bridge (closed arrow), lagging chromosomes (open arrow); panel iii, telophase bridge (closed arrow) and micronuclei (open arrow). B, percent of anaphases showing lagging chromosomes and chromatin bridges after DST treatment. C, immunofluorescence showing a DST-treated cell with a lagging chromosome that binds anti-centromere serum CREST6 (green). Spindle microtubule formation is shown with anti-tubulin antibody (red). Chromosomes are stained with DAPI (blue). D, quantitative analysis of centromeric/pericentric proteins as described in Ref. 42. The values shown are the means ± S.D. of centromeric fluorescence intensities. Asterisk indicates a significant difference (p < 0.05) after treatment.

 
The mean immunofluorescence intensities of a number of centromere and scaffold/matrix proteins were quantified at the native centromeres of metaphase chromosomes before and after DST treatment (Fig. 2D). The kinetochore proteins CENP-A and CENP-C, scaffold/matrix protein Topo II{alpha}, and heterochromatin protein Hp1{alpha} were all found to be significantly reduced at the centromeres of the DST-treated cells. In contrast, the checkpoint protein BubR1 was higher in the treated cells, suggesting the activation of mitotic checkpoints.

DST Contracts the Domains of CENP-A Binding and Enhanced S/MAR—The effects of treatment with 75 µg/ml DST on the CENP-A binding domain were determined. We have previously defined the binding domain of the kinetochore protein CENP-A by ChIP and genomic array analysis, and shown that its size and position is affected by TSA (7, 35). Here, the effects of DST on the CENP-A-binding domain was determined using the ChIP/array technique (Fig. 3A). Prior to drug treatment, the CENP-A-binding domain encompassed the four BACs bA87P3, bA153G5, bA359H22, and bA87E14 (7, 35). Following drug treatment, only two of the BACs bA153G5 and bA359H22 showed significant binding, indicating first that the size of the CENP-A-binding domain was reduced by 40%, from ~330 to ~196 kb, and second that, unlike the effects of TSA, the core position of the CENP-A-binding domain has remained unchanged.



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  		FIG. 3.
Effects of DST on neocentromeric scaffold/matrix attachment and CENP-A binding. A, scaffold/matrix attachment across the 10q25 neocentromere region after distamycin A treatment. The y-axis represents the difference in scaffold/matrix attachment between the mardel (10) and normal control cell lines. Mean and standard deviation of the mean from six independent experiments are shown. For both sets of results the x-axis represents the position of BACs shown at the top of the graph along the 10q25 array spanning the neocentromeric region, and thepreviously reported domains are indicated at the bottom the graph with dotted lines while the size of the domain after distamycin A treatment is indicated by gray shading. B, CENP-A binding across the 10q25 region. Data were collected using a previously reported ChIP array analysis (35, 37). Percent difference in CENP-A binding ratios between the values of mardel (10) and the normal chromosome (10) of three independent experiments are plotted across the 10q25 BAC contig. C, Southern blot scaffold/matrix attachment analysis of native human chromosome 10 centromere. Hybridization of chromosome 10-specific probe PZ10–1.3 on EcoRI-digested total genomic, S/MAR, and loop DNA fractions before and after DST treatment are shown. The position of the 342-bp 2-mer repeat unit of genomic PZ10–1.3 is indicated.

 
The effects of DST on centromere scaffold/matrix attachment were also determined. S/MAR array analysis at the 10q25 neocentromere for cells incubated 75 µg/ml DST for 72 h showed a significant reduction in the overall size of the S/MAR-enriched domain of the 10q25 neocentromere by approximately two-thirds, from ~3.5 Mb to ~1.2 Mb (Fig. 3B). The region of significantly increased S/MARs after DST treatment was defined by the 9 BACs between bA206G17 and bA326H7, inclusive.

To determine whether DST had a similar effect on S/MARs in native human centromeres, we performed Southern blot analysis on the human chromosome 10 centromere. In untreated cells, the chromosome 10 {alpha}-satellite repeat unit was strongly scaffold/matrix-attached, with a relative strength of 84% (Fig. 3C). After DST treatment, the relative strength of the chromosome 10 {alpha}-satellite was reduced to 46%, which corresponded to a reduction of scaffold/matrix attachment of the chromosome 10 centromere by about 50% (Fig. 3C).

Transcription Is Not Differentially Regulated at the Neocentromere by DST—The effects of 75 µg/ml DST on the expression activity for each of the 47 genes residing at the 10q25 neocentromere region were investigated. The results indicated that DST did not activate any of the 36 previously inactive genes in either the M10 or N10 cell lines (Fig. 1B). Furthermore, of the 11 genes that were previously shown to be transcriptionally active at the neocentromere, 10 that have been tested were found not to be significantly up- or down-regulated by DST in either cell lines (Table I). These results provided further support that transcription within the neocentromeric chromatin is not differentially regulated compared with that of the corresponding normal genomic chromatin.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The chromosomal scaffold/matrix has been implicated in the packaging of chromatin above the 30-nm fiber level (19, 20). We described previously that, like the native {alpha}-satellite DNA-based centromeres (19, 20), two different neocentromeres at 10q25 and 20p12 also contain a greatly increased densities of S/MARs (21, 22). In the present study, we demonstrate that partial histone hyperacetylation induced by 33 nM TSA significantly reduced the expanse of the domain of enriched S/MARs at the 10q25 neocentromere from ~3.5 Mb to ~1.7 Mb, with the reduced S/MAR domain continuing to fully encompass the CENP-A-binding region. A significant contraction of the region of enriched scaffold/matrix attachment was also seen in the native centromere of human chromosome 10 following similar TSA treatment.

The reduction in size of the enriched S/MAR domain by one-half at the 10q25 neocentromere and one-fifth at the normal chromosome 10 centromere presumably results from destabilization of the binding of proteins that contribute to the formation of this domain following neocentromere activation. One such protein is Topo II{alpha}, which is a major component of the chromosomal scaffold/matrix (43) that has been shown to be enriched at the 10q25 neocentromere (5). A direct interaction between Topo II{alpha} and the histone deacetylases HDAC-1 and HDAC-2 has been described in mammalian cells, with protein complexes containing these proteins demonstrating both deacetylation and topoisomerase activities (44, 45). Furthermore, TSA has been shown to disrupt the association of other scaffold/matrix proteins (46–48). It is therefore reasonable to speculate that partial inhibition of HDAC activities by 33 nM TSA has directly disrupted the association of Topo II{alpha} and other scaffold/matrix proteins at the centromeres. As no deleterious effects on chromosomal segregation functions were observed at this concentration of TSA (7), the present data indicate that a substantial reduction (by up to 50% in the neocentromere and 20% in the normal chromosome 10 centro-mere) in the size of the original enhanced S/MAR domain does not noticeably affect centromere function.

Compared with 33 nM TSA, the inhibition of scaffold/matrix attachment by the naturally occurring DNA-intercalating antibiotic DST at a concentration of 75 µg/ml results in a greater reduction of the S/MAR domain, by approximately two-thirds, from ~3.5 Mb to ~1.2 Mb at the 10q25 neocentromere, and by approximately one-half at the chromosome 10 centromere. This reduction is accompanied by a decrease in the size of the CENP-A-binding domain at the neocentromere by ~40%, from ~330 to ~196 kb; however, no lateral shift in the position of the CENP-A-binding domain is observed, which contrasts the previously described lateral shift of this domain by 320 Mb following 33 nM TSA treatment (7).

DST inhibits the binding of a number of scaffold/matrix proteins such as Topo II, SAF-A, SATB1, Erp60, and P230 (49–53). By quantitative immunofluorescence, 75 µg/ml DST causes a general reduction in the level of centromeric Topo II{alpha}, as well as the levels for CENP-A, CENP-C, and Hp1{alpha}. CENP-A is a histone H3 variant that binds specifically to centromeric nucleosomes. CENP-C belongs to a group of kinetochore proteins that includes CENP-B, CENP-E, CENP-F, and CENP-G, which have been shown to be associated with the chromosome scaffold/matrix (54–59), whereas Hp1{alpha} is a major component of the heterochromatin nucleation protein complexes (60, 61). Notwithstanding the possible effects of DST on other cellular processes, the observed dramatic reduction in the size of the enhanced S/MAR domain and the binding levels of these essential centromeric/pericentric proteins presumably contribute to the observed impairment in mitotic segregation function. This impaired function provides an explanation for the observed increase in the level of the mitotic checkpoint protein BubR1 on the total centromere population following DST treatment. Other studies have also shown that the disruption of chromosomal scaffold/matrix proteins decreases mitotic chromosome condensation and sister chromatid resolution (62–64).

Transcriptional potential is in part regulated by histone acetylation. Transcriptionally active chromatin in the human genome contains hyperacetylated histones, whereas constitutive and facultative heterochromatin contain virtually no acetylated histones (65, 66). Hyperacetylation of histone H3 lysine 9 alters nucleosomal conformation resulting in the DNA being more accessible to transcription factors (67), whereas TSA-induced hyperacetylation has been shown to induce partial relief of transcriptional repression (68). Our results indicate that treatment with 33 nM TSA does not result in any significant up- or down-regulation of expressing genes, or the activation of non-expressing or inactivation of expressing genes within or immediately surrounding the 10q25 neocentromere. An exception is the HCG39837 gene, which is up-regulated at this TSA level; however since a similar derepressed state of this gene is seen in the normal chromosome 10, it does not appear to be neocentromere chromatin-related.

Other investigators have shown that transcriptional potential can be affected by DST through the inhibition of S/MAR elements within genes. For example, treatment with this drug has resulted in the repression of the mouse mammary tumor virus (MMTV) and {beta}-interferon (IFN-{beta}) gene promoters (50, 69). However, at 75 µg/ml DST, we have detected no significant alteration to the expression activities of any of the genes residing at the 10q25 neocentromere.

This study therefore demonstrates that moderate inhibition of histone deacetylation with TSA, and DNA intercalation with DST, both result in the substantial inhibition of scaffold/matrix attachment at 10q25 neocentromere and at the native centro-mere. While no detectable mitotic phenotype is observed at the TSA dosage used, the DST-induced effect is accompanied by chromosomal missegregation, reduced protein binding, and the up-regulation of mitotic checkpoint mechanisms, suggesting that scaffold/matrix attachment properties are closely linked to those of a wider network of scaffold/matrix and centromeric/pericentric proteins. The absence of detectable alteration in gene expression at the neocentromere compared with the corresponding normal genomic region (21), and the failure of gene expression levels to respond to the reorganization of the S/MAR domain following TSA and DST treatments, further suggest that despite the formation of specialized centromeric chromatin, transcriptional regulation at the neocentromere is indistinguishable from that of the corresponding normal genomic loci.


   FOOTNOTES
 
* This work was supported by National Health and Research Council and NIGMS, National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ Recipient of a Melbourne Research Scholarship from the Dept. of Pediatrics, University of Melbourne. Back

A Senior Principal Research Fellow of the National Health and Medical Research Council of Australia. To whom correspondence should be addressed: Murdoch Childrens Research Institute, Royal Children's Hospital, Flemington Rd., Parkville 3052, Australia. Tel.: 61-3-8341-6306; Fax: 61-3-9348-1391; E-mail: andy.choo{at}mcri.edu.au.

1 The abbreviations used are: TSA, trichostatin A; DST, distamycin A; S/MAR, scaffold/matrix attachment region; M10, mardel(10) chromosome; N10, normal chromosome 10; LIS, 3,5-diiodasalicylic acid; R, ratio of enhancement; {Delta}CT, change in PCR cycle threshold; ChIP, chromatin immunoprecipitation assay; DAPI, 4',6-diamidino-2-phenylindole; RT-PCR, reverse transcriptase-PCR; PMSF, phenylmethylsulfonyl fluoride. Back


   ACKNOWLEDGMENTS
 
We thank P. Kalitsis for anti-CENP-C and CENP-A, T. Yen for anti-BubR1, and P. Chambon for anti-HP1{alpha} antibodies.



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