CENP-A nucleosomes localize to transcription factor hotspots and subtelomeric sites in human cancer cells
© Athwal et al.; licensee BioMed Central. 2015
Received: 26 November 2014
Accepted: 1 December 2014
Published: 13 January 2015
The histone H3 variant CENP-A is normally tightly regulated to ensure only one centromere exists per chromosome. Native CENP-A is often found overexpressed in human cancer cells and a range of human tumors. Consequently, CENP-A misregulation is thought to contribute to genome instability in human cancers. However, the consequences of such overexpression have not been directly elucidated in human cancer cells.
To investigate native CENP-A overexpression, we sought to uncover CENP-A-associated defects in human cells. We confirm that CENP-A is innately overexpressed in several colorectal cancer cell lines. In such cells, we report that a subset of structurally distinct CENP-A-containing nucleosomes associate with canonical histone H3, and with the transcription-coupled chaperones ATRX and DAXX. Furthermore, such hybrid CENP-A nucleosomes localize to DNase I hypersensitive and transcription factor binding sites, including at promoters of genes across the human genome. A distinct class of CENP-A hotspots also accumulates at subtelomeric chromosomal locations, including at the 8q24/Myc region long-associated with genomic instability. We show this 8q24 accumulation of CENP-A can also be seen in early stage primary colorectal tumors.
Our data demonstrate that excess CENP-A accumulates at noncentromeric locations in the human cancer genome. These findings suggest that ectopic CENP-A nucleosomes could alter the state of the chromatin fiber, potentially impacting gene regulation and chromosome fragility.
Hallmarks of the cancer state include large-scale gene expression changes , chromosomal rearrangement, and aneuploidy [2–6]. While the mechanistic basis for these events remains under investigation, such events have been attributed to DNA methylation changes , telomere disruption , repair and DNA damage pathway protein defects , replication distress , and misregulation of the centromere-specific histone H3 variant, CENP-A [10–13]. CENP-A’s normal function is to serve as the sole structural marker for centromeric chromatin identity , by directly associating with a triad of inner kinetochore proteins CENP-C, CENP-N and CENP-B , which in turn recruit the rest of the kinetochore and microtubules to ensure faithful genome segregation during mitosis . Consequently, mislocalization of CENP-A to noncentromere regions is believed to be a prognostic marker for aneuploidies driven by chromosomal breakage and rearrangements, emanating from bicentric chromosomes [10, 11, 13, 17, 18]. Indeed, artificial overexpression studies in flies demonstrate that under certain conditions, CENP-A can seed neocentromeres [17, 19]. However, when moderately overexpressed to the levels similar to that previously seen in cancer cells [10, 11], CENP-A does not easily seed neocentromeres , but rather expands centromere domains . In related studies, overexpressed yeast or Drosophila CENP-A accumulates in the euchromatic arms, where it is continually targeted for proteolysis and subsequently degraded [22, 23]. Indeed, a recent study confirms this occurs also in human HeLa cells, wherein forced artificial overexpression of tagged CENP-A results in accumulation at ectopic locations . However, although CENP-A mRNA is innately overexpressed several fold in a number of human solid tumors, including colorectal tumors [10, 11, 18, 25–27], its behavior in cancer cells has not been investigated.
To elucidate consequences associated with CENP-A misregulation, we examined CENP-A mRNA and protein levels, partners, structure, and global nucleosome occupancy in human primary normal and colorectal cancers cells, as well as in primary tumors. We report that CENP-A is overexpressed at the mRNA and protein level in some human colorectal cancers. This excess CENP-A partners with histone H3, and associates with transcriptionally coupled chaperones ATRX and DAXX in colorectal cancer cell lines. This distinct class of noncentromeric CENP-A nucleosomes forms a stable octameric nucleosomal species as detected by atomic force microscopy (AFM) and confirmed by high-resolution DNA analysis, which demonstrates binding of 150 to 170 bp of DNA. These distinctive CENP-A nucleosomes localize to open regions of the genome as mapped by DNase I hypersensitivity (DHS), such as promoters of genes, and contain transcription factor binding motifs. In addition, we observe a correlation between large clusters of CENP-A and subtelomeric locations including the fragile region at 8q24. In this 8q24 region, we show that CENP-A is bound to CENP-C, a phenomena that also occurs in early human colorectal tumors, but not in normal human colon cells. Taken together, our data uncover a new role for a classical histone variant in human cancer cell lines.
CENP-A is overexpressed, and ectopic CENP-A nucleosomes associate with H3, ATRX, and DAXX in colorectal cancer cells
CENP-A, ATRX and DAXX are overexpressed in colorectal cell lines
0.57 ± 0.12
2.19 ± 0.73
9.40 ± 1.25
1.22 ± 0.12
1.26 ± 0.09
1.55 ± 0.55
5.22 ± 0.14
2.07 ± 0.07
1.32 ± 0.10
2.89 ± 0.95
13.94 ± 4.01
3.66 ± 0.15
1.35 ± 0.11
4.69 ± 1.80
19.05 ± 8.10
3.18 ± 0.15
1.75 ± 0.23
1.24 ± 0.25
22.40 ± 3.91
2.80 ± 0.22
Protein levels of CENP-A, CENP-B, H2A.Z, H3, ATRX, and DAXX in normal and cancer cell lines
CpB fold enrichment (CpB IP/seq. CpA IP) a
Ectopic CpA fold enrichment (Seq. CpA IP/CpB IP) a
H2A.Z fold enrichment (Seq. CpA IP/CpB IP) a
H3 fold enrichment (Seq. CpA IP/CpB IP) a
ATRX fold enrichment (Seq. CpA IP/CpB IP) a
DAXX fold enrichment (Seq. CpA IP/CpB IP) a
A recent study has reported that artificially overexpressed tagged CENP-A associates with the H3.3 chaperone DAXX in HeLa cells . Since both, ATRX and DAXX, were overexpressed in HeLa and SW480 cells relative to normal (Figure 1A), we next investigated association of these chaperones with centromeric and ectopic CENP-A IPs from normal colon, HeLa, and SW480 cells. We noted a strong association between these chaperones and CENP-A in colorectal cancer cells (Figure 1F, Table 2). In contrast to a recent report demonstrating that artificially overexpressed CENP-A relies on DAXX/ATRX to associate at ectopic locations, we were unable to conclude that there was specific enrichment exclusive to the ectopic CENP-A fraction, but rather noted both centromeric and ectopic CENP-A fractions associated with these transcription-coupled chaperones.
These results outline three distinguishing characteristics of the ‘high’ CENP-A state in human cells: increased association of CENP-A with H3.3 chaperones ATRX and DAXX, increased interaction of canonical H3 with ectopic CENP-A, and an abundance of the ectopic CENP-A fraction.
Ectopic CENP-A nucleosomes have altered conformations
In vivo, CENP-A and H3 do not mix within single nucleosomes . Given the association of ectopic CENP-A and H3 above, we were curious whether such nucleosomes, or their chromatin fibers, might present an alteration of nucleosomal features. To this end, we turned to high-resolution microscopy. In an extensive series of studies using AFM coupled to other biochemical assays, we have previously shown that in contrast to in vitro reconstituted recombinant CENP-A nucleosomes, which are octameric and generally indistinguishable from H3 nucleosomes [38–41], CENP-A nucleosomes purified from native human centromeres from HeLa or HEK cells, display smaller dimensions [42, 43], and attain a stable octameric height only at specific points of the human cell cycle . Therefore, we next used AFM to measure native nucleosomal dimensions of ectopic versus centromeric and recombinant CENP-A nucleosomes.
Ectopic CENP-A nucleosomes are stable octamers
SW480 bulk chromatin (n)
Recomb. H3 octamer (n)
Recomb. CENP-A octamer (n)
SW480 total CENP-A (n)
SW480 ectopic CENP-A (n)
Height a (nm)
2.53 ± 0.26 (401)
2.32 ± 0.32 (57)
2.37 ± 0.34 (57)
2.17 ± 0.35 (546)
2.46 ± 0.40 (135)
Diameter b (nm)
13.8 ± 1.4 (401)
12.1 ± 1.4 (20)
10.6 ± 1.4 (20)
14.1 ± 2.9 (546)
14.5 ± 3.0 (135)
These data indicate that two distinct populations of CENP-A nucleosomes co-exist in colorectal cancer cells: one that contains diminutive features similar to that previously reported from native centromeres, and another that closely mimics the stable H3 or CENP-A octameric nucleosome in vitro.
Ectopic CENP-A hotspots localize to DNase I hotspots and transcription factor binding sites
Genome coverage of chromatin input samples from normal colon, HeLa, and SW480
Number of reads
Percent genome coverage (%)
Normal colon a
HeLa - rep 1 a
HeLa - rep 2 a
SW480 - rep 1 a
SW480 - rep 2 a
To investigate the nature of ectopic CENP-A hotspots, we next classified them with respect to known genomic and epigenetic features. Irrespective of the difference in the total number of hotspots, a sizeable portion of ectopic CENP-A was found at gene loci, with 23%, 38%, and 44% of ectopic hotspots at genes in HeLa, SW480, and normal colon cells, respectively (Figure 4B, right panel for histogram, and Additional file 1 contains the dataset of all hotspots discovered). Thus, CENP-A presence at genes seems to be a common feature, as it was found in all cell lines examined, with a significant fraction of those sites present at promoters of genes (7%, 15%, and 34% in HeLa, SW480, and normal colon cells, respectively). Indeed, CENP-A enrichment at promoters is statistically significant in SW480 compared to HeLa cells (Fisher’s exact test P value: 0.0174), suggesting that colon cells tend to accumulate CENP-A at open chromatin regions (specific examples are shown in Figure 4C).
In the experiments above, we noted that the transcription-coupled chaperones ATRX and DAXX are overexpressed in SW480 cells (Figure 1A), whereas levels of the CENP-A chaperone HJURP, which normally restricts CENP-A to centromeres [23, 31, 32, 51, 52], generally did not correlate with increased CENP-A levels. We wondered whether ectopic CENP-A accumulation at promoters is linked to HJURP presence. Therefore, we performed HJURP IPs from cross-linked chromatin, using the CENP-B depletion strategy as above (Figure 1C), followed by high throughput sequencing analysis to unveil potential sites of ectopic HJURP localization. We were unable to obtain robust ectopic HJURP enrichment. Fewer than 300 HJURP hotspots were detected in SW480 cells (Figure 4D, Additional file 1 for list of HJURP hotspots). Although 36% of the 942 HeLa CENP-A hotspots correlate with HeLa HJURP sites, only 5% of SW480 CENP-A hotspots co-localize with SW480 HJURP sites (Figure 4D). Such paucity of noncentromeric HJURP sites overlapping with ectopic CENP-A sites in SW480 is consistent with HJURP’s primary documented role as a centromere-targeted chaperone, and would support the hypothesis that overexpressed CENP-A can co-opt alternative chaperone pathways to accumulate at genes, as has recently been shown for forced overexpression of CENP-A in human cells .
As expected, the vast majority of DHS enrich primarily at promoters in HeLa and SW480 cells (Figure 5B, right panel shows histograms, and Additional file 1 for a list of DHS), and overlap completely with the compendium of aggregated DHS clusters identified by the ENCODE project for 129 human cell lines (Figure 5C). DHS identified in our data sets included promoters of housekeeping genes, oncogenes, and tumor suppressor genes (Additional file 1 for list of all DHS, examples in Figure 5D). For example, the Myc gene, a known regulator atop a cascade of tumor effector proteins , has a large DHS site astride its promoter in SW480 cells (Figure 5D). Indeed, the gene encoding CENP-A itself has a strong DHS site upstream of its promoter specifically in SW480 cells but not in HeLa cells, providing a satisfying correlation between increased accessibility of the CENP-A gene promoter, and excess CENP-A mRNA (and subsequently, protein) present in SW480 cells (Figure 5D).
When comparing DHS hotspots to ectopic CENP-A sites, we observed that a large fraction of DHS tracks with ectopic CENP-A locations (Figure 5B, left and middle panels). Globally, about approximately 380 CENP-A sites overlap with DHS sites in HeLa (Figure 5B, left and middle panels), whereas twice that number, approximately 740 SW480 CENP-A hotspots align perfectly with SW480 DHS.
Ectopic CENP-A is enriched at DNase I hypersensitive (DHS) sites and transcription factor binding sites
CENP-A hotspots a
DNase clusters b
Txn Factor ChIP c
Analysis of total sequence tags obtained from normal colon, HeLa and SW480 CENP-A immunoprecipitations (IPs) demonstrates they are not enriched in repetitive elements
# total tags
Ectopic CENP-A nucleosomes cluster at subtelomeric sites, including 8q24/Myc, in colorectal cancer cells and tumors
The deep sequencing data uncovered a 30 MB region of CENP-A and DHS co-enrichment on the 8q24/Myc locus in SW480, but not HeLa or normal cells. This enrichment was apparent even after correcting for copy number amplification of this locus (Figure 8B, see input-adjusted hotspots below the tag density tracks). This result was surprising, because although this region has been extensively studied, there are no extant reports of it containing unusual histone variants. Furthermore, large domains of CENP-A usually exist only in active centromeres, wherein they attract inner kinetochore proteins such as CENP-C, which connect CENP-A to the outer kinetochore during mitosis . We tested whether CENP-C was enriched in the 8q24 region. Using CENP-A and CENP-C ChIP followed by quantitative PCR (qtPCR) for probes spanning this 30 MB locus (primer locations indicated in Figure 8A), we observed robust enrichment of both CENP-A and CENP-C within the domain spanning the 8q24 locus (Figure 8C, qtPCR graph). We reasoned that a CENP-A/CENP-C domain spanning 30 MB should be visible by immunofluorescence. Therefore, we used a combination of 8q24-FISH and CENP-A-IF to visualize this region. To ensure the accuracy of detection, we first tested the 8q24 FISH probe on metaphase spreads from normal human lymphocytes. As expected, we observed two discrete subtelomeric signals per chromosome, for a total of 4 N per mitotic cell (Figure 8D, upper left panel). We next tested whether 8q24 was amplified and translocated in the cancer cells, as would be expected for this locus. Co-FISH for 8q24 and the native centromere 8 demonstrates one set of 8q24 signals originating from a subtelomeric location on chromosome 8, as well as a number of additional signals emanating from translocated sites (Figure 8C, upper right panel).
CENP-A is enriched at the 8q24/ Myc locus in tumor-derived SW480 cells and in human tumors
Cells with co-localization a (%)
Cells counted (N)
We were intrigued by the presence of CENP-A/CENP-C at the 8q24 locus in the SW480 colorectal cancer SW480 cell line, which was derived from a late stage colorectal tumor nearly 30 years ago . We sought to understand how early in tumorigenesis CENP-A might mislocalize to 8q24. We acquired primary early and late stage colorectal tumors, as well as matched normal tissue from the same patients, and performed FISH/IF to test co-localization of CENP-A to 8q24. The co-IF/FISH data show that the 8q24/Myc locus is amplified in all four tumors, and that CENP-A domains are enriched on one of these 8q24 loci ranging from 33 to 78% of tumor cells, depending on the donor (Figure 8C, lowest set of panels for representative images of normal versus tumor, white arrow points to co-localization, quantification in Table 7). Thus, CENP-A occupancy of this locus is robust and occurs even in early stage tumors.
In this report, we present a comprehensive examination of the histone variant CENP-A in colorectal normal and cancer cells, finding that ectopic CENP-A exists outside centromeres in human cells. Ectopic CENP-A tracks to two distinct types of domains: small regions found at promoters and accessible chromatin; and large domains found at sites of common chromosomal rearrangements. Our report yields a number of specific findings. First, CENP-A, which is innately overexpressed in cancer cells (Figure 1A-B, Table 1), associates with histone H3 (Figure 1E, Table 2), and shows increased association with transcription-coupled chaperones DAXX and ATRX (Figure 1F, Table 2). Second, ectopic CENP-A nucleosomes are stable octamers in configuration (Figure 2, Table 3), containing 125 to 165 bp of DNA (Figure 3B-D). Third, ectopic CENP-A nucleosomal tags are depleted in centromeric consensus satellite sequences (Table 6), and localize instead to unique noncentromeric locations in normal and cancer cell lines (Figure 3A). These nucleosomes occupy genes and promoters (Figure 4B-C), are HJURP-free (Figure 4D), and correlate primarily with hyper-accessible (DHS) chromatin (Figure 5, Table 5). Fourth, CENP-A/DHS ectopic sites co-occupy regions containing known transcription factor binding motifs (Figure 6, Table 5). Lastly, large clusters of CENP-A hotspots exist in regions spanning pericentric and subtelomeric regions specifically in colorectal cancer cells (Figures 7 and 8, Table 7). An example of such a cluster is at a segment of the 8q24 locus spanning the Myc oncogene, which, even in relatively early stage tumors tested in this study, associates with CENP-A and CENP-C (Figure 8B-D, Table 7).
A number of avenues of investigation arise from our observations. Regardless of the absolute amount of ectopic CENP-A, in normal colon cells, and in the cancer cell lines examined, there is a connection between DHS/transcription factor binding sites and ectopic CENP-A (Figures 5 and 6). That CENP-A can compete for regions linked to transcription was initially demonstrated in budding yeast, wherein CENP-A is reported to exist at barely detectable levels in a handful of genic promoters , which increases when CENP-A is artificially overexpressed . Such CENP-A is continually targeted for subsequent proteolysis [23, 52]. Earlier work has also demonstrated that artificial constitutive overexpression of CENP-A in Drosophila cells results in a gradual accumulation and slow removal of CENP-A from chromosome arms , possibly via association with the common histone chaperone RbAp48/p55 . In vitro, common chaperones such as p55 and NAP-1 assemble CENP-A nucleosomes efficiently [41, 66]. However, generally it has not been thought that such phenomena could occur in human cells, with many laboratories publishing studies using tagged/overexpressed CENP-A as a marker for human centromeres. However, a recent report tracking artificially overexpressed human CENP-A has demonstrated that it can occupy ectopic sites, binds histone H3.3, contains octameric size DNA fragments, and is potentially chaperoned by ATRX and DAXX . Indeed, in worms, which form holocentric centromeres that line the edges of chromosomes, normal amounts of CENP-A seed centromeric domains using regions of low nucleosome turnover . Our report demonstrates that a subset of native human CENP-A binds H3, forms octameric height nucleosomes, which localize to accessible chromatin domains at promoters and transcription factor sites at low levels even in normal human colon cells. This process appears to be magnified in amplitude in colorectal cancer cell lines, where a significant fraction of ectopic CENP-A nucleosomes overlap with DHS and transcription factor binding sites (Figure 5 and 6). It is feasible that a default transcription-linked pathway exists to use trace amounts of CENP-A either promiscuously expressed at the wrong time (that is, not at the end of G2 ), or remnant after HJURP-dependent incorporation at centromeres is complete at mid-G1 . Not mutually exclusive to this explanation is the interesting possibility that defects in the timing of CENP-A expression, or promiscuous binding of CENP-A to other chaperones, coupled to defects in proteolysis, might cumulatively conspire to permit increased CENP-A accumulation at transcription factor binding sites in cells.
A functional implication of stable CENP-A occupancy of promoters/DHS and its correlation with transcription factor binding sites is the potential link to gene expression changes reported in cancer cells. It is currently unknown if CENP-A is recruited by, or competes for transcription factor binding sites, either of which would be predicted to impact gene expression. Indeed, the DHS data demonstrate that many of the sites that attract CENP-A are already DHS and transcription factor binding sites, that is, high nucleosome turnover regions in a number of human cell lines. At the vast majority of genes in vivo, octameric H3 nucleosomes, with specific N-terminal tail modifications, dominate the epigenetic regulatory landscape . Ectopic CENP-A nucleosomes would lack known H3 N-terminal tail modifications, and could potentially circumvent traditional epigenetic regulatory cascades. Thus, the functional impact of CENP-A nucleosomes on pre-existing DHS sites, or on promoter architecture, remains an exciting avenue of research. Ongoing studies are focused on whether recruitment of transcriptional activator or repressor complexes is altered in the presence of ectopic CENP-A nucleosomes, and whether such events influence gene expression patterns specifically in the cancer context.
Our study also provides support for a potential role for CENP-A in chromosomal instability. Whereas various artificial overexpression studies over the past decade have clearly established CENP-A’s ability to seed neocentromeres , this study provides a correlation between CENP-A and a defined chromosomal rearrangement at 8q24 in human cancer cells, which is absent in normal colon cells (Figure 8, Table 7). When normal human ES cells are challenged by induced DNA breaks, excess native CENP-A is rapidly mobilized, but does not localize to immediate break sites indicated by gamma-H2A.X staining . However, a recent study used osteosarcoma-derived U2OS cancer cells, in which an artificially induced break was shown to efficiently recruit overexpressed CENP-A:GFP . Thus, depending on the timing of the break, and availability of free histones, CENP-A might enrich during subsequent steps of chromatin re-establishment following repair or translocations of amplified regions in cancer cells. An avenue of research that arises from these findings is elucidating the timing of CENP-A enrichment at breakpoints during tumorigenesis, and investigating its potential role in structural rearrangements of chromosomes in subtelomeric sites such as 8q24.
Increased levels of CENP-A expression have been reported in metastatic prostate, breast, lymphoma, lung and colorectal tumors. Consequently, our observations, combined with other recently published studies on artificially induced hybrid CENP-A/H3 nucleosomes [24, 73], have implications for accumulation of downstream epigenetic defects that arise during tumorigenesis.
All cell culture medium except epithelial cell medium was supplemented with 10% fetal bovine serum and 1X penicillin and streptomycin. Cell culture media DMEM was used for HeLa cells, RPMI for SW480 and DLD1, and McCoy’s media for HCT116 and HT29. Epithelial cell medium was used to culture normal human colon epithelial cell (HcoEpiC). HcoEpiC cells are very slow growing, with the cell cycle lengths ranging from 36 to 90 hours depending on passage number.
Total nuclear proteins extraction
Nuclei were purified from cell lines: HcoEpiC, HeLa, HCT116, DLD1, HT29 and SW480 following published procedure [43, 44]. Total nuclear proteins extracts were prepared in RIPA-Buffer. Equal amount of nuclear proteins were fractioned on SDS page gels, stained and analyzed on Odyssey and amounts were adjusted to equal amount of histone H4 for further analysis. Samples containing equal amount of histone H4 were fractioned on SDS page gel and the amounts of CENP-A, HJURP, ATRX, DAXX and histone H4 were determined by quantitative western blot analysis. Relative concentration of CENP-A in different cell lines was calculated as ratio CENP-A/H4 in a cell line divided by ratio of CENP-A/H4 in normal colon cells. Relative concentrations of ATRX, DAXX, and HJURP were calculated similarly.
Quantitative western blot analysis
Quantitative infrared western blotting was performed using Odyssey Li-Cor CLx system (Lincoln, NE, USA). Briefly, infrared western blot (WB) signal was acquired with high dynamic range and analyzed using Image Studio software. Bands of interest were manually selected and their total intensity quantified with subtraction of median background signal from an area 3-pixels wide above and below the band in the same lane. The resulting total infrared signal values (arbitrary unit) were used for subsequent calculations as indicated.
CENP-A, and CENP-B chromatin immunoprecipitation (ChIP) for WB analysis was performed following published protocol [43, 44]. Briefly, cells were harvested, washed with PBS once, PBS containing 0.1% Tween 20 three times and nuclei were released with TM2 buffer (20 mM Tris-HCl, pH 8.0; 2 mM MgCl2) containing 0.5% Nonidet P40 (Sigma) and 0.5 mM PMSF. The nuclei were washed with TM2 buffer to remove detergent once. To release chromatin, nuclei were digested with 0.3 U/ml MNase (Sigma) for 8 mins and reaction was stopped with the addition of 10 mM EGTA. Nuclei were extracted in low salt buffer (0.5X PBS containing 5 mM EGTA and 0.5 mM PMSF) over night at 4°C. Chromatin IP was performed using Dynabeads-protein G (Life Technologies, Grand Island, NY, USA), Agarose protein A/G plus (Santa Cruz, Santa Cruz, CA, USA) or protein G sepharose 4 Fast flow (GE Healthcare, Laurel, MD, USA) and antibodies listed below.
Antibodies used for chromatin immunoprecipitation, western blot, and immunofluorescence
CENP-A: rabbit CENP-A (Santa Cruz; SC-22787, Santa Cruz, CA, USA); rabbit CENP-A (Millipore; 07-574, Billerica, MA, USA), custom CENP-A (CSEM laboratory); mouse CENP-A (Abcam, ab13939, Cambridge, MA, USA); goat CENP-A (Santa Cruz, sc-11277). HJURP: rabbit HJURP (Bethyl, A302-822A, Montgomery, TX, USA). H2A.Z: rabbit H2A.Z (Abcam, ab4174). CENP-C: goat CENP-C (Santa Cruz, sc11285). CENP-B: rabbit CENP-B (Santa Cruz, sc-22788), rabbit CENP-B (abcam, ac25743). ACA serum (Centromere Ab Positive Serum) BBI Solutions (Cat #SG140-2, Lot #3284-146-2, Cardiff, UK).
Chromatin IP for CHIP-Seq was performed similarly as above, except for the following modifications: MNase concentration was 0.6U/ml, digestion time was 10 min for cancer cells and 8 min for normal HcoEpiC cells, and nuclei were treated with 0.05 to 0.1% formaldehyde for gentle in situ crosslinking within intact nuclei for 30 min at RT, as indicated in ENCODE protocols, before extraction of chromatin in low salt buffer. IP-enriched chromatin was eluted with 100 mM NaHCO3 and 1% SDS at 65°C for 2 h. To reverse cross-linking, NaCl was added to the final 200 mM concentration and incubated at 67°C for additional 4 h followed by RNase A (150 μg/ml) treatment for 1 h and then proteins were digested with proteinase K (100 μg/ml) for 3 h. The DNA was purified by phenol extraction and ethanol precipitated. The DNA was repaired using PreCR Repair mix (New England Biolabs, Ipswich, MA, USA) following manufacturer’s instructions. DNA was purified using Chroma Spin columns (Clontech, Mountain View, CA, USA).
Nucleosome reconstitution in vitro
Lyophilized recombinant histones (a gift from Jennifer Ottesen) were unfolded in 7 M guanidinium HCl, mixed in equimolar amounts (Either H3 or CENP-A and one each H2A, H2B, H4), and refolded into 2 M NaCl according to the protocol by Luger et al. . Refolded nucleosomes were reconstituted onto a plasmid containing a ‘Widom 601’ positioning sequence (a gift from Carl Wu) using sequential salt dialysis adapted for low volumes. Briefly, histone octamers were mixed with plasmid DNA at 0.9:1 ratio in 2 M NaCl, 10 mM Tris-Cl pH = 8.0, 1 mM EDTA (0.18 mg/ml histones; 0.2 mg/ml DNA) and incubated on ice for 30 min. Next, 40 ul of histone/DNA mix was layered onto a dialysis disc (Millipore, 0.025um, Billerica, MA, USA) covered with a dialysis membrane (Thermo Scientific, 7000 MWCO, Waltham, MA, USA) and floated on the surface of 50 ml pre-chilled 1 M NaCl, 10 mM Tris-Cl pH = 8.0, 1 mM EDTA buffer. Sequential dialysis steps against 1 M, 0.8 M, 0.6 M, and 0.15 M NaCl (each with 10 mM Tris-Cl pH = 8.0, 1 mM EDTA) were carried out for 2 hours at 4°C (0.6 M dialysis was done overnight). Reconstituted chromatin was diluted one hundredfold in 1X PBS, 2 mM MgCl2 buffer and imaged on AP-mica .
Atomic force microscopy imaging and analysis
AFM imaging of bulk and immunoprecipitated CENP-A chromatin was performed essentially as described previously [43, 44] with some adaptations (see manual analysis below). Extracted or IP-eluted chromatin was deposited on APS-mica (prepared as described by Dimitriadis et al., 2010 ) in the presence of divalent magnesium ions. The sample was incubated for 10 minutes, briefly rinsed with MilliQ water, and dried in a vacuum chamber. The sample was imaged using AFM 5500 (Agilent Technologies, now Keysight Technologies, Santa Clara, CA, USA) operating in AC mode (noncontact/tapping), equipped with either OTESPA or TESP silicone tip (Bruker Nano, Santa Barbara, CA, USA) with a nominal radius of 3 to 7 nm. Images were captured at 4096x4096 resolution with an instrument operating at setpoint equivalent to 65% to 75% of free amplitude (typically 1.5 to 2.5 V).
Acquired images were processed using Gwyddion (gwyddion.net) software (flattening, line correction, and polynomial background subtraction) and analyzed either manually (see below), or, for bulk controls, using NIH Image J software (imagej.nih.gov/ij/) Particle Analysis function. Briefly, the images were limited by threshold (to remove tip convolution) and filtered to include only round or elliptical shapes. Max. height, total area, and volume information was collected. SigmaPlot software was used to statistically analyze the data and generate graphs. For ectopic and recombinant CENP-A and H3 nucleosomes, manual measurements were performed in Gwyddion software to ensure that strictly DNA-associated particles were included (diameter cutoff <20 nm).
BioAnalyzer analysis of DNA fragments obtained from chromatin immunofluorescence
DNA samples were prepared according to manufacturer’s recommendations and ran on High Sensitivity DNA Chips (Agilent Cat #5067-4626, Wilmington, DE, USA) on the Agilent 2100 BioAnalyzer system. Data with the control lower and upper limits were automatically called or manually aligned (see figure) with the Agilent 2100 Expert Software.
DNase I digestion of chromatin from HeLa and SW480
HeLa and SW480 cells were harvested with trypsin, washed twice with ice cold PBS containing 0.1% Tween 20, and resuspend in low sucrose buffer (15 mM Tris-HCl, PH 8.0, 15 mM NaCl, 60 mM KCl, 1 mM EDTA, 0.5 mM EGTA, 1 mM spermidine, EDTA free protease inhibitors). Cells were mixed (1:1) with same buffer containing 0.04% NP-40 and nuclei were released at 4°C. Nuclei were harvested by centrifugation, washed with low sucrose buffer and DNase I digestion was performed with 20 million nuclei as previously described [76, 77]. DNA fragments of 100 to 500 bp from a chromatin digestion with 60 U/ml DNase I (Sigma, St. Louis, MO, USA) were purified using sucrose gradient  and DNA was precipitated in 0.1 volume of sodium acetate and 0.7 volume of isopropanol.
Bioinformatic analysis of chromatin immunoprecipitation-seq, DNase-seq, and TOMTOM DNA motif enrichment
Purified DNA from ChIP or DNase I digested chromatin were used to prepare libraries for Illumina high-throughput sequencing as described in manufacturer’s protocol (Illumina Sequencing, San Diego, CA, USA). Libraries were sequenced to generate 35 bp single end reads using Illumina GAII sequencer at the Advanced Technology Center, NCI (Frederick, MD). Sequence reads were mapped to the reference genome hg19 by the CASAVA 1.8.2 pipeline.
Hotspot detection for DNase-seq
Hotspot detection and input adjustment for chromatin immunoprecipitation-seq
The hotspot detection algorithm was similarly applied to ChIP-seq data with the following modification. The sequencing data from matching input samples are used for the processing of ChIP data, as a measure of background signal that might be significant. After normalizing the input data to match the number of tags in the ChIP data, the number of input tags is subtracted from the number of ChIP tags in the target window before calculating its z-score.
DNA Motif discovery analysis
A motif discovery analysis on selected DNA sequences was performed using MEME  on a parallel cluster at the NIH Biowulf supercomputing facility (meme.nbcr.net/). DNA sequences for MEME input were from the top 2000 (by tag density) hotspots. To limit the computational load, only the 200-bp regions with the highest tag density were used instead of the entire width of hotspots in cases where the hotspots spanned greater than 200 bp. The width of motif for searching was set to 6 and 20 for minimum and maximum, respectively. To identify binding motifs for known transcription factors, we queried individual position-specific matrices against the Transfac database using the Tomtom software (http://meme.nbcr.net/meme/cgi-bin/tomtom.cgi). We retrieved statistically significant matches that share the majority of specific nucleotides in the sequence motifs.
Quantitative PCR analysis
Quantitative (real time) PCR was performed using the IQ-Sybr Green Supermix kit from BioRad (#170-8880, Hercules, CA, USA) in 25 μl reaction according to the manufacturer’s protocol and samples were amplified using I-cycler fitted with MyIQ Single color real time PCR detection system (BioRad, Hercules, CA, USA). In all experiments no template- and Mock IP (normal IgG IP; negative control) controls, and input chromatin DNA and IP samples (CENP-A & CENP-C) were included from same experiment. The qtPCR reactions were setup in triplicate thus giving three threshold cycle numbers (Ct) for each sample. Experiment was repeated three separate times. Enrichments and fold changes were calculated as follows:
Ct.i = average Ct of input
Ct.m = average Ct of mock IP
Ct.IP = average Ct of IP samples (CENP-A and CENP-C)
STDV.i = standard deviation of input
STDV.m = standard deviation of mock
STDV.IP = standard deviation of IP
Step 3 Fold Enrichment = FC of IP/ FC of Mock IP ± E
Identification and labeling of fluorescent in situhybridization probe for 8q24/Myc
Five overlapping bacterial artificial chromosomes (BAC) containing human chromosome 8q24 region (chr 8: 125,771,341-127,401,859) and a larger region spanning 8q24/Myc (location of both probes are depicted on Figure 8B) were selected and obtained from commercial source (Invitrogen, Grand Island, NY, USA). DNA was isolated from each BAC and labeled with biotin-dUTP and hybridized to normal blood lymphocytes metaphase-spread slides. Each BAC was evaluated for intensity and specificity of hybridization at target region. The BAC named RP11-150 N13 was selected to be used as a probe for 8q24 (chr 8:126,377,028 to 126,556,325), and a previously published Myc probe was used to confirm the results . For probes, 2 μg BAC DNA was labeled with biotin-dUTP by nick translation in the presence of 4 nmol/L labeled nucleotide. Approximately 100 to 200 ng of labeled BAC probe was ethanol precipitated in the presence of 20 μg each salmon sperm DNA and human Cot1 DNA. The dry pellet was dissolved in 5 to 6 μl of hybridization buffer. The hybridization buffer contained 50% deionized formamide, 20% dextran sulfate and 4X SSC. The probe was denatured for 5 min at 80°C and then pre-annealed for 1 h at 37°C before adding to the slides for hybridization.
Co-immunofluorescence and fluorescent in situ hybridization experiments
The IF on metaphase chromosomes, interphase cells from cell lines and tumor-normal patient sample cells, was performed on unfixed cells following published protocol with some modifications [80, 81]. Enrichment of mitotic cells was achieved by double thymidine block to arrest cells in G1 phase of cells cycle. Actively growing culture was treated 5 mM thymidine for 18 to 20 h. The cells were released from first block and grown in fresh medium for 10 h followed by second block with 5 mM thymidine for 12 h. Cells were released from second block and cultured further in fresh medium for 9 h. These cells were either harvested to make slides or treated with 100 μg/ml colcemid (Roche, Indianapolis, IN, USA) for 1 h to make metaphase chromosomes and then harvested to make slides. The cells were harvested with trypsin and washed with PBS, resuspended in 75 mM KCl, and incubated at 37°C for 13 min and then placed on ice. Cells were cytospun onto glass slides for 5 min at 600 rpm. After air drying, the slides were incubated in freshly prepared KCM buffer (120 mM KCl, 20 mM NaCl, 10 mM Tris-HCl, pH 8.0, 0.5 mM EDTA) containing 0.1% Triton X-100 and protease inhibitors (1 μg/ml aprotinin, pepstatin A, Leupeptin and antipain each) for 15 min at room temperature (RT) followed by blocking (KCM buffer containing 3% BSA, protease inhibitors and 1:100 dilution normal IgG) for 30 min and primary antibody (KCM buffer containing 1% BSA, protease inhibitors and 1:100 dilution normal IgG) for 1 h. The slides were washed with KCM three times 5 min each at RT followed by secondary antibody staining for 1 h at RT. The slides were washed with KCM buffer four times 5 min each at RT, fixed with 10% buffered formalin for 10 min at RT, washed with H2O three times 5 min each at RT, incubated in carnoy’s fixture for 30 min at RT followed by dehydration in ethanol series (70, 95 and 100% ethanol) 5 min each and air dried.
For FISH, slides were equilibrated in 2X SSC for 5 min and digested with pepsin (10 μg/ ml) for 3 min at 37°C. Pepsin digestion time varied for different samples based on amount of cytoplasm left after spinning cells on slides or age of slides. The slides were washed three times in 2X SSC and dehydrated in ethanol series. The DNA on slides was denatured in 70% formamide and 2X SSC at 80°C for 5 min. The slides were incubated in ice cold 70% and 95% ethanol for 3 min each followed by 100% ethanol for 5 min at RT. Then denatured slides were hybridized with pre-annealed for 20 to 24 h at 37°C. At the end of hybridization, the slides were washed in 50% formamide and 2X SSC three times for 5 min each at 45°C, 0.2X SSC four times for 5 min each at 65°C and 2X SSC at room temperature once for 5 min. After washing, slides were incubated with blocking buffer (4× SSC/0.1% Tween-20, 3% bovine albumin) containing normal sheep or goat IgG (1:100 dilution) for 1 h at 37°C. The slides were then incubated with 1:1000 dilution streptavidin alexa 488 (Invitrogen, Grand Island, NY, USA) in developing buffer (4X SSC/0.1% Tween 20, 1% BSA) containing normal IgG for 1 h. The slides were washed in 4X SSC/0.1% tween 20 solution four times at 45°C followed by two washes in 2X SSC at room temperature. Slides were air dried and mounted with aqueous mounting media containing DAPI (Vector Labs, Burlingame, CA, USA). The slides were observed with a DeltaVision RT system (Applied Precision, GE Healthcare, Issaquah, WA, USA) controlling an interline charge-coupled device camera (Coolsnap; Roper) mounted on inverted microscope (IX-70; Olympus America, Center Valley, PA, USA). Images were captured using the 100X objective at 0.06 μm z-sections, de-convolved, and 2D-projected using softWoRx (api.gehealthcare.com/api/softworx-suite.asp). One hundred interphase cells were analyzed for CENP-A and 8q24 for all cell lines. For tumors, the number of cells analyzed ranged from 70 to 85 except for one tumor in which fifty cells were analyzed due to insufficient material.
Tumor and matched normal tissue
Tumor/normal tissues were obtained from the CHTN network. The pathology report indicated Tumor 1, Tumor 2, and Tumor 3 were moderately differentiated stage three tumor with no metastasis, high grade poorly differentiated stage three tumor with metastasis to one lymph node, and low grade well differentiated stage three tumor with no metastasis, respectively. Tumor cells were minced in buffer containing 250 mM sucrose, 15 mM Tris- HCl PH 7.5, 15 mM NaCl, 60 mM KCl, 1 mM EDTA, 0.5 mM EGTA, 0.15 mM spermine, 0.5 mM Spermidine and protease inhibitors (adapted from Dalal et al., 2005 ). Cells were collected by centrifugation at 600 g (1500 rpm) for 10 min at 4°C. The cell pellet was washed twice with same buffer. The cell pellet was resuspended in buffer containing 2 M sucrose instead of 250 mM and spun at 16,000× g for 30 min at 4°C. The cells were washed with buffer containing no sucrose and cells were cytospun onto glass slides for 5 min at 600 rpm. The slides were air dried and processed for IF and FISH as above.
- CENP-A (-B/C/N):
centromere protein A (B/C/N)
DNase I hypersensitive site
Holliday junction recognition protein
We thank the NCI-ATP SAIC High Throughput Illumina Sequencing Facility for deep sequencing and subsidies; Dr. Carl Wu for the gift of 601 plasmid; Dr. Jennifer Otteson for the gift of recombinant histones; Dr. Sam John for expert advice on DHS assays; Drs. Andre Nussenzweig, Tom Misteli, Minh Bui, and Delphine Quenet for critical comments on the manuscript; Tatiana Karpova in the LRBGE Imaging Facility; and the Mid-Atlantic Cooperative Human Tissue Network for tumor samples.
The Intramural Research Program of the National Cancer Institute supported all authors in this study. SF volunteered as a guest researcher for the bulk of this study and was subsequently supported by the NCI post-baccalaureate program.
- Chan TA, Glockner S, Yi JM, Chen W, Van Neste L, Cope L, et al.: Convergence of mutation and epigenetic alterations identifies common genes in cancer that predict for poor prognosis. PLoS Med 2008, 5:e114. 10.1371/journal.pmed.0050114View ArticlePubMed CentralPubMedGoogle Scholar
- Thompson SL, Compton DA: Chromosomes and cancer cells. Chromosome Res 2011, 19:433–444. 10.1007/s10577-010-9179-yView ArticlePubMed CentralPubMedGoogle Scholar
- Ganem NJ, Pellman D: Linking abnormal mitosis to the acquisition of DNA damage. J Cell Biol 2012, 199:871–881. 10.1083/jcb.201210040View ArticlePubMed CentralPubMedGoogle Scholar
- Holland AJ, Cleveland DW: The deubiquitinase USP44 is a tumor suppressor that protects against chromosome missegregation. J Clin Invest 2012, 122:4325–4328. 10.1172/JCI66420View ArticlePubMed CentralPubMedGoogle Scholar
- Gordon DJ, Resio B, Pellman D: Causes and consequences of aneuploidy in cancer. Nat Rev Genet 2012, 13:189–203.PubMedGoogle Scholar
- Forment JV, Kaidi A, Jackson SP: Chromothripsis and cancer: causes and consequences of chromosome shattering. Nat Rev Cancer 2012, 12:663–670. 10.1038/nrc3352View ArticlePubMedGoogle Scholar
- Stimpson KM, Song IY, Jauch A, Holtgreve-Grez H, Hayden KE, Bridger JM, et al.: Telomere disruption results in non-random formation of de novo dicentric chromosomes involving acrocentric human chromosomes. PLoS Genet 2010., 6: doi:10.1371/journal.pgen.1001061Google Scholar
- Downs JA, Nussenzweig MC, Nussenzweig A: Chromatin dynamics and the preservation of genetic information. Nature 2007, 447:951–958. 10.1038/nature05980View ArticlePubMedGoogle Scholar
- Barlow JH, Faryabi RB, Callen E, Wong N, Malhowski A, Chen HT, et al.: Identification of early replicating fragile sites that contribute to genome instability. Cell 2013, 152:620–632. 10.1016/j.cell.2013.01.006View ArticlePubMed CentralPubMedGoogle Scholar
- Tomonaga T, Matsushita K, Yamaguchi S, Oohashi T, Shimada H, Ochiai T, et al.: Overexpression and mistargeting of centromere protein-A in human primary colorectal cancer. Cancer Res 2003, 63:3511–3516.PubMedGoogle Scholar
- Tomonaga T, Matsushita K, Ishibashi M, Nezu M, Shimada H, Ochiai T, et al.: Centromere protein H is up-regulated in primary human colorectal cancer and its overexpression induces aneuploidy. Cancer Res 2005, 65:4683–4689. 10.1158/0008-5472.CAN-04-3613View ArticlePubMedGoogle Scholar
- Sullivan LL, Boivin CD, Mravinac B, Song IY, Sullivan BA: Genomic size of CENP-A domain is proportional to total alpha satellite array size at human centromeres and expands in cancer cells. Chromosome Res 2011, 19:457–470. 10.1007/s10577-011-9208-5View ArticlePubMed CentralPubMedGoogle Scholar
- Amato A, Schillaci T, Lentini L, Di Leonardo A: CENPA overexpression promotes genome instability in pRb-depleted human cells. Mol Cancer 2009, 8:119.View ArticlePubMed CentralPubMedGoogle Scholar
- Verdaasdonk JS, Bloom K: Centromeres: unique chromatin structures that drive chromosome segregation. Nat Rev Mol Cell Biol 2011, 12:320–332. 10.1038/nrm3107View ArticlePubMed CentralPubMedGoogle Scholar
- Guse A, Carroll CW, Moree B, Fuller CJ, Straight AF: In vitro centromere and kinetochore assembly on defined chromatin templates. Nature 2011, 477:354–358. 10.1038/nature10379View ArticlePubMed CentralPubMedGoogle Scholar
- Santaguida S, Musacchio A: The life and miracles of kinetochores. EMBO J 2009, 28:2511–2531. 10.1038/emboj.2009.173View ArticlePubMed CentralPubMedGoogle Scholar
- Heun P, Erhardt S, Blower MD, Weiss S, Skora AD, Karpen GH: Mislocalization of the Drosophila centromere-specific histone CID promotes formation of functional ectopic kinetochores. Dev Cell 2006, 10:303–315. 10.1016/j.devcel.2006.01.014View ArticlePubMed CentralPubMedGoogle Scholar
- McGovern SL, Qi Y, Pusztai L, Symmans WF, Buchholz TA, et al.: Centromere protein-A, an essential centromere protein, is a prognostic marker for relapse in estrogen receptor-positive breast cancer. Breast Cancer Res 2012, 14:R72. 10.1186/bcr3181View ArticlePubMed CentralPubMedGoogle Scholar
- Olszak AM, van Essen D, Pereira AJ, Diehl S, Manke T, Maiato H, et al.: Heterochromatin boundaries are hotspots for de novo kinetochore formation. Nat Cell Biol 2011, 13:799–808. 10.1038/ncb2272View ArticlePubMedGoogle Scholar
- Van Hooser AA, Ouspenski II, Gregson HC, Starr DA, Yen TJ, Goldberg ML, et al.: Specification of kinetochore-forming chromatin by the histone H3 variant CENP-A. J Cell Sci 2001, 114:3529–3542.PubMedGoogle Scholar
- Lam AL, Boivin CD, Bonney CF, Rudd MK, Sullivan BA: Human centromeric chromatin is a dynamic chromosomal domain that can spread over noncentromeric DNA. Proc Natl Acad Sci U S A 2006, 103:4186–4191. 10.1073/pnas.0507947103View ArticlePubMed CentralPubMedGoogle Scholar
- Moreno-Moreno O, Torras-Llort M, Azorin F: Proteolysis restricts localization of CID, the centromere-specific histone H3 variant of Drosophila, to centromeres. Nucleic Acids Res 2006, 34:6247–6255. 10.1093/nar/gkl902View ArticlePubMed CentralPubMedGoogle Scholar
- Hewawasam G, Shivaraju M, Mattingly M, Venkatesh S, Martin-Brown S, Florens L, et al.: Psh1 is an E3 ubiquitin ligase that targets the centromeric histone variant Cse4. Mol Cell 2010, 40:444–454. 10.1016/j.molcel.2010.10.014View ArticlePubMed CentralPubMedGoogle Scholar
- Lacoste N, Woolfe A, Tachiwana H, Garea AV, Barth T, Cantaloube S, et al.: Mislocalization of the centromeric histone variant CenH3/CENP-A in human cells depends on the chaperone DAXX. Mol Cell 2014, 53:631–644. 10.1016/j.molcel.2014.01.018View ArticlePubMedGoogle Scholar
- Qiu JJ, Guo JJ, Lv TJ, Jin HY, Ding JX, Feng WW, et al.: Prognostic value of centromere protein-A expression in patients with epithelial ovarian cancer. Tumour Biol 2013, 34:2971–2975. 10.1007/s13277-013-0860-6View ArticlePubMedGoogle Scholar
- Wu Q, Qian YM, Zhao XL, Wang SM, Feng XJ, Chen XF, et al.: Expression and prognostic significance of centromere protein A in human lung adenocarcinoma. Lung Cancer 2012, 77:407–414. 10.1016/j.lungcan.2012.04.007View ArticlePubMedGoogle Scholar
- Hu Z, Huang G, Sadanandam A, Gu S, Lenburg ME, Pai M, et al.: The expression level of HJURP has an independent prognostic impact and predicts the sensitivity to radiotherapy in breast cancer. Breast Cancer Res 2010, 12:R18. 10.1186/bcr2487View ArticlePubMed CentralPubMedGoogle Scholar
- Earnshaw W, Bordwell B, Marino C, Rothfield N: Three human chromosomal autoantigens are recognized by sera from patients with anti-centromere antibodies. J Clin Invest 1986, 77:426–430. 10.1172/JCI112320View ArticlePubMed CentralPubMedGoogle Scholar
- Palmer DK, O’Day K, Wener MH, Andrews BS, Margolis RL: A 17-kD centromere protein (CENP-A) copurifies with nucleosome core particles and with histones. J Cell Biol 1987, 104:805–815. 10.1083/jcb.104.4.805View ArticlePubMedGoogle Scholar
- Bernad R, Sanchez P, Rivera T, Rodriguez-Corsino M, Boyarchuk E, Vassias I, et al.: Xenopus HJURP and condensin II are required for CENP-A assembly. J Cell Biol 2011, 192:569–582. 10.1083/jcb.201005136View ArticlePubMed CentralPubMedGoogle Scholar
- Foltz DR, Jansen LE, Bailey AO, Yates JR 3rd, Bassett EA, Wood S, et al.: Centromere-specific assembly of CENP-a nucleosomes is mediated by HJURP. Cell 2009, 137:472–484. 10.1016/j.cell.2009.02.039View ArticlePubMed CentralPubMedGoogle Scholar
- Dunleavy EM, Roche D, Tagami H, Lacoste N, Ray-Gallet D, Nakamura Y, et al.: HJURP is a cell-cycle-dependent maintenance and deposition factor of CENP-A at centromeres. Cell 2009, 137:485–497. 10.1016/j.cell.2009.02.040View ArticlePubMedGoogle Scholar
- Greaves IK, Rangasamy D, Ridgway P, Tremethick DJ: H2A.Z contributes to the unique 3D structure of the centromere. Proc Natl Acad Sci U S A 2007, 104:525–530. 10.1073/pnas.0607870104View ArticlePubMed CentralPubMedGoogle Scholar
- Jin C, Felsenfeld G: Nucleosome stability mediated by histone variants H3.3 and H2A.Z. Genes Dev 2007, 21:1519–1529. 10.1101/gad.1547707View ArticlePubMed CentralPubMedGoogle Scholar
- Masumoto H, Masukata H, Muro Y, Nozaki N, Okazaki T: A human centromere antigen (CENP-B) interacts with a short specific sequence in alphoid DNA, a human centromeric satellite. J Cell Biol 1989, 109:1963–1973. 10.1083/jcb.109.5.1963View ArticlePubMedGoogle Scholar
- Hasson D, Panchenko T, Salimian KJ, Salman MU, Sekulic N, Alonso A, et al.: The octamer is the major form of CENP-A nucleosomes at human centromeres. Nat Struct Mol Biol 2013, 20:687–695. 10.1038/nsmb.2562View ArticlePubMed CentralPubMedGoogle Scholar
- Shelby RD, Vafa O, Sullivan KF: Assembly of CENP-A into centromeric chromatin requires a cooperative array of nucleosomal DNA contact sites. J Cell Biol 1997, 136:501–513. 10.1083/jcb.136.3.501View ArticlePubMed CentralPubMedGoogle Scholar
- Bui M, Walkiewicz MP, Dimitriadis EK, Dalal Y: The CENP-A nucleosome: a battle between Dr Jekyll and Mr Hyde. Nucleus 2013, 4:37–42. 10.4161/nucl.23588View ArticlePubMed CentralPubMedGoogle Scholar
- Tachiwana H, Kagawa W, Shiga T, Osakabe A, Miya Y, Saito K, et al.: Crystal structure of the human centromeric nucleosome containing CENP-A. Nature 2011, 476:232–235. 10.1038/nature10258View ArticlePubMedGoogle Scholar
- Walkiewicz MP, Dimitriadis EK, Dalal Y: CENP-A octamers do not confer a reduction in nucleosome height by AFM. Nat Struct Mol Biol 2014, 21:2–3. 10.1038/nsmb.2742View ArticlePubMedGoogle Scholar
- Yoda K, Ando S, Morishita S, Houmura K, Hashimoto K, Takeyasu K, et al.: Human centromere protein A (CENP-A) can replace histone H3 in nucleosome reconstitution in vitro. Proc Natl Acad Sci U S A 2000, 97:7266–7271. 10.1073/pnas.130189697View ArticlePubMed CentralPubMedGoogle Scholar
- Dalal Y, Wang H, Lindsay S, Henikoff S: Tetrameric structure of centromeric nucleosomes in interphase Drosophila cells. PLoS Biol 2007, 5:e218. 10.1371/journal.pbio.0050218View ArticlePubMed CentralPubMedGoogle Scholar
- Dimitriadis EK, Weber C, Gill RK, Diekmann S, Dalal Y: Tetrameric organization of vertebrate centromeric nucleosomes. Proc Natl Acad Sci U S A 2010, 107:20317–20322. 10.1073/pnas.1009563107View ArticlePubMed CentralPubMedGoogle Scholar
- Bui M, Dimitriadis EK, Hoischen C, An E, Quenet D, Giebe S, et al.: Cell-cycle-dependent structural transitions in the human CENP-A nucleosome in vivo. Cell 2012, 150:317–326. 10.1016/j.cell.2012.05.035View ArticlePubMed CentralPubMedGoogle Scholar
- Luger K, Mader AW, Richmond RK, Sargent DF, Richmond TJ: Crystal structure of the nucleosome core particle at 2.8 A resolution. Nature 1997, 389:251–260. 10.1038/38444View ArticlePubMedGoogle Scholar
- Tachiwana H, Kagawa W, Kurumizaka H: Comparison between the CENP-A and histone H3 structures in nucleosomes. Nucleus 2012, 3:6–11. 10.4161/nucl.18372View ArticlePubMedGoogle Scholar
- Henikoff S, Ramachandran S, Krassovsky K, Bryson TD, Codomo CA, Brogaard K, et al.: The budding yeast Centromere DNA Element II wraps a stable Cse4 hemisome in either orientation in vivo. Elife 2014, 3:e01861.View ArticlePubMed CentralPubMedGoogle Scholar
- Krassovsky K, Henikoff JG, Henikoff S: Tripartite organization of centromeric chromatin in budding yeast. Proc Natl Acad Sci U S A 2012, 109:243–248. 10.1073/pnas.1118898109View ArticlePubMed CentralPubMedGoogle Scholar
- Cole HA, Howard BH, Clark DJ: The centromeric nucleosome of budding yeast is perfectly positioned and covers the entire centromere. Proc Natl Acad Sci U S A 2011, 108:12687–12692. 10.1073/pnas.1104978108View ArticlePubMed CentralPubMedGoogle Scholar
- Blower MD, Sullivan BA, Karpen GH: Conserved organization of centromeric chromatin in flies and humans. Dev Cell 2002, 2:319–330. 10.1016/S1534-5807(02)00135-1View ArticlePubMed CentralPubMedGoogle Scholar
- Shuaib M, Ouararhni K, Dimitrov S, Hamiche A: HJURP binds CENP-A via a highly conserved N-terminal domain and mediates its deposition at centromeres. Proc Natl Acad Sci U S A 2010, 107:1349–1354. 10.1073/pnas.0913709107View ArticlePubMed CentralPubMedGoogle Scholar
- Ranjitkar P, Press MO, Yi X, Baker R, MacCoss MJ, Biggins S: An E3 ubiquitin ligase prevents ectopic localization of the centromeric histone H3 variant via the centromere targeting domain. Mol Cell 2010, 40:455–464. 10.1016/j.molcel.2010.09.025View ArticlePubMed CentralPubMedGoogle Scholar
- Groudine M, Weintraub H: Propagation of globin DNAase I-hypersensitive sites in absence of factors required for induction: a possible mechanism for determination. Cell 1982, 30:131–139. 10.1016/0092-8674(82)90019-8View ArticlePubMedGoogle Scholar
- Wu C, Bingham PM, Livak KJ, Holmgren R, Elgin SC: The chromatin structure of specific genes: I. Evidence for higher order domains of defined DNA sequence. Cell 1979, 16:797–806. 10.1016/0092-8674(79)90095-3View ArticlePubMedGoogle Scholar
- John S, Sabo PJ, Thurman RE, Sung MH, Biddie SC, Johnson TA, et al.: Chromatin accessibility pre-determines glucocorticoid receptor binding patterns. Nat Genet 2011, 43:264–268. 10.1038/ng.759View ArticlePubMedGoogle Scholar
- John S, Sabo PJ, Canfield TK, Lee K, Vong S, Weaver M, et al.: Genome-Scale Mapping of DNase I Hypersensitivity. Curr Protoc Mol Biol 2013, Chapter 27:Unit 21.27.PubMedGoogle Scholar
- Nie Z, Hu G, Wei G, Cui K, Yamane A, Resch W, et al.: c-Myc is a universal amplifier of expressed genes in lymphocytes and embryonic stem cells. Cell 2012, 151:68–79. 10.1016/j.cell.2012.08.033View ArticlePubMed CentralPubMedGoogle Scholar
- Rudd MK, Friedman C, Parghi SS, Linardopoulou EV, Hsu L, Trask BJ: Elevated rates of sister chromatid exchange at chromosome ends. PLoS Genet 2007, 3:e32. 10.1371/journal.pgen.0030032View ArticlePubMed CentralPubMedGoogle Scholar
- Beroukhim R, Mermel CH, Porter D, Wei G, Raychaudhuri S, Donovan J, et al.: The landscape of somatic copy-number alteration across human cancers. Nature 2010, 463:899–905. 10.1038/nature08822View ArticlePubMed CentralPubMedGoogle Scholar
- Boerma EG, Siebert R, Kluin PM, Baudis M: Translocations involving 8q24 in Burkitt lymphoma and other malignant lymphomas: a historical review of cytogenetics in the light of todays knowledge. Leukemia 2009, 23:225–234. 10.1038/leu.2008.281View ArticlePubMedGoogle Scholar
- Popescu NC, Zimonjic DB: Chromosome-mediated alterations of the MYC gene in human cancer. J Cell Mol Med 2002, 6:151–159. 10.1111/j.1582-4934.2002.tb00183.xView ArticlePubMedGoogle Scholar
- Feo S, Di Liegro C, Jones T, Read M, Fried M: The DNA region around the c-myc gene and its amplification in human tumour cell lines. Oncogene 1994, 9:955–961.PubMedGoogle Scholar
- Huppi K, Pitt JJ, Wahlberg BM, Caplen NJ: The 8q24 gene desert: an oasis of non-coding transcriptional activity. Front Genet 2012, 3:69.View ArticlePubMed CentralPubMedGoogle Scholar
- Camps J, Nguyen QT, Padilla-Nash HM, Knutsen T, McNeil NE, Wangsa D, et al.: Integrative genomics reveals mechanisms of copy number alterations responsible for transcriptional deregulation in colorectal cancer. Genes Chromosomes Cancer 2009, 48:1002–1017. 10.1002/gcc.20699View ArticlePubMedGoogle Scholar
- Screpanti E, De Antoni A, Alushin GM, Petrovic A, Melis T, Nogales E, et al.: Direct binding of cenp-C to the mis12 complex joins the inner and outer kinetochore. Curr Biol 2011, 21:391–398.View ArticlePubMed CentralPubMedGoogle Scholar
- Furuyama T, Dalal Y, Henikoff S: Chaperone-mediated assembly of centromeric chromatin in vitro. Proc Natl Acad Sci U S A 2006, 103:6172–6177. 10.1073/pnas.0601686103View ArticlePubMed CentralPubMedGoogle Scholar
- Steiner FA, Henikoff S: Holocentromeres are dispersed point centromeres localized at transcription factor hotspots. Elife 2014, 3:e02025.View ArticlePubMed CentralPubMedGoogle Scholar
- Shelby RD, Monier K, Sullivan KF: Chromatin assembly at kinetochores is uncoupled from DNA replication. J Cell Biol 2000, 151:1113–1118. 10.1083/jcb.151.5.1113View ArticlePubMed CentralPubMedGoogle Scholar
- Jansen LE, Black BE, Foltz DR, Cleveland DW: Propagation of centromeric chromatin requires exit from mitosis. J Cell Biol 2007, 176:795–805. 10.1083/jcb.200701066View ArticlePubMed CentralPubMedGoogle Scholar
- Strahl BD, Allis CD: The language of covalent histone modifications. Nature 2000, 403:41–45. 10.1038/47412View ArticlePubMedGoogle Scholar
- Ambartsumyan G, Gill RK, Perez SD, Conway D, Vincent J, Dalal Y, et al.: Centromere protein A dynamics in human pluripotent stem cell self-renewal, differentiation and DNA damage. Hum Mol Genet 2010, 19:3970–3982. 10.1093/hmg/ddq312View ArticlePubMed CentralPubMedGoogle Scholar
- Zeitlin SG, Baker NM, Chapados BR, Soutoglou E, Wang JY, Berns MW, et al.: Double-strand DNA breaks recruit the centromeric histone CENP-A. Proc Natl Acad Sci U S A 2009, 106:15762–15767. 10.1073/pnas.0908233106View ArticlePubMed CentralPubMedGoogle Scholar
- Arimura Y, Shirayama K, Horikoshi N, Fujita R, Taguchi H, Kagawa W, et al.: Crystal structure and stable property of the cancer-associated heterotypic nucleosome containing CENP-A and H3.3. Sci Rep 2014, 4:7115.View ArticlePubMed CentralPubMedGoogle Scholar
- Luger K, Rechsteiner TJ, Richmond TJ: Expression and purification of recombinant histones and nucleosome reconstitution. Methods Mol Biol 1999, 119:1–16.PubMedGoogle Scholar
- Lyubchenko YL, Gall AA, Shlyakhtenko LS: Atomic force microscopy of DNA and protein-DNA complexes using functionalized mica substrates. Methods Mol Biol 2001, 148:569–578.PubMedGoogle Scholar
- Crawford GE, Davis S, Scacheri PC, Renaud G, Halawi MJ, Erdos MR, et al.: DNase-chip: a high-resolution method to identify DNase I hypersensitive sites using tiled microarrays. Nat Methods 2006, 3:503–509. 10.1038/nmeth888View ArticlePubMed CentralPubMedGoogle Scholar
- John S, Sabo PJ, Johnson TA, Sung MH, Biddie SC, Lightman SL, et al.: Interaction of the glucocorticoid receptor with the chromatin landscape. Mol Cell 2008, 29:611–624. 10.1016/j.molcel.2008.02.010View ArticlePubMedGoogle Scholar
- Bailey TL, Gribskov M: Methods and statistics for combining motif match scores. J Comput Biol 1998, 5:211–221. 10.1089/cmb.1998.5.211View ArticlePubMedGoogle Scholar
- Heselmeyer-Haddad K, Berroa Garcia LY, Bradley A, Ortiz-Melendez C, Lee WJ, Christensen R, et al.: Single-cell genetic analysis of ductal carcinoma in situ and invasive breast cancer reveals enormous tumor heterogeneity yet conserved genomic imbalances and gain of MYC during progression. Am J Pathol 2012, 181:1807–1822. 10.1016/j.ajpath.2012.07.012View ArticlePubMed CentralPubMedGoogle Scholar
- Gill RK, Vazquez MF, Kramer A, Hames M, Zhang L, Heselmeyer-Haddad K, et al.: The use of genetic markers to identify lung cancer in fine needle aspiration samples. Clin Cancer Res 2008, 14:7481–7487. 10.1158/1078-0432.CCR-07-5242View ArticlePubMed CentralPubMedGoogle Scholar
- Van Hooser A, Brinkley WR: Methods for in situ localization of proteins and DNA in the centromere-kinetochore complex. Methods Cell Biol 1999, 61:57–80.View ArticlePubMedGoogle Scholar
- Dalal Y, Fleury TJ, Cioffi A, Stein A: Long-range oscillation in a periodic DNA sequence motif may influence nucleosome array formation. Nucleic Acids Res 2005, 33:934–945. 10.1093/nar/gki224View ArticlePubMed CentralPubMedGoogle Scholar
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