The HACs we formed were highly penetrant within a clonal cell population ( Figure 1 F), likely due to the pulse of mCherry-LacI-HJURP driving efficient and rapid centromere acquisition that can then be propagated independently of the initial HJURP-mediated seeding of CENP-A nucleosome assembly. Alternatively, we considered that low, leaky expression of mCherry-LacI-HJURP continues to drive CENP-A nucleosome assembly on the HAC, thereby stabilizing the HAC in the cell. Therefore, we tested if genetically ablating mCherry-LacI-HJURP expression via CRISPR/Cas9-mediated gene editing affects HAC stability ( Figure 1 A). Choosing a cell line in which the chr11 α-satellite BACHAC is present in ≥95% of cells, we derived three monoclonal cell lines in which mCherry-LacI-HJURP expression has been disrupted ( Figure 1 G). Using the standard approach for measuring HAC maintenance (), wherein all clones were cultured without antibiotic selection (G418-S) for 60 days, we found that the absence of mCherry-LacI-HJURP did not affect the daily HAC loss rate ( Figure 1 H). These daily HAC loss rates are similarly low as those reported for “conventional” HACs ( Figure 1 H, the range is shaded in gray ()). Thus, the action of seeding CENP-A nucleosome assembly is limited to centromere establishment. After that, the centromere on the HAC is epigenetically maintained in the same manner as on natural chromosomes.

We first generated BAC constructs containing α-satellite sequences that are deemed nonfunctional in natural chromosomes due to a low density of CENP-B boxes (). A successful strategy to make these sequences functional to form a HAC is to first manipulate the constructs to increase the density of CENP-B boxes (). We devised an alternative strategy to avoid manipulation of the α-satellite sequences, themselves, by artificially driving an initial round of CENP-A chromatin assembly on an adjacent site on the construct. Our general strategy was to assemble constructs consisting of BACs harboring an array of LacO repeats immediately adjacent to human genomic DNA sequences (hereafter termed BAC) ( Figure 1 A). Then, to the LacO array, we targeted mCherry-LacI-HJURP, inducibly expressed from a genomically integrated transgene. This targeting would potentially initiate the assembly of CENP-A nucleosomes directly onto the BAC and facilitate the spreading of CENP-A nucleosomes to the neighboring sequences (). We engineered two BACvectors containing α-satellite DNA coming from CENP-A-poor regions of the centromere on chromosomes (chr) 7 and 11 ( Figure 1 A). Our cloning strategy positioned the LacO repeats within 300 bp of the α-satellite sequence, keeping this distance small to potentially permit efficient spreading of centromeric chromatin. We isolated α-satellite BACconstructs that successfully recombined ( Figure 1 B) and retained both the repetitive α-satellite and LacO arrays ( Figure 1 C). Using established methodologies to isolate and identify HACs (), we found that a pulse of mCherry-LacI-HJURP expression was sufficient to stimulate HAC formation ( Figures 1 D and 1E). Because we obtained nearly identical results on two independent α-satellite sequences ( Figures 1 D–1F), we conclude that our strategy would stimulate HAC formation on broad classes of α-satellite higher order repeats. As expected, there was no HAC formation in the absence of the round of CENP-A chromatin assembly directed by the pulse of mCherry-LacI-HJURP ( Figure 1 D), indicating that the presence of the LacO array, itself, does not drive centromere formation on BACconstructs.

(H) Quantification of the daily HAC loss rate in WT or mCherry-LacI-HJURP KO cells after culturing without G418-S for 60 days. The mean daily loss rate (± SEM) is shown. n = 60 WT cells and 180 mCherry-LacI-HJURP KO cells, pooled from three independent experiments for each indicated cell type. The WT cells are from a chr11 α-satellite BAC LacO clone, and the mCherry-LacI-HJURP KO cells are pooled from three derivative chr11 α-satellite BAC LacO clones. n.s., not significant.

(F) Quantification of the percentage of cells containing an α-satellite BAC LacO HAC within each HAC-positive clone. The mean value (± SEM) is shown for each BAC LacO construct.

Because prior efforts with conventional HACs failed to form any functional centromeres in the absence of CENP-B (), there are no data to indicate whether or not CENP-B is also important for HAC maintenance. To address this issue, we performed a HAC maintenance assay with a cell line containing a chr11 α-satellite BACHAC and three monoclonal cell line derivatives of it in which we disrupted the CENP-B gene ( Figures 2 H and 2I). We found that the absence of CENP-B did not affect the daily HAC loss rate of the α-satellite HACs ( Figure 2 J). Further, CENP-A was retained at the HAC in the absence of CENP-B through our 60-day assay ( Figure S1 ). Thus, we conclude that CENP-B is also dispensable for the maintenance of a HAC.

Representative images of a chr11 α-satellite BAC LacO HAC in CENP-B KO cells at the beginning of the HAC maintenance assay (Day 0) and after 60 days of culturing in the absence of G418-S (Day 60). Insets are 2.5× magnification. Bar, 10 μm.

We next directly tested whether CENP-B expression—one of the universal requirements for conventional HAC formation ()—could be bypassed by seeding CENP-A nucleosome assembly. To do so, we disrupted the CENP-B gene prior to performing a new set of HAC formation assays ( Figures 2 A–2C). We found that chr11 α-satellite BACHACs form in the absence of CENP-B ( Figure 2 D,E). Because HAC formation on this construct is dependent on induction of mCherry-LacI-HJURP ( Figure 2 D,E), we conclude that seeding CENP-A nucleosomes onto the α-satellite DNA bypasses the requirement of CENP-B for centromere formation. Further, the absence of CENP-B did not affect the high number of cells containing a HAC ( Figure 2 F) (82% ± 7% in CENP-B knockout [KO] cells versus 73% ± 8% in wild type [WT] cells, shown in Figure 1 F) or substantially alter the amount of CENP-A on the centromere of the HAC relative to those on natural chromosomes ( Figure 2 G) (note there is a small but measurable increase in the CENP-B KO cells). Thus, our experiments indicate that the absence of CENP-B has no detectable negative effect upon forming a HAC via seeding of CENP-A nucleosomes.

(J) Quantification of the daily HAC loss rate in WT or mCherry-Lac-HJURP KO cells after culturing without G418-S for 60 days (shading as in Figure 1 H). The mean daily loss rate (± SEM) is shown. n = 60 WT cells and 180 CENP-B KO cells, pooled from 3 independent experiments for each indicated cell type. The WT cells are from a chr11 α-satellite BACclone, and the CENP-B KO cells are pooled from three derivative chr11 α-satellite BACclones. n.s., not significant.

(G) Quantification of CENP-A intensity at HACs formed in WT and CENP-B KO cells relative to the intensity at centromeres on endogenous chromosomes. The mean ratio (± SEM) is shown. n = 50 WT cells and 57 CENP-B KO cells, pooled from 3 independent clones for each indicated cell type. An asterisk indicates p < 0.05.

(F) Quantification of the percentage of CENP-B KO cells containing a HAC within each clone. The mean value (± SEM) is shown.

The most prominent proposal for the role of α-satellite DNA in HAC formation is that a high density of CENP-B boxes facilitates early steps in centromere formation (). Because seeding CENP-A nucleosome assembly bypasses the requirement of CENP-B for centromere formation ( Figure 2 ), we hypothesized that, likewise, the requirement for α-satellite DNA might be bypassed. To test this, we built and performed a small-scale HAC formation screen with a set of BACs containing an array of LacO repeats adjacent to non-α-satellite human genomic sequences ( Figures S2 A and S2B). We chose sequences for our initial screening based on proximity to known neocentromeres (), and we also included a clone several Mbp distal to a well-studied neocentromere (PD-NC4) ( Table S1 ). One construct in the screen, 4q21 BAC, formed several HACs ( Figures 3 A–3C and S2 C–S2E). In stark contrast to the α-satellite versions that we tested ( Figures 1 and 2 ), we found that 4q21 BACalso reproducibly formed HACs in the absence of the induction of mCherry-LacI-HJURP expression ( Figures 3 A–3C). We considered that non-α-satellite sequences might be particularly sensitive to leaky expression of mCherry-LacI-HJURP in the absence of doxycycline. Thus, we generated a version of the 4q21 BAC that is identical to 4q21 BACbut lacks the LacO array ( Figure S2 F) and found that it also forms HACs ( Figures 3 A, 3D, and 3E). This eliminated the possibility of a dependence on any leaky mCherry-LacI-HJURP expression or on any other property imparted by the LacO array itself. Because the only sequences to-date to form a HAC in the absence of seeding CENP-A nucleosomes require CENP-B (), we also considered the possibility that 4q21 BACHACs somehow form via a CENP-B-dependent centromere formation pathway. To directly test this, we performed HAC formation assays with 4q21 BACin our cell line where the CENP-B gene had been disrupted ( Figures 2 B and 2C) and found that HAC formation occurred in the absence of CENP-B ( Figures 3 A, 3F, and 3G). Thus, we conclude that the non-repetitive, non-centromeric 4q21 BACconstruct forms a HAC in a CENP-B-independent manner. Taken together, this series of HAC formation assays with non-α-satellite DNA constructs clearly indicate that centromere formation must be different from the CENP-B-dependent pathway used by traditional HACs () or our new CENP-B-independent HACs that require seeding CENP-A nucleosome assembly ( Figures 1 and 2 ).

(F) Restriction digest analysis with NotI on the 4q21 BAC construct showing that the 4q21 sequence and vector backbone are present in the BAC, while the LacO repeats have been removed.

(E) Illustration of the repeat abundance and position along the 4q21 sequence. The repeat elements are dispersed along the 4q21 sequence and do not appear to cluster in regions enriched with CENP-A in the 4q21 HAC clones ( Figures 5 A and 5B), indicating that there is no strong correlation between repeat element and CENP-A location.

(A) PCR analysis to confirm construction of the non-α-satellite BAC LacO constructs. The red and black primer pair amplifies a 458 bp fragment only in the parental non-α-satellite BAC vector, and the green and black primer pair amplifies a 558 bp fragment only in the non-α-satellite BAC LacO vector.

Multiple Pathways for HAC Formation on a Non-repetitive DNA Template

LacO in WT cells; clones 7–10 from 4q21 BAC in WT cells; clones 11–17 from 4q21 BACLacO in CENP-B KO cells). Figure 4 Seeding CENP-A Nucleosome Assembly Dictates the Pathway to Centromere Formation Show full caption (A) Steps to test whether the 4q21 HACs have acquired CENP-B protein or functional α-satellite DNA. (B) Quantification of the intensity of CENP-B at chr11 α-satellite BACLacO and 4q21 HACs relative to the intensity at endogenous centromeres. Each data point represents a measurement taken at a single HAC. The mean ratio (± SEM) is shown. n = 20, 19, 20, 20, 20, 19, 21, 18, 13, 22, and 18 HACs for the clones shown, in order. p is < 0.0001, 0.8566, 0.0019, 0.6401, 0.2215, 0.0343, < 0.0001, 0.6269, < 0.0001, < 0.0001, < 0.0001 for the clones shown, in order, based on a one-sample t test with a hypothetical mean of 0. Clones with a p value < 0.05 are marked with an asterisk; clones with a p value ≥ 0.05 are marked as not significant (n.s.). (C) Representative images of a 4q21 HAC that has acquired CENP-B-bound sequences (clone 8) and one that has not (clone 1). (D) Quantification of the intensity of a CENP-A ChIP probe at chr11 α-satellite BACLacO and 4q21 HACs relative to the intensity at endogenous centromeres. Each data point represents a measurement taken at a single HAC. The mean ratio (± SEM) is shown. n = 20, 18, 20, 22, 22, 20, 18, 19, 19, 18, and 19 HACs for the clones shown, in order. p is < 0.0001, 0.5642, 0.0005, 0.1028, 0.9098, 0.9602, 0.4708, 0.7553, < 0.0001, < 0.0001, 0.7278 for the clones shown, in order, based on a one-sample t test with a hypothetical mean of 0. Clones with a p value 0.05 are marked with an asterisk; clones with a p value ≥ 0.05 are marked as not significant (n.s.). (E) Representative images of a 4q21 HAC that has acquired CENP-A-associated sequences (clone 16) and one that has not (clone 1). The HACs are detected with HA-LacI, which binds the LacO repeats present in the HACs. Insets: 2.5× magnifications. Bar, 10 μm (C and E). (F) Summary of the quantitative analysis of all 4q21 HAC clones. See also Figure S3 and Table S2 We developed a tripartite strategy ( Figure 4 A) to investigate the pathway for centromere formation for each of the 17 clones isolated through our collection of 4q21-based HAC experiments ( Figure 3 ) (clones 1–6 from 4q21 BACin WT cells; clones 7–10 from 4q21 BAC in WT cells; clones 11–17 from 4q21 BACin CENP-B KO cells).

Figure S3 LacO HAC Clones, Related to Analysis of the Centromeric Protein and Sequence Abundance, as well as the Organization, of Various 4q21 BACHAC Clones, Related to Figure 4 Show full caption A,B) Plot with an expanded y axis of the ratio of CENP-B (A) and CENP-A ChIP FISH probe (B) intensity at the HAC relative to endogenous centromeres for clones with a mean below 0.2; related to Figures 4 B and 4D. Clones with a p value < 0.05 are marked with an asterisk; clones with a p value ≥ 0.05 are marked as not significant (n.s.). LacO sequence one time; therefore, if the HAC had undergone a simple amplification of the 4q21 BACLacO sequence, multiples of a 203 kb band should be observed. However, we observed varying band sizes (C), indicating that each HAC had undergone structural rearrangements during HAC formation, which has been previously observed with α-satellite HACs ( Kouprina et al., 2012 Kouprina N.

Samoshkin A.

Erliandri I.

Nakano M.

Lee H.-S.

Fu H.

Iida Y.

Aladjem M.

Oshimura M.

Masumoto H.

et al. Organization of synthetic alphoid DNA array in human artificial chromosome (HAC) with a conditional centromere. C,D) Southern blot analysis of the indicated cell lines showing variable sequence organization within the 4q21 HACs. Genomic DNA from each cell line was digested with the indicated restriction enzyme, separated by pulsed-field gel electrophoresis, transferred to a membrane, and hybridized with a LacO-specific probe. The FseI restriction enzyme digests the 4q21 BACsequence one time; therefore, if the HAC had undergone a simple amplification of the 4q21 BACsequence, multiples of a 203 kb band should be observed. However, we observed varying band sizes (C), indicating that each HAC had undergone structural rearrangements during HAC formation, which has been previously observed with α-satellite HACs (). In all HACs assessed, the LacO array was largely intact (D), indicating that the rearrangements occurred in the 4q21 and backbone sequences within each HAC and not within the LacO array. (E) Restriction enzyme map of the FseI cut site and the fragment produced by BamHI enzyme digestion of the 4q21 BACLacO construct. BamHI cuts 26 other times throughout the 4q21 sequence and backbone (not shown), but these fragments are largely not detected by the LacO-specific probe (as shown in Panel D). First, using immunofluorescence to detect CENP-B protein and fluorescence in situ hybridization (FISH) to detect the HACs, we found that four of the ten clones that formed in the WT (CENP-B-positive) background had no detectable CENP-B protein ( Figures 4 B, 4C, and S3 A) (clones 1, 3, 4, and 7). The other six of the ten clones had detectable CENP-B, with widely varying levels of acquired native centromere sequences likely housing some or all of the functional centromeric chromatin.

LacO had no detectable acquisition of functional centromeric chromatin ( Second, using FISH to detect functional centromeric chromatin on HACs detected with the expression of HA epitope-tagged LacI, we found that seven of the ten remaining clones generated with 4q21 BAChad no detectable acquisition of functional centromeric chromatin ( Figures 4 D, 4E, and S3 B) (clones 1, 4, 11–14, and 17; note that clone 7 was generated with a 4q21 BAC construct that lacks a binding site for the HA epitope-tagged LacI, so it could not be included in the second step of our analysis). Two other HACs appeared to form with the acquisition of high levels of functional centromeric chromatin (clones 15 and 16) and another HAC formed with the acquisition of only very little functional centromeric chromatin (clone 3) ( Figures 4 D, 4E, and S3 B).

Hasson et al., 2013 Hasson D.

Panchenko T.

Salimian K.J.

Salman M.U.

Sekulic N.

Alonso A.

Warburton P.E.

Black B.E. The octamer is the major form of CENP-A nucleosomes at human centromeres. LacO ( Third, eight out of the original seventeen 4q21-based HACs whose formation could not be attributed to the acquisition of functional centromeric chromatin in the first two steps of our analysis were subjected to CENP-A chromatin immunoprecipitation sequencing (ChIP-seq) ( Figure 4 F). By comparing the reads in each HAC-containing cell line to the parental cell line lacking a HAC, we assigned all of the reads coming from the HAC to either the 4q21-containing BAC sequences or the rest of the human reference genome ( Table S2 ). As with prior analysis of human neocentromeres (), there is a massive increase in CENP-A ChIP-seq reads from the functional centromere on the HAC relative to what is observed in parental cells lacking a HAC. Thus, we assigned all 4q21 CENP-A ChIP reads to the HAC. Using this strategy, we found that four of the HACs (clones 1, 11, 12, and 14) have centromeres residing on DNA essentially entirely comprised of 4q21-containing BAC sequences, while the other four (clones 4, 7, 13, and 17) have acquired genomic sequences upon which at least a portion of the functional centromere (defined by the presence of CENP-A nucleosomes) resides ( Figure 4 F; Table S2 ). Both types of HACs (those with centromeres exclusively on the 4q21 sequence and those with acquired genomic sequences) multimerized with rearrangements at unique locations relative to one another but always within non-repetitive regions (i.e., outside of the LacO array) of 4q21 BAC Figures S3 C–S3E).