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DNA replication acts as an error correction mechanism to maintain centromere identity by restricting CENP-A to centromeres

Abstract

Chromatin assembled with the histone H3 variant CENP-A is the heritable epigenetic determinant of human centromere identity. Using genome-wide mapping and reference models for 23 human centromeres, CENP-A binding sites are identified within the megabase-long, repetitive α-satellite DNAs at each centromere. CENP-A is shown in early G1 to be assembled into nucleosomes within each centromere and onto 11,390 transcriptionally active sites on the chromosome arms. DNA replication is demonstrated to remove ectopically loaded, non-centromeric CENP-A. In contrast, tethering of centromeric CENP-A to the sites of DNA replication through the constitutive centromere associated network (CCAN) is shown to enable precise reloading of centromere-bound CENP-A onto the same DNA sequences as in its initial prereplication loading. Thus, DNA replication acts as an error correction mechanism for maintaining centromere identity through its removal of non-centromeric CENP-A coupled with CCAN-mediated retention and precise reloading of centromeric CENP-A.

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Data availability

ChIP-seq and Repli-seq datasets generated during the current study were deposited at GEO under primary accession number GSE111381. Figures 1, 2, 3, 4, 5 and 6 and Supplementary Figs. 1, 2, 3 and 5 have associated raw mapping data. Mass spectrometry data were deposited at the ProteomeXchange database under accession number PXD013385. Mass spectrometry data are found in Table 1 and Supplementary Fig. 5.

Previously published ChIP-seq data that were reanalysed in this study include HT1080b (ref. 44) CENP-AFLAG ChIP-seq (GSM2808187 and corresponding input dataset GSM2808190) and HuRef (ref. 45) CENP-A native ChIP-seq (GSM1494428, GSM1494429, GSM1494430, GSM1494431, and corresponding input datasets GSM1494424, GSM1494425, GSM1494426, GSM1494427). Hela CENP-ATAP, Hela CENP-ALAP, HT1080b CENP-AFLAG (ref. 44) and HuRef (ref. 45) endogenous CENP-A ChIP-seq datasets were intersected with the following publicly available ENCODE HeLa S3 datasets: DNase I (GSM736564 and GSM736510), H3K4me1 (GSM798322), H3K4me2 (GSM733734), H3K4me3 (GSM733682), H2A.z (GSM1003483), H3K9me3 (GSM1003480), H3K27ac (GSM733684), H3K27me3 (GSM945208), H3K36me3 (GSM733711), H3K79me2 (GSM733669), H3K9ac (GSM733756) and H4K20me1 (GSM733689). CENP-ATAP and CENP-ALAP SICER peaks were also intersected with ENCODE CTCF datasets (GSM749729 and GSM749739), and high-occupancy target region datasets (GSE54296).

Source data for Figs. 1, 3, 5, 6 and 7 and Supplementary Figs. 3, 4 and 5 have been provided as Supplementary Table 4. All other data supporting the findings of this study are available from the corresponding authors on reasonable request.

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Acknowledgements

We thank A. Desai, P. Ly and C. Eissler for critical discussion, L.E.T. Jansen (Gulbenkian Institute, Portugal) for reagents and D.-H. Kim for productive discussions and technical help. This work was supported by grants (R01 GM-074150 and R35 GM-122476) from the NIH to D.W.C., who receives salary support from Ludwig Cancer Research.

Author information

Y.N.-A. and D.W.C. conceived and designed experiments and wrote the manuscript. Y.N.-A. performed experiments. K.H.M. analysed the sequencing data. M.A.M. and O.S. analysed data and performed experiments. D.F. suggested experiments and provided key experimental input. A.Y.L. and B.R. prepared sequencing libraries and provided resources. A.A. and J.R.Y. performed mass spectrometry experiments and provided resources.

Correspondence to Yael Nechemia-Arbely or Don W. Cleveland.

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Supplementary Figure 1 Identification of peaks enriched for CENP-A binding.

(a) Scheme showing experimental design for tagging an endogenous CENP-A locus to produce CENP-A+/LAP HeLa cells. These cells were then adapted to suspension growth. (b) Scheme showing the experimental design for obtaining increased levels of CENP-ATAP expression. CENP-ATAP is expressed in these cells at 4.5-fold the level of CENP-A in the parental HeLa cells1. (c, d) Localization of endogenously tagged CENP-ALAP (c) and CENP-ATAP (d) determined with indirect immunofluorescence using anti-GFP antibody (c) or rabbit IgG (d). Scale bar, 5 μm. The experiment was repeated independently three times with similar results for both (c) and (d). (e) FACS analysis of DNA content showing the synchronization efficiency of CENP-A+/LAP and CENP-ATAP HeLa cell lines. The experiment was repeated independently five times with similar results. (f, g) Examples of centromeric regions of chromosome 7 (f) and 5 (g) showing increased occupancy of overexpressed CENP-ATAP (compare CENP-ATAP with CENP-ALAP). The experiment was repeated independently twice with similar results. (h) Overlap between G1 and G2 CENP-ATAP binding peaks at α-satellite sequences. (i) Top, overlap between G1 and G2 CENP-A single-mapping binding sites at α-satellite HOR sequences. Bottom, peak overlap between G1 CENP-ATAP (increased expression) and CENP-ALAP (endogenous level) single-mapping binding sites at α-satellite HOR sequences.

Supplementary Figure 2 CENP-A ChIP-seq identifies CENP-A binding at reference centromeres of 23 human chromosomes.

CENP-ALAP bound DNAs at G1 and G2 were sequenced, with 2 replicates per condition, and mapped to the centromeric reference models in the hg38 assembly2,3. Shown are the raw mapping data (coloured) for every human centromere (except for the centromere of chromosome 19 that shares almost all of its α-satellites arrays with α-satellites arrays of chromosomes 1 and 5) and CENP-A binding called as SICER peaks (black lines, underneath) for one replicate for each time point. The experiment was repeated twice independently with similar results. Centromere reference location, red. CENP-B box, orange.

Supplementary Figure 3 Ectopic deposition of CENP-A into open and active chromatin at G1 does not function as a seeding hotspot for neocentromere formation.

(a) Number of non-α-satellite CENP-A SICER binding sites called at G1 or G2 at different fold thresholds (above background). (b) Human DLD1 cells with auxin degradable CENP-AAID and a doxycycline-inducible CENP-AWT4, were synchronized at G1 using the CDK4/6 inhibitor PD-0332991 (also known as Palbociclib) or at mitosis using nocodazole, following addition of doxycycline. The experiment was repeated independently three times with similar results. (c) Read mapping data of CENP-ATAP ChIP-seq at G1 (red) and G2 (blue), at the chromosomal location of a known patient derived neocentromere5 found in chromosome 13. The experiment was repeated twice independently with similar results. A third human neocentromere, identified in line MS4221, has been identified to lie within a 400 kb neocentromere at position 86.5 to 86.9 Mb on chromosome 8 in hg195,6 (corresponding to 85.78–85.88 Mb in hg38). However, a gap and segmental duplications that appear in this region precluded precise analysis of CENP-A mapping at this neocentromere. (d-g) Fold enrichment of CENP-ATAP chromatin in randomly cycling cells or at G1 (d, e) and CENP-ALAP chromatin (f, g) at G1 at different genomic locations. SICER peaks ≥ 5-fold supported between two replicates were analysed for their enrichment level at different genomic locations, compared to the level of enrichment at these sites by chance. (h, i) Number of CENP-ATAP (h) and CENP-ALAP (i) SICER peaks ≥ 5-fold that overlap with ‘HOT’ regions in the human genome in G1 and G2 synchronized cells. Data shown are from two biologically independent experiments. Source data for d-i can be found in Supplementary Table 4.

Supplementary Figure 4 Non-centromeric CENP-A binding peaks overlap with active transcription marks.

(a,b) The chromatin features of CENP-ATAP (a) and CENP-ALAP (b) non-centromeric preferential sites were analysed by intersecting SICER peaks ≥ 5-fold supported between two replicates with publicly available ENCODE datasets for histone modification profiles in HeLa S3, that represent modifications typically associated with transcription activation or repression. The experiment was performed one time, except for DNase I and H3K27me3 for which there are 2 ENCODE datasets available, and therefore for DNase I and H3K27me3 the experiment was repeated twice independently with similar results. Statistics source data for Supplementary Fig. 4a, b can be found in Supplementary Table 4. (The sum of ectopic CENP-ATAP sites at active or repression marks is more than 100%, the result of overlap between H3K9me3 and active transcription marks.) (c,d) The chromatin features of sites of preferential, non-centromeric CENP-A binding were analysed for histone modification profiles associated with transcription activation or repression in HeLa S3 cells by intersecting SICER peaks ≥ 5-fold found in previously published CENP-A ChIP-seq datasets in HT10807 (c) and HuRef8 (d) cell lines with publicly available ENCODE datasets for histone modification profiles in HeLa S3. For HT1080b (c) the experiment was performed one time, except for DNase I and H3K27me3 for which there are 2 ENCODE datasets available, and therefore for DNase I and H3K27me3 the experiment was repeated twice independently with similar results. For HuRef (d), The experiment was performed four times, except for DNase I and H3K27me3 for which there are 2 ENCODE datasets available, and therefore for DNase I and H3K27me3 the experiment was repeated independently eight times with similar results. Source data for a-d can be found in Supplementary Table 4.

Supplementary Figure 5 Centromeres are late replicating with CENP-A remaining tethered locally by continued binding to the CCAN complex.

(a) FACS analysis of DNA content showing the synchronization efficiency of CENP-ATAP HeLa cell line across S phase. The experiment was repeated independently twice with similar results. (b) Genomic DNA of cells labelled for 1 hour with BrdU was sonicated prior to the BrdU immunoprecipitation and fragments of 200–800 bp were obtained. The experiment was repeated independently twice with similar results. Unprocessed images of DNA gels can be found in Supplementary Fig. 6. (c) Quantitative real-time PCR for MRGPRE and MMP15 genes, previously reported to replicate early (ref9 and ENCODE Repli-seq). (d) Quantitative real-time PCR for HBE1 and Sat210 genes, previously reported to replicate late (ref11 and ENCODE Repli-seq). (e) Quantitative real-time PCR for α-satellite DNA. Data shown in c-e are from two biologically independent experiments. Source data for c-e can be found in Supplementary Table 4. (f) MNase digestion profile showing the nucleosomal DNA length distributions of bulk input mononucleosomes (upper panel) and purified CENP-ATAP following native ChIP at early S and mid-S phase. The experiment was repeated twice independently with similar results. (g) CENP-A ChIP-seq raw mapping data spanning the whole of cen18 at G1, mid-S phase and G2, and BrdU repli-seq at early S (S1), mid-S (S4) and late S/G2 (S7). SICER peaks are denoted as black lines underneath the raw mapping data. The experiment was repeated twice independently with similar results. Centromere reference location, red. CENP-B boxes, orange. Scale bar, 2Mb. (h) Overlap degree between CENP-A G1 and mid-S at α-satellite HORs single copy variants. (i) Ethidium Bromide stained DNA agarose gel showing MNase digestion profile of bulk chromatin used for mass spectrometry identification of proteins associating with CENP-ATAP chromatin (left panel) and for CENP-ATAP co-immunoprecipitation experiment (right panel). Mass spectrometry was performed once and co-IP was performed twice with similar results. Unprocessed images of DNA gels can be found in Supplementary Fig. 6. (j-n) CENP-ATAP immunopurification followed by mass spectrometry identifies association with CENP-A chromatin of DNA replication related proteins (j,k), chromatin remodelling factors and nuclear chaperones (l), histones (m) and centromere and kinetochore proteins (n).

Supplementary Figure 6

Unprocessed film scans of all immunoblots and DNA gels with corresponding protein and DNA size markers.

Supplementary information

Supplementary Information

Supplementary Figs. 1–6 and their legends, and legends for Supplementary Video 1 and Supplementary Tables 1–4

Reporting Summary

Supplementary Table 1

Read statistics for ChIP-seq and Repli-seq experiments.

Supplementary Table 2

Endogenous CENP-A sequence mapping onto α-satellite DNAs in human centromere reference models for each autosome and the X chromosome.

Supplementary Table 3

Antibodies used in the study.

Supplementary Table 4

Statistical source data for graphical representations.

Supplementary Video 1

Rapid CENP-CAE/AE depletion following IAA treatment.

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Fig. 1: CENP-A ChIP-seq identifies CENP-A binding at reference centromeres of 23 human chromosomes.
Fig. 2: Retention of centromeric CENP-A through DNA replication.
Fig. 3: Sites of CENP-A assembly onto chromosome arms in early G1 are removed by G2.
Fig. 4: Ectopic CENP-A is removed following DNA replication from the arms of all 23 human chromosomes.
Fig. 5: Neocentromeres are not positioned at sites of preferential CENP-A loading on chromosome arms.
Fig. 6: Ectopic CENP-A is removed contemporaneously with replication fork progression, while centromeric CENP-A is retained.
Fig. 7: CENP-C and the CCAN complex are essential for the epigenetic maintenance of human centromeres during DNA replication.
Supplementary Figure 1: Identification of peaks enriched for CENP-A binding.
Supplementary Figure 2: CENP-A ChIP-seq identifies CENP-A binding at reference centromeres of 23 human chromosomes.
Supplementary Figure 3: Ectopic deposition of CENP-A into open and active chromatin at G1 does not function as a seeding hotspot for neocentromere formation.
Supplementary Figure 4: Non-centromeric CENP-A binding peaks overlap with active transcription marks.
Supplementary Figure 5: Centromeres are late replicating with CENP-A remaining tethered locally by continued binding to the CCAN complex.
Supplementary Figure 6