Next-Generation Sequencing Enables Spatiotemporal Resolution of Human Centromere Replication Timing

Abstract

Centromeres serve a critical function in preserving genome integrity across sequential cell divisions, by mediating symmetric chromosome segregation. The repetitive, heterochromatic nature of centromeres is thought to be inhibitory to DNA replication, but has also led to their underrepresentation in human reference genome assemblies. Consequently, centromeres have been excluded from genomic replication timing analyses, leaving their time of replication unresolved. However, the most recent human reference genome, hg38, included models of centromere sequences. To establish the experimental requirements for achieving replication timing profiles for centromeres, we sequenced G1- and S-phase cells from five human cell lines, and aligned the sequence reads to hg38. We were able to infer DNA replication timing profiles for the centromeres in each of the five cell lines, which showed that centromere replication occurs in mid-to-late S phase. Furthermore, we found that replication timing was more variable between cell lines in the centromere regions than expected, given the distribution of variation in replication timing genome-wide. These results suggest the potential of these, and future, sequence models to enable high-resolution studies of replication in centromeres and other heterochromatic regions.

CENP-A chromatin prevents replication stress at centromeres to avoid structural aneuploidy

“…Since DNA replication stress can induce recombination, we assessed if replication fork dynamics were altered by CENP-A depletion using DNA combing for single molecule analysis (33). We performed sequential labeling of nascent DNA with nucleotide analogs, CldU and IdU (Figure 2A) during early S when mostly euchromatin is replicated (34), and in late S when centromeres are replicated (18,19) as confirmed by BrdU-Immunoprecipitation ( Figure S2A). A change in CIdU + IdU tracks length was used as indicator of a change in replication fork progression.…”

“…While pericentromeric regions form heterochromatin, the core centromere harbors euchromatic characteristics with active transcription throughout the cell cycle, where long non-coding RNAs act in cis to contribute to centromere functions (16,17). To date, it is unknown how a) transcription (17), b) recombination (10), c) late replication (18,19)…”

Comparing DNA replication programs reveals large timing shifts at centromeres of endocycling cells in maize roots

“…These seven centromeres, which are well assembled in the maize B73 RefGen_v4 genome, contain satellite repeats interspersed with retrotransposons [38,47], enabling almost 50% of our sequencing reads that map to these centromeres to be uniquely positioned. In most species, in which centromeres contain large numbers of tandemly arrayed satellite repeats, it is difficult to map centromeric sequence reads to unique positions and, thus, to fully assess centromeric RT patterns [76]. Though yeast centromeres replicate in early S phase [77][78][79][80], most higher eukaryotes replicate centromeres asynchronously through mid to late S phase [54,[81][82][83][84][85][86] The presence of distinct peaks of early replication in or adjacent to functional centromeres (arrowheads in Fig 3 and S12) is noteworthy because they signify a population preference for initiation in early S phase at these loci.…”

New, easy, quick and efficient DNA replication timing analysis by high-throughput approaches

“…We wanted to know if START-R suite can run the correct analyses with this type of data and also with other organisms than mouse and human. We performed exactly the same pipeline used for early-late Repli-seq data described above for Drosophila, zebrafish and human S/G1 data (Armstrong et al, 2018;Siefert et al, 2017;Massey et al, 2019), in order to be sure that the integration into the START-R pipeline was correct. Then and as expected,…”

“…Different laboratories analyze variations of DNA copy number between G1 and S phase cells (S/G1 ratio) to study the replication timing program with Repli-seq. We used data obtained from different organisms such as Drosophila, zebrafish and human (Armstrong et al, 2018;Siefert et al, 2017;Massey et al, 2019), to validate the START-R suite (GEO accession numbers: GSM3154888 and GSM3154890 for HWT Drosophila melanogaster female larvae wing disc cells in S and G1 phase, respectively; GSM2282090 for 28hpf Danio rerio embryos; SRX3413939-40 for HEK293T human cells in S and G1 phase, respectively). As previously, reads from G1 and S fractions are mapped with Bowtie2, then PCR duplicates are removed by RmDUP tool, and Bamcoverage is used to obtain the coverage with RPKM.…”