Identification and characterization of genetic and epigenetic factors important in de novo centromere formation in caenorhabditis elegans

Abstract

During cell division, kinetochores are assembled on centromeres to connect the chromosomes to mitotic spindles. The centromere is a special chromatin region that is marked by the conserved centromeric protein A (CENP-A). The centromere should be precisely propagated and inherited. Ectopic centromere (neocentromere) formation on chromosomes, in addition to the original centromere, will cause chromosome instability, aneuploidy and even trigger tumorigenesis. Neocentromeres were commonly found in cancer cells and patients with rearranged chromosomes. However, the cellular mechanism of neocentromere formation is not fully understood. As de novo centromeres can form rapidly on artificial chromosomes (ACs) after injection of foreign DNA in C. elegans germline, C. elegans artificial chromosomes were used as a model to determine the cellular mechanism of de novo centromere formation. In this study, I demonstrate that RbAp46/48 (LIN-53)-HAT-1 complex is essential for CENP-A (HCP-3) and Mis18BP1 (KNL-2) deposition on exogenously introduced DNA and facilitates de novo centromere formation on ACs in C. elegans embryos. De novo centromere formation on exogenous DNA is also dependent on Mis18BP1 (KNL-2) and condensin subunit, SMC-4, but is independent of the DNA replication process and MCM DNA helicase complex subunit, MCM-2. To reveal the relationship between DNA features and de novo centromere formation, I compared the segregation rates of ACs with different AT-contents or repetitiveness. AC segregation results suggest that high AT-content (74%) sequences are preferred for de novo centromeric formation when compared with lower AT-content (52% and 66%) sequences. For sequence repetitiveness, highly repetitive ACs do not take precedence over non-repetitive "complex" ACs in centromere formation. To investigate the organization of de novo centromere on ACs, a C. elegans strain carrying a “complex” AC was constructed by co-injection of restriction enzyme-digested yeast genomic DNA and fluorescent markers under a somatic, germline or ubiquitous promoter. Whole genome sequencing (WGS) of this strain was carried out using MinION and Illumina Mi-Seq platforms. By combining short, high-accurate Mi-Seq reads and long, error-prone MinION sequencing reads, de novo genome assembly of this strain with high continuity (N50 of 1.48 Mb) and accuracy (99.86 % identity to C. elegans reference genome) was achieved. I demonstrate that NHEJ is the dominant pathway for joining the injected linearized DNA fragments into a megabase-sized AC in C. elegans. Furthermore, CENP-A (HCP-3) chromatin immunoprecipitation followed by sequencing (ChIP-seq) shows that CENP-A (HCP-3) was excluded from the transcription active regions on ACs, consistent with the reported ChIP-chip results on endogenous chromosomes, suggesting that the non-expressed regions are preferred for de novo centromere formation. The sequence of the AC and its CENP-A domains provide insights in the AC formation and DNA element preferred for de novo centromere formation, respectively.