Synthetic chromosomes to understand genome instability

Genome instability is a foundational hallmark of cancer, which results in increased genetic diversity, thereby promoting tumour evolution. However, as a multitude of complex factors cause genome instability, it is difficult to understand the underlying mechanisms. One key stage at which genome instability can arise is during chromosome segregation. Typically, the presence of centromeres ensures faithful chromosome segregation however in cancers, aberrant centromere formation can lead to inefficient segregation resulting in aneuploidy, whereas endogenous centromere deletion can lead to the formation of neocentromeres that maintains genomically unstable karyotypes. Although the centromere's role in genome stability has long been known, the investigation into factors which determine centromere formation and maintenance has proven difficult due to its location on repetitive heterochromatic regions of the DNA. The Gilbert lab has isolated a chromosome 3 harbouring a neocentromere at 3q24 (Ventura et al., 2004), named Neo3, within a human-hamster hybrid cell line, HybNeo3 (Naughton et al., 2021). As the Neo3 is located within non-repetitive, it will facilitate investigation into the centromere.
Previous studies in model organisms have indicated that the stability of centromeres depends on transcription. Experiments performed by the Gilbert lab show Neo3 in a decompacted chromosomal region compared to the same loci on normal chromosome 3 and this decompaction was transcription-dependent. Although Neo3 has active marks and RNA polymerase II localisation, there were no detectable transcripts (Naughton et al., 2021). Therefore, I will investigate the role of transcription in regulating chromosome stability on the Neo3 centromere, with the use of Cas9-based transactivators. This may reveal the mechanistic background of whether transcription causes instability, how the transcription affects chromatin architecture at the centromere, and primary constriction determination.
As centromeres can shift and form aberrant neocentromeres, it is important to identify genomic locations at which centromeres may form. To investigate this, the HybNeo3 will be genetically engineered to incorporate loxP sites flanking the Neo3 to allow for inducible deletion using CRISPR/Cas9, upon Cre-dependent recombination. We will additionally incorporate selective markers to help screen for cells where a neocentromere has formed and we will characterise this library of new neocentromeres by deep sequencing. Recent publications have shown that neocentromere formation by these means is possible (Murillo-Pineda et al., 2021). Deep sequencing and FISH analysis of the new neocentromeres throughout their passages may permit the identification of consensus or repetitive sequence formation at the neocentromere.
The Kinetochore is an essential organisation of proteins that localise at the centromere and potentiate faithful segregation of chromosomes during cell division. Neo3 along with the new neocentromere library will also be investigated for their kinetochore function and organisation.
Finally, a CRISPRi library will be developed by identifying the top 1000 most mutated genes in cancer from the cancer genome atlas (TCGA). The library will be used to transfect HybNeo3 and the inducible Neo3 deletion system, additionally cells will be scored for whether the synthetic chromosome is lost or maintained. If lost, it can be deemed that the candidate gene is essential for centromere formation and or maintenance, therefore its repression inhibits the formation and maintenance of neocentromeres. To corroborate the role of genes in centromere formation and maintenance, they may be overexpressed within an induced Neo3-deletion cell and assessed on whether a higher proportion of cells form centromeres. Such investigation may identify driver genes for genome instability, which could be novel targets for future drug development.