The ends of linear chromosomes are protected from being recognized as DNA damage by a nucleo-protein complex called telomere (Figure 1). Telomere is composed of repetitive DNA sequences (TTAGGG in all vertebrates) and a protein complex called shelterin that directly binds to telomeric DNA. The end of telomeric DNA has protruding 3'-overhang that can loop back into double-strand repeat sequences to form a protective structure (T-loop) (Figure 1).
Telomeric DNA sequences get shorter during each human somatic cell division cycle until they eventually reach a critical length – upon which telomeric ends no longer serve their protective function. This leads to permanent cell cycle arrest, a phenomenon called replicative senescence, through activation of various DNA damage response (DDR) mechanisms. When cells possess defects in DDR, they can bypass senescence and keep dividing while telomeres get further shortened. This final stage, called telomere crisis, is characterized by massive cell death and accumulation of chromosome instability, the latter of which is a well-accepted driving force of tumorigenesis (Figure 2).
Figure 2 Involvement of telomeres in cellular aging and tumorigenesis.
The length of individual telomeres (there are 92 telomeres on 46 chromosomes!) in a given cell varies (green circles). In a young, healthy somatic cell, telomeres take a loop form called "t-loop," which protects chromosome ends from being recognized as DNA damage ("closed"-state). Due to the end replication problem, telomeres get shorter and shorter until some telomeres hit the "intermediate" threshold (orange circles). The intermediate-state telomeres are recognized as DNA damage while still being protected from telomere-to-telomere fusion due to residual telomere repeats and TRF2. This intermediate-state allows persistent DDR signaling from telomeric ends, leading to permanent cell cycle arrest, called "replicative senescence". If a cell possesses mutations in the DDR pathway, the intermediate state telomeres cannot halt the cell cycle. Such cell keeps dividing and loses telomeric DNA and TRF2 at the end of chromosomes (red circles), resulting in "open-state" telomeres, which are free to fuse with another DNA double-strand end. Chromosome fusions cause bridge formation in mitosis, which triggers chromosome abnormalities and most cells die during this telomere crisis stage. On the other hand, if a cell possesses a mutation that allows expression of telomerase catalytic subunit, such cell may outgrow from the crisis population and become tumor.
Chromosome fusions have been proposed to be the underlying cause of both cell death and chromosome instabilities during telomere crisis. However, the detailed mechanisms remain elusive. The goal of our current projects is to understand how chromosome fusions can result in diverse cellular fates during telomere crisis. Figure 3 shows the mitotic cell death pathway hypothesis during the telomere crisis. Telomere fusions somehow cause cell cycle arrest in mitosis in the spindle assembly checkpoint (SAC)-dependent manner. Mitotic arrest causes telomere deprotection (mitotic telomere deprotection), which then leads to cell death.
Project 1 the fate of a distinct chromosome fusion
We have recently developed a new cellular tool, called Fusion Visualization system for the Xp sister chromatid fusion (FuVis-XpSIS). The FuVis relies on an artificial DNA cassette integrated into the Xp subtelomere. The cassette has been designed so that the CRISPR/Cas9-mediated DSB of the cassette generates a single sister chromatid fusion concomitantly with mCitrine expression (Figure 4). With this new technique, we are analyzing the fate of a specific single sister chromatid fusion in cancer cells.
Figure 4 Summary of FuVis-XpSIS system
In the sister cassette, the YFP gene is separated into two exons. Exon 1 is driven by a promoter, while exon 2 is located upstream of the promoter in opposite direction. Exon 1 is connected to the p2a-neomycin resistant gene by splicing dinner (SD) and acceptor (SA). The sister cassette was integrated into a subtelomere region on the short arm of chromosome X. This cell becomes neomycin resistant without expressing YFP.
When the "Cas9 target" region is cut by CRISPR/Cas9, in most cases, the damage is repaired with indels that mutate the original Cas9 target sequence. However, in a rare case, two sister chromatids are fused through erroneous repair, resulting in sister-chromatid fusion. This sister-chromatid fusion allows expression of full-length YFP through splicing.
Project 2 the molecular mechanism of mitotic telomere deprotection
As shown in Figure 3, mitotic telomere deprotection is an important cell death pathway during telomere crisis, which can be both pro-and anti-tumorigenesis pathways. We are focusing on the molecular mechanism that induces telomere deprotection during mitotic arrest with a hope to manipulate mitotic telomere deprotection to combat pre-cancerous cells.