Biology subjects

Tijhuis, A.

Publications and source records attributed to Tijhuis, A..

4 recordsLinked to original sources

bioRxiv2025

BTRR complex deficiency is a driver for genomic instability in Bloom syndrome

Biallelic loss-of-function (LoF) variants in the BTRR complex members BLM, TOP3A, RMI1, and RMI2 cause Bloom syndrome (BSyn). The BTRR complex mainly acts on DNA replication and DNA repair processes, and dysfunction of this complex underlies the increased genomic instability and cancer predisposition associated with the BSyn phenotype. Here, we report CRISPR/Cas9-based genome-edited isogenic induced pluripotent stem cell (iPSC) models with compound heterozygous LoF variants in BLM, TOP3A, and RMI1. The cellular phenotype of all three BTRR-deficient iPSC lines included chromosome segregation defects, increased sister chromatid exchange rates, and impaired DNA single-strand template repair. Using single-cell whole genome sequencing, we showed that BTRR complex deficiency causes increased genome copy number alterations (CNAs) and, therefore, is a driver for genomic instability. CNA load was further induced by applying replication stress, and we observed that BTRRKO iPSCs acquired fewer de novo CNA events compared to wild-type cells, suggesting a possible selection loss against cells with high levels of DNA damage. Importantly, stress-induced and non-stress-induced CNAs in single-cell genomes were not stochastically distributed throughout the genome, but instead enriched at fragile sites. This finding might offer an opportunity for the development of novel NGS-based approaches to measure rates of genomic instability in disease conditions.

genetics↗

bioRxiv2023

Chronic deregulation of the spindle assembly checkpoint triggers myelosuppression and gastrointestinal atrophy

Interference with microtubule dynamics in mitosis activates the spindle assembly checkpoint (SAC) to prevent chromosome segregation errors. The SAC induces mitotic arrest by inhibiting the anaphase-promoting complex (APC) via the mitotic checkpoint complex (MCC). The MCC component MAD2 neutralises the critical APC cofactor, CDC20, preventing mitotic exit. In cancer cell lines, this can provoke apoptosis involving pro-apoptotic BCL2 family members BIM and NOXA while BCL2 overexpression blocks mitotic cell death, facilitating SAC adaptation. However, the consequences of apoptosis after SAC perturbation in vivo are unclear. By conditional MAD2 overexpression across tissues in mice, we observed that chronic SAC activation triggers bone marrow aplasia and intestinal atrophy. While myelosuppression was tolerated, gastrointestinal atrophy was detrimental. Remarkably, co-deletion of Bim/Bcl2l11, but not Bid, Puma/Bbc3 or Noxa/Pmaip, prevented developing gastrointestinal syndrome caused by chronic SAC activity, identifying BIM as rate-limiting for mitotic cell death in the gastrointestinal epithelium. In contrast, only BCL2 overexpression but none of the BH3-only protein deficiencies tested could mitigate myelosuppression, highlighting tissue and cell type-specific survival dependencies in response to SAC perturbation in vivo.

cell biology↗

bioRxiv2023

Tunable DNMT1 degradation reveals cooperation of DNMT1 and DNMT3B in regulating DNA methylation dynamics and genome organization

DNA methylation (DNAme) is a key epigenetic mark that regulates critical biological processes maintaining overall genome stability. Given its pleiotropic function, studies of DNAme dynamics are crucial, but currently available tools to interfere with DNAme have limitations and major cytotoxic side effects. Here, we present untransformed and cancer cell models that allow inducible and reversible global modulation of DNAme through DNMT1 depletion. By dynamically assessing the effects of induced passive demethylation through cell divisions at both the whole genome and locus-specific level, we reveal a cooperative activity between DNMT1 and DNMT3B to maintain and control DNAme. Moreover, we show that gradual loss of DNAme is accompanied by progressive and reversible changes in heterochromatin abundance, compartmentalization, and peripheral localization. DNA methylation loss coincided with a gradual reduction of cell fitness due to G1 arrest, but with minor level of mitotic failure. Altogether, this powerful system allows DNMT and DNA methylation studies with fine temporal resolution, which may help to reveal the etiologic link between DNA methylation dysfunction and human disease.

molecular biology↗

bioRxiv2021

Mechanisms of genetic instability in a single S-phase following whole genome doubling

Diploid and stable karyotypes are associated with health and fitness in animals. In contrast, whole genome duplications (WGDs) - doubling full chromosome content - are linked to genetic instability (GIN) and frequently found in human cancers 1-3. It has been established that WGDs fuel chromosome instability through abnormal mitosis 4-8, however, the immediate consequences of tetraploidy in the first interphase are not known. This is an essential question because single WGD events such as cytokinesis failure can promote tumorigenesis 9. Here, we found that newly born tetraploid human cells undergo high rates of DNA damage during DNA replication in the first S-phase. Using DNA combing and single cell sequencing, we show that DNA replication dynamics is perturbed, generating under- and over-replicated regions. Mechanistically, we found that these defects result from the lack of protein mass scaling up at the G1/S transition, which impairs the fidelity of DNA replication. This work shows that within a single interphase, unscheduled tetraploid cells can acquire highly abnormal karyotypes. These findings provide an explanation for the GIN landscape that favors tumorigenesis after tetraploidization.

cell biology↗