Biology subjects

Wolthuis, R. M. F.

Publications and source records attributed to Wolthuis, R. M. F..

8 recordsLinked to original sources

bioRxiv2026

The SKA complex is a mitotic vulnerability of chromosomally unstable cancers

Aneuploidy and chromosomal instability are common characteristics of cancer but remain underexploited therapeutically. Because chromosomally unstable cells exhibit altered mitotic control, they are selectively vulnerable to inhibition of the kinesin KIF18A. Here, we identify the spindle and kinetochore-associated (SKA) complex as a mitotic regulator that establishes a similar dependency. SKA loss is tolerated in cells with a defective mitotic checkpoint, but when mitosis is prolonged, it leads to mitotic errors and loss of viability. Genetic analysis reveals that SKA and KIF18A are epistatic, although they function through mechanistically distinct mitotic pathways. SKA depletion selectively impacts chromosomally unstable cancer cells and mirrors the effects of KIF18A inhibition. We thus identify a cellular vulnerability associated with prolonged mitosis in chromosomally unstable cancers.

cell biology↗

bioRxiv2025

Breaking Bad: Exploring & Complementing the Effects of the DNASE1L3 p.Arg206Cys Variant on Cell-Free DNA from an Isogenic Cell Line Model

DNASE1L3 is a key endonuclease, essential for proper fragmentation and clearance of cell-free DNA (cfDNA). The p.R206C common variant impairs DNASE1L3 secretion and activity, causing aberrant cfDNA fragmentation and therefore affecting liquid biopsy-based screening and diagnostics. Existing studies on DNASE1L3 relied on resource-intensive murine models or plasmid-based overexpression, which do not accurately represent native expression. To address this, we developed an isogenic HEK293T cell line model by using CRISPR Prime Editing for endogenous expression of DNASE1L3R206C. We analyzed the cfDNA composition directly from conditioned culture medium and found that fragment size distributions in mutant cells mimics the hypofragmented profiles previously observed in plasma samples from p.R206C carriers. We also showed that in vitro treatment of hypofragmented cfDNA with recombinant wildtype DNASE1L3 could enrich for mononucleosomal fragments, with fragment end-motifs characteristic of DNASE1L3 cleavage activity. This could open avenues for DNASE1L3 as a candidate pre-treatment agent to improve the accuracy and efficiency of cfDNA sequencing-based diagnostics in hypofragmented liquid biopsies. These findings demonstrate that our isogenic cell line model provides a controlled system to study cfDNA fragmentation biology and DNASE1L3 function.

molecular biology↗

bioRxiv2024

Finding novel vulnerabilities of hypomorphic BRCA1 alleles

With the recent rise in CRISPR/Cas9-mediated genome-wide synthetic lethality screens, many new synthetic lethal targets have been identified for diseases with underlying genetic causes such as tumours with BRCA1 mutations. Such screens often use full deficiency of a protein to identify novel vulnerabilities. However, patient-derived mutations not only result in loss of the protein but often also concern missense mutations with hypomorphic phenotypes. Here we study the genetic vulnerabilities of two previously described hypomorphic BRCA1 missense mutations and compare these to a BRCA1-depleted setting to study whether this affects screening for synthetic lethal interactions. Our research showed that BRCA1I26A mutated cells have very similar vulnerabilities to BRCA1 wildtype cells, confirming its low tumorigenic effect. In contrast, the BRCA1R1699Q mutation induced a more similar phenotype to BRCA1-deficient cells. For this mutation, we also unveiled a unique vulnerability to the loss of NDE1. Specifically in BRCA1R1699Q mutated cells, and not BRCA1-proficient or -deficient cells, NDE1 loss leads to increased genomic instability. Altogether our findings highlight the importance to differentiate between patient-derived mutations when assessing novel treatment targets.

cancer biology↗

bioRxiv2022

CRISPR screens in sister chromatid cohesion defective cells reveal PAXIP1-PAGR1 as regulator of chromatin association of cohesin

The cohesin complex regulates higher order chromosome architecture through maintaining sister chromatid cohesion and folding chromatin by active DNA loop extrusion. Impaired cohesin function underlies a heterogeneous group of genetic syndromes and is associated with cancer. Here, by using synthetic lethality CRISPR screens in isogenic human cell lines defective of specific cohesion regulators, we mapped the genetic dependencies induced by absence of DDX11 or ESCO2. The obtained high confidence synthetic lethality networks are strongly enriched for genes involved in DNA replication and mitosis and support the existence of parallel sister chromatid cohesion establishment pathways. Among the hits, we identified the chromatin binding, BRCT-domain containing protein PAXIP1 as a novel cohesin regulator. Depletion of PAXIP1 severely aggravated cohesion defects in ESCO2 defective cells, leading to mitotic cell death. PAXIP1 promoted the global chromatin association of cohesin, independent of DNA replication, a function that could not be explained by indirect effects of PAXIP1 on transcription or the DNA damage response. Cohesin regulation by PAXIP1 required its binding partner PAGR1 and a conserved FDF motif in PAGR1. Similar motifs were previously found in multiple cohesin regulators, including CTCF, to mediate physical interactions with cohesin. PAXIP1 co-localizes with cohesin on multiple genomic loci, including at active gene promoters and enhancers. Together, this study identifies the PAXIP1-PAGR1 complex as a novel regulator of cohesin occupancy on chromatin. Possibly, this role in cohesin regulation is also relevant for previously described functions of PAXIP1 in transcription, immune cell maturation and DNA repair.

molecular biology↗

bioRxiv2022

Evaluation of folliculin detection by immunohistochemistry in Birt-Hogg-Dube associated kidney tumors

Germline inactivating mutations in folliculin (FLCN) cause Birt-Hogg-Dube (BHD) syndrome, a rare autosomal dominant disorder predisposing to kidney tumors. Kidney tumors associated with BHD typically lack FLCN expression due to loss of heterozygosity. In this study we assessed the potential of four commercial anti-FLCN antibodies for immunohistochemistry, as currently no routine diagnostic FLCN stainings are performed in the clinic. Despite comprehensive testing, we could not identify a commercial anti-FLCN antibody that is reproducibly effective in immunohistochemical analyses of formalin-fixed paraffin-embedded tissue material. We propose that dedicated future efforts are required to develop a suitable antibody for diagnostic immunohistochemical stainings. The inclusion of FLCN expression status as part of standard renal tumor pathology may contribute to better analyses of the molecular pathology of BHD tumors and facilitate identification of BHD patients, improve their (genetic and clinical) counseling, and enable genetic testing of at risk relatives.

genetics↗

bioRxiv2022

ShrinkCRISPR : A flexible method for differential fitness analysis of CRISPR-Cas9 screen data.

CRISPR screens provide large-scale assessment of cellular gene functions. Pooled libraries typically consist of several single guide RNAs (sgRNAs) per gene, for a large number of genes, which are transduced in such a way that every cell receives at most one sgRNA, resulting in the disruption of a single gene in that cell. This approach is often used to investigate effects on cellular fitness, by measuring sgRNA abundance at different time points. Comparing gene knockout effects between different cell populations is challenging due to variable cell-type specific parameters and between replicates variation. Failure to take those into account can lead to inflated or false discoveries. We propose a new, flexible approach called ShrinkCRISPR that can take into account multiple sources of variation. Impact on cellular fitness between conditions is inferred by using a mixed-effects model, which allows to test for gene-knockout effects while taking into account sgRNA-specific variation. Estimates are obtained using an empirical Bayesian approach. ShrinkCRISPR can be applied to a variety of experimental designs, including multiple factors. In simulation studies, we compared ShrinkCRISPR results with those of drugZ and MAGeCK, common methods used to detect differential effect on cell fitness. ShrinkCRISPR yielded as many true discoveries as drugZ using a paired screen design, and outperformed both drugZ and MAGeCK for an independent screen design. Although conservative, ShrinkCRISPR was the only approach that kept false discoveries under control at the desired level, for both designs. Using data from several publicly available screens, we showed that ShrinkCRISPR can take data for several time points into account simultaneously, helping to detect early and late differential effects. ShrinkCRISPR is a robust and flexible approach, able to incorporate different sources of variations and to test for differential effect on cell fitness at the gene level. These improve power to find effects on cell fitness, while keeping multiple testing under the correct control level and helping to improve reproducibility. ShrinkCrispr can be applied to different study designs and incorporate multiple time points, making it a complete and reliable tool to analyze CRISPR screen data.

bioinformatics↗

bioRxiv2021

ELOF1 is a transcription-coupled DNA repair factor that directs RNA polymerase II ubiquitylation

Cells employ transcription-coupled repair (TCR) to eliminate transcription-blocking DNA lesions. The binding of the TCR-specific repair factor CSB triggers DNA damage-induced ubiquitylation of RNA polymerase II (RNAPII) at a single lysine (K1268) by the CRL4CSA ubiquitin ligase. However, how the CRL4CSA ligase is specifically directed toward the K1268 site is unknown. Here, we identify ELOF1 as the missing link that facilitates RNAPII ubiquitylation, a key signal for the assembly of downstream repair factors. This function requires its constitutive interaction with RNAPII close to the K1268 site, revealing ELOF1 as a specificity factor that positions CRL4CSA for optimal RNAPII ubiquitylation. Furthermore, drug-genetic interaction screening reveals an unanticipated compensatory TCR pathway in which ELOF1 together with known factors DOT1L and HIRA protect CSB-deficient cells from collisions between transcription and replication machineries. Our study provides a genetic framework of the transcription stress response and reveals key insights into the molecular mechanism of TCR.

molecular biology↗

bioRxiv2019

Non-Redundant Roles in Sister Chromatid Cohesion of the DNA Helicase DDX11 and the SMC3 Acetyl Transferases ESCO1/2

In a process linked to DNA replication, duplicated chromosomes are co-entrapped into large, circular cohesin complexes and functional sister chromatid cohesion (SCC) is established by acetylation of the SMC3 cohesin subunit. Several rare human developmental syndromes are characterized by defective SCC. Roberts Syndrome (RBS) is caused by mutations in the SMC3 acetyl transferase ESCO2, whereas mutations in the DNA helicase DDX11 lead to Warsaw Breakage Syndrome (WABS). We found that WABS-derived cells predominantly rely on ESCO2, not ESCO1, for residual SCC, growth and survival. Reciprocally, RBS-derived cells depend on DDX11 to maintain low levels of SCC. Synthetic lethality between DDX11 and ESCO2 correlated with a prolonged delay in mitosis, and was rescued by knock down of the cohesion remover WAPL. Rescue experiments using mouse or human cDNAs revealed that DDX11, ESCO1 and ESCO2 act on differential aspects of DNA replication-coupled SCC establishment. Importantly, DDX11 is required for normal DNA replication fork speed without clearly affecting SMC3 acetylation. We propose that DDX11 and ESCO2 control spatially separated fractions of cohesin, with supportive roles for DDX11 in replication-associated cohesion establishment, and for ESCO2 in cohesin complexes located around centromeres, respectively.

cell biology↗