Biology subjects

Medema, R. H.

Publications and source records attributed to Medema, R. H..

At least 19 recordsLinked to original sources

bioRxiv2026

Gene context shapes spatial organization and transcriptional heterogeneity of extrachromosomal DNA in cancer cells

Extrachromosomal DNA (ecDNA) promotes tumor adaptability by making oncogenic driver genes variably abundant, spatially mobile, and heterogeneously expressed. Transcriptional hyperactivation and spatial mobility of ecDNA-amplified genes is associated with aberrant regulatory states that could create targetable vulnerabilities in ecDNA-driven cancers. However, how gene context shapes their nuclear positioning and aberrant transcription remains unclear. To explore spatio-transcriptional behavior across ecDNA-amplified genes, we developed FLAGx, a multimodal imaging method that quantitatively resolves transcriptional activity and nuclear position of individual ecDNA genes in situ. Surprisingly, we find that gene context affects subnuclear distribution and transcriptional deregulation of ecDNA. Further, ecDNA clustering, cell cycle regulation, and drug responses depend on gene context, while transcriptional shutdown in mitosis persists across ecDNA species, arguing against pervasive mitotic transcription as a common feature. These findings show that driver gene context shapes ecDNA behavior and establish FLAGx as a useful approach to separate gene-specific from shared ecDNA features.

cancer biology↗

bioRxiv2026

Transcription controls chromatin-nuclear lamina contacts through distinct Lamin A and LBR tethering mechanisms

Lamina-associated domains (LADs) are large genomic regions that interact with the nuclear lamina (NL). Much of the underlying "grammar" governing their positioning at the nuclear periphery remains unclear. LADs are composed of heterochromatin and typically harbor repressed genes, and their association with the NL is generally incompatible with strong transcriptional activity. The extent to which transcription globally shapes chromatin-NL interactions is not fully understood. Here, we combined acute transcription inhibition using Flavopiridol or Triptolide with genome-wide mapping of chromatin-NL contacts. We found that chromatin-NL interactions are rapidly rewired upon transcription inhibition. Changes in chromatin-NL contacts upon transcription shutdown are predictable based on transcriptional activity and the presence of H3K9me3-marked heterochromatin. This rewiring is reversible, as genome-NL interactions quickly return to baseline levels following drug wash-off. Notably, gain and loss of chromatin-NL interactions upon transcription shutdown reflect two distinct tethering mechanisms. Inter-LADs genomic regions (iLADs) enriched in highly active genes and located near stable LADs, which are tethered by Lamin A (LMNA/C), become re-attached to the NL following transcription inhibition. In parallel, H3K9-methylated regions tethered to the nuclear envelope by the Lamin B receptor (LBR) undergo extensive detachment from the NL. Strikingly, LMNA/C and LBR oppositely regulate transcription-sensitive LADs and are required for transcriptional control of chromatin-NL contacts. Together, our findings highlight the plasticity and dynamic nature of chromatin-NL interactions and provide the first evidence that LMNA/C- and LBR-mediated tethering mechanisms exhibit distinct sensitivities to transcription inhibition. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=155 SRC="FIGDIR/small/738400v1_ufig1.gif" ALT="Figure 1"> View larger version (46K): org.highwire.dtl.DTLVardef@16781a2org.highwire.dtl.DTLVardef@6febd1org.highwire.dtl.DTLVardef@1e3220forg.highwire.dtl.DTLVardef@d137f7_HPS_FORMAT_FIGEXP M_FIG C_FIG HIGHLIGHTSO_LITranscription inhibition alters chromatin-NL contacts rapidly and reversibly C_LIO_LIActive transcription prevents inter-LADs located near LMNA/C-tethered LADs from associating with the nuclear lamina. C_LIO_LILBR-tethered heterochromatin is repositioned away from the NL C_LIO_LITranscription-dependent modulation of chromatin-NL contacts is dependent on LMNA/C and partially on LBR C_LI

genomics↗

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

Megabase-scale detachment of genome-nuclear lamina interactions is required for efficient repair of double stranded breaks

Lamina-associated domains (LADs) are large genomic regions that contact the nuclear lamina (NL). Double-strand breaks (DSBs) in LADs are known to be repaired more slowly and with different pathway preferences compared to other chromatin contexts. However, little is known about the chromatin changes at LADs that occur during DSB repair. Here, we report that a single DSB inside a LAD can cause detachment from the NL over several megabases. This profound spatial rearrangement is transient and reverts within 48 hours. Preventing this detachment slows down repair kinetics and renders repair incomplete, indicating that NL detachment is required for efficient repair of DSBs in LADs. NL detachment is dependent on {psi}H2AX and ATM, while it is antagonized by DNAPKcs activity. Remarkably, {psi}H2AX also antagonizes NL interactions at chromosome ends. Taken together, our data indicate that {psi}H2AX accumulation in LADs induces large scale rewiring of genome-NL interactions, allowing for efficient repair of DSBs.

cell biology↗

bioRxiv2025

Visualizing homology search in living cells

Homologous recombination (HR) is a DNA repair process that requires a homologous sequence for the repair of DNA double-stranded breaks. The search for homology is the rate limiting step for HR, but the dynamics of homology search in vivo are currently poorly understood. This is largely due to a lack of tools to visualize homology search in action in living human cells. Here, we show that RAD51 and MND1, two proteins operating in homology search, become visible in long filamentous structures several hours after double-stranded break formation. Using GFP-MND1 we can visualize these filaments in living human cells and find that they are highly dynamic, explore the nuclear space, and can resolve over time. We show that the resolution of these filaments depends on RAD54L, known for its role in RAD51-driven homology search and strand-invasion. In addition, we find that loss of cohesin also inhibits the resolution of these filaments, in accordance with a role for cohesin in HR. Thus, our data suggest that these filaments are visible intermediates of an active DNA repair process, and that MND1-GFP can be used as a tool to study the dynamics of homology search in living human cells.

cell biology↗

bioRxiv2025

Identification of critical factors of the replication stress response in human cells

High fidelity of replication is important to preserve genomic integrity and ensure healthy progeny. Perturbations of replication, also known as replication stress, is frequently observed in cancer cells and is considered a cancer cell-specific trait. Although replication stress drives genomic instability and tumor progression, it also generates a targetable cancer-specific vulnerability. In order to identify potential therapeutic targets in cancer cells that experience replication stress, we performed a genome wide genetic screen in human HAP1 cells challenged with low doses of replication stress-inducing drugs. We identified a large set of genes that specifically hamper cell survival in the context of replication stress. In addition to well-known players in the replication stress response and DNA repair, such as RNASEH1, BRIP1, and MDC1, we identified several genes with no prior described role in DNA replication, damage tolerance or repair. We validated that the loss of GIGYF2, HNRNPA2B1, and SUMO2 renders cells more vulnerable to replication stress. For GIGYF2 and SUMO2, we could implicate a role in homologous recombination. Taken together, our replication stress screen identified several known as well as some novel factors that protect against the toxic implications of replication stress. These factors could entail potential therapeutic targets for cancer cells experiencing replication stress.

cell biology↗

bioRxiv2024

Coordinated control of genome-nuclear lamina interactions by Topoisomerase 2B and Lamin B receptor

Lamina-associated domains (LADs) are megabase-sized genomic regions anchored to the nuclear lamina (NL). Factors controlling the interactions of the genome with the NL have largely remained elusive. Here, we identified DNA topoisomerase 2 beta (TOP2B) as a regulator of these interactions. TOP2B binds predominantly to inter-LAD (iLAD) chromatin and its depletion results in a partial loss of genomic partitioning between LADs and iLADs, suggesting that its activity might protect specific iLADs from interacting with the NL. TOP2B depletion affects LAD interactions with lamin B receptor (LBR) more than with lamins. LBR depletion phenocopies the effects of TOP2B depletion, despite the different positioning of the two proteins in the genome. This suggests a complementary mechanism for organising the genome at the NL. Indeed, co-depletion of TOP2B and LBR causes partial LAD/iLAD inversion, reflecting changes typical of oncogene-induced senescence. We propose that a coordinated axis controlled by TOP2B in iLADs and LBR in LADs maintains the partitioning of the genome between the NL and the nuclear interior. HighlightsO_LILADs and iLADs differ in supercoiling state C_LIO_LITOP2B controls genome partitioning between nuclear lamina and nuclear interior C_LIO_LITOP2B depletion preferentially affects genome interactions with LBR C_LIO_LISimilar impact of TOP2B depletion and LBR depletion on genome-NL interactions C_LIO_LICo-depletion of TOP2B and LBR recapitulates LAD reshaping typical of oncogene-induced senescence. C_LI

genomics↗

bioRxiv2023

Reduction of chromosomal instability and inflammation is a common aspect of adaptation to aneuploidy

Aneuploidy, while detrimental to untransformed cells, is notably prevalent in cancer cells. This indicates that cancer cells have the ability to surmount the initial stress responses associated with aneuploidy, enabling rapid proliferation despite aberrant karyotypes. To generate more insight into key processes and requirements underlying the adaptation to aneuploidy, we generated a panel of aneuploid clones in p53-deficient RPE-1 cells and studied their behavior over time. As expected, de novo generated aneuploid clones initially displayed reduced fitness, enhanced levels of chromosomal instability and an upregulated inflammatory response. Intriguingly, after a prolonged period of culturing, aneuploid clones exhibited increased proliferation rates while maintaining aberrant karyotypes, indicative of an adaptive response to the aneuploid state. Interestingly, all adapted clones displayed reduced chromosomal instability (CIN) and reduced inflammatory signaling, suggesting that these are common aspects of adaptation to aneuploidy. Collectively, our data suggests that CIN and concomitant inflammation are key processes that require correction to allow for fast growth. Finally, we provide evidence that amplification of oncogenic KRAS can promote adaptation.

cell biology↗

bioRxiv2023

A compendium of Amplification-Related Gain Of Sensitivity (ARGOS) genes in human cancer

Chromosomal gains are among the most frequent somatic genetic alterations occurring in cancer. While the effect of sustained oncogene expression has been characterized, the impact of copy-number gains affecting collaterally-amplified "bystander" genes on cellular fitness remains less understood. To investigate this, we built a comprehensive map of dosage compensations across human cancers by integrating expression and copy number profiles from over 8,000 TCGA tumors and CCLE cell lines. Further, we analyzed the effect of gene overexpression across 17 human cancer ORF screens to provide an overview of genes that prove toxic to cancer cells when overexpressed. Combining these two independent approaches we propose a class of Amplification-Related Gain Of Sensitivity (ARGOS) genes. These genes are located in commonly amplified regions of the genome, have lower expression levels than expected by their copy-number status, and are toxic to cancer cells when overexpressed. We experimentally validated CDKN1A and RBM14 as high-confidence pan-cancer ARGOS genes in lung and breast cancer cell line models. We additionally suggest that RBM14s mechanism of toxicity involves altered DNA damage response and innate immune signaling processes following gene overexpression. Finally, we provide a comprehensive catalog of compensated, toxic, and ARGOS genes as a community resource.

cancer biology↗

bioRxiv2023

Paradoxical activation of oncogenic signaling as a cancer treatment strategy

Cancer homeostasis depends on a balance between activated oncogenic pathways driving tumorigenesis and engagement of stress-response programs that counteract the inherent toxicity of such aberrant signaling. While inhibition of oncogenic signaling pathways has been explored extensively, there is increasing evidence that overactivation of the same pathways can also disrupt cancer homeostasis and cause lethality. We show here that inhibition of Protein Phosphatase 2A (PP2A) hyperactivates multiple oncogenic pathways and engages stress responses in colon cancer cells. Genetic and compound screens identify combined inhibition of PP2A and WEE1 as synergistic in multiple cancer models by collapsing DNA replication and triggering premature mitosis followed by cell death. This combination also suppressed the growth of patient-derived tumors in vivo. Remarkably, acquired resistance to this drug combination suppressed the ability of colon cancer cells to form tumors in vivo. Our data suggest that paradoxical activation of oncogenic signaling can result in tumor suppressive resistance.

cancer biology↗

bioRxiv2022

Widespread chromatin context-dependencies ofDNA double-strand break repair proteins

DNA double-strand breaks are repaired by multiple pathways, including non-homologous end-joining (NHEJ) and microhomology-mediated end-joining (MMEJ). The balance of these pathways is dependent on the local chromatin context, but the underlying mechanisms are poorly understood. By combining knockout screening with a dual MMEJ:NHEJ reporter inserted in 19 different chromatin environments, we identified dozens of DNA repair proteins that modulate pathway balance dependent on the local chromatin state. Proteins that favor NHEJ mostly synergize with euchromatin, while proteins that favor MMEJ generally synergize with distinct types of heterochromatin. BRCA2 is an example of the former, which is corroborated by chromatin-dependent shifts in mutation patterns of BRCA2-/- cancer genomes. These results uncover a complex network of proteins that regulate MMEJ:NHEJ balance in a chromatin context-dependent manner. ONE SENTENCE SUMMARYA multiplexed screen reveals how dozens of proteins sense the local chromatin context to tune the balance between two DNA repair pathways.

molecular biology↗

bioRxiv2022

MND1 enables repair of two-ended DNA double-strand breaks

Faithful and timely repair of DNA double-strand breaks (DSBs) is fundamental for the maintenance of genomic integrity. Here, we demonstrate that the meiotic recombination co-factor MND1 facilitates the repair of DSBs in somatic cells. We show that MND1 localizes to DSBs, where it stimulates DNA repair through homologous recombination (HR). Importantly, MND1 is not involved in the response to replication-associated DSBs, implying that it is dispensable for HR-mediated repair of one-ended DSBs. Instead, we find that MND1 specifically plays a role in the response to two-ended DSBs that are induced by IR or various chemotherapeutic drugs, specifically in G2. MND1 localization to DSBs is dependent on resection of the DNA ends, and seemingly occurs through direct binding of MND1 to RAD51-coated ssDNA. Importantly, the lack of MND1-driven HR repair directly potentiates the toxicity of IR-induced damage, which could open new possibilities for therapeutic intervention, specifically in HR-proficient tumors.

cell biology↗

bioRxiv2021

Cancer tolerance to chromosomal instability is driven by Stat1 inactivation in vivo

Chromosomal instability is a hallmark of cancer, but also an instigator of aneuploidy-induced stress, reducing cellular fitness. To better understand how cells with CIN adjust to aneuploidy and adopt a malignant fate in vivo, we performed a genome-wide mutagenesis screen in mice. We find that specifically aneuploid tumors inactivate Stat1 signaling in combination with increased Myc activity. By contrast, loss of p53 is common, but not enriched in CIN tumors. Validation in another tissue type confirmed that CIN promotes immune cell infiltration, which is alleviated by Stat1 loss combined with Myc activation, but not with p53 inactivation, or Myc activation alone. Importantly, we find that this mechanism is preserved in human aneuploid cancers. We conclude that aneuploid cancers inactivate Stat1 signaling to circumvent immune surveillance.

cancer biology↗

bioRxiv2021

Indisulam synergizes with palbociclib to induce senescence through inhibition of CDK2 kinase activity

Inducing senescence in cancer cells is emerging as a new therapeutic strategy. In order to find ways to enhance senescence induction by palbociclib, a CDK4/6 inhibitor approved for treatment of metastatic breast cancer, we performed functional genetic screens in palbociclib-resistant cells. Using this approach, we found that loss of CDK2 results in strong senescence induction in palbociclib-treated cells. Treatment with the CDK2 inhibitor indisulam, which phenocopies genetic CDK2 inactivation, led to sustained senescence induction when combined with palbociclib in various cell lines and lung cancer xenografts. Treating cells with indisulam led to downregulation of cyclin H, which prevented CDK2 activation. Combined treatment with palbociclib and indisulam induced a senescence program and sensitized cells to senolytic therapy. Our data indicate that inhibition of CDK2 through indisulam treatment can enhance senescence induction by CDK4/6 inhibition.

cancer biology↗

bioRxiv2021

Monosomies, trisomies and segmental aneuploidies differentially affect chromosomal stability

Aneuploidy and chromosomal instability are both commonly found in cancer. Chromosomal instability leads to karyotype heterogeneity in tumors and is associated with therapy resistance, metastasis and poor prognosis. It has been hypothesized that aneuploidy per se is sufficient to drive CIN, however due to limited models and heterogenous results, it has remained controversial which aspects of aneuploidy can drive CIN. In this study we systematically tested the impact of different types of aneuploidies on the induction of CIN. We generated a plethora of isogenic aneuploid clones harboring whole chromosome or segmental aneuploidies in human p53-deficient RPE-1 cells. We observed increased segregation errors in cells harboring trisomies that strongly correlated to the number of gained genes. Strikingly, we found that clones harboring only monosomies do not induce a CIN phenotype. Finally, we found that an initial chromosome breakage event and subsequent fusion can instigate breakage-fusion-bridge cycles in segmental aneuploidies. This suggests that monosomies, trisomies and segmental aneuploidies have fundamentally different effects on chromosomal instability and these results help us to decipher the complex relationship between aneuploidy and CIN.

cell biology↗

bioRxiv2021

Optimized Cas9 expression improves performance of large-scale CRISPR screening

BackgroundThe widespread application of CRISPR/Cas9 technology has yielded numerous findings in biomedical research in recent years, making it an invaluable tool for gene knockout and for high-throughput screening studies. In (low-throughput) gene knockout studies, editing efficiency is not a major concern because only a few edited clones are necessary for a successful assay. However, in large scale pooled screening studies, editing efficiency is a major concern because each sgRNA has to knockout its target gene in a large cell population in a short period of time. Therefore, a thorough understanding of the role that key factors play in determining CRISPR knockout efficiency is essential to improve the performance of pooled CRISPR screening. MethodsIn this study, cell lines with different expression levels of CAS9 were generated and used to determine gene-editing efficiency. Collections of sgRNAs targeting essential genes were used to study their depletion in the different cell line models. ResultsUsing cell lines with variable expression of Cas9, we confirmed that editing efficiency and speed are mostly dependent on the sgRNA sequence and Cas9 expression, respectively. Importantly, we show that the strategy employed for delivering sgRNAs and Cas9 to cells impacts the performance of high-throughput screens, which is improved in conditions with higher Cas9 expression. ConclusionsOur findings highlight the importance of optimizing Cas9 expression levels when performing gene editing experiments and provide guidance on the necessary decisions for implementing optimal pooled CRISPR screening strategies.

genomics↗

bioRxiv2021

Unexpected gene activation following CRISPR-Cas9-mediated genome editing

The discovery of the Clustered Regularly-Interspaced Short Palindromic Repeats (CRISPR) and its development as a genome editing tool has revolutionized the field of molecular biology. In the DNA damage field, CRISPR has brought an alternative to induce endogenous double-strand breaks (DSB) at desired genomic locations and study the DNA damage response and its consequences. Many systems for sgRNA delivery have been reported in order to efficiency generate this DSB, including lentiviral vectors. However, some of the consequences of these systems are yet not well understood. Here we report that lentiviral-based sgRNA vectors can integrate into the endogenous genomic target location, leading to undesired activation of the target gene. By generating a DSB in the regulatory region of the ABCB1 gene using a lentiviral sgRNA vector, we can induce the formation of taxol-resistant colonies. We show that these colonies upregulated ABCB1 via integration of the EEF1A1 and the U6 promoters from the sgRNA vector. We believe that this is an unreported CRISPR/Cas9 artefact that researchers need to be aware of when using lentiviral vectors for genome editing.

genomics↗

bioRxiv2021

Perturbations in 3D genome organization can promote acquired drug resistance

Acquired drug resistance is a major problem in the treatment of cancer. hTERT-immortalized, untransformed RPE-1 (RPE) cells can acquire resistance to taxol by derepressing the ABCB1 gene, encoding for the multidrug transporter P-gP. Here we have investigated how the ABCB1 gene is derepressed. We show that activation of the ABCB1 gene is associated with reduced DNA methylation, reduced H3K9 trimethylation and increased H3K27 acetylation at the ABCB1 promoter. In addition, we find that the ABCB1 locus has moved away from the nuclear lamina in the taxol-resistant cells. This raises the question which of these alterations were causal to derepression. Directly modifying DNA methylation or H3K27 methylation had neither significant effect on ABCB1 expression, nor did it promote drug resistance. In contrast, the disruption of Lamin B Receptor (LBR), a component of the nuclear lamina involved in genome organization, did promote the acquisition of a taxol-resistant phenotype in a subset of cells. Using CRISPRa-mediated gene activation, we could further substantiate a model in which disruption of lamina association renders the ABCB1 gene permissive to derepression. Based on these data we propose a model in which nuclear lamina dissociation of a repressed gene allows for its activation, implying that deregulation of the 3D genome topology could play an important role in tumor evolution and the acquisition of drug resistance.

molecular biology↗