Biology subjects

Wei, P.-C.

Publications and source records attributed to Wei, P.-C..

6 recordsLinked to original sources

bioRxiv2026

An integrated genome-wide resource reveals distinct replication environments of DNA breakage in human cancer cell lines

Replication stress is a major source of genome instability in cancer, yet the genomic features that determine where DNA double-strand breaks (DSBs) arise remain incompletely defined. Here, we establish an integrated genome-wide resource of DSBs, DNA replication, replication timing, transcription, and R-loops in two widely used cancer cell lines, U2-OS and HeLa, under steady-state conditions and following prolonged low-dose DNA polymerase inhibition. We combine these datasets with systematic statistical testing and comparative analytical approaches to define the replication environments associated with genome fragility. Endogenous DSBs preferentially accumulated at origin-rich initiation zones, where R-loops were enriched, whereas prolonged DNA polymerase inhibition redirected DSB formation toward origin-poor, late-replicating regions. Among the replication features examined, replication initiation zones and late-replicating areas were most sensitive to prolonged replication stress. R-loops were specifically enriched at initiation zones but depleted from late-replicating regions and recurrent DNA break clusters (RDCs), demonstrating that their association with genome fragility is context dependent. Replication stress further induced RDCs within long, actively transcribed genes, while their locations only partially overlapped with common fragile sites. Functional analyses identified MUS81 as a major regulator of RDC formation. MUS81 loss increased RDCs, whereas restoration of its catalytic activity suppressed them, indicating that MUS81 resolves replication intermediates before they persist into late-replicating fragile regions. Together, this resource and analytical framework provide a systematic basis for dissecting how replication architecture, transcription, and DNA processing shape genome fragility in cancer cells.

genomics↗

bioRxiv2025

Recurrent DNA Break Clusters Regulated by Polymerase Theta Are Essential for Replication Stress Induced Copy Number Variation

Copy number variations (CNVs) are a form of genetic alteration strongly implicated in numerous neurological and psychiatric disorders, as well as brain cancer. Replication stress is a common cause of CNVs. Despite the prevailing model that CNVs arise from DNA double strand breaks (DSBs), there has been no assay that directly perturbs presumed DSB sources and measures CNV output. Here, we identified a subset of recurrent DNA break clusters (RDCs) as a causal factor for CNV formation. In murine neural progenitor cells under replication stress, mapping the formation of CNVs revealed their location in RDC regions that contain actively transcribed genes. CRISPR/Cas9-mediated transcriptional suppression abrogated both RDC and CNV formation, but does not alter their replication timing. We found that DNA polymerase theta (Pol {theta}), a protector against CNV formation, plays a critical but context dependent role upstream of RDC formation. Chemically inhibiting the activity of Pol {theta} reduced end filling and micro-homology-mediated end joining in XRCC4/P53-deficient cells. Conversely, Pol {theta} inhibition led to elevated DSB density detection at RDC-containing loci in wild-type neural stem and progenitor cells, suggesting its role in preventing transcription-replication conflicts. Our data identify RDCs as contributors to genomic heterogeneity with plausible downstream effects on brain disorders and malignancy.

molecular biology↗

bioRxiv2025

Adaptive Clonal Expansion Shapes Brain Development

Embryonic neural stem/progenitor cells (NSPCs) exhibit remarkable proliferative plasticity, allowing them to fully recover neuronal populations even after substantial cell loss 1,2. However, it remains unclear whether all embryonic NSPCs respond to brain lesions. To address this, we developed a mouse model to investigate NSPC proliferation dynamics, hypothesizing that the loss of progenitor cells would induce fitness competition among NSPCs. In this model, half of the founder NSPCs were ablated using diphtheria toxin A at the onset of neurogenesis, yet the surviving cells regenerated a brain containing all neuronal types within five of the total twenty embryonic days. Analysis of allelic variants revealed overrepresented somatic variants, indicating that only a small fraction of NSPCs underwent significant clonal expansion during early neurogenesis. Modelling proliferation dynamics predicted that as few as 10% of NSPCs could produce 83% of neurons by the time of birth. Single nucleotide substitution analysis suggested a potential link to oxidative metabolism in some of the expanded clones. Moreover, single-cell transcriptomics showed delayed development and a reduced NSPC pool as consequences of adaptive clonal expansion. Our findings highlight that NSPC exhibit varying expansion potential and that adaptive clonal expansion indirectly altered neuronal cell composition in the brain.

developmental biology↗

bioRxiv2023

Transcription-Replication Conflict Shapes DNA Break Dynamics

Recurrent DNA break clusters (RDCs) are replication-transcription collision hotspots; many are unique to neural progenitor cells. Through high-resolution replication sequencing and a capture-ligation assay in mouse neural progenitor cells experiencing replication stress, we unraveled the replication features dictating RDC location and orientation. Most RDCs occur at the replication forks traversing timing transition regions (TTRs), where sparse replication origins connect unidirectional forks. Leftward-moving forks generate telomere-connected DNA double-strand breaks (DSBs), while rightward-moving forks lead to centromere-connected DSBs. Strand-specific mapping for DNA-bound RNA revealed co-transcriptional dual-strand DNA:RNA hybrids present at a higher density in RDC than in other actively transcribed long genes. In addition, mapping RNA polymerase activity revealed that head-to-head interactions between replication and transcription machinery resulted in 60% DSB contribution to the head-on compared to 40% for co-directional. Our findings revealed TTR as a novel fragile class and highlighted how the linear interaction between transcription and replication impacts genome stability.

genomics↗

bioRxiv2019

Induction of Recurrent Break Cluster Genes in Neural Progenitor Cells Differentiated from Embryonic Stem Cells In Culture

Mild replication stress enhances appearance of dozens of robust recurrent genomic break clusters, termed RDCs, in cultured primary mouse neural stem and progenitor cells (NSPCs). Robust RDCs occur within genes ("RDC-genes") that are long and have roles in neural cell communications and/or have been implicated in neuropsychiatric diseases or cancer. We sought to develop an in vitro approach to determine whether specific RDC formation is associated with neural development. For this purpose, we adapted a system to induce neural progenitor cell (NPC) development from mouse embryonic stem cell (ESC) lines deficient for XRCC4 plus p53, a genotype that enhances DNA double-strand break (DSB) persistence to enhance detection. We tested for RDCs by our genome wide DSB identification approach that captures DSBs genome-wide via their ability to join to specific genomic Cas9/sgRNA-generated bait DSBs. In XRCC4/p53-deficient ES cells, we detected 7 RDCs, which were in genes, with two RDCs being robust. In contrast, in NPCs derived from these ES cell lines, we detected 29 RDCs, a large fraction of which were robust and associated with long, transcribed neural genes that were also robust RDC-genes in primary NSPCs. These studies suggest that many RDCs present in NSPCs are developmentally influenced to occur in this cell type and indicate that induced development of NPCs from ES cells provides an approach to rapidly elucidate mechanistic aspects of NPC RDC formation. SIGNIFICANCE STATEMENTWe previously discovered a set of long neural genes susceptible to frequent DNA breaks in primary mouse brain progenitor cells. We termed these genes RDC-genes. RDC-gene breakage during brain development might alter neural gene function and contribute to neurological diseases and brain cancer. To provide an approach to characterize the unknown mechanism of neural RDC-gene breakage, we asked whether RDC-genes appear in neural progenitors differentiated from embryonic stem cells in culture. Indeed, robust RDC-genes appeared in neural progenitors differentiated in culture and many overlapped with robust RDC-genes in primary brain progenitors. These studies indicate that in vitro development of neural progenitors provides a model system for elucidating how RDC-genes are formed.

neuroscience↗

bioRxiv2018

Defective DNA damage repair leads to frequent catastrophic genomic events in murine and human tumors

Chromothripsis and chromoanasynthesis are catastrophic events leading to clustered genomic rearrangements. Whole-genome sequencing revealed frequent chromothripsis or chromoanasynthesis (n= 16/26) in brain tumors developing in mice deficient for factors involved in homologous-recombination-repair or non-homologous-end-joining. Catastrophic events were tightly linked to Myc/Mycn amplification, with increased DNA damage and inefficient apoptotic response already observable at early postnatal stages. Inhibition of repair processes and comparison of the mouse tumors with human medulloblastomas (n=68) and glioblastomas (n=32) identified chromothripsis as associated with MYC/MYCN gains and with DNA repair deficiencies, pointing towards therapeutic opportunities to target DNA repair defects in tumors with complex genomic rearrangements.

cancer biology↗