Biology subjects

Norman, R. X.

Publications and source records attributed to Norman, R. X..

3 recordsLinked to original sources

bioRxiv2025

ER-tethering directs TREX1 penetration of a BAF-dependent barrier at micronuclei

Micronuclei are membrane-encapsulated nuclear aberrations that form following chromosome segregation errors. Micronuclear membrane collapse permits access of the pattern recognition receptor cGAS and its antagonist, the TREX1 exonuclease. TREX1 carboxy-terminal domain mediated endoplasmic reticulum tethering association is essential for invasion into ruptured micronuclei, however the mechanisms underlying this dependency are unknown. Here, we identify barrier-to-autointegration nuclear assembly factor 1 (BAF) as a key regulator of TREX1 activity at micronuclei. BAF accumulates on micronuclei following membrane collapse and augments TREX1 recruitment in a manner that depends on BAF interactions with membrane-associated LEM-domain proteins. Despite delayed entry, TREX1 exhibits enhanced micronuclear DNA degradation and independence from ER-tethering in BAF-deficient cells. In accordance, recombinant BAF protein inhibits TREX1-mediated DNA degradation in vitro in a manner that depends on BAF DNA-binding. BAF similarly outcompetes cGAS for micronuclear DNA interaction and reduces cGAS activation at micronuclei. These findings reveal, a BAF-dependent protective barrier to diffusive entry of DNA binding proteins at ruptured micronuclei, explaining the requirement of TREX1 ER-tethering for micronuclear localization and suppression of productive cGAS DNA substrate interactions that activate innate immune responses in chromosomally unstable cells.

molecular biology↗

bioRxiv2025

Mitotic transcription ensures ecDNA inheritance through chromosomal tethering

Extrachromosomal DNA (ecDNA) are circular DNA bodies that play critical roles in tumor progression and treatment resistance by amplifying oncogenes across a wide range of cancer types. ecDNA lack centromeres and are thus not constrained by typical Mendelian segregation, enabling their unequal accumulation within daughter cells and associated increases in copy number. Despite intrinsic links to their oncogenic potential, the fidelity and mechanisms of ecDNA inheritance are poorly understood. Here, we show that ecDNA are protected against cytosolic mis-segregation through mitotic clustering and by tethering to the telomeric and subtelomeric regions of mitotic chromosomes. ecDNA-chromosome tethering depends on BRD4 transcriptional co-activation and mitotic transcription of the long non-coding RNA PVT1, which is co-amplified with MYC in colorectal and prostate cancer cell lines. Disruption of ecDNA-chromosome tethering through BRD4 inhibition, PVT1 depletion, or inhibiting mitotic transcription results in cytosolic mis-segregation, ecDNA reintegration, and the formation of homogeneously staining regions (HSRs). We propose that nuclear inheritance of ecDNA is facilitated by an RNA-mediated physical tether that links ecDNA to mitotic chromosomes and thus protects against cytosolic mis-segregation and chromosomal integration.

molecular biology↗

bioRxiv2024

Dynamic Plk1 recruitment to the inner centromere

Mitosis is carefully orchestrated by reversible phosphorylation events. Polo-like kinase 1 (Plk1) regulates multiple functions across the kinetochore during mitotic progression. Recently, Bub1 (outer kinetochore) and CENP-U (inner kinetochore) were described as two major sites of Plk1 recruitment to the kinetochore. Here, we report an additional dynamic site of Plk1 recruitment to the inner centromere. Inner centromere docking occurs during late prometaphase and metaphase, exhibiting transient residency at multiple chromosomes. Chromosomes with inner centromere-localized Plk1 have end-on attached microtubules, diminished Spindle Assembly Checkpoint (SAC) components, and low Shugoshin 1 (Sgo1) levels at the inner centromere. Mechanistically, recruitment is driven by Cdk1 activity and requires Plk1s Polo-Box Domain (PBD). Moreover, inhibition of Bub1 or Protein Phosphatase 2A (PP2A) increases Plk1 recruitment and residency at the inner centromere. Collectively, our data identify a novel pathway for Plk1 recruitment to the inner centromere that is dynamically regulated by counteracting activities of Cdk1 and Bub1/PP2A.

cell biology↗