Doxorubicin is a chemotherapeutic agent used for the treatment of solid and hematopoietic malignancies. Doxorubicin acts as a DNA intercalator and inhibits the topoisomerase II enzyme, leading to double-stranded breaks. We hypothesize that doxorubicin mutagenesis leads to a nonrandom pattern of somatic mutations on the genome, termed a mutational signature. We expose cells to doxorubicin using two different methodologies: (1) continuous exposure of a constant dose of doxorubicin and whole-genome sequencing of single-cell clones (previously published methodology) and (2) pulsed exposure of escalating doses of doxorubicin and whole-genome sequencing of a bulk population. We first confirm that the methodologies do not yield differing results through previously published cisplatin-exposed single-cell clone genome data compared with the dose escalation and bulk genome data. We then derive two doxorubicin signatures. The first, SBS-doxorubicin-1, is defined by an abundance of C > A mutations. The second, SBS-doxorubicin-2, is defined by a higher occurrence of T > C mutations. We then assess for the presence of our signatures within the genomes of patients treated with a doxorubicin-containing regimen. Across 10 bulk immune sorted samples from three patients with follicular lymphoma/diffuse large B cell lymphoma, as well as two metastatic samples from one patient with renal medullary carcinoma, we are able to detect both signatures in 83% of samples, with remaining samples harboring either signature. In summary, we experimentally derive two doxorubicin mutational signatures and demonstrate their presence in patient samples.
RNA Polymerase II (Pol II) transcribes all protein-coding and many nonprotein-coding genes in the genome. Pol II transcription termination is crucial for RNA maturation and, when disrupted, can lead to altered RNA processing and RNA export. Termination involves two intertwined processes: RNA cleavage and Pol II release from the DNA (displacement). Despite its importance, the exact mechanisms underlying Pol II displacement from the DNA remain poorly understood. Moreover, under certain cellular stress conditions, there is a partial failure of cleavage, leading to a shift of the position of displacement further downstream, a phenomenon known as readthrough transcription. We present a systematic analysis of Pol II termination across cell types and species and provide novel insights into the mechanism of displacement. Using a probabilistic mixture model to quantify Pol II dynamics across entire gene bodies from nascent RNA sequencing data, we find that genes fall into two classes defined by conserved base composition patterns near the displacement site. Both classes share a T-rich region upstream of displacement, and one class additionally carries a GC-rich region surrounding displacement. We find that GC-rich predisplacement regions have more accessible chromatin and higher levels of phospho-threonine 4 on the CTD of Pol II. Additionally, we find that displacement at genes with only the upstream T-rich pattern are more likely to be affected by perturbations that alter displacement, including heat shock, viral infection, kinase inhibition, and arsenic treatment. Thus, our work identifies two types of Pol II displacement regions, which are differentially affected by perturbations of cellular homeostasis.
