Elucidating chromatin structure and function of double minutes through cell cycles

St. Jude Children'S Research Hospital, Memphis TN

Investigators

Abstract

ABSTRACT Double minute chromosomes (DMs), also known as extrachromosomal DNAs (ecDNAs), are acentric minichromosomal bodies that have been documented in cytogenetic studies since the 1960s. Early studies revealed that drug-resistant genes and oncogenes could be amplified in these abnormal chromosomes, suggesting they had roles in tumorigenesis and tumor development. Recent work has revealed the striking involvement of DMs in human cancers, with DMs being present in nearly half of cancer types and in almost a third of samples from patients with cancer. There is accumulating evidence that DMs are associated with poor clinical outcomes and tumor aggressiveness. The lack of innovative tools with which to study and manipulate DMs is a major barrier to progress in the field. Early karyotypic investigations suggested that DMs had a “euchromatin-like” structure, but the underlying mechanisms of oncogene regulation on DMs remain uncharted. Recent studies revealed enhancer hijacking models and ecDNA hub–enabled interchromosomal regulation; however, the chromatin structure and associated protein networks of oncogenes on DMs during interphase are underexplored. Furthermore, during mitosis, DMs cannot use the spindle apparatus for proper segregation because they lack centromeres. Instead, DMs tether directly to the chromosomes to reach the daughter nuclei. This mode of transportation might resemble viral hitchhiking, in which trans-acting tethering proteins bridge viral genomes and host chromosomes. The tethering factors of DMs and their roles in mediating mitotic chromosome attachment are unknown. The proposed research aims to develop a suite of novel biochemical and chemical strategies to investigate the chromatin structure of DMs during the cell cycle to elucidate the mechanisms of DM-specific gene expression and chromosome maintenance. First, we will devise single-molecule sequencing strategies to elucidate the chromatin accessibility and histone landscapes of DMs. This will provide a novel means of evaluating gene regulation at the level of the individual DM. Next, we will develop a new approach to chemical proximity cross- linking (ProX) to enable selective capture of native chromatin loci at DM elements. We envision integrating ProX with established genomic, proteomic, and bioinformatic pipelines to gain a holistic view of DM chromatin and its associated partners. Finally, we will create libraries of synthetic miniaturized DMs and use them in cell-based assays to uncover DNA elements that confer chromosome hitchhiking properties. In parallel, we will combine the biochemical purification of metaphase DMs with quantitative proteomics to reveal hitchhiking factors. Our multi-pronged approach will establish a framework for characterizing the chromatin structure of native circular DMs and for understanding its role in mediating DM maintenance and gene expression. We anticipate that mechanistic insights gained will provide therapeutic frameworks for directly targeting DMs in human cancers.