Amplification and Quantitation of Telomeric Extrachromosomal Circles

Background

To avoid the loss of genetic information and the acquisition of potentially lethal genomic aberrations during DNA replication, linear chromosomes possess nucleoprotein cap structures known as telomeres. Conversely, active telomere extension imparts cells with replicative immortality, which is essential during embryonic development (L. Liu et al., 2007), as well as during malignant transformation and cancer progression (Robinson and Schiemann, 2016; Robinson et al., 2019). In most developmental and tumorigenic contexts, telomere extension is carried out by the reverse transcriptase telomerase. However, telomeres are also extended in a telomerase-independent fashion through a mechanism known as alternative lengthening of telomeres (ALT), which is active in embryonic stem cells and early embryogenesis (L. Liu et al., 2007; Pickett and Reddel, 2015). Moreover, approximately 15% of tumors are reliant upon ALT for telomere synthesis, with some cancer types showing evidence of ALT in more than half of patients (Sung et al., 2020). Given the growing recognition of the prevalence and functional significance of ALT in both development and disease, establishing robust methods for monitoring ALT activity is of critical importance.

Telomere lengthening via ALT is mediated by homologous recombination (HR) between adjacent telomere templates, followed by DNA synthesis through a mechanism similar to break-induced replication (BIR) (Dilley et al., 2016; Roumelioti et al., 2016). Importantly, telomere HR and BIR generate small circular DNAs containing telomeric DNA sequences (T. Zhang et al., 2019).These telomeric extrachromosomal circles, termed C-circles, act as specific markers that can be used to quantitate ALT activity in biological samples (Henson et al., 2009). More broadly, multiple methods exist to assay telomere maintenance mechanism (TMM) identity. For example, HR of contiguous telomeres results in telomere sister chromatid exchange (T-SCE), a hallmark of ALT (Londono-Vallejo et al., 2004) that can be visualized using chromosome orientation fluorescence in situ hybridization (CO-FISH) (Cesare et al., 2015). In addition, ALT-associated HR and DNA synthesis are facilitated by the promyelocytic leukemia protein (PML) within structures known as ALT-associated PML bodies (ABPs) (Yeager et al., 1999; J. M. Zhang et al., 2019). APBs can be detected and quantified using a combined immunofluorescence/FISH approach (Henson et al., 2005). Lastly, mutational loss (Lovejoy et al., 2012) or downregulation (Robinson et al., 2020) of the ATRX/DAXX chromatin remodeling complex is widespread in ALT-driven cell lines and tumors and can be easily ascertained using sequencing or quantitative gene expression methods. Despite the established relationships between these molecular markers and ALT, they suffer from drawbacks that restrict their experimental utility. For instance, T-SCE can occur during telomeric DNA damage repair (Mao et al., 2016; H. Liu et al., 2018). Moreover, loss of ATRX and DAXX is not uniformly distributed in ALT-positive specimens (Lovejoy et al., 2012) and can be found in ALT-negative specimens as well (de Nonneville and Reddel, 2021). The variable nature of these markers necessitates a combinatorial approach to TMM classification that includes a sensitive and reliable method for assessing ALT.

Because of the high degree of specificity of C-circles for ALT (Henson et al., 2009) and their relationship to clinical features of ALT-driven cancers (Grandin et al., 2019), measuring and quantifying C-circle abundance has become the preferred method for assessing ALT activity, motivating the continual development of improved methods for C-circle quantitation. Furthermore, the diversity of biological contexts in which telomeres are actively maintained and the differences in telomere dynamics across organisms (Monaghan et al., 2018; Wright and Shay, 2000) create demand for generalized approaches to the interrogation of TMM identity. Here, we describe a protocol for C-circle quantitation, based upon previous reports (Lau et al., 2013; Henson et al., 2017), consisting of (i) rapid isolation and spectroscopic quantification of genomic and extrachromosomal DNA using the Quick C-circle Preparation (QCP) method, (ii) enrichment of extrachromosomal circular DNAs via rolling circle amplification (RCA), and (iii) determination of C-circle abundance using quantitative polymerase chain reaction (qPCR). We have applied this protocol across an array of biological and experimental contexts (Robinson et al., 2020 and 2021), asserting it as a generalizable platform for C-circle detection and, when paired with additional approaches, the assignment of TMM identity.