Chan, Kui Ming

Abstract Extrachromosomal DNA (ecDNA) drives the evolution of cancer cells by conferring survival advantages over normal cells. However, the functional importance of ecDNA replication and maintenance is yet to be evaluated, and the molecular components that contributed to these processes remain largely unknown. Here, using CRISPR-C (circularization of genes and chromosomes by CRISPR) technology and synthetic circular DNA mimicking ecDNA identified in tumors, we generated ecDNA-positive (ecDNA+) cell models. Using these models, we identified the proteins involved in ecDNA replication and maintenance and found DNA damage response (DDR)-related pathways were significantly enriched. We further demonstrated that ecDNA replication led to increased DDR and genome instability and DDR was also essential for ecDNA maintenance, consistent with observations that ecDNA+ tumors exhibited higher DDR scores than ecDNA-negative (ecDNA–) tumors in patients. ATM, MDC1, and CHK2, but not ATR, were activated in ecDNA+ cells compared to ecDNA– cells. Moreover, ecDNA+ cells were more sensitive to ATM and CHK2 inhibition. We further found that type II topoisomerase TOP2B and DNA ligase III (LIG3) were critical regulators for ecDNA replication and maintenance and related DDR. In summary, our findings of the reciprocal interactions between ecDNA and DDR provide new insights into detection and therapy for ecDNA+ tumors.

Abstract Proximity labeling catalyzed by promiscuous enzymes, such as APEX2, has emerged as a powerful approach to characterize multiprotein complexes and protein–protein interactions. However, current methods depend on the expression of exogenous fusion proteins and cannot be applied to identify proteins surrounding post-translationally modified proteins. To address this limitation, we developed a new method to label proximal proteins of interest by antibody-mediated protein A-ascorbate peroxidase 2 (pA-APEX2) labeling (AMAPEX). In this method, a modified protein is bound in situ by a specific antibody, which then tethers a pA-APEX2 fusion protein. Activation of APEX2 labels the nearby proteins with biotin; the biotinylated proteins are then purified using streptavidin beads and identified by mass spectrometry. We demonstrated the utility of this approach by profiling the proximal proteins of histone modifications including H3K27me3, H3K9me3, H3K4me3, H4K5ac, and H4K12ac, as well as verifying the co-localization of these identified proteins with bait proteins by published ChIP-seq analysis and nucleosome immunoprecipitation. Overall, AMAPEX is an efficient method to identify proteins that are proximal to modified histones.

X chromosome inactivation (XCI) is the most dramatic example of epigenetic silencing in eukaryotes. Once established, the inactivated X chromosome (Xi) remains silenced throughout subsequent cell divisions. Though the initiation of XCI has been studied extensively, the protein factors involved in Xi silencing and maintenance are largely unknown. Here we report the discovery of a diverse set of 32 proteins involved in maintenance of Xi silencing through a genome-wide RNAi screen. In addition, we describe the mechanistic roles of two proteins—origin recognition complex 2 (Orc2) and heterochromatin protein 1 (HP1α)—in Xi silencing. Immunofluorescence studies indicate that Orc2 and HP1α localize on Xi in mouse cells. Depletion of Orc2 by shRNA leads to the loss of both Orc2 and HP1α localization on Xi. Furthermore, the silencing of genes on Xi is disrupted in both Orc2- and HP1α-depleted cells. Finally, we show, using ChIP assay, that the localization of HP1α and Orc2 to the promoter regions of Xi-silenced genes is interdependent. These findings reveal a diverse set of proteins involved in Xi silencing, show how Orc2 and HP1α impact Xi silencing, and provide a basis for future studies on the maintenance of Xi silencing.