The versatility of Ascorbate Peroxidase-aided mapping uncovers insights of the nuclear lamina interactions and function

Abstract

The nuclear lamina (NL) is a proteinaceous network found beneath the inner nuclear membrane. The NL is linked to a number of dynamic cellular activities including chromatin organization, transcription and RNA/protein trafficking through nuclear pores. Our understanding of the NL has been hindered in part by the general insolubility and low extractability of proteins from this region. This has spurred the development of proximity ligation methods that label proteins and/or DNA near the NL for systematic identification (Bar et al., 2018; Chen et al., 2018b; Guelen et al., 2008; Roux et al., 2012). To simplify labeling and improve temporal resolution, we fused APEX2 (Hung et al., 2014; Lam et al., 2015) to the nuclear lamina protein lamin-B1 to map proteins, RNA and DNA associated with the NL. We show that APEX2 labeling of the NL is robust and requires as little as 20 seconds. In addition to identifying the NL proteome, this method revealed NL-proximal RNA species that were largely spliced. These NL-proximal RNAs show a bias toward long 3’ UTRs, suggesting an RNA-regulatory role of the NL. This is further supported by the finding of a bias toward longer 3’ UTRs in genes deregulated in lamin-null cells. Interestingly, these RNAs share a sequence motif in their 3’ UTRs. Finally, we demonstrate that the APEX2 method can reliably map lamina-associated domains (LADs) at different stages of the cell cycle, revealing a variability of short LADs regions enriched for histone lysine 27 trimethylation (H3K27me3). Thus the APEX2 method report here is a useful addition to the molecular toolbox for the study of the NL and permits the identification of proteome, transcriptome, and genome elements associated with this nuclear substructure.

High quality mapping of chromatin at or near the nuclear lamina for small numbers of cells

“…As previously described (Tran et al, 2020;van Schaik et al, 2019), the vast majority of LADs mapped by cTSA-seq here were stable across the G1, S and G2 stages of the cell cycle ( Figure 3A, 3B). This was also observed using both the GM12878 cell line and wild-type mouse embryonic stem cells (mESCs) ( Figure S3C).…”

“…Recently, we and others have mapped G1, S, and G2/M phase LADs from cells FACS sorted by their DNA content (Tran et al, 2020;van Schaik et al, 2019). These maps revealed fairly small variations in LADs among the three cell cycle stages, and indicated that LADs in G1, S, and G2 phases are largely stable (Tran et al, 2020;van Schaik et al, 2019).…”

“…Most studies of LADs have been performed using asynchronously dividing cell cultures, and represents an averaged NL-chromatin interaction across all phases of the cell cycle. Recently, we and others have mapped G1, S, and G2/M phase LADs from cells FACS sorted by their DNA content (Tran et al, 2020;van Schaik et al, 2019). These maps revealed fairly small variations in LADs among the three cell cycle stages, and indicated that LADs in G1, S, and G2 phases are largely stable (Tran et al, 2020;van Schaik et al, 2019).…”

“…However, some limitations of these methods, such as insolubility of NL and temporal resolution of labeling, preclude their use for efficient mapping of LADs in early G1. Recently, we adapted the Ascorbate Peroxide identification (APEX2-ID) method, which APEX2 was expressed as a fusion protein with lamin-B1, to temporally map LADs during the cell cycle (Tran et al, 2020).…”

“…However, the APEX2-ID method has its own limitations, such as variable transfection efficiency in different cells and unwelcomed overexpression effects. The van Steensel group recently developed a technology to map cell cycle LADs using an antibodytargeted DamID approach highlighting a convergence toward antibody-based methods (van Schaik et al, 2019).…”