Shieldin Complex Research Articles

EZH2 Inhibition Sensitizes CARM1-High, Homologous Recombination Proficient Ovarian Cancers to PARP Inhibition

In response to DNA double-strand breaks, MAD2L2-containing shieldin complex plays a critical role in the choice between homologous recombination (HR) and non-homologous end-joining (NHEJ)-mediated repair. Here we show that EZH2 inhibition upregulates MAD2L2 and sensitizes HR-proficient epithelial ovarian cancer (EOC) to poly(adenosine diphosphate-ribose) polymerase (PARP) inhibitor in a CARM1-dependent manner. CARM1 promotes MAD2L2 silencing by driving the switch from the SWI/SNF complex to EZH2 through methylating the BAF155 subunit of the SWI/SNF complex on the MAD2L2 promoter. EZH2 inhibition upregulates MAD2L2 to decrease DNA end resection, which increases NHEJ and chromosomal abnormalities, ultimately causing mitotic catastrophe in PARP inhibitor treated HR-proficient cells. Significantly, EZH2 inhibitor sensitizes CARM1-high, but not CARM-low, EOCs to PARP inhibitors in both orthotopic and patient-derived xenografts.

Structural basis for shieldin complex subunit 3–mediated recruitment of the checkpoint protein REV7 during DNA double-strand break repair

Shieldin complex subunit 3 (SHLD3) is the apical subunit of a recently-identified shieldin complex and plays a critical role in DNA double-strand break repair. To fulfill its function in DNA repair, SHLD3 interacts with the mitotic spindle assembly checkpoint protein REV7 homolog (REV7), but the details of this interaction remain obscure. Here, we present the crystal structures of REV7 in complex with SHLD3's REV7-binding domain (RBD) at 2.2-2.3 Å resolutions. The structures revealed that the ladle-shaped RBD in SHLD3 uses its N-terminal loop and C-terminal α-helix (αC-helix) in its interaction with REV7. The N-terminal loop exhibited a structure similar to those previously identified in other REV7-binding proteins, and the less-conserved αC-helix region adopted a distinct mode for binding REV7. In vitro and in vivo binding analyses revealed that the N-terminal loop and the αC-helix are both indispensable for high-affinity REV7 binding (with low-nanomolar affinity), underscoring the crucial role of SHLD3 αC-helix in protein binding. Moreover, binding kinetics analyses revealed that the REV7 "safety belt" region, which plays a role in binding other proteins, is essential for SHLD3-REV7 binding, as this region retards the dissociation of the RBD from the bound REV7. Together, the findings of our study reveal the molecular basis of the SHLD3-REV7 interaction and provide critical insights into how SHLD3 recognizes REV7.

The Shieldin Complex Protects DNA Ends for 53BP1-Mediated DNA Repair

Abstract The shieldin complex, comprised of SHLD1, SHLD2, SHLD3, and REV7, is a 53BP1 effector complex.

The shieldin complex mediates 53BP1-dependent DNA repair.

53BP1 is a chromatin-binding protein that regulates DNA double-strand break (DSB) repair by suppressing the nucleolytic resection of DNA termini1,2. This function of 53BP1 requires interactions with PTIP3 and RIF14–9, the latter also recruiting REV7/MAD2L2 to break sites10,11. How 53BP1-pathway proteins shield DNA ends is unknown but two models best explain their action: in one model, the 53BP1 complex strengthens the nucleosomal barrier to end-resection nucleases12,13, whereas in a second model, 53BP1 recruits effector proteins with end-protection activity. Here we describe the identification of such a 53BP1 effector complex, Shieldin, which includes C20orf196 (SHLD1), FAM35A (SHLD2), CTC-534A2.2 (SHLD3) and REV7. Shieldin localizes to DSB sites in a 53BP1- and RIF1-dependent manner and its SHLD2 subunit binds to ssDNA via OB-fold domains analogous to those of RPA1 and POT1. Loss of Shieldin impairs non-homologous end-joining (NHEJ), leads to defective immunoglobulin class switching and causes hyper-resection. Mutations in Shieldin subunit genes also cause resistance to poly(ADP-ribose) polymerase (PARP) inhibition in BRCA1-deficient cells and tumours due to restoration of homologous recombination (HR). Finally, we show that ssDNA binding by SHLD2 is critical for Shieldin function, consistent with a model where Shieldin protects DNA ends to mediate 53BP1-dependent DNA repair.

Shieldin complex promotes DNA end-joining and counters homologous recombination in BRCA1-null cells.

BRCA1 deficiencies cause breast, ovarian, prostate and other cancers, and render tumours hypersensitive to PARP inhibitors. To understand resistance mechanisms, we conducted whole-genome CRISPR-Cas9 synthetic-viability/resistance screens in BRCA1-deficient breast cancer cells treated with PARP inhibitors. We identified two previously uncharacterized proteins, C20orf196 and FAM35A, whose inactivation confers strong PARP-inhibitor resistance. Mechanistically, we show C20orf196 and FAM35A form a complex, “Shieldin” (SHLD1/2), with FAM35A interacting with single-stranded DNA via its C-terminal OB fold region. We establish that Shieldin acts as the downstream effector of 53BP1/RIF1/MAD2L2 to promote DNA double-strand break (DSB) end-joining through restricting DSB resection and counteract homologous recombination by antagonising BRCA2/RAD51 loading in BRCA1-deficient cells. Notably, Shieldin inactivation further sensitises BRCA1-deficient cells to cisplatin, suggesting how defining the SHLD1/2 status of BRCA1-deficient tumours might aid patient stratification and yield new treatment opportunities. Highlighting this potential, we document reduced SHLD1/2 expression in human breast cancers displaying intrinsic or acquired PARP-inhibitor resistance.