Structures, functions and adaptations of the human LINE-1 ORF2 protein.

The LINE-1 (L1) retrotransposon is an ancient genetic parasite that has written around one-third of the human genome through a 'copy and paste' mechanism catalysed by its multifunctional enzyme, open reading frame 2 protein (ORF2p)1. ORF2p reverse transcriptase (RT) and endonuclease activities have been implicated in the pathophysiology of cancer2,3, autoimmunity4,5 and ageing6,7, making ORF2p a potential therapeutic target. However, a lack of structural and mechanistic knowledge has hampered efforts to rationally exploit it. We report structures of the human ORF2p 'core' (residues 238-1061, including the RT domain) by X-ray crystallography and cryo-electron microscopy in several conformational states. Our analyses identified two previously undescribed folded domains, extensive contacts to RNA templates and associated adaptations that contribute to unique aspects of the L1 replication cycle. Computed integrative structural models of full-length ORF2p show a dynamic closed-ring conformation that appears to open during retrotransposition. We characterize ORF2p RT inhibition and reveal its underlying structural basis. Imaging and biochemistry show that non-canonical cytosolic ORF2p RT activity can produce RNA:DNA hybrids, activating innate immune signalling through cGAS/STING and resulting in interferon production6-8. In contrast to retroviral RTs, L1 RT is efficiently primed by short RNAs and hairpins, which probably explains cytosolic priming. Other biochemical activities including processivity, DNA-directed polymerization, non-templated base addition and template switching together allow us to propose a revised L1 insertion model. Finally, our evolutionary analysis demonstrates structural conservation between ORF2p and other RNA- and DNA-dependent polymerases. We therefore provide key mechanistic insights into L1 polymerization and insertion, shed light on the evolutionary history of L1 and enable rational drug development targeting L1.

LINE-1 retrotransposition and its deregulation in cancers: implications for therapeutic opportunities.

Long interspersed element 1 (LINE-1) is the only protein-coding transposon that is active in humans. LINE-1 propagates in the genome using RNA intermediates via retrotransposition. This activity has resulted in LINE-1 sequences occupying approximately one-fifth of our genome. Although most copies of LINE-1 are immobile, ∼100 copies are retrotransposition-competent. Retrotransposition is normally limited via epigenetic silencing, DNA repair, and other host defense mechanisms. In contrast, LINE-1 overexpression and retrotransposition are hallmarks of cancers. Here, we review mechanisms of LINE-1 regulation and how LINE-1 may promote genetic heterogeneity in tumors. Finally, we discuss therapeutic strategies to exploit LINE-1 biology in cancers.

Arginine regulates HSPA5/BiP translation through ribosome pausing in triple-negative breast cancer cells.

BACKGROUND: Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer with a high mortality rate due to a lack of therapeutic targets. Many TNBC cells are reliant on extracellular arginine for survival and express high levels of binding immunoglobin protein (BiP), a marker of metastasis and endoplasmic reticulum (ER) stress response. METHODS: In this study, the effect of arginine shortage on BiP expression in the TNBC cell line MDA-MB-231 was evaluated. Two stable cell lines were generated in MDA-MB-231 cells: the first expressed wild-type BiP, and the second expressed a mutated BiP free of the two arginine pause-site codons, CCU and CGU, termed G-BiP. RESULTS: The results showed that arginine shortage induced a non-canonical ER stress response by inhibiting BiP translation via ribosome pausing. Overexpression of G-BiP in MDA-MB-231 cells promoted cell resistance to arginine shortage compared to cells overexpressing wild-type BiP. Additionally, limiting arginine led to decreased levels of the spliced XBP1 in the G-BiP overexpressing cells, potentially contributing to their improved survival compared to the parental WT BiP overexpressing cells. CONCLUSION: In conclusion, these findings suggest that the downregulation of BiP disrupts proteostasis during arginine shortage-induced non-canonical ER stress and plays a key role in cell growth inhibition, indicating BiP as a target of codon-specific ribosome pausing upon arginine shortage.

Methylcellulose colony assay and single-cell micro-manipulation reveal progenitor-like cells in adult human pancreatic ducts.

Progenitor cells capable of self-renewal and differentiation in the adult human pancreas are an under-explored resource for regenerative medicine. Using micro-manipulation and three-dimensional colony assays we identify cells within the adult human exocrine pancreas that resemble progenitor cells. Exocrine tissues were dissociated into single cells and plated into a colony assay containing methylcellulose and 5% Matrigel. A subpopulation of ductal cells formed colonies containing differentiated ductal, acinar, and endocrine lineage cells, and expanded up to 300-fold with a ROCK inhibitor. When transplanted into diabetic mice, colonies pre-treated with a NOTCH inhibitor gave rise to insulin-expressing cells. Both colonies and primary human ducts contained cells that simultaneously express progenitor transcription factors SOX9, NKX6.1, and PDX1. In addition, in silico analysis identified progenitor-like cells within ductal clusters in a single-cell RNA sequencing dataset. Therefore, progenitor-like cells capable of self-renewal and tri-lineage differentiation either pre-exist in the adult human exocrine pancreas, or readily adapt in culture.

LINE-1 ORF1p as a candidate biomarker in high grade serous ovarian carcinoma.

Long interspersed element 1 (LINE-1) open reading frame 1 protein (ORF1p) expression is a common feature of many cancer types, including high-grade serous ovarian carcinoma (HGSOC). Here, we report that ORF1p is not only expressed but also released by ovarian cancer and primary tumor cells. Immuno-multiple reaction monitoring-mass spectrometry assays showed that released ORF1p is confidently detectable in conditioned media, ascites, and patients' plasma, implicating ORF1p as a potential biomarker. Interestingly, ORF1p expression is detectable in fallopian tube (FT) epithelial precursors of HGSOC but not in benign FT, suggesting that ORF1p expression in an early event in HGSOC development. Finally, treatment of FT cells with DNA methyltransferase inhibitors led to robust expression and release of ORF1p, validating the regulatory role of DNA methylation in LINE-1 repression in non-tumorigenic tissue.

Frequency and mechanisms of LINE-1 retrotransposon insertions at CRISPR/Cas9 sites.

CRISPR/Cas9-based genome editing has revolutionized experimental molecular biology and entered the clinical world for targeted gene therapy. Identifying DNA modifications occurring at CRISPR/Cas9 target sites is critical to determine efficiency and safety of editing tools. Here we show that insertions of LINE-1 (L1) retrotransposons can occur frequently at CRISPR/Cas9 editing sites. Together with PolyA-seq and an improved amplicon sequencing, we characterize more than 2500 de novo L1 insertions at multiple CRISPR/Cas9 editing sites in HEK293T, HeLa and U2OS cells. These L1 retrotransposition events exploit CRISPR/Cas9-induced DSB formation and require L1 RT activity. Importantly, de novo L1 insertions are rare during genome editing by prime editors (PE), cytidine or adenine base editors (CBE or ABE), consistent with their reduced DSB formation. These data demonstrate that insertions of retrotransposons might be a potential outcome of CRISPR/Cas9 genome editing and provide further evidence on the safety of different CRISPR-based editing tools.

LINE-1 expression in cancer correlates with p53 mutation, copy number alteration, and S phase checkpoint.

Retrotransposons are genomic DNA sequences that copy themselves to new genomic locations via RNA intermediates; LINE-1 is the only active and autonomous retrotransposon in the human genome. The mobility of LINE-1 is largely repressed in somatic tissues but is derepressed in many cancers, where LINE-1 retrotransposition is correlated with p53 mutation and copy number alteration (CNA). In cell lines, inducing LINE-1 expression can cause double-strand breaks (DSBs) and replication stress. Reanalyzing multiomic data from breast, ovarian, endometrial, and colon cancers, we confirmed correlations between LINE-1 expression, p53 mutation status, and CNA. We observed a consistent correlation between LINE-1 expression and the abundance of DNA replication complex components, indicating that LINE-1 may also induce replication stress in human tumors. In endometrial cancer, high-quality phosphoproteomic data allowed us to identify the DSB-induced ATM-MRN-SMC S phase checkpoint pathway as the primary DNA damage response (DDR) pathway associated with LINE-1 expression. Induction of LINE-1 expression in an in vitro model led to increased phosphorylation of MRN complex member RAD50, suggesting that LINE-1 directly activates this pathway.

RNF8 has both KU-dependent and independent roles in chromosomal break repair.

Chromosomal double strand breaks (DSBs) can initiate several signaling events, such as ubiquitination, however the precise influence of such signaling on DSB repair outcomes remains poorly understood. With an RNA interference screen, we found that the E3 ubiquitin ligase RNF8 suppresses a deletion rearrangement mediated by canonical non-homologous end joining (C-NHEJ). We also found that RNF8 suppresses EJ without insertion/deletion mutations, which is a hallmark of C-NHEJ. Conversely, RNF8 promotes alternative EJ (ALT-EJ) events involving microhomology that is embedded from the edge of the DSB. These ALT-EJ events likely require limited end resection, whereas RNF8 is not required for single-strand annealing repair involving extensive end resection. Thus, RNF8 appears to specifically facilitate repair events requiring limited end resection, which we find is dependent on the DSB end protection factor KU. However, we also find that RNF8 is important for homology-directed repair (HDR) independently of KU, which appears linked to promoting PALB2 function. Finally, the influence of RNF8 on EJ is distinct from 53BP1 and the ALT-EJ factor, POLQ. We suggest that RNF8 mediates both ALT-EJ and HDR, but via distinct mechanisms, since only the former is dependent on KU.

The RAD52 S346X variant reduces risk of developing breast cancer in carriers of pathogenic germline BRCA2 mutations.

Women who carry pathogenic mutations in BRCA1 and BRCA2 have a lifetime risk of developing breast cancer of up to 80%. However, risk estimates vary in part due to genetic modifiers. We investigated the association of the RAD52 S346X variant as a modifier of the risk of developing breast and ovarian cancers in BRCA1 and BRCA2 mutation carriers from the Consortium of Investigators of Modifiers of BRCA1/2. The RAD52 S346X allele was associated with a reduced risk of developing breast cancer in BRCA2 carriers [per-allele hazard ratio (HR) = 0.69, 95% confidence interval (CI) 0.56-0.86; P = 0.0008] and to a lesser extent in BRCA1 carriers (per-allele HR = 0.78, 95% CI 0.64-0.97, P = 0.02). We examined how this variant affected DNA repair. Using a reporter system that measures repair of DNA double-strand breaks (DSBs) by single-strand annealing (SSA), expression of hRAD52 suppressed the loss of this repair in Rad52-/- mouse embryonic stem cells. When hRAD52 S346X was expressed in these cells, there was a significantly reduced frequency of SSA. Interestingly, expression of hRAD52 S346X also reduced the stimulation of SSA observed upon depletion of BRCA2, demonstrating the reciprocal roles for RAD52 and BRCA2 in the control of DSB repair by SSA. From an immunofluorescence analysis, we observed little nuclear localization of the mutant protein as compared to the wild-type; it is likely that the reduced nuclear levels of RAD52 S346X explain the diminished DSB repair by SSA. Altogether, we identified a genetic modifier that protects against breast cancer in women who carry pathogenic mutations in BRCA2 (P = 0.0008) and to a lesser extent BRCA1 (P = 0.02). This RAD52 mutation causes a reduction in DSB repair by SSA, suggesting that defects in RAD52-dependent DSB repair are linked to reduced tumor risk in BRCA2-mutation carriers.

BLM has Contrary Effects on Repeat-Mediated Deletions, based on the Distance of DNA DSBs to a Repeat and Repeat Divergence.

Repeat-mediated deletions (RMDs) often involve repetitive elements (e.g., short interspersed elements) with sequence divergence that is separated by several kilobase pairs (kbps). We have examined RMDs induced by DNA double-strand breaks (DSBs) under varying conditions of repeat sequence divergence (identical versus 1% and 3% divergent) and DSB/repeat distance (16 bp-28.4 kbp). We find that the BLM helicase promotes RMDs with long DSB/repeat distances (e.g., 28.4 kbp), which is consistent with a role in extensive DSB end resection, because the resection nucleases EXO1 and DNA2 affect RMDs similarly to BLM. In contrast, BLM suppresses RMDs with sequence divergence and intermediate (e.g., 3.3 kbp) DSB/repeat distances, which supports a role in heteroduplex rejection. The role of BLM in heteroduplex rejection is not epistatic with MSH2 and is independent of the annealing factor RAD52. Accordingly, the role of BLM on RMDs is substantially affected by DSB/repeat distance and repeat sequence divergence.

53BP1 Enforces Distinct Pre- and Post-resection Blocks on Homologous Recombination.

53BP1 activity drives genome instability and lethality in BRCA1-deficient mice by inhibiting homologous recombination (HR). The anti-recombinogenic functions of 53BP1 require phosphorylation-dependent interactions with PTIP and RIF1/shieldin effector complexes. While RIF1/shieldin blocks 5'-3' nucleolytic processing of DNA ends, it remains unclear how PTIP antagonizes HR. Here, we show that mutation of the PTIP interaction site in 53BP1 (S25A) allows sufficient DNA2-dependent end resection to rescue the lethality of BRCA1Δ11 mice, despite increasing RIF1 "end-blocking" at DNA damage sites. However, double-mutant cells fail to complete HR, as excessive shieldin activity also inhibits RNF168-mediated loading of PALB2/RAD51. As a result, BRCA1Δ1153BP1S25A mice exhibit hallmark features of HR insufficiency, including premature aging and hypersensitivity to PARPi. Disruption of shieldin or forced targeting of PALB2 to ssDNA in BRCA1D1153BP1S25A cells restores RNF168 recruitment, RAD51 nucleofilament formation, and PARPi resistance. Our study therefore reveals a critical function of shieldin post-resection that limits the loading of RAD51.

Evidence for interaction between Hsp90 and the ER membrane complex.

Numerous putative heat shock protein 90 (Hsp90)-interacting proteins, which could represent novel folding clients or co-chaperones, have been identified in recent years. Two separate high-throughput screens in yeast uncovered genetic effects between Hsp90 and components of the ER membrane complex (EMC), which is required for tolerance to unfolded protein response stress in yeast. Herein, we provide the first experimental evidence supporting that there is a genuine interaction of Hsp90 with the EMC. We demonstrate genetic interactions between EMC2 and the known Hsp90 co-chaperone encoded by STI1, as well as Hsp90 point mutant allele-specific differences in inherent growth and Hsp90 inhibitor tolerance in the absence and presence of EMC2. In co-precipitation experiments, Hsp90 interacts with Emc2p, whether or not Emc2p contains amino acid sequences designated as a tetratricopeptide repeat motif. Yeast with multiple EMC gene deletions exhibit increased sensitivity to Hsp90 inhibitor as well as defective folding of the well-established Hsp90 folding client, the glucocorticoid receptor. Altogether, our evidence of physical, genetic, and functional interaction of Hsp90 with the EMC, as well as bioinformatic analysis of shared interactors, supports that there is a legitimate interaction between them in vivo.

Repeat-mediated deletions can be induced by a chromosomal break far from a repeat, but multiple pathways suppress such rearrangements.

Chromosomal deletion rearrangements mediated by repetitive elements often involve repeats separated by several kilobases and sequences that are divergent. While such rearrangements are likely induced by DNA double-strand breaks (DSBs), it has been unclear how the proximity of DSBs relative to repeat sequences affects the frequency of such events. We generated a reporter assay in mouse cells for a deletion rearrangement involving repeats separated by 0.4 Mb. We induced this repeat-mediated deletion (RMD) rearrangement with two DSBs: the 5' DSB that is just downstream from the first repeat and the 3' DSB that is varying distances upstream of the second repeat. Strikingly, we found that increasing the 3' DSB/repeat distance from 3.3 kb to 28.4 kb causes only a modest decrease in rearrangement frequency. We also found that RMDs are suppressed by KU70 and RAD51 and promoted by RAD52, CtIP, and BRCA1. In addition, we found that 1%-3% sequence divergence substantially suppresses these rearrangements in a manner dependent on the mismatch repair factor MSH2, which is dominant over the suppressive role of KU70. We suggest that a DSB far from a repeat can stimulate repeat-mediated rearrangements, but multiple pathways suppress these events.