Didier Trono

Cryo-EM structures and binding of mouse and human ACE2 to SARS-CoV-2 variants of concern indicate that mutations enabling immune escape could expand host range

Kelvin Ka Ching Lau, Alexander Myasnikov, Sergey Nazarov, Dongchun Ni, Florence Pojer, Henning Paul-Julius Stahlberg, Didier Trono, Priscilla Turelli, Emiko Uchikawa

nvestigation of potential hosts of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is crucial to understanding future risks of spillover and spillback. SARS-CoV-2 has been reported to be transmitted from humans to various animals after requiring relatively few mutations. There is significant interest in describing how the virus interacts with mice as they are well adapted to human environments, are used widely as infection models and can be infected. Structural and binding data of the mouse ACE2 receptor with the Spike protein of newly identified SARS-CoV-2 variants are needed to better understand the impact of immune system evading mutations present in variants of concern (VOC). Previous studies have developed mouse-adapted variants and identified residues critical for binding to heterologous ACE2 receptors. Here we report the cryo-EM structures of mouse ACE2 bound to trimeric Spike ectodomains of four different VOC: Beta, Omicron BA.1, Omicron BA.2.12.1 and Omicron BA.4/5. These variants represent the oldest to the newest variants known to bind the mouse ACE2 receptor. Our high-resolution structural data complemented with bio-layer interferometry (BLI) binding assays reveal a requirement for a combination of mutations in the Spike protein that enable binding to the mouse ACE2 receptor.

2023

De novo design of protein interactions with learned surface fingerprints

Michael Bronstein, Stephen M Buckley, Bruno Emanuel Ferreira De Sousa Correia, Beat Fierz, Pablo Gainza Cirauqui, Sandrine Madeleine Suzanne Georgeon, Casper Alexander Goverde, Zander Harteveld, Anthony Marchand, Jane Marsden, Dongchun Ni, Elisa Oricchio, Aaron Simone Petruzzella, Charlène Mireille Raymonde Raclot, Stéphane Rosset, Andreas Scheck, Henning Paul-Julius Stahlberg, Freyr Sverrisson, Alexandra Teslenko, Didier Trono, Priscilla Turelli, Alexandra Krina Van Hall-Beauvais, Sarah Wehrle

Physical interactions between proteins are essential for most biological processes governing life. However, the molecular determinants of such interactions have been challenging to understand, even as genomic, proteomic and structural data increase. This knowledge gap has been a major obstacle for the comprehensive understanding of cellular protein–protein interaction networks and for the de novo design of protein binders that are crucial for synthetic biology and translational applications. Here we use a geometric deep-learning framework operating on protein surfaces that generates fingerprints to describe geometric and chemical features that are critical to drive protein–protein interactions10. We hypothesized that these fingerprints capture the key aspects of molecular recognition that represent a new paradigm in the computational design of novel protein interactions. As a proof of principle, we computationally designed several de novo protein binders to engage four protein targets: SARS-CoV-2 spike, PD-1, PD-L1 and CTLA-4. Several designs were experimentally optimized, whereas others were generated purely in silico, reaching nanomolar affinity with structural and mutational characterization showing highly accurate predictions. Overall, our surface-centric approach captures the physical and chemical determinants of molecular recognition, enabling an approach for the de novo design of protein interactions and, more broadly, of artificial proteins with function.

2023

Efficient and sensitive profiling of RNA–protein interactions using TLC-CLIP

Julien Léonard Duc, Christina Ernst, Didier Trono

RNA-binding proteins are instrumental for post-transcriptional gene regulation, controlling all aspects throughout the lifecycle of RNA molecules. However, transcriptome-wide methods to profile RNA-protein interactions in vivo remain technically challenging and require large amounts of starting material. Herein, we present an improved library preparation strategy for crosslinking and immunoprecipitation (CLIP) that is based on tailing and ligation of cDNA molecules (TLC). TLC involves the generation of solid-phase cDNA, followed by ribotailing to significantly enhance the efficiency of subsequent adapter ligation. These modifications result in a streamlined, fully bead-based library preparation strategy, which eliminates time-consuming purification procedures and drastically reduces sample loss. As a result, TLC-CLIP displays unparalleled sensitivity, enabling the profiling of RNA-protein interactions from as few as 1000 cells. To demonstrate the effectiveness of TLC-CLIP, we profiled four endogenous RNA-binding proteins, showcasing its reproducibility and improved precision resulting from a higher occurrence of crosslinking-induced deletions. These deletions serve as an intrinsic quality metric and increase both specificity and nucleotide-resolution.

2023

A cis-acting structural variation at the ZNF558 locus controls a gene regulatory network in human brain development

Johan Jakobsson, Julien Paul André Pontis, Didier Trono

The human forebrain has expanded in size and complexity compared to chimpanzees despite limited changes in protein-coding genes, suggesting that gene expression regulation is an important driver of brain evolution. Here, we identify a KRAB-ZFP transcription factor, ZNF558, that is expressed in human but not chimpanzee forebrain neural progenitor cells. ZNF558 evolved as a suppressor of LINE-1 transposons but has been co-opted to regulate a single target, the mitophagy gene SPATA18. ZNF558 plays a role in mitochondrial homeostasis, and loss-of-function experiments in cerebral organoids suggests that ZNF558 influences developmental timing during early human brain development. Expression of ZNF558 is controlled by the size of a variable number tandem repeat that is longer in chimpanzees compared to humans, and variable in the human population. Thus, this work provides mechanistic insight into how a cis-acting structural variation establishes a regulatory network that affects human brain evolution.

2022

A satellite DNA array barcodes chromosome 7 and regulates totipotency via ZFP819

Gabriela Ecco, Helen Mary Rowe, Didier Trono

Mammalian genomes are a battleground for genetic conflict between repetitive elements and KRAB-zinc finger proteins (KZFPs). We asked whether KZFPs can regulate cell fate by using ZFP819, which targets a satellite DNA array, ZP3AR. ZP3AR coats megabase regions of chromosome 7 encompassing genes encoding ZSCAN4, a master transcription factor of totipotency. Depleting ZFP819 in mouse embryonic stem cells (mESCs) causes them to transition to a 2-cell (2C)–like state, whereby the ZP3AR array switches from a poised to an active enhancer state. This is accompanied by a global erosion of heterochromatin roadblocks, which we link to decreased SETDB1 stability. These events result in transcription of active LINE-1 elements and impaired differentiation. In summary, ZFP819 and TRIM28 partner up to close chromatin across Zscan4, to promote exit from totipotency. We propose that satellite DNAs may control developmental fate transitions by barcoding and switching off master transcription factor genes.

2022

Cell-free method for the quantitative measurement of virus neutralizing antibodies

Didier Trono, Priscilla Turelli

The invention provides an in vitro method for the cell-free quantification of virus neutralizing activity, such as the SARS-CoV-2 neutralizing activity, of biological samples and kit for carrying out the method. The invention also provides a method of screening biological samples or collections of molecules to identify neutralizing antibodies or antiviral drugs.

2022

Human reproduction is regulated by retrotransposons derived from ancient Hominidae-specific viral infections

Julien Paul André Pontis, Yu Tao, Didier Trono

The transcription factor network required for primordial germ cell (PGC) specification is known to diverge in mammals. Here the authors show that hominidae-specific transposable element (TE) LTR5Hs becomes transcriptionally active during PGC specification, and LTR5Hs inactivation abrogates human PGC specification Germ cells are essential to pass DNA from one generation to the next. In human reproduction, germ cell development begins with the specification of primordial germ cells (PGCs) and a failure to specify PGCs leads to human infertility. Recent studies have revealed that the transcription factor network required for PGC specification has diverged in mammals, and this has a significant impact on our understanding of human reproduction. Here, we reveal that the Hominidae-specific Transposable Elements (TEs) LTR5Hs, may serve as TEENhancers (TE Embedded eNhancers) to facilitate PGC specification. LTR5Hs TEENhancers become transcriptionally active during PGC specification both in vivo and in vitro with epigenetic reprogramming leading to increased chromatin accessibility, localized DNA demethylation, enrichment of H3K27ac, and occupation of key hPGC transcription factors. Inactivation of LTR5Hs TEENhancers with KRAB mediated CRISPRi has a significant impact on germ cell specification. In summary, our data reveals the essential role of Hominidae-specific LTR5Hs TEENhancers in human germ cell development.

NATURE PORTFOLIO2022

Humoral Responses Against Variants of Concern by COVID-19 mRNA Vaccines in Immunocompromised Patients

Céline Pellaton, Madeleine Suffiotti, Didier Trono

IMPORTANCE There are limited comparative data on the durability of neutralizing antibody (nAb) responses elicited by messenger RNA (mRNA) vaccines against the SARS-CoV-2 variants of concern (VOCs) in immunocompromised patients and healthy controls. OBJECTIVE To assess the humoral responses after vaccination with BNT162b2 (Pfizer- BioNTech) or mRNA-1273 (Moderna) vaccines. DESIGN, SETTING, AND PARTICIPANTS In this prospective, longitudinal monocentric comparative effectiveness study conducted at the Lausanne University Hospital, binding IgG anti-spike antibody and nAb levels were measured at 1 week, 1 month, 3 months, and 6 months after vaccination with mRNA-1273 (24.6% of participants) or BNT162b2 (753% of participants). INTERVENTIONS All participants received 2 doses of either mRNA-1273 or BNT162b2 vaccines 4 to 6 weeks apart. MAIN OUTCOMES AND MEASURES The primary outcome of the study was the persistence of nAb responses against the original, nonvariant SARS-CoV-2 (2019-nCoV) and different VOCs at 6 months after vaccination. Key secondary outcomes were associations of the type of mRNA vaccine, the underlying disease, and the treatment with the response to vaccination. RESULTS Among the 841 participants enrolled between January 14 and August 8, 2021, the patient population comprised 637 participants (mean [SD] age, 61.8 [13.7] years; 386 [60.6%] female), and the healthy control population comprised 204 participants (mean [SD] age, 45.9 [12.0] years; 144 [70.6%] female). There were 399 patients with solid cancers, 101 with hematologic cancers. 38 with solid organ transplants, 99 with autoimmune diseases, and 204 healthy controls. More than 15 000 nAb determinations were performed against the original, nonvariant 2019-nCoV and the Alpha, Beta, Gamma, and Delta variants. The proportions of nAbs and their titers decreased in all study groups at 6 months after vaccination, with the greatest decreases for the Beta and Delta variants. For Beta, the proportion decreased to a median (SE) of 392% (5.5%) in those with hematologic cancers, 44,8% (2.7%) in those with solid cancers, 23.1% (8.3%) in those with solid organ transplants, and 22.7% (4.8%) in those with autoimmune diseases compared with 52.1% (4.2 degrees A.) in healthy controls. For Delta, the proportions decreased to 41.8% (5.6%) in participants with hematologic cancer, 51.9% (2.7%) in those with solid cancers, 26.9% (8.7%) in those with solid organ transplants, and 30.7% (5.3%) in those with autoimmune diseases compared with 56.9% (4.1%) healthy controls. Neutralizing antibody titers decreased 3.5- to 5-fold between month 1 and month 6, and the estimated duration of response was greater and more durable among those participants vaccinated with mRNA-1273. In participants with solid cancers, the estimated duration of nAbs against the Beta variant was 221 days with mRNA-1273 and 146 days with BNT162b2, and against the Delta variant, it was 226 days with mRNA-1273 and 161 with BNT162b2. The estimated duration of nAbs in participants with hematologic cancers was 113 and 127 days against Beta and Delta variants, respectively. CONCLUSIONS AND RELEVANCE This comparative effectiveness study suggests that approximately half of patients with hematologic cancers and solid cancers, about 70% of patients with solid organ transplants or autoimmune diseases, and 40% of healthy controls have lost nAbs against the circulating VOCs at 6 months after vaccination. These findings may be helpful for developing the best boosting vaccination schedule especially in immunocompromised patients.

AMER MEDICAL ASSOC2022

Krab-containing zinc finger protein and cancer

Didier Trono, Priscilla Turelli

The present invention provides methods for detecting cancer as well as agents and compositions for treating cancer by modulating the expression and/or activity of one or more i) KRAB-containing zinc finger protein (KZFP), ii) mRNA encoding a KZFP, and/or iii) KZFP gene.

2022