Robert Jan Lebbink

A Systematic Mammalian Genetic Interaction Map Reveals Pathways Underlying Ricin Susceptibility

Genetic interaction (GI) maps, comprising pairwise measures of how strongly the function of one gene depends on the presence of a second, have enabled the systematic exploration of gene function in microorganisms. Here, we present a two-stage strategy to construct high-density GI maps in mammalian cells. First, we use ultracomplex pooled shRNA libraries (25 shRNAs/gene) to identify high-confidence hit genes for a given phenotype and effective shRNAs. We then construct double-shRNA libraries from these to systematically measure GIs between hits. A GI map focused on ricin susceptibility broadly recapitulates known pathways and provides many unexpected insights. These include a noncanonical role for COPI, a previously uncharacterized protein complex affecting toxin clearance, a specialized role for the ribosomal protein RPS25, and functionally distinct mammalian TRAPP complexes. The ability to rapidly generate mammalian GI maps provides a potentially transformative tool for defining gene function and designing combination therapies based on synergistic pairs.

Collagens are functional, high affinity ligands for the inhibitory immune receptor LAIR-1

Collagens are the most abundant proteins in the human body, important in maintenance of tissue structure and hemostasis. Here we report that collagens are high affinity ligands for the broadly expressed inhibitory leukocyte-associated immunoglobulin-like receptor-1 (LAIR-1). The interaction is dependent on the conserved Gly-Pro-Hyp collagen repeats. Antibody cross-linking of LAIR-1 is known to inhibit immune cell function in vitro. We now show that collagens are functional ligands for LAIR-1 and directly inhibit immune cell activation in vitro. Thus far, all documented ligands for immune inhibitory receptors are membrane molecules, implying a regulatory role in cell–cell interaction. Our data reveal a novel mechanism of peripheral immune regulation by inhibitory immune receptors binding to extracellular matrix collagens.

Up-regulation of miR-21 by HER2/neu signaling promotes cell invasion

The cell surface receptor tyrosine kinase HER2/neu enhances tumor metastasis. Recent studies suggest that deregulated microRNA (miRNA) expression promotes invasion and metastasis of cancer cells; we therefore explored the possibility that HER2/neu signaling induces the expression of specific miRNAs involved in this process. We identified a putative oncogenic miRNA, miR-21, whose expression is correlated with HER2/neu up-regulation and is functionally involved in HER2/neu-induced cell invasion. We show that miR-21 is up-regulated via the MAPK (ERK1/2) pathway upon stimulation of HER2/neu signaling in breast cancer cells, and overexpression of other ERK1/2 activators such as RASV12 or ID-1 is sufficient to induce miR-21 up-regulation in HER2/neu-negative breast cancer cells. Furthermore, the metastasis suppressor protein PDCD4 (programmed cell death 4) is down-regulated by miR-21 in breast cancer cells expressing HER2/neu. Our data reveal a mechanism for HER2/neu-induced cancer cell invasion via miRNA deregulation. In addition, our results identify miR-21 as a potential therapeutic target for the prevention of breast cancer invasion and metastasis.

Efficient Intracellular Delivery of Native Proteins

Modulation of protein function is used to intervene in cellular processes but is often done indirectly by means of introducing DNA or mRNA encoding the effector protein. Thus far, direct intracellular delivery of proteins has remained challenging. We developed a method termed iTOP, for induced transduction by osmocytosis and propanebetaine, in which a combination of NaCl hypertonicity-induced macropinocytosis and a transduction compound (propanebetaine) induces the highly efficient transduction of proteins into a wide variety of primary cells. We demonstrate that iTOP is a useful tool in systems in which transient cell manipulation drives permanent cellular changes. As an example, we demonstrate that iTOP can mediate the delivery of recombinant Cas9 protein and short guide RNA, driving efficient gene targeting in a non-integrative manner.

Rapid Creation and Quantitative Monitoring of High Coverage shRNA Libraries

Short hairpin RNA libraries are limited by low efficacy of many shRNAs and by off-target effects, which give rise to false negatives and false positives, respectively. Here we present a strategy for rapidly creating expanded shRNA pools (∼30 shRNAs per gene) that are analyzed by deep sequencing (EXPAND). This approach enables identification of multiple effective target-specific shRNAs from a complex pool, allowing a rigorous statistical evaluation of true hits.

The Soluble Leukocyte-Associated Ig-Like Receptor (LAIR)-2 Antagonizes the Collagen/LAIR-1 Inhibitory Immune Interaction

Leukocyte-associated Ig-like receptor (LAIR)-1 is a collagen-receptor that inhibits immune cell function upon collagen binding. Next to LAIR-1, the human genome encodes LAIR-2, a putative soluble homolog. In this study we show, for the first time, that the LAIR-2 gene is broadly transcribed in human PBMC, mirroring the expression profile of LAIR-1. LAIR-2 protein is expressed as a soluble receptor exhibiting high affinity for various collagen molecules to which it binds in a hydroxyproline-dependent manner. In vitro stimulation of PBMC induces secretion of LAIR-2. We detect high amounts of LAIR-2 in urine of pregnant women, indicating that the soluble receptor is indeed produced in vivo and can be cleared from the body via urine. Furthermore, LAIR-2 levels are increased in synovial fluid of patients with rheumatoid arthritis as compared with osteoarthritis patients. We hypothesize that soluble LAIR-2 may function as a natural competitor for LAIR-1, thereby regulating its inhibitory potential. Indeed, LAIR-2 prevents binding of human LAIR-1 to collagens and LAIR-1 cross-linking in vitro, suggesting that the protein has an immunoregulatory function in vivo. Hence, we reveal a novel mechanism of immune regulation by a soluble LAIR receptor regulating the inhibitory potential of the membrane-bound LAIR-1 via competition for ligands.

CRISPR/Cas9-Mediated Genome Editing of Herpesviruses Limits Productive and Latent Infections

Herpesviruses infect the majority of the human population and can cause significant morbidity and mortality. Herpes simplex virus (HSV) type 1 causes cold sores and herpes simplex keratitis, whereas HSV-2 is responsible for genital herpes. Human cytomegalovirus (HCMV) is the most common viral cause of congenital defects and is responsible for serious disease in immuno-compromised individuals. Epstein-Barr virus (EBV) is associated with infectious mononucleosis and a broad range of malignancies, including Burkitt’s lymphoma, nasopharyngeal carcinoma, Hodgkin’s disease, and post-transplant lymphomas. Herpesviruses persist in their host for life by establishing a latent infection that is interrupted by periodic reactivation events during which replication occurs. Current antiviral drug treatments target the clinical manifestations of this productive stage, but they are ineffective at eliminating these viruses from the infected host. Here, we set out to combat both productive and latent herpesvirus infections by exploiting the CRISPR/Cas9 system to target viral genetic elements important for virus fitness. We show effective abrogation of HCMV and HSV-1 replication by targeting gRNAs to essential viral genes. Simultaneous targeting of HSV-1 with multiple gRNAs completely abolished the production of infectious particles from human cells. Using the same approach, EBV can be almost completely cleared from latently infected EBV-transformed human tumor cells. Our studies indicate that the CRISPR/Cas9 system can be effectively targeted to herpesvirus genomes as a potent prophylactic and therapeutic anti-viral strategy that may be used to impair viral replication and clear latent virus infection.

Systematic Identification of Barriers to Human iPSC Generation

Reprogramming of somatic cells to induced pluripotent stem cells (iPSCs) holds enormous promise for regenerative medicine. To elucidate endogenous barriers limiting this process, we systematically dissected human cellular reprogramming by combining a genome-wide RNAi screen, innovative computational methods, extensive single-hit validation, and mechanistic investigation of relevant pathways and networks. We identify reprogramming barriers, including genes involved in transcription, chromatin regulation, ubiquitination, dephosphorylation, vesicular transport, and cell adhesion. Specific a disintegrin and metalloproteinase (ADAM) proteins inhibit reprogramming, and the disintegrin domain of ADAM29 is necessary and sufficient for this function. Clathrin-mediated endocytosis can be targeted with small molecules and opposes reprogramming by positively regulating TGF-β signaling. Genetic interaction studies of endocytosis or ubiquitination reveal that barrier pathways can act in linear, parallel, or feedforward loop architectures to antagonize reprogramming. These results provide a global view of barriers to human cellular reprogramming.

The Mouse Homologue of the Leukocyte-Associated Ig-Like Receptor-1 Is an Inhibitory Receptor That Recruits Src Homology Region 2-Containing Protein Tyrosine Phosphatase (SHP)-2, but Not SHP-1

We report the molecular cloning and characterization of the first leukocyte-associated Ig-like receptor 1 (LAIR-1) homologue in mice that we have named mouse LAIR-1 (mLAIR-1). The mLAIR-1 gene maps to the proximal end of mouse chromosome 7 in a region syntenic with human chromosome 19q13.4 where the leukocyte receptor cluster is located. The protein shares 40% sequence identity with human LAIR-1, has a single Ig-like domain, and contains two immunoreceptor tyrosine-based inhibitory motif-like structures in its cytoplasmic tail. Mouse LAIR-1 is broadly expressed on various immune cells, and cross-linking of the molecule on stably transfected RBL-2H3 and YT.2C2 cells results in strong inhibition of their degranulation and cytotoxic activities, respectively. Upon pervanadate stimulation, the mLAIR-1 cytoplasmic tail becomes phosphorylated, thereby recruiting Src homology region 2-containing tyrosine phosphatase-2. Interestingly, unlike human LAIR-1, Src homology region 2-containing tyrosine phosphatase-1 is not recruited to the mLAIR-1 cytoplasmic tail. Screening human and mouse cell lines for mLAIR-1 and human LAIR-1 binding partners identified several lines expressing putative ligand(s) for both receptors.

Identification of multiple potent binding sites for human leukocyte associated Ig-like receptor LAIR on collagens II and III.

Immune responses are tightly controlled by the opposing actions of activating and inhibitory immune receptors. Previously we identified collagens as ligands for the inhibitory leukocyte-associated Ig-like receptor-1 (LAIR-1), revealing a novel mechanism of peripheral immune regulation by inhibitory immune receptors binding to extracellular matrix collagens. This interaction can be blocked by LAIR-2, a secreted member of the LAIR-1 family. LAIR-1 specifically interacts with synthetic trimeric peptides containing 10 repeats of glycine-proline-hydroxyproline (GPO) residues which can directly inhibit immune cell activation in vitro. Here we studied the interaction of human LAIR-1 and LAIR-2 with collagen in more detail by using novel overlapping synthetic trimeric peptides (Toolkits) encompassing the entire triple-helical domain of human collagens II and III. LAIR-1 and LAIR-2 bind several of these collagen-like peptides, with LAIR-2 being able to bind more than LAIR-1. LAIR binding to trimeric collagen peptides was influenced by GPO content of the peptide, although additional non-GPO triplets contributed to the interaction. Furthermore, we identified several trimeric peptides that were potent LAIR-1 ligands and could efficiently induce inhibition of T cell activation and FceRI-induced degranulation of RBL-2H3 cells through binding to LAIR-1. A detailed understanding of the LAIR recognition motifs within collagen may lead to the development of potent reagents that can be used in in vitro, ex vivo, and in vivo functional studies to dissect the biology and function of the collagen/LAIR-1 interaction.