Integration of Pathological Criteria and Immunohistochemical Evaluation for Invasive Lobular Carcinoma Diagnosis: Recommendations From the European Lobular Breast Cancer Consortium.

Invasive lobular carcinoma (ILC) is the second most frequent type of breast cancer (BC) and its peculiar morphology is mainly driven by inactivation of CDH1, the gene coding for E-cadherin cell adhesion protein. ILC-specific therapeutic and disease-monitoring approaches are gaining momentum in the clinic, increasing the importance of accurate ILC diagnosis. Several essential and desirable morphologic diagnostic criteria are currently defined by the World Health Organization, the routine use of immunohistochemistry (IHC) for E-cadherin is not recommended. Disagreement in the diagnosis of ILC has been repeatedly reported, but interpathologist agreement increases with the use of E-cadherin IHC. In this study, we aimed to harmonize the pathological diagnosis of ILC by comparing 5 commonly used E-cadherin antibody clones (NCH-38, EP700Y, Clone 36, NCL-L-E-cad [Clone 36B5], and ECH-6). We determined their biochemical specificity for the E-cadherin protein and IHC staining performance according to type and location of mutation on the CDH1 gene. Western blot analysis on mouse cell lines with conditional E-cadherin expression revealed a reduced specificity of EP700Y and NCL-L-E-cad for E-cadherin, with cross-reactivity of Clone 36 to P-cadherin. The use of IHC improved interpathologist agreement for ILC, lobular carcinoma in situ, and atypical lobular hyperplasia. The E-cadherin IHC staining pattern was associated with variant allele frequency and likelihood of nonsense-mediated RNA decay but not with the type or position of CDH1 mutations. Based on these results, we recommend the indication for E-cadherin staining, choice of antibodies, and their interpretation to standardize ILC diagnosis in current pathology practice.

Microenvironment-induced restoration of cohesive growth associated with focal activation of P-cadherin expression in lobular breast carcinoma metastatic to the colon.

Invasive lobular carcinoma (ILC) is a special breast cancer type characterized by noncohesive growth and E-cadherin loss. Focal activation of P-cadherin expression in tumor cells that are deficient for E-cadherin occurs in a subset of ILCs. Switching from an E-cadherin deficient to P-cadherin proficient status (EPS) partially restores cell-cell adhesion leading to the formation of cohesive tubular elements. It is unknown what conditions control EPS. Here, we report on EPS in ILC metastases in the large bowel. We reviewed endoscopic colon biopsies and colectomy specimens from a 52-year-old female (index patient) and of 18 additional patients (reference series) diagnosed with metastatic ILC in the colon. EPS was assessed by immunohistochemistry for E-cadherin and P-cadherin. CDH1/E-cadherin mutations were determined by next-generation sequencing. The index patient's colectomy showed transmural metastatic ILC harboring a CDH1/E-cadherin p.Q610* mutation. ILC cells displayed different growth patterns in different anatomic layers of the colon wall. In the tunica muscularis propria and the tela submucosa, ILC cells featured noncohesive growth and were E-cadherin-negative and P-cadherin-negative. However, ILC cells invading the mucosa formed cohesive tubular elements in the intercryptal stroma of the lamina propria mucosae. Inter-cryptal ILC cells switched to a P-cadherin-positive phenotype in this microenvironmental niche. In the reference series, colon mucosa infiltration was evident in 13 of 18 patients, one of which showed intercryptal EPS and conversion to cohesive growth as described in the index patient. The large bowel is a common metastatic site in ILC. In endoscopic colon biopsies, the typical noncohesive growth of ILC may be concealed by microenvironment-induced EPS and conversion to cohesive growth.

Rapid autopsies to enhance metastatic research: the UPTIDER post-mortem tissue donation program.

Research on metastatic cancer has been hampered by limited sample availability. Here we present the breast cancer post-mortem tissue donation program UPTIDER and show how it enabled sampling of a median of 31 (range: 5-90) metastases and 5-8 liquids per patient from its first 20 patients. In a dedicated experiment, we show the mild impact of increasing time after death on RNA quality, transcriptional profiles and immunohistochemical staining in tumor tissue samples. We show that this impact can be counteracted by organ cooling. We successfully generated ex vivo models from tissue and liquid biopsies from distinct histological subtypes of breast cancer. We anticipate these and future findings of UPTIDER to elucidate mechanisms of disease progression and treatment resistance and to provide tools for the exploration of precision medicine strategies in the metastatic setting.

Canonical Kaiso target genes define a functional signature that associates with breast cancer survival and the invasive lobular carcinoma histological type.

Invasive lobular carcinoma (ILC) is a low- to intermediate-grade histological breast cancer type caused by mutational inactivation of E-cadherin function, resulting in the acquisition of anchorage independence (anoikis resistance). Most ILC cases express estrogen receptors, but options are limited in relapsed endocrine-refractory disease as ILC tends to be less responsive to standard chemotherapy. Moreover, ILC can relapse after >15 years, an event that currently cannot be predicted. E-cadherin inactivation leads to p120-catenin-dependent relief of the transcriptional repressor Kaiso (ZBTB33) and activation of canonical Kaiso target genes. Here, we examined whether an anchorage-independent and ILC-specific transcriptional program correlated with clinical parameters in breast cancer. Based on the presence of a canonical Kaiso-binding consensus sequence (cKBS) in the promoters of genes that are upregulated under anchorage-independent conditions, we defined an ILC-specific anoikis resistance transcriptome (ART). Converting the ART genes into human orthologs and adding published Kaiso target genes resulted in the Kaiso-specific ART (KART) 33-gene signature, used subsequently to study correlations with histological and clinical variables in primary breast cancer. Using publicly available data for ERPOS Her2NEG breast cancer, we found that expression of KART was positively associated with the histological ILC breast cancer type (p < 2.7E-07). KART expression associated with younger patients in all invasive breast cancers and smaller tumors in invasive ductal carcinoma of no special type (IDC-NST) (<2 cm, p < 6.3E-10). We observed associations with favorable long-term prognosis in both ILC (hazard ratio [HR] = 0.51, 95% CI = 0.29-0.91, p < 3.4E-02) and IDC-NST (HR = 0.79, 95% CI = 0.66-0.93, p < 1.2E-04). Our analysis thus defines a new mRNA expression signature for human breast cancer based on canonical Kaiso target genes that are upregulated in E-cadherin deficient ILC. The KART signature may enable a deeper understanding of ILC biology and etiology. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

The laminin-keratin link shields the nucleus from mechanical deformation and signalling.

The mechanical properties of the extracellular matrix dictate tissue behaviour. In epithelial tissues, laminin is a very abundant extracellular matrix component and a key supporting element. Here we show that laminin hinders the mechanoresponses of breast epithelial cells by shielding the nucleus from mechanical deformation. Coating substrates with laminin-111-unlike fibronectin or collagen I-impairs cell response to substrate rigidity and YAP nuclear localization. Blocking the laminin-specific integrin ß4 increases nuclear YAP ratios in a rigidity-dependent manner without affecting the cell forces or focal adhesions. By combining mechanical perturbations and mathematical modelling, we show that ß4 integrins establish a mechanical linkage between the substrate and keratin cytoskeleton, which stiffens the network and shields the nucleus from actomyosin-mediated mechanical deformation. In turn, this affects the nuclear YAP mechanoresponses, chromatin methylation and cell invasion in three dimensions. Our results demonstrate a mechanism by which tissues can regulate their sensitivity to mechanical signals.

TLN1 contains a cancer-associated cassette exon that alters talin-1 mechanosensitivity.

Talin-1 is the core mechanosensitive adapter protein linking integrins to the cytoskeleton. The TLN1 gene is comprised of 57 exons that encode the 2,541 amino acid TLN1 protein. TLN1 was previously considered to be expressed as a single isoform. However, through differential pre-mRNA splicing analysis, we discovered a cancer-enriched, non-annotated 51-nucleotide exon in TLN1 between exons 17 and 18, which we refer to as exon 17b. TLN1 is comprised of an N-terminal FERM domain, linked to 13 force-dependent switch domains, R1-R13. Inclusion of exon 17b introduces an in-frame insertion of 17 amino acids immediately after Gln665 in the region between R1 and R2 which lowers the force required to open the R1-R2 switches potentially altering downstream mechanotransduction. Biochemical analysis of this isoform revealed enhanced vinculin binding, and cells expressing this variant show altered adhesion dynamics and motility. Finally, we showed that the TGF-ß/SMAD3 signaling pathway regulates this isoform switch. Future studies will need to consider the balance of these two TLN1 isoforms.

Loss of E-cadherin leads to Id2-dependent inhibition of cell cycle progression in metastatic lobular breast cancer.

Invasive lobular breast carcinoma (ILC) is characterized by proliferative indolence and long-term latency relapses. This study aimed to identify how disseminating ILC cells control the balance between quiescence and cell cycle re-entry. In the absence of anchorage, ILC cells undergo a sustained cell cycle arrest in G0/G1 while maintaining viability. From the genes that are upregulated in anchorage independent ILC cells, we selected Inhibitor of DNA binding 2 (Id2), a mediator of cell cycle progression. Using loss-of-function experiments, we demonstrate that Id2 is essential for anchorage independent survival (anoikis resistance) in vitro and lung colonization in mice. Importantly, we find that under anchorage independent conditions, E-cadherin loss promotes expression of Id2 in multiple mouse and (organotypic) human models of ILC, an event that is caused by a direct p120-catenin/Kaiso-dependent transcriptional de-repression of the canonical Kaiso binding sequence TCCTGCNA. Conversely, stable inducible restoration of E-cadherin expression in the ILC cell line SUM44PE inhibits Id2 expression and anoikis resistance. We show evidence that Id2 accumulates in the cytosol, where it induces a sustained and CDK4/6-dependent G0/G1 cell cycle arrest through interaction with hypo-phosphorylated Rb. Finally, we find that Id2 is indeed enriched in ILC when compared to other breast cancers, and confirm cytosolic Id2 protein expression in primary ILC samples. In sum, we have linked mutational inactivation of E-cadherin to direct inhibition of cell cycle progression. Our work indicates that loss of E-cadherin and subsequent expression of Id2 drive indolence and dissemination of ILC. As such, E-cadherin and Id2 are promising candidates to stratify low and intermediate grade invasive breast cancers for the use of clinical cell cycle intervention drugs.

Spatial collagen stiffening promotes collective breast cancer cell invasion by reinforcing extracellular matrix alignment.

The tumor micro-environment often contains stiff and irregular-bundled collagen fibers that are used by tumor cells to disseminate. It is still unclear how and to what extent, extracellular matrix (ECM) stiffness versus ECM bundle size and alignment dictate cancer cell invasion. Here, we have uncoupled Collagen-I bundling from stiffness by introducing inter-collagen crosslinks, combined with temperature induced aggregation of collagen bundling. Using organotypic models from mouse invasive ductal and invasive lobular breast cancers, we show that increased collagen bundling in 3D induces a generic increase in breast cancer invasion that is independent of migration mode. However, systemic collagen stiffening using advanced glycation end product (AGE) crosslinking prevents collective invasion, while leaving single cell invasion unaffected. Collective invasion into collagen matrices by ductal breast cancer cells depends on Lysyl oxidase-like 3 (Loxl3), a factor produced by tumor cells that reinforces local collagen stiffness. Finally, we present clinical evidence that collectively invading cancer cells at the invasive front of ductal breast carcinoma upregulate LOXL3. By uncoupling the mechanical, chemical, and structural cues that control invasion of breast cancer in three dimensions, our data reveal that spatial control over stiffness and bundling underlie collective dissemination of ductal-type breast cancers.

Atlas of Lobular Breast Cancer Models: Challenges and Strategic Directions.

Invasive lobular carcinoma (ILC) accounts for up to 15% of all breast cancer (BC) cases and responds well to endocrine treatment when estrogen receptor α-positive (ER+) yet differs in many biological aspects from other ER+ BC subtypes. Up to 30% of patients with ILC will develop late-onset metastatic disease up to ten years after initial tumor diagnosis and may experience failure of systemic therapy. Unfortunately, preclinical models to study ILC progression and predict the efficacy of novel therapeutics are scarce. Here, we review the current advances in ILC modeling, including cell lines and organotypic models, genetically engineered mouse models, and patient-derived xenografts. We also underscore four critical challenges that can be addressed using ILC models: drug resistance, lobular tumor microenvironment, tumor dormancy, and metastasis. Finally, we highlight the advantages of shared experimental ILC resources and provide essential considerations from the perspective of the European Lobular Breast Cancer Consortium (ELBCC), which is devoted to better understanding and translating the molecular cues that underpin ILC to clinical diagnosis and intervention. This review will guide investigators who are considering the implementation of ILC models in their research programs.

Uncovering mutation-specific morphogenic phenotypes and paracrine-mediated vessel dysfunction in a biomimetic vascularized mammary duct platform.

The mammary gland is a highly vascularized tissue capable of expansion and regression during development and disease. To enable mechanistic insight into the coordinated morphogenic crosstalk between the epithelium and vasculature, we introduce a 3D microfluidic platform that juxtaposes a human mammary duct in proximity to a perfused endothelial vessel. Both compartments recapitulate stable architectural features of native tissue and the ability to undergo distinct forms of branching morphogenesis. Modeling HER2/ERBB2 amplification or activating PIK3CA(H1047R) mutation each produces ductal changes observed in invasive progression, yet with striking morphogenic and behavioral differences. Interestingly, PI3KαH1047R ducts also elicit increased permeability and structural disorganization of the endothelium, and we identify the distinct secretion of IL-6 as the paracrine cause of PI3KαH1047R-associated vascular dysfunction. These results demonstrate the functionality of a model system that facilitates the dissection of 3D morphogenic behaviors and bidirectional signaling between mammary epithelium and endothelium during homeostasis and pathogenesis.

Host interactors of effector proteins of the lettuce downy mildew Bremia lactucae obtained by yeast two-hybrid screening.

Plant pathogenic bacteria, fungi and oomycetes secrete effector proteins to manipulate host cell processes to establish a successful infection. Over the last decade the genomes and transcriptomes of many agriculturally important plant pathogens have been sequenced and vast candidate effector repertoires were identified using bioinformatic analyses. Elucidating the contribution of individual effectors to pathogenicity is the next major hurdle. To advance our understanding of the molecular mechanisms underlying lettuce susceptibility to the downy mildew Bremia lactucae, we mapped physical interactions between B. lactucae effectors and lettuce candidate target proteins. Using a lettuce cDNA library-based yeast-two-hybrid system, 61 protein-protein interactions were identified, involving 21 B. lactucae effectors and 46 unique lettuce proteins. The top ten interactors based on the number of independent colonies identified in the Y2H and two interactors that belong to gene families involved in plant immunity, were further characterized. We determined the subcellular localization of the fluorescently tagged lettuce proteins and their interacting effectors. Importantly, relocalization of effectors or their interactors to the nucleus was observed for four protein-pairs upon their co-expression, supporting their interaction in planta.

Functional Dissection of C. elegans bZip-Protein CEBP-1 Reveals Novel Structural Motifs Required for Axon Regeneration and Nuclear Import.

The basic leucine-zipper (bZIP) domain transcription factors CCAAT/enhancer-binding proteins (C/EBP) have a variety of roles in cell proliferation, differentiation, and stress response. In the nervous system, several isoforms of C/EBP function in learning and memory, neuronal plasticity, neuroinflammation, and axon regeneration. We previously reported that the Caenorhabditis elegans C/EBP homolog, CEBP-1, is essential for axon regeneration. CEBP-1 consists of 319 amino acids, with its bZIP domain at the C-terminus and a long N-terminal fragment with no known protein motifs. Here, using forward genetic screening with targeted genome editing, we have identified a unique domain in the N-terminus that is critical for its in vivo function. Additionally, we characterized three nuclear localization signals (NLS) in CEBP-1 that act together to mediate CEBP-1's nuclear import. Moreover, the Importin-α, IMA-3, can bind to CEBP-1 via one of the NLS. ima-3 is ubiquitously expressed in all somatic cells, and ima-3 null mutants are larval lethal. Using Cre-lox dependent neuron-specific deletion strategy, we show that ima-3 is not critical for axon development, but is required for axon regeneration in adults. Together, these data advance our understanding of CEBP-1's function, and suggest new regulators that remain to be identified to expand the CEBP-1 protein interactome.

Merlin/ERM proteins regulate growth factor-induced macropinocytosis and receptor recycling by organizing the plasma membrane:cytoskeleton interface.

The architectural and biochemical features of the plasma membrane are governed by its intimate association with the underlying cortical cytoskeleton. The neurofibromatosis type 2 (NF2) tumor suppressor merlin and closely related membrane:cytoskeleton-linking protein ezrin organize the membrane:cytoskeleton interface, a critical cellular compartment that both regulates and is regulated by growth factor receptors. An example of this poorly understood interrelationship is macropinocytosis, an ancient process of nutrient uptake and membrane remodeling that can both be triggered by growth factors and manage receptor availability. We show that merlin deficiency primes the membrane:cytoskeleton interface for epidermal growth factor (EGF)-induced macropinocytosis via a mechanism involving increased cortical ezrin, altered actomyosin, and stabilized cholesterol-rich membranes. These changes profoundly alter EGF receptor (EGFR) trafficking in merlin-deficient cells, favoring increased membrane levels of its heterodimerization partner, ErbB2; clathrin-independent internalization; and recycling. Our work suggests that, unlike Ras transformed cells, merlin-deficient cells do not depend on macropinocytic protein scavenging and instead exploit macropinocytosis for receptor recycling. Finally, we provide evidence that the macropinocytic proficiency of NF2-deficient cells can be used for therapeutic uptake. This work provides new insight into fundamental mechanisms of macropinocytic uptake and processing and suggests new ways to interfere with or exploit macropinocytosis in NF2 mutant and other tumors.

A tissue-specific protein purification approach in Caenorhabditis elegans identifies novel interaction partners of DLG-1/Discs large.

BACKGROUND: Affinity purification followed by mass spectrometry (AP/MS) is a widely used approach to identify protein interactions and complexes. In multicellular organisms, the accurate identification of protein complexes by AP/MS is complicated by the potential heterogeneity of complexes in different tissues. Here, we present an in vivo biotinylation-based approach for the tissue-specific purification of protein complexes from Caenorhabditis elegans. Tissue-specific biotinylation is achieved by the expression in select tissues of the bacterial biotin ligase BirA, which biotinylates proteins tagged with the Avi peptide. RESULTS: We generated N- and C-terminal tags combining GFP with the Avi peptide sequence, as well as four BirA driver lines expressing BirA ubiquitously and specifically in the seam and hyp7 epidermal cells, intestine, or neurons. We validated the ability of our approach to identify bona fide protein interactions by identifying the known LGL-1 interaction partners PAR-6 and PKC-3. Purification of the Discs large protein DLG-1 identified several candidate interaction partners, including the AAA-type ATPase ATAD-3 and the uncharacterized protein MAPH-1.1. We have identified the domains that mediate the DLG-1/ATAD-3 interaction, and show that this interaction contributes to C. elegans development. MAPH-1.1 co-purified specifically with DLG-1 purified from neurons, and shared limited homology with the microtubule-associated protein MAP1A, a known neuronal interaction partner of mammalian DLG4/PSD95. A CRISPR/Cas9-engineered GFP::MAPH-1.1 fusion was broadly expressed and co-localized with microtubules. CONCLUSIONS: The method we present here is able to purify protein complexes from specific tissues. We uncovered a series of DLG-1 interactors, and conclude that ATAD-3 is a biologically relevant interaction partner of DLG-1. Finally, we conclude that MAPH-1.1 is a microtubule-associated protein of the MAP1 family and a candidate neuron-specific interaction partner of DLG-1.

A combined binary interaction and phenotypic map of C. elegans cell polarity proteins.

The establishment of cell polarity is an essential process for the development of multicellular organisms and the functioning of cells and tissues. Here, we combine large-scale protein interaction mapping with systematic phenotypic profiling to study the network of physical interactions that underlies polarity establishment and maintenance in the nematode Caenorhabditis elegans. Using a fragment-based yeast two-hybrid strategy, we identified 439 interactions between 296 proteins, as well as the protein regions that mediate these interactions. Phenotypic profiling of the network resulted in the identification of 100 physically interacting protein pairs for which RNAi-mediated depletion caused a defect in the same polarity-related process. We demonstrate the predictive capabilities of the network by showing that the physical interaction between the RhoGAP PAC-1 and PAR-6 is required for radial polarization of the C. elegans embryo. Our network represents a valuable resource of candidate interactions that can be used to further our insight into cell polarization.

Axon injury triggers EFA-6 mediated destabilization of axonal microtubules via TACC and doublecortin like kinase.

Axon injury triggers a series of changes in the axonal cytoskeleton that are prerequisites for effective axon regeneration. In Caenorhabditis elegans the signaling protein Exchange Factor for ARF-6 (EFA-6) is a potent intrinsic inhibitor of axon regrowth. Here we show that axon injury triggers rapid EFA-6-dependent inhibition of axonal microtubule (MT) dynamics, concomitant with relocalization of EFA-6. EFA-6 relocalization and axon regrowth inhibition require a conserved 18-aa motif in its otherwise intrinsically disordered N-terminal domain. The EFA-6 N-terminus binds the MT-associated proteins TAC-1/Transforming-Acidic-Coiled-Coil, and ZYG-8/Doublecortin-Like-Kinase, both of which are required for regenerative growth cone formation, and which act downstream of EFA-6. After injury TAC-1 and EFA-6 transiently relocalize to sites marked by the MT minus end binding protein PTRN-1/Patronin. We propose that EFA-6 acts as a bifunctional injury-responsive regulator of axonal MT dynamics, acting at the cell cortex in the steady state and at MT minus ends after injury.

The C. elegans Crumbs family contains a CRB3 homolog and is not essential for viability.

Crumbs proteins are important regulators of epithelial polarity. In C. elegans, no essential role for the two described Crumbs homologs has been uncovered. Here, we identify and characterize an additional Crumbs family member in C. elegans, which we termed CRB-3 based on its similarity in size and sequence to mammalian CRB3. We visualized CRB-3 subcellular localization by expressing a translational GFP fusion. CRB-3::GFP was expressed in several polarized tissues in the embryo and larval stages, and showed apical localization in the intestine and pharynx. To identify the function of the Crumbs family in C. elegans development, we generated a triple Crumbs deletion mutant by sequentially removing the entire coding sequence for each crumbs homolog using a CRISPR/Cas9-based approach. Remarkably, animals lacking all three Crumbs homologs are viable and show normal epithelial polarity. Thus, the three C. elegans Crumbs family members do not appear to play an essential role in epithelial polarity establishment.

Controlled sumoylation of the mevalonate pathway enzyme HMGS-1 regulates metabolism during aging.

Many metabolic pathways are critically regulated during development and aging but little is known about the molecular mechanisms underlying this regulation. One key metabolic cascade in eukaryotes is the mevalonate pathway. It catalyzes the synthesis of sterol and nonsterol isoprenoids, such as cholesterol and ubiquinone, as well as other metabolites. In humans, an age-dependent decrease in ubiquinone levels and changes in cholesterol homeostasis suggest that mevalonate pathway activity changes with age. However, our knowledge of the mechanistic basis of these changes remains rudimentary. We have identified a regulatory circuit controlling the sumoylation state of Caenorhabditis elegans HMG-CoA synthase (HMGS-1). This protein is the ortholog of human HMGCS1 enzyme, which mediates the first committed step of the mevalonate pathway. In vivo, HMGS-1 undergoes an age-dependent sumoylation that is balanced by the activity of ULP-4 small ubiquitin-like modifier protease. ULP-4 exhibits an age-regulated expression pattern and a dynamic cytoplasm-to-mitochondria translocation. Thus, spatiotemporal ULP-4 activity controls the HMGS-1 sumoylation state in a mechanism that orchestrates mevalonate pathway activity with the age of the organism. To expand the HMGS-1 regulatory network, we combined proteomic analyses with knockout studies and found that the HMGS-1 level is also governed by the ubiquitin-proteasome pathway. We propose that these conserved molecular circuits have evolved to govern the level of mevalonate pathway flux during aging, a flux whose dysregulation is associated with numerous age-dependent cardiovascular and cancer pathologies.

Identification of human protein interaction domains using an ORFeome-based yeast two-hybrid fragment library.

Physical interactions between proteins are essential for biological processes. Hence, there have been major efforts to elucidate the complete networks of protein-protein interactions, or "interactomes", of various organisms. Detailed descriptions of protein interaction networks should include information on the discrete domains that mediate these interactions, yet most large-scale efforts model interactions between whole proteins only. We previously developed a yeast two-hybrid-based strategy to systematically map interaction domains and generated a domain-based interactome network for 750 proteins involved in C. elegans early embryonic development. Here, we expand the concept of Y2H-based interaction domain mapping to the genome-wide level. We generated a human fragment library by randomly fragmenting the full-length open reading frames (ORFs) present in the human ORFeome collection. Screens using several proteins required for cell division or polarity establishment as baits demonstrate the ability to accurately identify interaction domains for human proteins using this approach, while the experimental quality of the Y2H data was independently verified in coaffinity purification assays. The library generation strategy can easily be adapted to generate libraries from full-length ORF collections of other organisms.