Deciphering the complex interplay of obesity, epithelial barrier dysfunction, and tight junction remodeling: Unraveling potential therapeutic avenues.

Obesity stands as a formidable global health challenge, predisposing individuals to a plethora of chronic illnesses such as cardiovascular disease, diabetes, and cancer. A confluence of genetic polymorphisms, suboptimal dietary choices, and sedentary lifestyles significantly contribute to the elevated incidence of obesity. This multifaceted health issue profoundly disrupts homeostatic equilibrium at both organismal and cellular levels, with marked alterations in gut permeability as a salient consequence. The intricate mechanisms underlying these alterations have yet to be fully elucidated. Still, evidence suggests that heightened inflammatory cytokine levels and the remodeling of tight junction (TJ) proteins, particularly claudins, play a pivotal role in the manifestation of epithelial barrier dysfunction in obesity. Strategic targeting of proteins implicated in these pathways and metabolites such as short-chain fatty acids presents a promising intervention for restoring barrier functionality among individuals with obesity. Nonetheless, recognizing the heterogeneity among affected individuals is paramount; personalized medical interventions or dietary regimens tailored to specific genetic backgrounds and allergy profiles may prove indispensable. This comprehensive review delves into the nexus of obesity, tight junction remodeling, and barrier dysfunction, offering a critical appraisal of potential therapeutic interventions.

The link between glycemic control measures and eye microvascular complications in a clinical cohort of type 2 diabetes with microRNA-223-3p signature.

BACKGROUND: Type 2 diabetes (T2D) is a critical healthcare challenge and priority in Qatar which is listed amongst the top 10 countries in the world, with its prevalence presently at 17% double the global average. MicroRNAs (miRNAs) are implicated in the pathogenesis of (T2D) and long-term microvascular complications including diabetic retinopathy (DR). METHODS: In this study, a T2D cohort that accurately matches the characteristics of the general population was employed to find microRNA (miRNA) signatures that are correlated with glycemic and ß cell function measurements. Targeted miRNA profiling was performed in (471) T2D individuals with or without DR and (491) (non-diabetic) healthy controls from the Qatar Biobank. Discovery analysis identified 20 differentially expressed miRNAs in T2D compared to controls, of which miR-223-3p was significantly upregulated (fold change:5.16, p = 3.6e-02) and positively correlated with glucose and hemoglobin A1c (HbA1c) levels (p-value = 9.88e-04 and 1.64e-05, respectively), but did not show any significant associations with insulin or C-peptide. Accordingly, we performed functional validation using a miR-223-3p mimic (overexpression) under control and hyperglycemia-induced conditions in a zebrafish model. RESULTS: Over-expression of miR-223-3p alone was associated with significantly higher glucose (42.7 mg/dL, n = 75 vs 38.7 mg/dL, n = 75, p = 0.02) and degenerated retinal vasculature, and altered retinal morphology involving changes in the ganglion cell layer and inner and outer nuclear layers. Assessment of retinal angiogenesis revealed significant upregulation in the expression of vascular endothelial growth factor and its receptors, including kinase insert domain receptor. Further, the pancreatic markers, pancreatic and duodenal homeobox 1, and the insulin gene expressions were upregulated in the miR-223-3p group. CONCLUSION: Our zebrafish model validates a novel correlation between miR-223-3p and DR development. Targeting miR-223-3p in T2D patients may serve as a promising therapeutic strategy to control DR in at-risk individuals.

A GHRHR founder mutation causes isolated growth hormone deficiency type IV in a consanguineous Pakistani family.

Background: Isolated growth hormone deficiency (IGHD) is caused by a severe shortage or absence of growth hormone (GH), which results in aberrant growth and development. Patients with IGHD type IV (IGHD4) have a short stature, reduced serum GH levels, and delayed bone age. Objectives: To identify the causative mutation of IGHD in a consanguineous family comprising four affected patients with IGHD4 (MIM#618157) and explore its functional impact in silico. Methods: Clinical and radiological studies were performed to determine the phenotypic spectrum and hormonal profile of the disease, while whole-exome sequencing (WES) and Sanger sequencing were performed to identify the disease-causing mutation. In-silico studies involved protein structural modeling and docking, and molecular dynamic simulation analyses using computational tools. Finally, data from the Qatar Genome Program (QGP) were screened for the presence of the founder variant in the Qatari population. Results: All affected individuals presented with a short stature without gross skeletal anomalies and significantly reduced serum GH levels. Genetic mapping revealed a homozygous nonsense mutation [NM_000823:c.G214T:p.(Glu72*)] in the third exon of the growth-hormone-releasing hormone receptor gene GHRHR (MIM#139191) that was segregated in all patients. The substituted amber codon (UAG) seems to truncate the protein by deleting the C-terminus GPCR domain, thus markedly disturbing the GHRHR receptor and its interaction with the growth hormone-releasing hormone. Conclusion: These data support that a p.Glu72* founder mutation in GHRHR perturbs growth hormone signaling and causes IGHD type IV. In-silico and biochemical analyses support the pathogenic effect of this nonsense mutation, while our comprehensive phenotype and hormonal profiling has established the genotype-phenotype correlation. Based on the current study, early detection of GHRHR may help in better therapeutic intervention.

Disruption of polyhomeotic polymerization decreases nucleosome occupancy and alters genome accessibility.

Chromatin attains its three-dimensional (3D) conformation by establishing contacts between different noncontiguous regions. Sterile Alpha Motif (SAM)-mediated polymerization of the polyhomeotic (PH) protein regulates subnuclear clustering of Polycomb Repressive Complex 1 (PRC1) and chromatin topology. The mutations that perturb the ability of the PH to polymerize, disrupt long-range chromatin contacts, alter Hox gene expression, and lead to developmental defects. To understand the underlying mechanism, we combined the experiments and theory to investigate the effect of this SAM domain mutation on nucleosome occupancy and accessibility on a genome wide scale. Our data show that disruption of PH polymerization because of SAM domain mutation decreases nucleosome occupancy and alters accessibility. Polymer simulations investigating the interplay between distant chromatin contacts and nucleosome occupancy, both of which are regulated by PH polymerization, suggest that nucleosome density increases when contacts between different regions of chromatin are established. Taken together, it appears that SAM domain-mediated PH polymerization biomechanically regulates the organization of chromatin at multiple scales from nucleosomes to chromosomes and we suggest that higher order organization can have a top-down causation effect on nucleosome occupancy.

Network-based identification and prioritization of key transcriptional factors of diabetic kidney disease.

Diabetic nephropathy (DN) is one of the most established microvascular complications of diabetes and a key cause of end-stage renal disease. It is well established that gene susceptibility to DN plays a critical role in disease pathophysiology. Therefore, many genetic studies have been performed to categorize candidate genes in prominent diabetic cohorts, aiming to investigate DN pathogenesis and etiology. In this study, we performed a meta-analysis on the expression profiles of GSE1009, GSE30122, GSE96804, GSE99340, GSE104948, GSE104954, and GSE111154 to identify critical transcriptional factors associated with DN progression. The analysis was conducted for all individual datasets for each kidney tissue (glomerulus, tubules, and kidney cortex). We identified distinct clusters of susceptibility genes that were dysregulated in a renal compartment-specific pattern. Further, we recognized a small but a closely connected set of these susceptibility genes enriched for podocyte differentiation, several of which were characterized as genes encoding critical transcriptional factors (TFs) involved in DN development and podocyte function. To validate the role of identified TFs in DN progression, we functionally validated the three main TFs (DACH1, LMX1B, and WT1) identified through differential gene expression and network analysis using the hyperglycemic zebrafish model. We report that hyperglycemia-induced altered gene expression of the key TF genes leads to morphological abnormalities in zebrafish glomeruli, pronephric tubules, proximal and distal ducts. This study demonstrated that altered expression of these TF genes could be associated with hyperglycemia-induced nephropathy and, thus, aids in understanding the molecular drivers, essential genes, and pathways that trigger DN initiation and development.

Human leukocyte antigen class II gene diversity tunes antibody repertoires to common pathogens.

Allelic diversity of human leukocyte antigen (HLA) class II genes may help maintain humoral immunity against infectious diseases. In this study, we investigated germline genetic variation in classical HLA class II genes and employed a systematic, unbiased approach to explore the relative contribution of this genetic variation in the antibody repertoire to various common pathogens. We leveraged a well-defined cohort of 800 adults representing the general Arab population in which genetic material is shared because of the high frequency of consanguineous unions. By applying a high-throughput method for large-scale antibody profiling to this well-defined cohort, we were able to dissect the overall effect of zygosity for classical HLA class II genes, as well as the effects associated with specific HLA class II alleles, haplotypes and genotypes, on the antimicrobial antibody repertoire breadth and antibody specificity with unprecedented resolution. Our population genetic studies revealed that zygosity of the classical HLA class II genes is a strong predictor of antibody responses to common human pathogens, suggesting that classical HLA class II gene heterozygosity confers a selective advantage. Moreover, we demonstrated that multiple HLA class II alleles can have additive effects on the antibody repertoire to common pathogens. We also identified associations of HLA-DRB1 genotypes with specific antigens. Our findings suggest that HLA class II gene polymorphisms confer specific humoral immunity against common pathogens, which may have contributed to the genetic diversity of HLA class II loci during hominine evolution.

Sib-pair subgroup familial type 1 diabetes mellitus in children in the state of Qatar.

BACKGROUND: Type 1 diabetes is the most common type of diabetes mellitus (DM) in children. It can be sporadic in onset or cluster in families, which comprises parent-offspring and sib-pair subgroups. The risk of developing DM in first-degree relatives of affected individuals is 8-15 fold higher. There is limited data about familial DM from the Gulf region. This study aims to describe the clinical, biochemical and genetic characteristics of sib-pair familial type 1 diabetes in Qatar. METHODS: Every child with DM following up at Sidra Medicine was recruited. Data was collected regarding clinical features, family history, type 1 diabetes autoantibodies and whole genome sequencing was performed. Genetic analysis for MODY genes and HLA association analysis was conducted. RESULTS: 44 families with sib-pair familial diabetes were identified. Of these, 2 families had 4 affected siblings and 5 families had 3 affected siblings. The majority are of Qatari ethnicity and the most common autoantibody was GAD65. The most common age of onset in the proband was 5-9 years while it was 10-14 years in subsequent siblings. The occurrence of DKA & HbA1c levels were lower in the second affected sibling. No relevant MODY gene variants were found. HLA analysis found 15 variants in at least 50% of the subjects. Most common were HLA-F*01*01*01G, HLA- DPA1*01*03*01G, HLA- DRB3*02*02*01G, HLA- E*01*01*01G & DRB4*03*01N. CONCLUSIONS: The prevalence of sib-pair diabetes is 3.64%. The second affected siblings were older. MODY is unlikely and Class I and II HLA genes was present in sib-pair diabetes.

Qatar genome: Insights on genomics from the Middle East.

Despite recent biomedical breakthroughs and large genomic studies growing momentum, the Middle Eastern population, home to over 400 million people, is underrepresented in the human genome variation databases. Here we describe insights from Phase 1 of the Qatar Genome Program with whole genome sequenced 6047 individuals from Qatar. We identified more than 88 million variants of which 24 million are novel and 23 million are singletons. Consistent with the high consanguinity and founder effects in the region, we found that several rare deleterious variants were more common in the Qatari population while others seem to provide protection against diseases and have shaped the genetic architecture of adaptive phenotypes. These results highlight the value of our data as a resource to advance genetic studies in the Arab and neighboring Middle Eastern populations and will significantly boost the current efforts to improve our understanding of global patterns of human variations, human history, and genetic contributions to health and diseases in diverse populations.

A population study of clinically actionable genetic variation affecting drug response from the Middle East.

Clinical implementation of pharmacogenomics will help in personalizing drug prescriptions and alleviate the personal and financial burden due to inefficacy and adverse reactions to drugs. However, such implementation is lagging in many parts of the world, including the Middle East, mainly due to the lack of data on the distribution of actionable pharmacogenomic variation in these ethnicities. We analyzed 6,045 whole genomes from the Qatari population for the distribution of allele frequencies of 2,629 variants in 1,026 genes known to affect 559 drugs or classes of drugs. We also performed a focused analysis of genotypes or diplotypes of 15 genes affecting 46 drugs, which have guidelines for clinical implementation and predicted their phenotypic impact. The allele frequencies of 1,320 variants in 703 genes affecting 299 drugs or class of drugs were significantly different between the Qatari population and other world populations. On average, Qataris carry 3.6 actionable genotypes/diplotypes, affecting 13 drugs with guidelines for clinical implementation, and 99.5% of the individuals had at least one clinically actionable genotype/diplotype. Increased risk of simvastatin-induced myopathy could be predicted in ~32% of Qataris from the diplotypes of SLCO1B1, which is higher compared to many other populations, while fewer Qataris may need tacrolimus dosage adjustments for achieving immunosuppression based on the CYP3A5 diplotypes compared to other world populations. Distinct distribution of actionable pharmacogenomic variation was also observed among the Qatari subpopulations. Our comprehensive study of the distribution of actionable genetic variation affecting drugs in a Middle Eastern population has potential implications for preemptive pharmacogenomic implementation in the region and beyond.

Melanocortin-4 receptor complexity in energy homeostasis,obesity and drug development strategies.

The melanocortin-4 receptor (MC4R) has been critically investigated for the past two decades, and novel findings regarding MC4R signalling and its potential exploitation in weight loss therapy have lately been emphasized. An association between MC4R and obesity is well established, with disease-causing mutations affecting 1% to 6% of obese patients. More than 200 MC4R variants have been reported, although conflicting results as to their effects have been found in different cohorts. Most notably, some MC4R gain-of-function variants seem to rescue obesity and related complications via specific pathways such as beta-arrestin (ß-arrestin) recruitment. Broadly speaking, however, dysfunctional MC4R dysregulates satiety and induces hyperphagia. The picture at the mechanistic level is complicated as, in addition to the canonical G stimulatory pathway, the ß-arrestin signalling pathway and ions (particularly calcium) seem to interact with MC4R signalling to contribute to or alleviate obesity pathogenesis. Thus, the overall complexity of the MC4R signalling spectra has broadened considerably, indicating there is great potential for the development of new drugs to manage obesity and its related complications. Alpha-melanocyte-stimulating hormone is the major endogenous MC4R agonist, but structure-based ligand discovery studies have identified possible superior and selective agonists that can improve MC4R function. However, some of these agonists characterized in vitro and in vivo confer adverse effects in patients, as demonstrated in clinical trials. In this review, we provide a comprehensive insight into the genetics, function and regulation of MC4R and its contribution to obesity. We also outline new approaches in drug development and emerging drug candidates to treat obesity.

Clinical features, epidemiology, autoantibody status, HLA haplotypes and genetic mechanisms of type 1 diabetes mellitus among children in Qatar.

To describe the clinical features, epidemiology, autoantibody status, HLA haplotypes and genetic mechanisms of type 1 diabetes mellitus (T1DM). Patients (0-18 years) with diabetes were recruited. Clinical data was collected, autoantibodies and c-peptide were measured. Whole Genome Sequencing was performed. Genomic data analysis was compared with the known genes linked with T1DM and HLA alleles were studied. 1096 patients had one or more antibody positivity. The incidence of T1DM in 2020 was 38.05 per 100,000 children and prevalence was 249.73. GADA was the most common autoantibody followed by IAA. Variants in GSTCD, SKAP2, SLC9B1, BANK1 were most prevalent. An association of HLA haplotypes DQA1*03:01:01G (OR = 2.46, p value = 0.011) and DQB1*03:02:01G (OR = 2.43, p value = 0.022) was identified. The incidence of T1DM in Qatar is the fourth highest in the world, IA2 autoantibody was the most specific with some patients only having ZnT8 or IA2 autoantibodies thus underlining the necessity of profiling all 4 autoantibodies. The genes associated with T1DM in the Arab population were different from those that are common in the Caucasian population. HLA-DQ was enriched in the Qatari patients suggesting that it can be considered a major risk factor at an early age.

Regulation of Circular RNA CircNFATC3 in Cancer Cells Alters Proliferation, Migration, and Oxidative Phosphorylation.

Circular RNAs were once considered artifacts of transcriptome sequencing but have recently been identified as functionally relevant in different types of cancer. Although there is still no clear main function of circRNAs, several studies have revealed that circRNAs are expressed in various eukaryotic organisms in a regulated manner often independent of their parental linear isoforms demonstrating conservation across species. circNFATC3, an abundant and uncharacterized circular RNA of exon 2 and 3 from NFATC3, was identified in transcriptomic data of solid tumors. Here we show that circNFATC3 gain- and loss-of-function experiments using RNAi-mediated circRNA silencing and circular mini vector-mediated overexpression of circularized constructs in breast and ovarian cancer cell lines affect molecular phenotypes. The knockdown of circNFATC3 induces a reduction in cell proliferation, invasion, migration, and oxidative phosphorylation. Gain-of-function of circNFATC3 in MDA-MB-231 and SK-OV-3 cells show a significant increase in cell proliferation, migration, and respiration. The above results suggest that circNFATC3 is a functionally relevant circular RNA in breast and ovarian cancer.

Insights Into the Role of CircRNAs: Biogenesis, Characterization, Functional, and Clinical Impact in Human Malignancies.

Circular RNAs (circRNAs) are an evolutionarily conserved novel class of non-coding endogenous RNAs (ncRNAs) found in the eukaryotic transcriptome, originally believed to be aberrant RNA splicing by-products with decreased functionality. However, recent advances in high-throughput genomic technology have allowed circRNAs to be characterized in detail and revealed their role in controlling various biological and molecular processes, the most essential being gene regulation. Because of the structural stability, high expression, availability of microRNA (miRNA) binding sites and tissue-specific expression, circRNAs have become hot topic of research in RNA biology. Compared to the linear RNA, circRNAs are produced differentially by backsplicing exons or lariat introns from a pre-messenger RNA (mRNA) forming a covalently closed loop structure missing 3' poly-(A) tail or 5' cap, rendering them immune to exonuclease-mediated degradation. Emerging research has identified multifaceted roles of circRNAs as miRNA and RNA binding protein (RBP) sponges and transcription, translation, and splicing event regulators. CircRNAs have been involved in many human illnesses, including cancer and neurodegenerative disorders such as Alzheimer's and Parkinson's disease, due to their aberrant expression in different pathological conditions. The functional versatility exhibited by circRNAs enables them to serve as potential diagnostic or predictive biomarkers for various diseases. This review discusses the properties, characterization, profiling, and the diverse molecular mechanisms of circRNAs and their use as potential therapeutic targets in different human malignancies.

Role of non-coding RNA networks in leukemia progression, metastasis and drug resistance.

Early-stage detection of leukemia is a critical determinant for successful treatment of the disease and can increase the survival rate of leukemia patients. The factors limiting the current screening approaches to leukemia include low sensitivity and specificity, high costs, and a low participation rate. An approach based on novel and innovative biomarkers with high accuracy from peripheral blood offers a comfortable and appealing alternative to patients, potentially leading to a higher participation rate.Recently, non-coding RNAs due to their involvement in vital oncogenic processes such as differentiation, proliferation, migration, angiogenesis and apoptosis have attracted much attention as potential diagnostic and prognostic biomarkers in leukemia. Emerging lines of evidence have shown that the mutational spectrum and dysregulated expression of non-coding RNA genes are closely associated with the development and progression of various cancers, including leukemia. In this review, we highlight the expression and functional roles of different types of non-coding RNAs in leukemia and discuss their potential clinical applications as diagnostic or prognostic biomarkers and therapeutic targets.

Identification of human genetic variants controlling circular RNA expression.

Circular RNAs (circRNAs) are abundant in eukaryotic transcriptomes and have been linked to various human disorders. However, understanding genetic control of circular RNA expression is in the early stages. Here we present the first integrated analysis of circRNAs and genome sequence variation from lymphoblastoid cell lines of the 1000 Genomes Project. We identified thousands of circRNAs in the RNA-seq data and show their association with local single-nucleotide polymorphic sites, referred to as circQTLs, which influence the circRNA transcript abundance. Strikingly, we found that circQTLs exist independently of eQTLs with most circQTLs having no effect on mRNA expression. Only a fraction of the polymorphic sites are shared and linked to both circRNA and mRNA expression with mostly similar effects on circular and linear RNA. A shared intronic QTL, rs55928920, of HMSD gene drives the circular and linear expression in opposite directions, potentially modulating circRNA levels at the expense of mRNA. Finally, circQTLs and eQTLs are largely independent and exist in separate linkage disequilibrium (LD) blocks with circQTLs highly enriched for functional genomic elements and regulatory regions. This study reveals a previously uncharacterized role of DNA sequence variation in human circular RNA regulation.

Cell Type-Specific TGF-ß Mediated EMT in 3D and 2D Models and Its Reversal by TGF-ß Receptor Kinase Inhibitor in Ovarian Cancer Cell Lines.

Transcriptome profiling of 3D models compared to 2D models in various cancer cell lines shows differential expression of TGF-ß-mediated and cell adhesion pathways. Presence of TGF-ß in these cell lines shows an increased invasion potential which is specific to cell type. In the present study, we identified exogenous addition of TGF-ß can induce Epithelial to Mesenchymal Transition (EMT) in a few cancer cell lines. RNA sequencing and real time PCR were carried out in different ovarian cancer cell lines to identify molecular profiling and metabolic profiling. Since EMT induction by TGF-ß is cell-type specific, we decided to select two promising ovarian cancer cell lines as model systems to study EMT. TGF-ß modulation in EMT and cancer invasion were successfully depicted in both 2D and 3D models of SKOV3 and CAOV3 cell lines. Functional evaluation in 3D and 2D models demonstrates that the addition of the exogenous TGF-ß can induce EMT and invasion in cancer cells by turning them into aggressive phenotypes. TGF-ß receptor kinase I inhibitor (LY364947) can revert the TGF-ß effect in these cells. In a nutshell, TGF-ß can induce EMT and migration, increase aggressiveness, increase cell survival, alter cell characteristics, remodel the Extracellular Matrix (ECM) and increase cell metabolism favorable for tumor invasion and metastasis. We concluded that transcriptomic and phenotypic effect of TGF-ß and its inhibitor is cell-type specific and not cancer specific.

Silencing of ANKRD12 circRNA induces molecular and functional changes associated with invasive phenotypes.

BACKGROUND: Circular RNAs (circRNAs) that form through non-canonical backsplicing events of pre-mRNA transcripts are evolutionarily conserved and abundantly expressed across species. However, the functional relevance of circRNAs remains a topic of debate. METHODS: We identified one of the highly expressed circRNA (circANKRD12) in cancer cell lines and characterized it validated it by Sanger sequencing, Real-Time PCR. siRNA mediated silencing of the circular junction of circANKRD12 was followed by RNA Seq analysis of circANKRD12 silenced cells and control cells to identify the differentially regulated genes. A series of cell biology and molecular biology techniques (MTS assay, Migration analysis, 3D organotypic models, Real-Time PCR, Cell cycle analysis, Western blot analysis, and Seahorse Oxygen Consumption Rate analysis) were performed to elucidate the function, and underlying mechanisms involved in circANKRD12 silenced breast and ovarian cancer cells. RESULTS: In this study, we identified and characterized a circular RNA derived from Exon 2 and Exon 8 of the ANKRD12 gene, termed here as circANKRD12. We show that this circRNA is abundantly expressed in breast and ovarian cancers. The circANKRD12 is RNase R resistant and predominantly localized in the cytoplasm in contrast to its source mRNA. We confirmed the expression of this circRNA across a variety of cancer cell lines and provided evidence for its functional relevance through downstream regulation of several tumor invasion genes. Silencing of circANKRD12 induces a strong phenotypic change by significantly regulating cell cycle, increasing invasion and migration and altering the metabolism in cancer cells. These results reveal the functional significance of circANKRD12 and provide evidence of a regulatory role for this circRNA in cancer progression. CONCLUSIONS: Our study demonstrates the functional relevance of circANKRD12 in various cancer cell types and, based on its expression pattern, has the potential to become a new clinical biomarker.

High-resolution protein-protein interaction mapping using all-versus-all sequencing (AVA-Seq).

Two-hybrid systems can be used for investigating protein-protein interactions and may provide important information about gene products with unknown function. Despite their success in mapping protein interactions, two-hybrid systems have remained mostly untouched by improvements in next-generation DNA sequencing. The two-hybrid systems rely on one-versus-all methods in which each bait is sequentially screened against an entire library. Here, we developed a screening method that joins both bait and prey as a convergent fusion into one bacterial plasmid vector that can then be amplified and paired-end sequencing by next-generation sequencing (NGS). Our method enables all-versus-all sequencing (AVA-Seq) and utilizes NGS to remove multiple bottlenecks of the two-hybrid system. AVA-Seq allows for high-resolution protein-protein interaction mapping of a small set of proteins and has the potential for lower-resolution mapping of entire proteomes. Features of the system include ORF selection to improve efficiency, high bacterial transformation efficiency, a convergent fusion vector to allow paired-end sequencing of interactors, and the use of protein fragments rather than full-length proteins to better resolve specific protein contact points. We demonstrate the system's strengths and limitations on a set of proteins known to interact in humans and provide a framework for future large-scale projects.