Finding Needles in the Haystack: Strategies for Uncovering Noncoding Regulatory Variants.

Despite accumulating evidence implicating noncoding variants in human diseases, unraveling their functionality remains a significant challenge. Systematic annotations of the regulatory landscape and the growth of sequence variant data sets have fueled the development of tools and methods to identify causal noncoding variants and evaluate their regulatory effects. Here, we review the latest advances in the field and discuss potential future research avenues to gain a more in-depth understanding of noncoding regulatory variants.

Disease Model of GATA4 Mutation Reveals Transcription Factor Cooperativity in Human Cardiogenesis.

Mutation of highly conserved residues in transcription factors may affect protein-protein or protein-DNA interactions, leading to gene network dysregulation and human disease. Human mutations in GATA4, a cardiogenic transcription factor, cause cardiac septal defects and cardiomyopathy. Here, iPS-derived cardiomyocytes from subjects with a heterozygous GATA4-G296S missense mutation showed impaired contractility, calcium handling, and metabolic activity. In human cardiomyocytes, GATA4 broadly co-occupied cardiac enhancers with TBX5, another transcription factor that causes septal defects when mutated. The GATA4-G296S mutation disrupted TBX5 recruitment, particularly to cardiac super-enhancers, concomitant with dysregulation of genes related to the phenotypic abnormalities, including cardiac septation. Conversely, the GATA4-G296S mutation led to failure of GATA4 and TBX5-mediated repression at non-cardiac genes and enhanced open chromatin states at endothelial/endocardial promoters. These results reveal how disease-causing missense mutations can disrupt transcriptional cooperativity, leading to aberrant chromatin states and cellular dysfunction, including those related to morphogenetic defects.

A Proteome-wide Fission Yeast Interactome Reveals Network Evolution Principles from Yeasts to Human.

Here, we present FissionNet, a proteome-wide binary protein interactome for S. pombe, comprising 2,278 high-quality interactions, of which ∼ 50% were previously not reported in any species. FissionNet unravels previously unreported interactions implicated in processes such as gene silencing and pre-mRNA splicing. We developed a rigorous network comparison framework that accounts for assay sensitivity and specificity, revealing extensive species-specific network rewiring between fission yeast, budding yeast, and human. Surprisingly, although genes are better conserved between the yeasts, S. pombe interactions are significantly better conserved in human than in S. cerevisiae. Our framework also reveals that different modes of gene duplication influence the extent to which paralogous proteins are functionally repurposed. Finally, cross-species interactome mapping demonstrates that coevolution of interacting proteins is remarkably prevalent, a result with important implications for studying human disease in model organisms. Overall, FissionNet is a valuable resource for understanding protein functions and their evolution.

In vivo versus in silico assessment of potentially pathogenic missense variants in human reproductive genes.

Infertility is a heterogeneous condition, with genetic causes thought to underlie a substantial fraction of cases. Genome sequencing is becoming increasingly important for genetic diagnosis of diseases including idiopathic infertility; however, most rare or minor alleles identified in patients are variants of uncertain significance (VUS). Interpreting the functional impacts of VUS is challenging but profoundly important for clinical management and genetic counseling. To determine the consequences of these variants in key fertility genes, we functionally evaluated 11 missense variants in the genes ANKRD31, BRDT, DMC1, EXO1, FKBP6, MCM9, M1AP, MEI1, MSH4 and SEPT12 by generating genome-edited mouse models. Nine variants were classified as deleterious by most functional prediction algorithms, and two disrupted a protein-protein interaction (PPI) in the yeast two hybrid (Y2H) assay. Though these genes are essential for normal meiosis or spermiogenesis in mice, only one variant, observed in the MCM9 gene of a male infertility patient, compromised fertility or gametogenesis in the mouse models. To explore the disconnect between predictions and outcomes, we compared pathogenicity calls of missense variants made by ten widely used algorithms to 1) those annotated in ClinVar and 2) those evaluated in mice. All the algorithms performed poorly in terms of predicting the effects of human missense variants modeled in mice. These studies emphasize caution in the genetic diagnoses of infertile patients based primarily on pathogenicity prediction algorithms and emphasize the need for alternative and efficient in vitro or in vivo functional validation models for more effective and accurate VUS description to either pathogenic or benign categories.

Structural basis of TRAPPIII-mediated Rab1 activation.

The GTPase Rab1 is a master regulator of the early secretory pathway and is critical for autophagy. Rab1 activation is controlled by its guanine nucleotide exchange factor, the multisubunit TRAPPIII complex. Here, we report the 3.7 Å cryo-EM structure of the Saccharomyces cerevisiae TRAPPIII complex bound to its substrate Rab1/Ypt1. The structure reveals the binding site for the Rab1/Ypt1 hypervariable domain, leading to a model for how the complex interacts with membranes during the activation reaction. We determined that stable membrane binding by the TRAPPIII complex is required for robust activation of Rab1/Ypt1 in vitro and in vivo, and is mediated by a conserved amphipathic α-helix within the regulatory Trs85 subunit. Our results show that the Trs85 subunit serves as a membrane anchor, via its amphipathic helix, for the entire TRAPPIII complex. These findings provide a structural understanding of Rab activation on organelle and vesicle membranes.

A full-proteome, interaction-specific characterization of mutational hotspots across human cancers.

Rapid accumulation of cancer genomic data has led to the identification of an increasing number of mutational hotspots with uncharacterized significance. Here we present a biologically informed computational framework that characterizes the functional relevance of all 1107 published mutational hotspots identified in approximately 25,000 tumor samples across 41 cancer types in the context of a human 3D interactome network, in which the interface of each interaction is mapped at residue resolution. Hotspots reside in network hub proteins and are enriched on protein interaction interfaces, suggesting that alteration of specific protein-protein interactions is critical for the oncogenicity of many hotspot mutations. Our framework enables, for the first time, systematic identification of specific protein interactions affected by hotspot mutations at the full proteome scale. Furthermore, by constructing a hotspot-affected network that connects all hotspot-affected interactions throughout the whole-human interactome, we uncover genome-wide relationships among hotspots and implicate novel cancer proteins that do not harbor hotspot mutations themselves. Moreover, applying our network-based framework to specific cancer types identifies clinically significant hotspots that can be used for prognosis and therapy targets. Overall, we show that our framework bridges the gap between the statistical significance of mutational hotspots and their biological and clinical significance in human cancers.

Functional genomic assays to annotate enhancer-promoter interactions genome wide.

Enhancers are pivotal for regulating gene transcription that occurs at promoters. Identification of the interacting enhancer-promoter pairs and understanding the mechanisms behind how they interact and how enhancers modulate transcription can provide fundamental insight into gene regulatory networks. Recently, advances in high-throughput methods in three major areas-chromosome conformation capture assay, such as Hi-C to study basic chromatin architecture, ectopic reporter experiments such as self-transcribing active regulatory region sequencing (STARR-seq) to quantify promoter and enhancer activity, and endogenous perturbations such as clustered regularly interspaced short palindromic repeat interference (CRISPRi) to identify enhancer-promoter compatibility-have further our knowledge about transcription. In this review, we will discuss the major method developments and key findings from these assays.

A 3D structural SARS-CoV-2-human interactome to explore genetic and drug perturbations.

Emergence of new viral agents is driven by evolution of interactions between viral proteins and host targets. For instance, increased infectivity of SARS-CoV-2 compared to SARS-CoV-1 arose in part through rapid evolution along the interface between the spike protein and its human receptor ACE2, leading to increased binding affinity. To facilitate broader exploration of how pathogen-host interactions might impact transmission and virulence in the ongoing COVID-19 pandemic, we performed state-of-the-art interface prediction followed by molecular docking to construct a three-dimensional structural interactome between SARS-CoV-2 and human. We additionally carried out downstream meta-analyses to investigate enrichment of sequence divergence between SARS-CoV-1 and SARS-CoV-2 or human population variants along viral-human protein-interaction interfaces, predict changes in binding affinity by these mutations/variants and further prioritize drug repurposing candidates predicted to competitively bind human targets. We believe this resource ( http://3D-SARS2.yulab.org ) will aid in development and testing of informed hypotheses for SARS-CoV-2 etiology and treatments.

Progranulin-derived granulin E and lysosome membrane protein CD68 interact to reciprocally regulate their protein homeostasis.

Progranulin (PGRN) is a glycoprotein implicated in several neurodegenerative diseases. It is highly expressed in microglia and macrophages and can be secreted or delivered to the lysosome compartment. PGRN comprises 7.5 granulin repeats and is processed into individual granulin peptides within the lysosome, but the functions of these peptides are largely unknown. Here, we identify CD68, a lysosome membrane protein mainly expressed in hematopoietic cells, as a binding partner of PGRN and PGRN-derived granulin E. Deletion analysis of CD68 showed that this interaction is mediated by the mucin-proline-rich domain of CD68. While CD68 deficiency does not affect the lysosomal localization of PGRN, it results in a specific decrease in the levels of granulin E but no other granulin peptides. On the other hand, the deficiency of PGRN, and its derivative granulin peptides, leads to a significant shift in the molecular weight of CD68, without altering CD68 localization within the cell. Our results support that granulin E and CD68 reciprocally regulate each other's protein homeostasis.

Structure-based validation can drastically underestimate error rate in proteome-wide cross-linking mass spectrometry studies.

Thorough quality assessment of novel interactions identified by proteome-wide cross-linking mass spectrometry (XL-MS) studies is critical. Almost all current XL-MS studies have validated cross-links against known three-dimensional structures of representative protein complexes. Here, we provide theoretical and experimental evidence demonstrating that this approach can drastically underestimate error rates for proteome-wide XL-MS datasets, and propose a comprehensive set of four data-quality metrics to address this issue.

In-depth and 3-dimensional exploration of the budding yeast phosphoproteome.

Phosphorylation is one of the most dynamic and widespread post-translational modifications regulating virtually every aspect of eukaryotic cell biology. Here, we assemble a dataset from 75 independent phosphoproteomic experiments performed in our laboratory using Saccharomyces cerevisiae. We report 30,902 phosphosites identified from cells cultured in a range of DNA damage conditions and/or arrested in distinct cell cycle stages. To generate a comprehensive resource for the budding yeast community, we aggregate our dataset with the Saccharomyces Genome Database and another recently published study, resulting in over 46,000 budding yeast phosphosites. With the goal of enhancing the identification of functional phosphorylation events, we perform computational positioning of phosphorylation sites on available 3D protein structures and systematically identify events predicted to regulate protein complex architecture. Results reveal hundreds of phosphorylation sites mapping to or near protein interaction interfaces, many of which result in steric or electrostatic "clashes" predicted to disrupt the interaction. With the advancement of Cryo-EM and the increasing number of available structures, our approach should help drive the functional and spatial exploration of the phosphoproteome.

Modulating the organic photovoltaic properties of non-fullerene acceptors by molecular modification based on Y6: a theoretical study.

Developing non-fullerene acceptors (NFAs) by modifying the backbone, side chains and end groups is the most important strategy to improve the power conversion efficiency of organic solar cells (OSCs). Among numerous developed NFAs, Y6 and its derivatives are famous NFAs in the OSC field due to their good performance. Herein, in order to understand the mechanism of tuning the photovoltaic performance by modifying the Y6's center backbone, π-spacer and side-chains, we selected the PM6:Y6 OSC as a reference and systematically studied PM6:AQx-2, PM6:Y6-T, PM6:Y6-2T, PM6:Y6-O, PM6:Y6-1O and PM6:Y6-2O OSC systems based on extensive quantum chemistry calculations. The results indicate that introducing quinoxaline to substitute thiadiazole in the backbone induces a blue-shift of absorption spectra, reduces the charge transfer (CT) distance (Δd) and average electrostatic potential (ESP), and increases the singlet-triplet energy gap (ΔEST), CT excitation energy and the number of CT states in low-lying excitations. Inserting thienyl and dithiophenyl as π spacers generates a red-shift of absorption spectra, enlarges Δd and average ESP, and reduces ΔEST and the number of CT states. Introducing furo[3,2-b]furan for substituting one thieno[3,2-b]thiophene unit in the Y6's backbone causes a red-shift of absorption spectra and increases ΔEST, Δd and average ESP as well as CT excitation energy. Introducing alkoxyl as a side chain results in a blue-shift of absorption spectra, and increases ΔEST, Δd, average ESP, CT excitation energy and the number of CT states. The rate constants calculated using Marcus theory suggest that all the molecular modifications of Y6 reduce the exciton dissociation and charge recombination rates at the heterojunction interface, while introducing furo[3,2-b]furan and alkoxyl enlarges CT rates.

Phosphoproteomics reveals distinct modes of Mec1/ATR signaling during DNA replication.

The Mec1/Tel1 kinases (human ATR/ATM) play numerous roles in the DNA replication stress response. Despite the multi-functionality of these kinases, studies of their in vivo action have mostly relied on a few well-established substrates. Here we employed a combined genetic-phosphoproteomic approach to monitor Mec1/Tel1 signaling in a systematic, unbiased, and quantitative manner. Unexpectedly, we find that Mec1 is highly active during normal DNA replication, at levels comparable or higher than Mec1's activation state induced by replication stress. This "replication-correlated" mode of Mec1 action requires the 9-1-1 clamp and the Dna2 lagging-strand factor and is distinguishable from Mec1's action in activating the downstream kinase Rad53. We propose that Mec1/ATR performs key functions during ongoing DNA synthesis that are distinct from their canonical checkpoint role during replication stress.

Handcuffing intrinsically disordered regions in Mlh1-Pms1 disrupts mismatch repair.

The DNA mismatch repair (MMR) factor Mlh1-Pms1 contains long intrinsically disordered regions (IDRs) whose exact functions remain elusive. We performed cross-linking mass spectrometry to identify interactions within Mlh1-Pms1 and used this information to insert FRB and FKBP dimerization domains into their IDRs. Baker's yeast strains bearing these constructs were grown with rapamycin to induce dimerization. A strain containing FRB and FKBP domains in the Mlh1 IDR displayed a complete defect in MMR when grown with rapamycin. but removing rapamycin restored MMR functions. Strains in which FRB was inserted into the IDR of one MLH subunit and FKBP into the other subunit were also MMR defective. The MLH complex containing FRB and FKBP domains in the Mlh1 IDR displayed a rapamycin-dependent defect in Mlh1-Pms1 endonuclease activity. In contrast, linking the Mlh1 and Pms1 IDRs through FRB-FKBP dimerization inappropriately activated Mlh1-Pms1 endonuclease activity. We conclude that dynamic and coordinated rearrangements of the MLH IDRs both positively and negatively regulate how the MLH complex acts in MMR. The application of the FRB-FKBP dimerization system to interrogate in vivo functions of a critical repair complex will be useful for probing IDRs in diverse enzymes and to probe transient loss of MMR on demand.

A massively parallel barcoded sequencing pipeline enables generation of the first ORFeome and interactome map for rice.

Systematic mappings of protein interactome networks have provided invaluable functional information for numerous model organisms. Here we develop PCR-mediated Linkage of barcoded Adapters To nucleic acid Elements for sequencing (PLATE-seq) that serves as a general tool to rapidly sequence thousands of DNA elements. We validate its utility by generating the ORFeome for Oryza sativa covering 2,300 genes and constructing a high-quality protein-protein interactome map consisting of 322 interactions between 289 proteins, expanding the known interactions in rice by roughly 50%. Our work paves the way for high-throughput profiling of protein-protein interactions in a wide range of organisms.

Trimethylation of Lys36 on H3 restricts gene expression change during aging and impacts life span.

Functional data indicate that specific histone modification enzymes can be key to longevity in Caenorhabditis elegans, but the molecular basis of how chromatin structure modulates longevity is not well understood. In this study, we profiled the genome-wide pattern of trimethylation of Lys36 on histone 3 (H3K36me3) in the somatic cells of young and old Caenorhabditis elegans. We revealed a new role of H3K36me3 in maintaining gene expression stability through aging with important consequences on longevity. We found that genes with dramatic expression change during aging are marked with low or even undetectable levels of H3K36me3 in their gene bodies irrespective of their corresponding mRNA abundance. Interestingly, 3' untranslated region (UTR) length strongly correlates with H3K36me3 levels and age-dependent mRNA expression stability. A similar negative correlation between H3K36me3 marking and mRNA expression change during aging was also observed in Drosophila melanogaster, suggesting a conserved mechanism for H3K36me3 in suppressing age-dependent mRNA expression change. Importantly, inactivation of the methyltransferase met-1 resulted in a decrease in global H3K36me3 marks, an increase in mRNA expression change with age, and a shortened life span, suggesting a causative role of the H3K36me3 marking in modulating age-dependent gene expression stability and longevity.

Variants in RABL2A causing male infertility and ciliopathy.

Approximately 7% of men worldwide suffer from infertility, with sperm abnormalities being the most common defect. Though genetic causes are thought to underlie a substantial fraction of idiopathic cases, the actual molecular bases are usually undetermined. Because the consequences of most genetic variants in populations are unknown, this complicates genetic diagnosis even after genome sequencing of patients. Some patients with ciliopathies, including primary ciliary dyskinesia and Bardet-Biedl syndrome, also suffer from infertility because cilia and sperm flagella share several characteristics. Here, we identified two deleterious alleles of RABL2A, a gene essential for normal function of cilia and flagella. Our in silico predictions and in vitro assays suggest that both alleles destabilize the protein. We constructed and analyzed mice homozygous for these two single-nucleotide polymorphisms, Rabl2L119F (rs80006029) and Rabl2V158F (rs200121688), and found that they exhibit ciliopathy-associated disorders including male infertility, early growth retardation, excessive weight gain in adulthood, heterotaxia, pre-axial polydactyly, neural tube defects and hydrocephalus. Our study provides a paradigm for triaging candidate infertility variants in the population for in vivo functional validation, using computational, in vitro and in vivo approaches.

SAAMBE-SEQ: a sequence-based method for predicting mutation effect on protein-protein binding affinity.

MOTIVATION: Vast majority of human genetic disorders are associated with mutations that affect protein-protein interactions by altering wild-type binding affinity. Therefore, it is extremely important to assess the effect of mutations on protein-protein binding free energy to assist the development of therapeutic solutions. Currently, the most popular approaches use structural information to deliver the predictions, which precludes them to be applicable on genome-scale investigations. Indeed, with the progress of genomic sequencing, researchers are frequently dealing with assessing effect of mutations for which there is no structure available. RESULTS: Here, we report a Gradient Boosting Decision Tree machine learning algorithm, the SAAMBE-SEQ, which is completely sequence-based and does not require structural information at all. SAAMBE-SEQ utilizes 80 features representing evolutionary information, sequence-based features and change of physical properties upon mutation at the mutation site. The approach is shown to achieve Pearson correlation coefficient (PCC) of 0.83 in 5-fold cross validation in a benchmarking test against experimentally determined binding free energy change (ΔΔG). Further, a blind test (no-STRUC) is compiled collecting experimental ΔΔG upon mutation for protein complexes for which structure is not available and used to benchmark SAAMBE-SEQ resulting in PCC in the range of 0.37-0.46. The accuracy of SAAMBE-SEQ method is found to be either better or comparable to most advanced structure-based methods. SAAMBE-SEQ is very fast, available as webserver and stand-alone code, and indeed utilizes only sequence information, and thus it is applicable for genome-scale investigations to study the effect of mutations on protein-protein interactions. AVAILABILITY AND IMPLEMENTATION: SAAMBE-SEQ is available at http://compbio.clemson.edu/saambe_webserver/indexSEQ.php#started. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Loss of TMEM106B and PGRN leads to severe lysosomal abnormalities and neurodegeneration in mice.

Haploinsufficiency of progranulin (PGRN) is a leading cause of frontotemporal lobar degeneration (FTLD). Loss of PGRN leads to lysosome dysfunction during aging. TMEM106B, a gene encoding a lysosomal membrane protein, is the main risk factor for FTLD with PGRN haploinsufficiency. But how TMEM106B affects FTLD disease progression remains to be determined. Here, we report that TMEM106B deficiency in mice leads to accumulation of lysosome vacuoles at the distal end of the axon initial segment in motor neurons and the development of FTLD-related pathology during aging. Ablation of both PGRN and TMEM106B in mice results in severe neuronal loss and glial activation in the spinal cord, retina, and brain. Enlarged lysosomes are frequently found in both microglia and astrocytes. Loss of both PGRN and TMEM106B results in an increased accumulation of lysosomal vacuoles in the axon initial segment of motor neurons and enhances the manifestation of FTLD phenotypes with a much earlier onset. These results provide novel insights into the role of TMEM106B in the lysosome, in brain aging, and in FTLD pathogenesis.

MaXLinker: Proteome-wide Cross-link Identifications with High Specificity and Sensitivity.

Protein-protein interactions play a vital role in nearly all cellular functions. Hence, understanding their interaction patterns and three-dimensional structural conformations can provide crucial insights about various biological processes and underlying molecular mechanisms for many disease phenotypes. Cross-linking mass spectrometry (XL-MS) has the unique capability to detect protein-protein interactions at a large scale along with spatial constraints between interaction partners. The inception of MS-cleavable cross-linkers enabled the MS2-MS3 XL-MS acquisition strategy that provides cross-link information from both MS2 and MS3 level. However, the current cross-link search algorithm available for MS2-MS3 strategy follows a "MS2-centric" approach and suffers from a high rate of mis-identified cross-links. We demonstrate the problem using two new quality assessment metrics ["fraction of mis-identifications" (FMI) and "fraction of interprotein cross-links from known interactions" (FKI)]. We then address this problem, by designing a novel "MS3-centric" approach for cross-link identification and implementing it as a search engine named MaXLinker. MaXLinker outperforms the currently popular search engine with a lower mis-identification rate, and higher sensitivity and specificity. Moreover, we performed human proteome-wide cross-linking mass spectrometry using K562 cells. Employing MaXLinker, we identified a comprehensive set of 9319 unique cross-links at 1% false discovery rate, comprising 8051 intraprotein and 1268 interprotein cross-links. Finally, we experimentally validated the quality of a large number of novel interactions identified in our study, providing a conclusive evidence for MaXLinker's robust performance.