Biology subjects

Major, M.

Publications and source records attributed to Major, M..

10 recordsLinked to original sources

bioRxiv2026

Pioneer and Altimeter: Fast Analysis of DIA Proteomics Data Optimized for Narrow Isolation Windows

Advances in protein mass spectrometry have enabled a single instrument to acquire data for hundreds of samples per day, but the speed of analysis software has not kept pace. Here we introduce Pioneer and Altimeter for rapid analysis of proteomics data acquired by data-independent acquisition (DIA). Altimeter generates in silico spectral libraries, which Pioneer uses to identify and quantify peptides and proteins. Pioneer exceeds peptide coverage by up to 10%, improves quantitative accuracy, and runs four to eight times faster compared with state-of-the-art tools. Pioneer and Altimeter also model isolation-window effects on fragment isotopes to take advantage of narrow isolation windows deployed on fast-scanning time-of-flight instruments. We benchmarked Pioneer and validated its false-discovery-rate and false-transfer-rate control across diverse instruments, sample types, and acquisition settings. Altimeter and Pioneer are open source and cross-platform, with a graphical interface for streamlined analysis on personal computers and a command-line interface for compute-cluster workflows.

bioinformatics↗

bioRxiv2025

Distinct Neurogenic Dynamics of Cortico-cortical Neuronal Subtypes in Layer 2/3 of the Mouse Visual Cortex

In the mammalian cerebral cortex, the birthdates of excitatory projection neurons are closely linked to their laminar positions, which are often associated with distinct long-range projection targets. Although broad relationships between neurogenic timing, laminar position, and projection patterns are well established, the degree to which birthdate specifies projection identity within the same cortical layer remains unclear. In the mouse primary visual cortex (V1), neurons projecting to lateral higher visual areas are relatively evenly distributed throughout layer 2/3, whereas those projecting to medial areas are biased toward more superficial sublayers. To determine whether these projection identities are linked to neurogenic timing, we combined EdU birthdating with retrograde viral tracing. Notably, we found that V1 layer 2/3 neurons projecting to lateral higher visual areas are preferentially born at embryonic day 15.5 (E15.5) compared to E16.5, whereas V1 neurons projecting to medial higher visual areas show no significant birthdate bias between E15.5 and E16.5. These findings suggest that distinct cortico-cortical projection subtypes in layer 2/3 are generated on different temporal schedules, linking neurogenic timing to fine-scale projection identity.

neuroscience↗

bioRxiv2024

Proximity proteomics provides a new resource for exploring the function of Afadin and the complexity of cell-cell adherens junctions

The network of proteins at the interface between cell-cell adherens junctions and the actomyosin cytoskeleton provides robust yet dynamic connections that facilitate cell shape change and motility. While this was initially thought to be a simple linear connection via classic cadherins and their associated catenins, we now have come to appreciate that many more proteins are involved, providing robustness and mechanosensitivity. Defining the full network of proteins in this network remains a key objective in our field. Proximity proteomics provides a means to define these networks. Mammalian Afadin and its Drosophila homolog Canoe are key parts of this protein network, facilitating diverse cell shape changes during gastrulation and other events of embryonic morphogenesis. Here we report results of several proximity proteomics screens, defining proteins in the neighborhood of both the N- and C-termini of mammalian Afadin in the premier epithelial model, MDCK cells. We compare our results with previous screens done in other cell types, and with proximity proteomics efforts with other junctional proteins. These reveal the value of multiple screens in defining the full network of neighbors and offer interesting insights into the overlap in protein composition between different epithelial cell junctions. Summary StatementAfadin BioID reveals new adherens junction proteins.

cell biology↗

bioRxiv2024

The small MAF transcription factor MAFG co-opts MITF to promote melanoma progression

Transcription factor deregulation potently drives melanoma progression by dynamically and reversibly controlling gene expression programs. We previously identified the small MAF family transcription factor MAFG as a putative driver of melanoma progression, prompting an in-depth evaluation of its role in melanoma. MAFG expression increases with human melanoma stages and ectopic MAFG expression enhances the malignant behavior of human melanoma cells in vitro, xenograft models, and genetic mouse models of spontaneous melanoma. Moreover, MAFG induces a melanoma phenotype switch from a melanocytic state to a more dedifferentiated state. Mechanistically, MAFG interacts with the lineage transcription factor MITF which is required for the pro-tumorigenic effects of MAFG. MAFG and MITF co-occupy numerous genomic sites and MAFG overexpression influences the expression of genes harboring binding sites for the MAFG[~]MITF complex. These results establish MAFG as a potent driver of melanomagenesis through dimerization with MITF and uncover an unappreciated mechanism of MITF regulation. Significance statementMITF is critically involved in melanoma progression and phenotype switching. We discovered that MAFG interacts with MITF to influence expression of MITF target genes and facilitate a shift toward a dedifferentiated melanoma cell state. This study demonstrates that MAFG promotes melanomagenesis by influencing MITF activity, an unappreciated mechanism of MITF regulation.

cancer biology↗

bioRxiv2022

Small Molecule Screen Identifies Pyrimethamine as an Inhibitor of NRF2-driven Esophageal Hyperplasia

ObjectiveNRF2 is a master transcription factor that regulates the stress response. NRF2 is frequently mutated and activated in human esophageal squamous cell carcinoma (ESCC), which drives resistance to chemotherapy and radiation therapy. Therefore, a great need exists for NRF2 inhibitors for targeted therapy of NRF2high ESCC. DesignWe performed high-throughput screening of two compound libraries from which hit compounds were further validated in human ESCC cells and a genetically modified mouse model. The mechanism of action of one compound was explored by biochemical assays. ResultsUsing high-throughput screening of two small molecule compound libraries, we identified 11 hit compounds as potential NRF2 inhibitors with minimal cytotoxicity at specified concentrations. We then validated two of these compounds, pyrimethamine and mitoxantrone, by demonstrating their dose- and time-dependent inhibitory effects on the expression of NRF2 and its target genes in two NRF2Mut human ESCC cells (KYSE70 and KYSE180). RNAseq and qPCR confirmed the suppression of global NRF2 signaling by these two compounds. Mechanistically, pyrimethamine reduced NRF2 half-life by promoting NRF2 ubiquitination and degradation in KYSE70 and KYSE180 cells. Expression of an Nrf2E79Q allele in mouse esophageal epithelium (Sox2CreER;LSL-Nrf2E79Q/+) resulted in an NRF2high phenotype, which included squamous hyperplasia, hyperkeratinization, and hyperactive glycolysis. Treatment with pyrimethamine (30mg/kg/day, p.o.) suppressed the NRF2high esophageal phenotype with no observed toxicity. ConclusionWe have identified and validated pyrimethamine as an NRF2 inhibitor that may be rapidly tested in the clinic as a radiation and chemotherapy sensitizer for NRF2high ESCC. SummaryO_ST_ABSWhat is already known on this topic - summarise the state of scientific knowledge on this subject before you did your study and why this study needed to be doneC_ST_ABSO_LIMutational activation of the NRF2 transcription factor drives ESCC progression and therapeutic resistance. Targeted therapies to block NRF2 have not yet been realized, despite great needs. C_LI What this study adds - summarise what we now know as a result of this study that we did not know beforeO_LIA screen of >35,000 small molecules identified eleven potential NRF2 inhibitors. Pyrimethamine and mitoxantrone were validated to inhibit the expression of NRF2 and NQO1 in human ESCC cells in both dose- and time-dependent manners. C_LIO_LIPyrimethamine enhanced NRF2 protein ubiquitination and degradation, resulting a decreased half-life. C_LIO_LIA genetically modified mouse model was established to express the Nrf2E79Q mutant allele in the mouse esophageal epithelium upon tamoxifen induction. Pyrimethamine suppressed the NRF2high esophageal phenotype induced by the mutant allele. C_LI How this study might affect research, practice or policy - summarise the implications of this studyO_LIAs an FDA-approved drug, Pyrimethamine has the potential for immediate translation to a clinical trial on NRF2high ESCC in humans. C_LIO_LIFurther exploration of its mechanisms of action may lead to more potent NRF2 inhibitors for future use. C_LI

cancer biology↗

bioRxiv2020

The TRIM9/TRIM67 neuronal interactome reveals novel activators of morphogenesis

TRIM9 and TRIM67 are neuronally-enriched E3 ubiquitin ligases essential for appropriate morphogenesis of cortical and hippocampal neurons and fidelitous responses to the axon guidance cue netrin-1. Deletion of murine Trim9 or Trim67 results in neuroanatomical defects and striking behavioral deficits, particularly in spatial learning and memory. TRIM9 and TRIM67 interact with cytoskeletal and exocytic proteins, but the full interactome is not known. Here we performed the unbiased proximity-dependent biotin identification (BioID) approach to define TRIM9 and TRIM67 protein-protein proximity network in developing cortical neurons and identified neuronal putative TRIM interaction partners. Candidates included cytoskeletal regulators, cytosolic protein transporters, exocytosis and endocytosis regulators, and proteins necessary for synaptic regulation. A subset of high priority candidates was validated, including Myo16, Coro1A, SNAP47, ExoC1, GRIP1, PRG-1, and KIF1A. For a subset of validated candidates, we utilized TIRF microscopy to demonstrate dynamic colocalization with TRIM proteins at the axonal periphery, including at the tips of filopodia. Further analysis demonstrated the RNAi-based knockdown of the unconventional myosin Myo16 in cortical neurons altered axonal branching patterns in a TRIM9 and netrin-1 dependent manner. Future analysis of other validated candidates will likely identify novel proteins and mechanisms by which TRIM9 and TRIM67 regulate neuronal form and function.

cell biology↗

bioRxiv2020

Leveraging expression from multiple tissues using sparse canonical correlation analysis (sCCA) and aggregate tests improves the power of transcriptome-wide association studies (TWAS)

Transcriptome-wide association studies (TWAS) test the association between traits and genetically predicted gene expression levels. The power of a TWAS depends in part on the strength of the correlation between a genetic predictor of gene expression and the causally relevant gene expression values. Consequently, TWAS power can be low when expression quantitative trait locus (eQTL) data used to train the genetic predictors have small sample sizes, or when data from causally relevant tissues are not available. Here, we propose to address these issues by integrating multiple tissues in the TWAS using sparse canonical correlation analysis (sCCA). We show that sCCA-TWAS combined with single-tissue TWAS using an aggregate Cauchy association test (ACAT) outperforms traditional single-tissue TWAS. In empirically motivated simulations, the sCCA+ACAT approach yielded the highest power to detect a gene associated with phenotype, even when expression in the causal tissue was not directly measured, while controlling the Type I error when there is no association between gene expression and phenotype. For example, when gene expression explains 2% of the variability in outcome, and the GWAS sample size is 20,000, the average power difference between the ACAT combined test of sCCA features and single-tissue, versus single-tissue combined with Generalized Berk-Jones (GBJ) method, single-tissue combined with S-MultiXcan or summarizing cross-tissue expression patterns using Principal Component Analysis (PCA) approaches was 5%, 8%, and 38%, respectively. The gain in power is likely due to sCCA cross-tissue features being more likely to be detectably heritable. When applied to publicly available summary statistics from 10 complex traits, the sCCA+ACAT test was able to increase the number of testable genes and identify on average an additional 400 additional gene-trait associations that single-trait TWAS missed. Our results suggest that aggregating eQTL data across multiple tissues using sCCA can improve the sensitivity of TWAS while controlling for the false positive rate.Author summary Transcriptome-wide association studies (TWAS) can improve the statistical power of genetic association studies by leveraging the relationship between genetically predicted transcript expression levels and an outcome. We propose a new TWAS pipeline that integrates data on the genetic regulation of expression levels across multiple tissues. We generate cross-tissue expression features using sparse canonical correlation analysis and then combine evidence for expression-outcome association across cross- and single-tissue features using the aggregate Cauchy association test. We show that this approach has substantially higher power than traditional single-tissue TWAS methods. Application of these methods to publicly available summary statistics for ten complex traits also identifies associations missed by single-tissue methods.View Full Text

genetics↗

bioRxiv2020

PKIS Deep Dive Yields a Chemical Starting Point for Dark Kinases and a Cell Active BRSK2 Inhibitor

The Published Kinase Inhibitor Set (PKIS) is a publicly-available chemogenomic library distributed to more than 300 laboratories by GlaxoSmithKline (GSK) between 2011–2015 and by SGC-UNC from 2015–2017. Screening this library of well-annotated, published kinase inhibitors has yielded a plethora of data in diverse therapeutic and scientific areas, funded applications, publications, and provided impactful pre-clinical results. Based on kinome-wide screening results, we report a thorough investigation of one PKIS compound, GW296115, as an inhibitor of several members of the Illuminating the Druggable Genome (IDG) list of understudied dark kinases. Specifically, GW296115 validates as a potent lead chemical tool that inhibits six IDG kinases with IC50 values less than 100nM. Focused studies establish that GW296115 is cell active, and directly engages BRSK2. Further evaluation showed that GW296115 downregulates BRSK2-driven phosphorylation and downstream signaling.Summary Statement GW296115 inhibits understudied kinases, including BRSK2, with IC50 values less than 100nM.Competing Interest StatementThe authors have declared no competing interest.View Full Text

pharmacology and toxicology↗

bioRxiv2019

Wnt Regulation: Exploring Axin-Disheveled interactions and defining mechanisms by which the SCF E3 ubiquitin ligase is recruited to the destruction complex

Wnt signaling plays key roles in embryonic development and adult stem cell homeostasis, and is altered in human cancer. Signaling is turned on and off by regulating stability of the effector {beta}-catenin. The multiprotein destruction complex binds and phosphorylates {beta}-catenin, and transfers it to the SCF-TrCP E3-ubiquitin ligase, for ubiquitination and destruction. Wnt signals act though Dishevelled to turn down the destruction complex, stabilizing {beta}-catenin. Recent work clarified underlying mechanisms, but important questions remain. We explore {beta}-catenin transfer from the destruction complex to the E3 ligase, and test models suggesting Dishevelled and APC2 compete for association with Axin. We find that Slimb/TrCP is a dynamic component of the destruction complex biomolecular condensate, while other E3 proteins are not. Recruitment requires Axin and not APC, and Axins RGS domain plays an important role. We find that elevating Dishevelled levels in Drosophila embryos has paradoxical effects, promoting the ability of limiting levels of Axin to turn off Wnt signaling. When we elevate Dishevelled levels, it forms its own cytoplasmic puncta, but these do not recruit Axin. SIM imaging in mammalian cells suggests that this may result by promoting Dishevelled: Dishevelled interactions at the expense of Dishevelled:Axin interactions when Dishevelled levels are high.

cell biology↗

bioRxiv2018

Integrative analysis of Dupuytren’s disease identifies novel risk locus and reveals a shared genetic etiology with BMI

Dupuytrens disease is the common inherited tissue-specific fibrotic disorder. Its characterized by progressive and irreversible fibroblastic proliferation affecting the palmar fascia of the hand, with an onset typically in the sixth decade of life. Although genome-wide association studies (GWAS) have identified 24 genomic regions associated with Dupuytrens risk, the biological mechanisms driving signal at these regions remain elusive. We identify potential biological mechanisms for Dupuytrens disease by integrating the most recent, largest GWAS (ncases = 3,871, ncontrols = 4,686) with eQTLs (47 tissue panels from five consortia, total n = 3,975) to perform a transcriptome-wide association study (TWAS). We identify 43 tissue-specific gene associations with Dupuytrens risk, one of which resides at least 0.5 Mb away from the 24 risk regions previously identified. We also estimate the genome-wide genetic correlation between Dupuytrens disease and 45 complex traits and find significant genetic correlations between Dupuytrens disease and body mass index ([Formula], P = 1.6 x 10-6), type II diabetes ([Formula], P = 1.7 x 10-4), triglycerides ([Formula], P = 3.5 x 10-4), and high-density lipoprotein ([Formula], P = 4.1 x 10-4), which suggests a shared genetic etiology. We further refine the genome-wide genetic correlation signal to identify 8 regions significantly negatively correlated with BMI and 3 regions significantly correlated (1 positively and 2 negatively correlated) with HDL; none of these regions contained the novel gene association identified by TWAS. Our results are consistent with previous epidemiological findings which show that lower BMI increases risk for Dupuytrens disease. These 12 novel risk regions provide new insight into the biological mechanisms of Dupuytrens disease and serve as a starting point for functional validation.

genetics↗