Biology subjects

Keegan, S.

Publications and source records attributed to Keegan, S..

18 recordsLinked to original sources

bioRxiv2026

Rethinking the Benign Nature of Class II Lupus Nephritis

Introduction: Class II lupus nephritis (LN) is considered clinically benign, yet up to 50% of patients progress to more severe disease and no molecular characterization exists. We aimed to define the single-cell landscape of Class II LN and identify cellular and transcriptional features associated with kidney outcome. Methods: We performed single-cell RNA sequencing on kidney biopsies from fifteen Class II LN patients (eight de novo, seven regressed from prior proliferative or membranous disease) and six healthy controls, integrated with 155 LN and 30 healthy-control samples from the Accelerating Medicines Partnership in SLE spanning proliferative, membranous, and mixed LN. Findings were correlated with 52-week renal response and supported by urinary proteomics. Results: Despite histologically minimal changes, Class II LN exhibited marked immune cell expansion comparable to proliferative and membranous LN, including CCL3+ CCL4+ intermediate monocytes producing TNF, FOLR2+ SIGLEC1+ macrophages producing TGF{beta} and PDGF, and CD69+ CD8+ effector memory T cells producing IFN{gamma}. Fibroblasts and myofibroblasts were significantly expanded and displayed divergent transcriptional programs: pro-fibrotic and inflammatory myofibroblast modules were associated with worse 52-week outcomes, while interferon-responsive and matrix-remodeling fibroblast programs were associated with improvement. Pathogenic and protective fibroblast populations received overlapping immune-derived signals, suggesting that fibroblast transcriptional state, rather than the identity of incoming signals, shapes the stromal response. Baseline chronicity index captured this stromal heterogeneity and was the clinical parameter most associated with outcome. Urinary proteomics aligned Class II with proliferative rather than membranous disease. Conclusions: Class II LN harbors substantial molecular activity not captured by routine histology. Fibroblast transcriptional programs and baseline chronicity index are candidate correlates of kidney outcome that warrant validation in larger, prospectively followed cohorts.

genomics↗

bioRxiv2026

Dual-inactivation of Regnase-1 and SOCS1 rewires exhausted CD8+ T cell fate to enhance anti-tumor functionality

The solid tumor microenvironment inhibits the functionality of tumor infiltrating T cells recognizing cognate tumor antigen, driving their differentiation towards terminal exhaustion. Interventions are sought to enhance the anti-tumor functionality of tumor-reactive T cells for clinical benefit. The functional genome regulating CD8+ T cell function against solid tumors was mapped by performing genome-wide, focused, and combination in vivo CRISPR/Cas9 screens using OT1 and PMEL TCR transgenic T cells in B16-OVA, MC38-gp100 and EG7-OVA syngeneic tumor models. The ability of the top single hits and combinations, which include Regnase-1 and SOCS1, to enhance CD8+ T cell anti-tumor function was evaluated in the OT1/B16-OVA model with large and established tumors, the disseminated PMEL/B16F10 tumor model, and in a novel murine TIL syngeneic model. The impact of Regnase-1 and SOCS1 single and dual-inactivation on the differentiation of exhausted CD8+ T cell subsets and on long-term persistent memory following tumor clearance was evaluated in OT1 CD8+ T cells in the B16-OVA model. The impact of single and dual-inactivation of Regnase-1 and SOCS1 on the anti-tumor function of experimental human T cell therapeutics was characterized in CRISPR/Cas9-engineered human TIL derived in vitro and in mesothelin-targeting CAR-Ts in vivo. NF-{kappa}B and cytokine signaling were identified as the top pathways regulating CD8+ T cell anti-tumor function, with Regnase-1 and Suppressor of Cytokine Signaling 1 (SOCS1) the top single and combination edits regulating the accumulation of tumor-specific TCR transgenic CD8+ T cells in syngeneic tumor models. Dual-inactivation of Regnase-1 and SOCS1 cooperated through non-redundant mechanisms to strongly expand intermediate (Texint) and effector (Texeff) exhausted CD8+ T cells within lymphoid tissues and tumor, with CD8+ T cells rewired to display an enhanced effector state and suppressed expression of TOX. Dual-edited persistent T effector memory cells (Tem) were formed following tumor clearance. Lastly, Regnase-1 and SOCS1 inactivation enhanced human Tumor Infiltrating Lymphocyte (TIL) and chimeric antigen receptor T cells (CAR-T) therapy functionality. Collectively, this study systematically mapped pathways regulating CD8+ T cell anti-tumor functionality, with Regnase-1 and SOCS1 dual-inactivation found to maximize anti-tumor function through non-redundant mechanisms.

immunology↗

bioRxiv2025

Transcriptional consequences of herpes simplex virus 1 ICP4 inducible expression in uninfected cells

Successful viral infections reflect the balanced outcome of a tightly regulated program of viral gene expression and manipulation of the host cell environment to favor production of new infectious particles. The productive (lytic) replication cycle of herpes simplex virus 1 (HSV-1) is dependent on the essential transcription factor ICP4 encoded by one of five immediate-early genes. At different steps in the HSV-1 temporal cascade, ICP4 either positively or negatively regulates transcription of immediate-early, early, and late HSV-1 genes, largely through sequence-specific binding to cis-acting regulatory elements. In contrast, the direct regulation of host transcription by ICP4 is less well understood. In this study we exogenously expressed doxycycline-inducible wild-type and mutant ICP4 proteins in uninfected primary human fibroblasts and performed RNA-Seq to identify ICP4-driven changes to the host transcriptome. Cross-referencing our findings to a published dataset of ICP4-dependent changes to the host transcriptome in HSV-1 infected cells provided validation for a subset of differentially-expressed genes regulated by ICP4. Furthermore, disrupting the ICP4 DNA-binding domain was sufficient to alter the cellular gene transcriptional program responsive to ICP4. This indicates that the DNA-binding domain of ICP4, which is required for site-specific DNA binding to the virus genome, may also regulate binding to the host genome. Together, these data provide a comprehensive transcriptomic analysis of how wild-type and mutant ICP4 expression impact cellular gene expression in uninfected cells. IMPORTANCEHerpes simplex virus 1 (HSV-1) is a widespread human pathogen responsible for a variety of mild to severe disease states. The viral lifecycle is driven by the essential viral transcription factor ICP4, which ensures the coordinated expression of most viral genes. Expression of ICP4-regulated genes and viral lifecycle progression impacts host gene expression; therefore, determining the specific impact of ICP4 on host gene expression has been challenging. Here, we provide a more direct analysis, examining changes in the transcriptome of primary fibroblasts when ICP4 is expressed alone. Differentially expressed genes were identified and cross-referenced to published data from infections with wild-type and ICP4-null viruses. We show that mutations in ICP4 that disrupt interactions with the host transcriptional machinery or interfere with site-specific DNA binding further modified this ICP4-driven remodeling of the host transcription. These data provide a comprehensive transcriptomic analysis of ICP4-driven dysregulation of host gene expression in uninfected cells.

microbiology↗

bioRxiv2025

Mapping cross-domain drivers of Alzheimer's disease risk through integrated network analysis

IntroductionAlzheimers Disease (AD) is a complex neurodegenerative disorder with numerous known risk factors. Identification of which genetic factors are causal drivers is difficult due to the long disease prodrome in an inaccessible organ. The application of integrative, systems-level approaches are crucial for addressing this complexity. MethodsSixteen biological domain specific interaction networks were derived from the top AD risk- enriched proteins within each domain. Weighted key driver analysis identified influential hub nodes within each network. ResultsDistinct processes and drivers were identified within each domains network. Domains including Structural Stabilization, Endolysosome, and Lipid Metabolism were especially influential. Integrating key drivers across domains identified consistent drivers such as CTNNB1, ACSL1, and ALDH3A2, suggesting fundamental roles contributing to AD risk. DiscussionThis highly integrative network-based approach identified context-dependent drivers and enabled the inference of interactions between domains. The identified drivers suggest potential targets for future therapeutic development.

systems biology↗

bioRxiv2025

Herpes simplex virus-1 fluidizes the nucleus enabling condensate formation

Molecular processes are profoundly influenced by the biophysical properties of the cell interior. However, the mechanisms that control these physical properties, and the processes they impact remain poorly understood, especially in the nucleus. We hypothesized that some viruses might change the biophysical properties of the nucleus to favor virus survival and replication and found that herpes simplex virus 1 (HSV-1) increases the mesoscale fluidity of the nucleus. The HSV-1 protein ICP4 caused fluidization and enabled growth of synthetic nuclear condensates. Conversely, conditions that decreased nuclear fluidity inhibited the growth of viral replication compartment condensates and reduced infectious virus production. Together, our data suggest that ICP4 increases nuclear fluidity to promote the formation of condensates that drive the progression of the HSV-1 life cycle. We speculate that a key function of ICP4 is to overcome the crowding and elastic confinement within cell nuclei that are a fundamental barrier to virus replication.

cell biology↗

bioRxiv2024

An explainable graph neural network approach for integrating multi-omics data with prior knowledge to identify biomarkers from interacting biological domains.

The rapid growth of multi-omics datasets, in addition to the wealth of existing biological prior knowledge, necessitates the development of effective methods for their integration. Such methods are essential for building predictive models and identifying disease-related molecular markers. We propose a framework for supervised integration of multi-omics data with biological priors represented as knowledge graphs. Our framework leverages graph neural networks (GNNs) to model the relationships among features from high-dimensional omics data and set transformers to integrate low-dimensional representations of omics features. Furthermore, our framework incorporates explainability methods to elucidate important biomarkers and extract interaction relationships between biological quantities of interest. We demonstrate the effectiveness of our approach by applying it to Alzheimers disease (AD) multi-omics data from the ROSMAP cohort, showing that the integration of transcriptomics and proteomics data with AD biological domain network priors improves the prediction accuracy of AD status and highlights functional AD biomarkers.

bioinformatics↗

bioRxiv2024

Machine Learning Classification of 53BP1 Foci

Background53BP1 foci are reflective of DNA double-strand break formation and have been used as radiation markers. Manual focus counting, while prone to bias and time constraints, remains the most accurate mode of detecting 53BP1 foci. Several studies have pursued automated focus detection to replace manual methods. Deep learning, spatial 3D images, and segmentation techniques are main components of the highest performing automated methods. While these approaches have achieved promising results regarding accurate focus detection and cell classification, they are not compatible with time-sensitive large-scale applications due to their demand for long run times, advanced microscopy, and computational resources. Further, segmentation of overlapping foci in 2D images has the potential to represent focus morphologies inaccurately. ResultsTo overcome these limitations, we developed a novel method to classify 2D fluorescence microscopy images of 53BP1 foci. Our approach consisted of three key features: (1) general 53BP1 focus classes, (2) varied parameter space composed of properties from individual foci and their respective Fourier transform, and (3) widely-available machine learning classifiers. We identified four main focus classes, which consisted of blurred foci and three levels of overlapping foci. Our parameter space for the training focus library, composed of foci formed by fluorescently-tagged BP1-2, showed a wide correlation range between variables which was validated using a publicly-available library of immunostained 53BP1 foci. Random forest achieved one of the highest and most stable performances for binary and multiclass problems, followed by a support vector machine and k-nearest neighbors. Specific metrics impacted the classification of blurred and low overlap foci for both train and test sets. ConclusionsOur method classified 53BP1 foci across separate fluorescent markers, resolutions, and damage-inducing methods, using off-the-shelf machine learning classifiers, a diverse parameter space, and well-defined focus classes.

bioinformatics↗

bioRxiv2024

D-type cyclins regulate DNA mismatch repair in the G1 and S phases of the cell cycle, maintaining genome stability

The large majority of oxidative DNA lesions occurring in the G1 phase of the cell cycle are repaired by base excision repair (BER) rather than mismatch repair (MMR) to avoid long resections that can lead to genomic instability and cell death. However, the molecular mechanisms dictating pathway choice between MMR and BER have remained unknown. Here, we show that, during G1, D-type cyclins are recruited to sites of oxidative DNA damage in a PCNA- and p21-dependent manner. D-type cyclins shield p21 from its two ubiquitin ligases CRL1SKP2 and CRL4CDT2 in a CDK4/6-independent manner. In turn, p21 competes through its PCNA-interacting protein degron with MMR components for their binding to PCNA. This inhibits MMR while not affecting BER. At the G1/S transition, the CRL4AMBRA1-dependent degradation of D-type cyclins renders p21 susceptible to proteolysis. These timely degradation events allow the proper binding of MMR proteins to PCNA, enabling the repair of DNA replication errors. Persistent expression of cyclin D1 during S-phase increases the mutational burden and promotes microsatellite instability. Thus, the expression of D-type cyclins inhibits MMR in G1, whereas their degradation is necessary for proper MMR function in S. One-Sentence SummaryTo maintain genome stability, D-type cyclins limit mismatch repair (MMR) in G1, whereas their degradation is necessary for proper MMR function in S phase.

cell biology↗

bioRxiv2023

Engineered transcription-associated Cas9 targeting in eukaryotic cells

DNA targeting Class 2 CRISPR-Cas effector nucleases, including the well-studied Cas9 proteins, evolved protospacer-adjacent motif (PAM) and guide RNA interactions that sequentially license their binding and cleavage activities at protospacer target sites. Both interactions are nucleic acid sequence specific but function constitutively; thus, they provide intrinsic spatial control over DNA targeting activities but naturally lack temporal control. Here we show that engineered Cas9 fusion proteins which bind to nascent RNAs near a protospacer can facilitate spatiotemporal coupling between transcription and DNA targeting at that protospacer: Transcription-associated Cas9 Targeting (TraCT). Engineered TraCT is enabled in eukaryotic yeast or human cells when suboptimal PAM interactions limit basal activity and when one or more nascent RNA substrates are still tethered to the actively transcribed target DNA in cis. Using yeast, we further show that this phenomenon can be applied for selective editing at one of two identical targets in distinct gene loci, or, in diploid allelic loci that are differentially transcribed. Our work demonstrates that temporal control over Cas9s targeting activity at specific DNA sites may be engineered without modifying Cas9s core domains and guide RNA components or their expression levels. More broadly, it establishes co-transcriptional RNA binding as a cis-acting mechanism that can conditionally stimulate CRISPR-Cas DNA targeting in eukaryotic cells.

bioengineering↗

bioRxiv2023

Rational design of a SOCS1-edited tumor infiltrating lymphocyte therapy for solid tumors using CRISPR/Cas9 screens

Cell therapies such as Tumor Infiltrating Lymphocyte (TIL) therapy have shown promise in the treatment of patients with refractory solid tumors, with improvement in response rates and durability of responses nevertheless sought. To identify targets capable of enhancing the anti-tumor activity of T cell therapies, large-scale in vitro and in vivo CRISPR/Cas9 screens were performed, with the suppressor of cytokine signaling 1 (SOCS1) gene identified as a top T cell-enhancing target. In murine CD8 T cell therapy models, SOCS1 served as a critical checkpoint in restraining the accumulation of T central memory cells in lymphoid organs as well as intermediate (Texint) and effector (Texeff) exhausted T cell subsets derived from progenitor exhausted T cell (Texprog) cells in tumors. A comprehensive CRISPR tiling screen of the SOCS1 coding region identified sgRNAs targeting the SH2 domain of SOCS1 as the most potent, with a sgRNA with minimal off-target cut sites used to manufacture KSQ-001, an engineered TIL therapy with SOCS1 inactivated by CRISPR/Cas9. KSQ-001 possessed increased responsiveness to cytokine signals and enhanced in vivo anti-tumor function in mouse models. These data demonstrate the use of CRISPR/Cas9 screens in the rational design of T cell therapies.

immunology↗

bioRxiv2023

GEMspa: a Napari plugin for analysis of single particle tracking data

The cellular environment is crowded with macromolecules and far from thermodynamic equilibrium. This active, crowded environment influences biochemical reactions and the formation of cellular structures such as membraneless organelles. These physical properties can change during normal physiology and in disease states such as neurodegenerative diseases and cancer, impacting cell behavior and function. Therefore, it is crucial to develop methods to characterize these properties. Microrheology is the inference of physical properties from the motion of tracer particles embedded within a material. This technique requires single particle tracking (SPT) and analysis of these tracks through the framework of soft-condensed matter physics. Analysis of SPT data can be challenging due to the lack of comprehensive user-friendly software tools. To address this, we introduce GEMspa, a software package implemented as a plugin for the open source image analysis platform, napari. GEMspa provides a GUI for a commonly used localization/tracking algorithm (via Trackpy), and a suite of methods to extract basic parameters describing particle motion. This platform aims to streamline the workflow of data analysis steps and allow researchers to visualize and optimize parameters for high-quality results, thereby making microrheology accessible to non-expert scientists.

biophysics↗

bioRxiv2022

KaryoCreate: a new CRISPR-based technology to generate chromosome-specific aneuploidy by targeting human centromeres

Aneuploidy, the presence of chromosome gains or losses, is a hallmark of cancer and congenital syndromes. Here, we describe KaryoCreate (Karyotype CRISPR Engineered Aneuploidy Technology), a system that enables generation of chromosome-specific aneuploidies by co-expression of a sgRNA targeting chromosome-specific CENPA-binding [a]-satellite repeats together with dCas9 fused to a mutant form of KNL1. We designed unique and highly specific sgRNAs for 19 out of 24 chromosomes. Expression of these sgRNAs with KNL1Mut-dCas9 leads to missegregation and induction of gains or losses of the targeted chromosome in cellular progeny with an average efficiency of 8% and 12% for gains and losses, respectively (up to 20%), tested and validated across 9 chromosomes. Using KaryoCreate in colon epithelial cells, we show that chromosome 18q loss, a frequent occurrence in gastrointestinal cancers, promotes resistance to TGF{beta}, likely due to synergistic hemizygous deletion of multiple genes. Altogether, we describe a novel technology to create and study chromosome missegregation and aneuploidy in the context of cancer and beyond. HighlightsO_LIWe designed sgRNAs targeting chromosome-specific centromeres across 19 human chromosomes C_LIO_LIKaryoCreate combines chromosome-specific centromeric sgRNAs with dCas9 fused to a mutant form of KNL1. C_LIO_LIKaryoCreate allows engineering gains and losses of specific human chromosomes. C_LIO_LIEngineered Chromosome 18q loss promotes tumor-associated phenotypes in colon-derived cells. C_LIO_LIKaryoCreate is a CRISPR-based technology to foster the study of centromere biology and aneuploidy. C_LI

cell biology↗

bioRxiv2021

nucGEMs probe the biophysical properties of the nucleoplasm

The cell interior is highly crowded and far from thermodynamic equilibrium. This environment can dramatically impact molecular motion and assembly, and therefore influence subcellular organization and biochemical reaction rates. These effects depend strongly on length-scale, with the least information available at the important mesoscale (10-100 nanometers), which corresponds to the size of crucial regulatory molecules such as RNA polymerase II. It has been challenging to study the mesoscale physical properties of the nucleoplasm because previous methods were labor-intensive and perturbative. Here, we report nuclear Genetically Encoded Multimeric nanoparticles (nucGEMs). Introduction of a single gene leads to continuous production and assembly of protein-based bright fluorescent nanoparticles of 40 nm diameter. We implemented nucGEMs in budding and fission yeast and in mammalian cell lines. We found differences in particle motility between the nucleus and the cytosol at the mesoscale, that mitotic chromosome condensation ejects nucGEMs from the nucleus, and that nucGEMs are excluded from heterochromatin and the nucleolus. nucGEMs enable hundreds of nuclear rheology experiments per hour, and allow evolutionary comparison of the physical properties of the cytosol and nucleoplasm.

biophysics↗

bioRxiv2021

Nanobody Repertoires for Exposing Vulnerabilities of SARS-CoV-2

Despite the great promise of vaccines, the COVID-19 pandemic is ongoing and future serious outbreaks are highly likely, so that multi-pronged containment strategies will be required for many years. Nanobodies are the smallest naturally occurring single domain antigen binding proteins identified to date, possessing numerous properties advantageous to their production and use. We present a large repertoire of high affinity nanobodies against SARS-CoV-2 Spike protein with excellent kinetic and viral neutralization properties, which can be strongly enhanced with oligomerization. This repertoire samples the epitope landscape of the Spike ectodomain inside and outside the receptor binding domain, recognizing a multitude of distinct epitopes and revealing multiple neutralization targets of pseudoviruses and authentic SARS-CoV-2, including in primary human airway epithelial cells. Combinatorial nanobody mixtures show highly synergistic activities, and are resistant to mutational escape and emerging viral variants of concern. These nanobodies establish an exceptional resource for superior COVID-19 prophylactics and therapeutics.

biochemistry↗

bioRxiv2018

The ULK1-FBXW5-SEC23B nexus controls autophagy

In response to nutrient deprivation, the cell needs to mobilize an extensive amount of membrane to form and grow the autophagosome, allowing the progression of autophagy. By providing membranes and a source for LC3 lipidation, COPII (Coat Protein Complex II) localizes to the endoplasmic reticulum (ER)-Golgi intermediate compartment (ERGIC) and promotes autophagosome biogenesis. However, the molecular mechanisms that, in response to starvation, divert COPII from the secretory pathway to the autophagic pathway are largely unknown. Here, we show that the F-box protein FBXW5 targets SEC23B, a component of COPII, for proteasomal degradation and that this event limits the autophagic flux in the presence of nutrients. In response to starvation, ULK1 phosphorylates SEC23B on Serine 186, preventing the interaction of SEC23B with FBXW5 and, therefore, inhibiting its degradation. Phosphorylated and stabilized SEC23B associates with SEC24A and SEC24B, but not SEC24C and SEC24D, and they re-localize to the ERGIC, promoting autophagic flux. Induction of autophagy and localization of both SEC23B and SEC24B to the ERGIC in response to nutrient deprivation are significantly reduced in SEC23B(S186A) knock-in cells. We propose that, in the presence of nutrients, FBXW5 limits COPII-mediated autophagosome biogenesis. Inhibition of this event by ULK1 ensures efficient execution of the autophagic cascade in response to nutrient starvation.

cell biology↗

bioRxiv2018

Transcription drives DNA replication initiation and termination in human cells

The locations of active DNA replication origins in the human genome, and the determinants of origin activation, remain controversial. Additionally, neither the predominant sites of replication termination nor the impact of transcription on replication-fork mobility have been defined. We demonstrate that replication initiation occurs preferentially in the immediate vicinity of the transcription start site of genes occupied by high levels of RNA polymerase II, ensuring co-directional replication of the most highly transcribed genes. Further, we demonstrate that dormant replication origin firing represents the global activation of pre-existing origins. We also show that DNA replication naturally terminates at the polyadenylation site of transcribed genes. During replication stress, termination is redistributed to gene bodies, generating a global reorientation of replication relative to transcription. Our analysis provides a unified model for the coupling of transcription with replication initiation and termination in human cells.

molecular biology↗

bioRxiv2017

A Method for Quantifying Molecular Interactions Using Stochastic Modelling and Super-Resolution Microscopy

We introduce the Interaction Factor (IF), a measure for quantifying the interaction of molecular clusters in super-resolution microscopy images. The IF is robust in the sense that it is independent of cluster density, and it only depends on the extent of the pair-wise interaction between different types of molecular clusters in the image. The IF for a single or a collection of images is estimated by first using stochastic modelling where the locations of clusters in the images are repeatedly randomized to estimate the distribution of the overlaps between the clusters in the absence of interaction (IF=0). Second, an analytical form of the relationship between IF and the overlap (which has the random overlap as its only parameter) is used to estimate the IF for the experimentally observed overlap. The advantage of IF compared to conventional methods to quantify interaction in microscopy images is that it is insensitive to changing cluster density and is an absolute measure of interaction, making the interpretation of experiments easier. We validate the IF method by using both simulated and experimental data and provide an ImageJ plugin for determining the IF of an image.

bioinformatics↗

bioRxiv2017

A toolbox of immunoprecipitation-grade monoclonal antibodies against human transcription factors.

A key component to overcoming the reproducibility crisis in biomedical research is the development of readily available, rigorously validated and renewable protein affinity reagents. As part of the NIH Protein Capture Reagents Program (PCRP), we have generated a collection of 1406 highly validated, immunoprecipitation (IP) and/or immunoblotting (IB) grade, mouse monoclonal antibodies (mAbs) to 736 human transcription factors. We used HuProt human protein microarrays to identify mAbs that recognize their cognate targets with exceptional specificity. Using an integrated production and validation pipeline, we validated these mAbs in multiple experimental applications, and have distributed them to the Developmental Studies Hybridoma Bank (DSHB) and several commercial suppliers. This study allowed us to perform a meta-analysis that identified critical variables that contribute to the generation of high quality mAbs. We find that using full-length antigens for immunization, in combination with HuProt analysis, provides the highest overall success rates. The efficiencies built into this pipeline ensure substantial cost savings compared to current standard practices.

biochemistry↗