Biology subjects

Spirohn-Fitzgerald, K.

Publications and source records attributed to Spirohn-Fitzgerald, K..

6 recordsLinked to original sources

bioRxiv2026

Dynamic engagement of dual-role regulators by the Sin3 complex

Gene expression is governed by dynamic switches between repressive and activating transcriptional states1,2. Among the molecules mediating these transitions, chromatin readers and transcription factors play pivotal roles3,4. However, how they assemble with regulatory machineries to enable crosstalk between gene repression and activation remains unknown. Here, we use an integrative structural dynamics approach - combining cryo-EM, crosslinking mass spectrometry, fragment-resolved protein interactome mapping and crystallography - to show how the dual-role chromatin reader Cti6 and transcription factors Ash1 and Ume6 engage the Sin3 deacetylase complex, a major regulatory hub in eukaryotes5. We find that Cti6 competes with Ash1 to drive its dynamic recruitment to a shared peripheral module, while Ume6 engages the Sin3 scaffold through a defined, minimal interface. Using high-throughput mutational scanning, we reveal deleterious and gain-of-function mutations in Sin3, identifying evolutionarily conserved residues essential for anchoring transcription factors. Together, these results provide structural and functional insights into how dual-role regulators engage the central Sin3 complex, revealing subtle assembly principles that may facilitate crosstalk between gene repression and activation. They also establish an integrative multidisciplinary framework to dissect the dynamics of macromolecular assemblies across biological systems.

molecular biology↗

bioRxiv2026

A scalable approach to resolving variants of uncertain significance

Over 90% of missense variants across [~]4,000 disease-associated genes are variants of uncertain significance (VUS). Experimental variant effect measurements provide critical evidence about pathogenicity and inform disease biology, but most variants lack data and clinical translation has been limited. The Impact of Genomic Variation on Function Consortium generated experimental data for 62,215 variants across ten genes using multiplexed assays and 1,407 variants across 163 genes using arrayed assays, curated 193,139 additional community-generated variant effect measurements across 30 additional genes, and developed automated calibration methods for translating experimental data and variant effect predictions into clinical evidence. To reduce current VUS, we developed a scalable workflow using only experimental and predictive evidence, enabling reclassification of 75% of the 16,115 VUS in these genes as pathogenic or benign with <1% error. To minimize future VUS, we analyzed >90,000 unobserved variants; 62% had enough evidence to be "preclassified" as pathogenic or benign. We validated our data, evidence and classifications using All of Us and created interactive resources to enable clinical use of the calibrated data. Thus, for 40 genes, representing 1% of the clinical genome, we resolve most existing VUS and future variants, illustrating how systematic use of scalable evidence can empower genomic medicine.

genomics↗

bioRxiv2025

Predicting interaction-specific protein-protein interaction perturbations by missense variants with MutPred-PPI

Disruption of protein-protein interactions (PPIs) is a major mechanism of a variants deleterious effect. Computational tools are needed to assess such variants at scale, yet existing predictors rarely consider loss of specific interactions, particularly when variants perturb binding interfaces without significantly affecting protein stability. To address this problem, we present MutPred-PPI, a graph attention network that predicts interaction-specific (edgetic) effects of missense variants by operating on AlphaFold 3-based protein complex contact graphs with protein language model embeddings imposed upon nodes. We systematically evaluated our model with stringent group cross-validation as well as benchmark data recently collected within the IGVF Consortium. MutPred-PPI outperformed all baseline methods across all evaluation criteria, achieving an AUC of 0.85 on seen proteins and 0.72 on previously unseen proteins in cross-validation, demonstrating strong generalizability despite scarce training data. To demonstrate biomedical relevance, we applied MutPred-PPI to variants from ClinVar, HGMD, COSMIC, gnomAD, and two de novo neurodevelopmental disorder-linked datasets. Disease-associated variants from Clin-Var and HGMD showed strong enrichment for both quasi-null and edgetic effects, whereas population variants from gnomAD increasingly preserved interactions with higher allele frequencies. Notably, we observed a strong edgetic disruption signature in highly recurrent cancer variants from both the full COSMIC dataset and a subset of variants from oncogenes. Recurrent tumor suppressor gene variants and autism spectrum disorder-associated variants exhibited moderate quasi-null enrichment, whilst neurodevelopmental disorder-linked variants showed a weak edgetic disruption signature. These results indicate distinct PPI perturbation mechanisms across disease types and show that MutPred-PPI captures functionally relevant molecular effects of pathogenic variants.

bioinformatics↗

bioRxiv2025

Viral transcriptional regulators extensively rewire host pathways through diverse mechanisms

Viral transcriptional regulators (vTRs) reprogram host gene regulatory networks to promote replication, persistence, and immune evasion. Despite the identification of hundreds of vTRs in human viruses, how they rewire host pathways remains unclear. Here, we systematically profiled 95 vTRs from diverse human viruses across multiple functional assays. vTRs perturb immune, cell proliferation/death, and signaling pathways through various mechanisms; some bind DNA directly, others cooperate or antagonize human transcription factors (hTFs), and some remodel chromatin. vTRs can act as activators or repressors and recruit similar but not identical repertoires of proteins as hTFs. These findings reveal vTRs as versatile transcriptional modulators that converge on conserved host "pressure points" while diversifying across pathways to promote viral replication and persistence. Notably, many vTR dysregulate genes within autoimmune, neurological, and cardiovascular risk loci, revealing mechanistic links to disease. Together, we provide a comprehensive resource for understanding and targeting viral control of human transcription.

genomics↗

bioRxiv2024

A comprehensive two-hybrid analysis to explore the L. pneumophila effector-effector interactome

Legionella pneumophila uses over 300 translocated effector proteins to rewire host cells during infection and create a replicative niche for intracellular growth. To date, several studies have identified L. pneumophila effectors that indirectly and directly regulate the activity of other effectors, providing an additional layer of regulatory complexity. Amongst these are "metaeffectors" - a special class of effectors that regulate the activity of other effectors once inside the host. A defining feature of metaeffectors is direct, physical interaction with a target effector. Metaeffector identification to date has depended on phenotypes in heterologous systems and experimental serendipity. Using a multiplexed, recombinant-barcode-based yeast two-hybrid technology we screened for protein-protein interactions amongst all L. pneumophila effectors and several components of the Dot/Icm type IV secretion system (>167,000 protein combinations). Of the 52 protein interactions identified by this approach, 44 are novel protein interactions, including ten novel effector-effector interactions (doubling the number of known effector-effector interactions).

microbiology↗

bioRxiv2024

Widespread variation in molecular interactions and regulatory properties among transcription factor isoforms

Most human Transcription factors (TFs) genes encode multiple protein isoforms differing in DNA binding domains, effector domains, or other protein regions. The global extent to which this results in functional differences between isoforms remains unknown. Here, we systematically compared 693 isoforms of 246 TF genes, assessing DNA binding, protein binding, transcriptional activation, subcellular localization, and condensate formation. Relative to reference isoforms, two-thirds of alternative TF isoforms exhibit differences in one or more molecular activities, which often could not be predicted from sequence. We observed two primary categories of alternative TF isoforms: "rewirers" and "negative regulators", both of which were associated with differentiation and cancer. Our results support a model wherein the relative expression levels of, and interactions involving, TF isoforms add an understudied layer of complexity to gene regulatory networks, demonstrating the importance of isoform-aware characterization of TF functions and providing a rich resource for further studies.

systems biology↗