<title>Abstract</title> Cancer shows aberrant alternative splicing (AS), which could functionally perturb proteins required for normal cellular behavior. Transcription factors (TFs) are proteins that regulate gene transcription. Some evidence about AS-driven perturbation of TFs in cancer has been documented. A comprehensive systematic analysis of how cancer-specific AS could affect functions of TFs is missing. Such an analysis could reveal the molecular mechanisms of cancer progression due to transcription misregulations, and thus could help identify therapeutic targets. Here, we systematically analyzed potential functional perturbations of TFs due to AS across 15 cancer types. By analyzing AS patterns, we identified 2 118 perturbed AS events (i.e., events showing significant AS pattern differences between normal and paired cancer samples) that affect 718 TFs across 14 cancer types. In 189 TFs, the perturbed AS events affected known functional domains of TFs. Indirect evidence for the functional impact of cancer-specific AS on TFs were also found: First, by relating AS patterns of the perturbed AS events with the DNA-binding and regulatory activity of TFs. Second, by using cancer dependency data to explore whether the affected TFs are essential for cancer cell line proliferation. Our findings show a large-scale functional perturbation of TFs due to cancer-specific AS. 

Aberrant splicing in human cancer has a large-scale functional impact on transcription factors

Abstract Transcription factors (TFs) bind specific DNA sequences to control gene expression. Modulating TF activity is of considerable therapeutic interest but very few TFs have been successfully drugged. TF DNA-binding interfaces are large and most TFs have no known allosteric ligands or allosteric sites. Here, we introduce TF-MAPs, a scalable and general platform to chart complete functional maps of sequence-specific DNA-binding proteins. We apply TF-MAPS to three human disease TFs: HNF1A, FOXG1 and FOXP1. Allostery in all three proteins is distance-dependent, anisotropic, and couples the DNA-binding interface to the protein surface. The allosteric surfaces of FOXG1 and FOXP1 are quite diverged, revealing a potential for protein-specific regulation in protein families. All three TFs have potentially druggable pockets where mutations have strong functional effects on binding, either within the binding interface or distant from it. Using TF-MAPS it should be possible to chart functional and allosteric maps for hundreds of human TFs and other DNA-binding proteins to understand DNA recognition and TF regulation, to mechanistically interpret clinical variants, and to guide the development of TF-modulating therapeutics. 

TF-MAPS: fast high-resolution functional and allosteric mapping of DNA-binding proteins

TF paralogs—Natural experiments in DNA binding

Biomolecular condensates are membraneless organelles that compartmentalize biological functions in living cells. Formed by the phase separation of biomolecules, condensates possess a wide range of mechanical responses. However, how condensate viscoelastic responses are encoded in the chemistries of their constituents—such as intrinsically disordered proteins (IDPs)—are not well understood. Here, we employ molecular dynamics simulations to connect measurable condensate viscoelasticity to the architectural heterogeneity and dynamic reconfigurability of associative networks formed by IDPs. Using a residue-resolution coarse-grained model, we characterize biologically relevant and synthetic condensates, demonstrating that their temperature sensitivity of elasticity is sequence dependent and modeled by exponential scaling laws. We interrogate condensate mesh heterogeneity via entanglement spacing, finding that entropy-driven structural heterogeneity and reduced IDP hydrophobicity favor condensate elasticity. Furthermore, we construct graph-theoretical representations of condensates and find that interaction network topologies with an abundance of redundant node pathways translates to more load-bearing paths for mechanical stress storage. Strikingly, we discover that elastic coupling of IDPs within condensates emerges when single-molecule shape memory timescales approach meshwork reconfiguration timescales. Akin to a condensate Deborah number, this interplay of timescales for molecular and microstructural processes dictates how restoring elastic forces propagate and are stored across IDP networks; linking condensate microstructure dynamics directly to mechanical responses. Taken together, our work provides a conceptual framework of how condensates act as stress-responsive biomaterials; helping illuminate how cells exploit condensate mechanics to sense and regulate their internal environment and opening avenues for the design of condensates with programmable material properties. SIGNIFICANCE Biomolecular condensates are soft materials that exhibit diverse viscoelastic responses. We show how microstructure dynamics dictate condensate mechanics by probing the heterogeneity of local structures and how their connectivity and persistence store elastic stress. Strikingly, we discover that the ratio of timescales of single-protein shape memory to that of the underlying network rearrangement is a robust indicator of whether a condensate is dominantly viscous or elastic. This finding makes condensate mechanics predictable from sequence-dependent “Deborah numbers,” which are experimentally accessible. Our work therefore provides a tangible framework for understanding condensate stress dissipation mechanisms and opens opportunities for designing condensates with programmable material properties. 

Biomolecular condensate viscoelasticity is dictated by the interplay between single-molecule shape memory and mesh reconfigurability

Abstract Alternative splicing massively expands the human proteome by generating multiple protein isoforms per gene. However, the functional properties of most protein isoforms are unknown, and it remains unclear whether most have distinct or redundant functions. The development of methods to address this question and characterise proteins at scale are needed. We hypothesized that high-content imaging combined with classifier-based analysis could provide a powerful and broadly applicable approach for characterizing subcellular localization and behavioural differences between protein isoforms. As a proof-of-concept, we examine 42 isoform pairs in HeLa cells, each pair consisting of two isoforms from the same gene that differ in the inclusion/exclusion of a tissue-regulated cassette exon, many differing by 10 or fewer amino acids. We identify localisation differences for ∼38% (16/42) of isoform pairs, and features describing these differences. We perform follow-up examination of two sorting nexin 2 (SNX2) isoforms that differ by four amino acids inserted into an α-helix, which drastically altered subcellular localisation. We find the localisation differences are caused by the presence of additional residues rather than their composition, which reduces vesicle association while enhancing interactions with the insulin receptor. Lastly, we generalise these findings by showing that microexons frequently overlap α-helices, are enriched in helix-stabilizing residues, have lengths that preserve helical register, and often modulate surface charge. Together, our findings support a broad role for alternative splicing in modulating protein subcellular localization and increasing functional proteomic diversity. 

High-content imaging reveals microexons’ ability to shape protein localisation

Nuclear Phase Separation Directs HOX Expression in Acute Myeloid Leukemia

The development of targeted therapies against drivers of malignancy requires a robust mechanistic understanding of the pathogenic axis underlying disease. Several subtypes of acute myeloid leukemias (AML), such as those driven by mutations in IDH1/2, FLT3, and KMT2A, have benefitted from the development of novel therapeutics that disrupt their transcriptional program. In contrast, there are no approved therapies that target the mutant version of Nucleophosmin (NPM1), which is the most common driver in adult AML. Mutations in NPM1 result in export of the mutant protein (NPM1c) to the cytoplasm, prompting the suggestion that aberrant localization leads to neomorphic and pathogenic functions of NPM1c in the cytoplasm. In parallel, recent data from several groups has suggested that NPM1c directly regulates gene expression within the nucleus. Despite this progress, a well-defined mechanism for NPM1c-mediated gene regulation is lacking, hindering the development of NPM1c-targeted therapies. To investigate the function of NPM1c, we generated over a dozen AML cell lines expressing unique NPM1c truncations and characterized their impact on the leukemic transcriptome and cell morphology. To our surprise, we found that although the majority of these truncated proteins reside in the cytoplasm, most were unable to recapitulate the transcriptional program driven by full-length NPM1c, and cells subsequently underwent differentiation and growth arrest. We further explored the chromatin binding ability of these truncated proteins to key genomic regions such as the HOXA cluster and MEIS1 through CUT&amp;RUN analysis. Next, we identified a region of the NPM1c protein critical for active nuclear import (NLS). Strikingly, disruption of this region enforces cytoplasmic sequestration of the truncated NPM1c protein. However, cells harboring this truncation fail to maintain leukemic gene expression and differentiate. Together these results suggest that aberrant localization and/or shuttling of NPM1c between cellular compartments is insufficient to maintain leukemic gene expression and cell-stemness. We next considered whether NPM1c had a de-novo function that may explain its role in gene regulation. We have recently shown that NPM1c forms phase-separated condensates in the nucleus that regulate HOX gene expression and are necessary for stem-ness. These NPM1c condensates recruit multiple gene regulatory factors including XPO1, NUP98, and KMT2A (Figure 1). Through selective degradation of NPM1c, or pharmacological inhibition of XPO1, we found that disruption of nuclear condensates abrogates recruitment of these protein co-factors. We further explore the effect of drug compounds known to inhibit NPM1-mutant AML on condensate behavior and chromatin binding. This condensate behavior is a defining feature of heterotypic phase separation, a process in which two or more biomolecules form multivalent interactions to create a condensate. Importantly, we found that significant overexpression of NPM1c causes an imbalance in the ratio between NPM1c and its binding partners, disrupting normal condensate formation, and causing subsequent differentiation and growth arrest. Collectively, these results suggest that therapeutic targeting of NPM1c, or a host of interacting partners, may disrupt condensate formation and the leukemic transcriptional program, providing an exciting new therapeutic vulnerability in NPM1-mutant AML. 

Nuclear Condensates Are a Therapeutic Vulnerability in NPM1-Mutant Acute Myeloid Leukemia

BPS2025 - Illuminating nanometer internal organization within condensates in live cells

Nanometer condensate organization in live cells derived from partitioning measurements

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

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