Biology subjects

Misteli, T.

Publications and source records attributed to Misteli, T..

At least 19 recordsLinked to original sources

bioRxiv2026

Attenuating LRRK2 activity ameliorates progerin-induced aging phenotypes in HGPS models and during physiological aging

Hutchinson-Gilford progeria syndrome (HGPS) is an ultra-rare premature aging disorder caused by progerin, a truncated lamin A variant generated by a silent de novo mutation activating a cryptic splice site in LMNA. The resulting morphological, epigenetic, genomic, and proteostasic defects closely recapitulate some hallmarks of cellular aging. Here, we identify the Parkinsons disease-associated kinase LRRK2 as a critical regulator of HGPS pathology and physiological aging. Rab29-mediated LRRK2 hyperactivation exacerbates progerin-induced cellular aging, whereas LRRK2 knockdown or overexpression of its opposing phosphatase, PPM1H, ameliorates progerin-induced defects. Progerin-expressing cells exhibit altered intracellular trafficking, which is regulated by LRRK2 and links diverse aging hallmarks. Consistent with these findings, reducing LRRK2 levels mitigates cellular aging phenotypes in physiologically aged cells, and loss of the C. elegans ortholog lrk-1 preserves aging-associated loss of motility and extends organismal lifespan. Together, our findings establish LRRK2 as a central node in cellular aging and position it as a potential therapeutic target for aging-related defects in both HGPS and physiological aging.

cell biology↗

bioRxiv2026

Re-establishment of TAD boundary organization during DNA replication

A ubiquitous feature of higher order genome organization is the presence of topologically associating domains (TADs). The chromatin architectural proteins cohesin and CTCF are known critical organizers of TADs, but the mechanisms of TAD establishment and maintenance, including their accurate duplication during genome replication, are not well characterized. To address this gap, we used high-throughput imaging-based CRISPR/Cas9 knock-out screening to discover chromatin factors involved in maintenance and establishment of TADs. Among the cellular factors that affect TAD organization, we found enrichment for cell cycle proteins, especially components of the DNA replication machinery. Accordingly, we demonstrate that TADs undergo temporary unfolding during S-phase DNA replication and that interference with progression through replication impedes restoration of normal TAD structure. Mechanistically, inhibition of the RPA complex prevents cohesin and CTCF binding, delays post-replication TAD re-folding and affects TAD folding in non-cycling cells. These results provide novel insights into how TAD structures are re-established during genome duplication.

cell biology↗

bioRxiv2026

Connecting multiway enhancer-promoter interactions to changes in gene expression in cancer

Hi-C, and more recently Micro-C, experiments suggest that genome organization is altered as normal cells become cancerous, often resulting in aberrant gene expression. In prostate cancer, there is evidence that large topologically associating domains in normal cells split and are accompanied by shifts in the epigenetic marks from inactive to active states. However, it is unclear whether changes in genome organization alone can account for the gene expression increase in cancer cells. By combining polymer physics concepts and data-driven modeling, we first calculated an ensemble of three-dimensional (3D) chromatin structures using only the 2D contact map as input. The enhancer-promoter (E-P) distance distributions for the overexpressed Androgen Receptor (AR) and Forkhead Box Protein A1 (FOXA1) are broad, with mean values exceeding the threshold for direct E-P contact. Importantly, the average E-P distances decrease in the cancer cells, compared to normal cells in the AR locus, whereas in FOXA1 they are roughly constant or increase modestly. Similarly, the number of multiway contacts increases as cancer progresses across cancer cell lines in the AR locus. In contrast, the number of multiway contacts in FOXA1 is similar in normal and cancer cells. Because the 3D characteristics do not explain the enhanced gene expression in cancer cells, we developed Activity-by-Multiway-Contact (AMC) Model that integrates the multiway contacts with enhancer biochemical activity. The AMC model provides a plausible mechanism for the overexpression of both AR and FOXA1 in prostate cancer. Moreover, using Micro-C data for breast cancer, we show that the number of multiway enhancer-promoter contacts increases in four of five genes studied. When multiway contacts are combined with biochemical activity, changes in gene expression found in the experiment, positively correlate with the AMC score. The predictions of the AMC model not only account for overexpression of genes in prostate and breast cancer but also provide a basis for understanding gene expression variations in other genes as well.

genomics↗

bioRxiv2026

Acute degron-mediated RUNX1 loss reprograms enhancer activity to epigenetically drive epithelial destabilization and initiate cancer hallmarks

The RUNX1 transcription factor mediates cell-type specific gene expression. RUNX1 suppression and perturbations are recurrently associated with breast tumor initiation and progression. However, the mechanisms governing the dual roles of RUNX1 in sustaining the mammary epithelial phenotype while epigenetically suppressing initiation of cancer-compromised gene expression are poorly understood. To address this, we used the power of degron-mediated acute, selective, and complete RUNX1 ablation in human mammary epithelial cells. RUNX1 mediates promoter and distal enhancer-driven expression of a gene cohort. Dynamic epigenomic responsiveness upon RUNX1 ablation reveals a rapid and selective decrease in chromatin accessibility and H3K27ac at RUNX1-bound enhancers, but not promoters. While differentially initiated and expressed genes contacted by RUNX1-bound enhancers are enriched in pathways involved in epithelial maintenance and stemness, genes with RUNX1-promoter occupancy support DNA damage responsiveness. Modified cell morphology, metabolic control, increased breast cancer stemness, plasticity, anchorage-independent survival, chemoresistance, and perturbed DNA damage reactivity are observed upon RUNX1 ablation. Together, these findings define RUNX1 as an epigenetic tumor suppressor that maintains epithelial cell state by preserving enhancer activity and preventing gene expression associated with hallmarks of cancer.

cancer biology↗

bioRxiv2025

TAD boundary architecture and gene activity are uncoupled

Topologically associating domains (TADs) are prominent features of genome organization. A proposed function of TADs is to contribute to gene regulation by promoting chromatin interactions within a TAD and by suppressing interactions between TADs. Here, we directly probe the structure-function relationship of TADs by simultaneously assessing the behavior of TAD boundaries and gene activity at the single-cell and -allele level using high-throughput imaging. We find that while TAD boundaries pair more frequently than non-boundary regions, these interactions are infrequent and are uncorrelated with transcriptional activity of genes within the TAD. Similarly, acute global transcriptional inhibition or gene-specific activation does not alter TAD boundary proximity. Furthermore, while loss of the cohesin component RAD21 alters gene activity, disruption of TAD boundaries by depletion of the architectural chromatin protein CTCF is insufficient to alter expression of genes within the TAD. These results suggest that TAD boundary architecture and gene activity are largely uncoupled.

cell biology↗

bioRxiv2025

OptoLoop: An optogenetic tool to probe the functional role of genome organization

The genome folds inside the cell nucleus into hierarchical architectural features, such as chromatin loops and domains. If and how this genome organization influences the regulation of gene expression remains only partially understood. The structure-function relationship of genomes has traditionally been probed by population-wide measurements after mutation of critical DNA elements or by perturbation of chromatin-associated proteins. To circumvent possible pleiotropic effects of such approaches, we have developed OptoLoop, an optogenetic system that allows direct manipulation of chromatin contacts by light in a controlled fashion. OptoLoop is based on the fusion between a nuclease-dead SpCas9 protein and the light-inducible oligomerizing protein CRY2. We demonstrate that OptoLoop can drive the ind uction of contacts between genomically distant, repetitive DNA loci. As a proof-of-principle application of OptoLoop, we probed the functional role of DNA looping in the regulation of the human telomerase gene TERT by long-range contacts with the telomere. By analyzing the extent of chromatin looping and nascent RNA production at individual alleles, we find evidence for looping-mediated repression of TERT. In sum, OptoLoop represents a novel means for the interrogation of structure-function relationships in the genome at single-allele resolution.

molecular biology↗

bioRxiv2025

Transcriptional profiling of Hutchinson-Gilford Progeria patients identifies primary target pathways of progerin

Hutchinson Gilford Progeria Syndrome (HGPS) is an ultra-rare pediatric premature aging disorder. The disease is caused by a point mutation in the LMNA gene leading to the production of the dominant-negative progerin isoform of the nuclear envelope protein lamin A. Disease severity and progression amongst the population of [~]140 known patients is variable. Most of the mechanistic insights into the disease have come from studies using cellular or mouse models of HGPS. To probe the clinical relevance of previously implicated cellular pathways and to address the extent of gene expression heterogeneity between patients, we have performed transcriptomic analysis of a comprehensive set of HGPS patients. We find misexpression of several cellular pathways across the patient population, particularly of multiple signaling pathways as well as the Unfolded Protein Response (UPR) and mesodermal cell fate specification. Variability amongst individual patients was limited, with misregulation of the major pathways observed in most patients. Comparing the transcriptome of patients with an inducible HGPS cell model, we distinguished immediate-early cellular response pathways from secondary adaptive pathways and identified mTORC1, the UPR, UV response, apoptosis and TNF signaling via NF-{kappa}B as primary targets of the disease-causing progerin protein.

cell biology↗

bioRxiv2025

Orderly mitosis shapes interphase genome organization

Genomes assume a complex 3D architecture in the interphase cell nucleus. Yet the molecular mechanisms that determine global genome architecture are only poorly understood. To identify mechanisms of higher order genome organization, we performed high-throughput imaging-based CRISPR knockout screens targeting 1064 genes encoding nuclear proteins in multiple human cell lines. We assessed changes in the distribution of centromeres at single cell resolution as surrogate markers for global genome organization. The screens revealed multiple major regulators of spatial distribution of centromeres including components of the nucleolus, kinetochore, cohesins, condensins, and the nuclear pore complex. Alterations in centromere distribution required progression through the cell cycle and acute depletion of mitotic factors with distinct functions altered centromere distribution in the subsequent interphase. These results identify molecular determinants of spatial centromere organization, and they show that orderly progression through mitosis shapes interphase genome architecture.

cell biology↗

bioRxiv2025

Genome reorganization and its functional impact during breast cancer progression

Cancer progression involves extensive alterations in epigenetic and gene expression programs, but the accompanying changes in higher-order genome organization remain less well understood. Using high-resolution Micro-C mapping in the MCF10 cell model of breast cancer, we profiled chromatin compartments, topologically associated domains, and chromatin loops. We find large-scale compartmental shifts occur predominantly in early stages of cancer development, with more fine-scale structural changes in TADs and looping accumulating during the later transition to metastasis. Relating these chromatin features to gene expression and enhancer-associated histone marks revealed that many differentially expressed genes are physically connected to distal regulatory elements. While enhancer-promoter contact frequency and distal enhancer activity correlated with gene expression, strong changes in chromatin looping were relatively infrequent during progression, suggesting that alterations in chromatin contacts are not globally necessary but may facilitate gene regulation at a subset of genes. These results elucidate the connection between gene regulation and genome remodeling in a cell-based cancer progression model.

genomics↗

bioRxiv2025

RNA-coupled CRISPR Screens Reveal ZNF207 as a Regulator of LMNA Aberrant Splicing in Progeria

Despite progress in understanding pre-mRNA splicing, the regulatory mechanisms controlling most alternative splicing events remain unclear. We developed CRASP-Seq, a method that integrates pooled CRISPR-based genetic perturbations with deep sequencing of splicing reporters, to quantitively assess the impact of all human genes on alternative splicing from a single RNA sample. CRASP-Seq identifies both known and novel regulators, enriched for proteins involved in RNA splicing and metabolism. As proof-of-concept, CRASP-Seq analysis of an LMNA cryptic splicing event linked to progeria uncovered ZNF207, primarily known for mitotic spindle assembly, as a regulator of progerin splicing. ZNF207 depletion enhances canonical LMNA splicing and decreases progerin levels in patient-derived cells. High-throughput mutagenesis further showed that ZNF207s zinc finger domain broadly impacts alternative splicing through interactions with U1 snRNP factors. These findings position ZNF207 as a U1 snRNP auxiliary factor and demonstrate the power of CRASP-Seq to uncover key regulators and domains of alternative splicing. Main PointsO_LICRASP-Seq: RNA-coupled CRISPR screen quantifying gene and domain impact on splicing C_LIO_LIProfiling of five events identified 370 genes influencing alternative splicing C_LIO_LIZNF207 regulates splicing by interacting with U1 snRNP via its zinc-finger domains C_LIO_LIZNF207 depletion corrects LMNA aberrant splicing causing progeria C_LI

genetics↗

bioRxiv2025

Simulation and quantitative analysis of spatial centromere distributionpatterns

A prominent feature of eukaryotic chromosomes are centromeres, which are specialized regions of repetitive DNA required for faithful chromosome segregation during cell division. In interphase cells centromeres are non-randomly positioned in the three-dimensional space of the nucleus in a cell-type specific manner. The functional relevance and the cellular mechanisms underlying this observation are unknown, and quantitative methods to measure distribution patterns of centromeres in 3D space are needed. Here we have developed an analytical framework that combines robust clustering metrics and advanced modeling techniques for the quantitative analysis of centromere distributions at the single cell level. To identify a robust quantitative measure for centromere clustering, we benchmarked six metrics for their ability to sensitively detect changes in centromere distribution patterns from high-throughput imaging data of human cells, both under normal conditions and upon experimental perturbation of centromere distribution. We find that Ripleys K Score has the highest accuracy with minimal sensitivity to variations in centromeres number, making it the most suitable metric for measuring centromere distributions. As a complementary approach, we also developed and validated spatial models to replicate centromere distribution patterns, and we show that a radially shifted Gaussian distribution best represents the centromere patterns seen in human cells. Our approach creates tools for the quantitative characterization of spatial centromere distributions with applications in both targeted studies of centromere organization as well as in unbiased screening approaches.

genomics↗

bioRxiv2024

Identification of molecular determinants of gene-specific bursting patterns by high-throughput imaging screens

Stochastic transcriptional bursting is a universal property of active genes. While different genes exhibit distinct bursting patterns, the molecular mechanisms for gene-specific stochastic bursting are largely unknown. We have developed and applied a high-throughput-imaging based screening strategy to identify cellular factors and molecular mechanisms that determine the bursting behavior of human genes. Focusing on epigenetic regulators, we find that protein acetylation is a strong acute modulator of burst frequency, burst size and heterogeneity of bursting. Acetylation globally affects the Off-time of genes but has gene-specific effects on the On-time. Yet, these effects are not strongly linked to promoter acetylation, which do not correlate with bursting properties, and forced promoter acetylation has variable effects on bursting. Instead, we demonstrate acetylation of the Integrator complex as a key determinant of gene bursting. Specifically, we find that elevated Integrator acetylation decreases bursting frequency. Taken together our results suggest a prominent role of non-histone proteins in determining gene bursting properties, and they identify histone-independent acetylation of a transcription cofactor as an allosteric modulator of bursting via a far-downstream bursting checkpoint.

molecular biology↗

bioRxiv2024

Allele-level visualization of transcription and chromatin by high-throughput imaging

The spatial arrangement of the genome within the nucleus is a pivotal aspect of cellular organization and function with implications for gene expression and regulation. While all genome organization features, such as loops, domains, and radial positioning, are non-random, they are characterized by a high degree of single-cell variability. Imaging approaches are ideally suited to visualize, measure, and study single-cell heterogeneity in genome organization. Here, we describe two methods for the detection of DNA and RNA of individual gene alleles by fluorescence in situ hybridization (FISH) in a high-throughput format. We have optimized combined DNA/RNA FISH approaches either using simultaneous or sequential detection. These optimized DNA and RNA FISH protocols, implemented in a 384-well plate format alongside automated image and data analysis, enable accurate detection of chromatin loci and their gene expression status across a large cell population with allele-level resolution. We successfully visualized MYC and EGFR DNA and RNA in multiple cell types, and we determined the radial position of active and inactive MYC and EGFR alleles. These optimized DNA/RNA detection approaches are versatile and sensitive tools for mapping of chromatin features and gene activity at the single-allele level and at high throughput.

cell biology↗

bioRxiv2023

HiTIPS: High-Throughput Image Processing Software for the Study of Nuclear Architecture and Gene Expression

High-throughput imaging (HTI) generates complex imaging datasets from a large number of experimental perturbations. Commercial HTI software for image analysis workflows does not allow full customization and adoption of new image processing algorithms in the analysis modules. While open-source HTI analysis platforms provide individual modules in the workflow, like nuclei segmentation, spot detection, or cell tracking, they are often limited in integrating novel analysis modules or algorithms. Here, we introduce the High-Throughput Image Processing Software (HiTIPS) to expand the range and customization of existing HTI analysis capabilities. HiTIPS incorporates advanced image processing and machine learning algorithms for automated cell and nuclei segmentation, spot signal detection, nucleus tracking, spot tracking, and quantification of spot signal intensity. Furthermore, HiTIPS features a graphical user interface that is open to integration of new algorithms for existing analysis pipelines and to adding new analysis pipelines through separate plugins. To demonstrate the utility of HiTIPS, we present three examples of image analysis workflows for high-throughput DNA FISH, immunofluorescence (IF), and live-cell imaging of transcription in single cells. Altogether, we demonstrate that HiTIPS is a user-friendly, flexible, and open-source HTI analysis platform for a variety of cell biology applications.

bioinformatics↗

bioRxiv2022

Nuclear position modulates long-range chromatin interactions

The human genome is non-randomly organized within the cell nucleus. Spatial mapping of genome folding by biochemical methods and imaging has revealed extensive variation in locus interaction frequencies between cells in a population and between homologs within an individual cell. Commonly used mapping approaches typically examine either the relative position of genomic sites to each other or the position of individual loci relative to nuclear landmarks. Whether the frequency of specific chromatin-chromatin interactions is affected by where in the nuclear space a locus is located is unknown. Here, we have simultaneously mapped at the single cell level the interaction frequencies and radial position of more than a hundred locus pairs using high-throughput imaging to ask whether the location within the nucleus affects interactions frequency. We find strong enrichment of many interactions at specific radial positions. Position-dependency of interactions was cell-type specific, correlated with local chromatin type, and cell-type-specific enriched associations were marked by increased variability, sometimes without a significant decrease in mean spatial distance. These observations demonstrate that genome organization relative to itself and relative to nuclear landmarks are closely interwoven. Significance StatementA genes nuclear environment is defined by its distance to other genes as well as its distance to nuclear structures such as the nuclear periphery. While both of these features have been shown to be important for gene function, they are often studied separately. We performed the first systematic analysis comparing these two features. We determined that at the level of single chromosomes they are correlated, suggesting that genome organization relative to itself and relative to nuclear landmarks are closely interwoven.

cell biology↗

bioRxiv2022

Identification of epigenetic modulators as determinants of nuclear size and shape

The shape and size of the human cell nucleus is highly variable amongst cell types and tissues. Changes in nuclear morphology are associated with disease, including cancer, as well as with premature and normal aging. Despite the very fundamental nature of nuclear morphology, the cellular factors that determine nuclear shape and size are not well understood. To identify regulators of nuclear architecture in a systematic and unbiased fashion, we performed a high-throughput imaging-based siRNA screen targeting 867 nuclear proteins including chromatin-associated proteins, epigenetic regulators, and nuclear envelope components. Using multiple morphometric parameters, we identified cell-type specific and general effectors of nuclear size and shape. Interestingly, most identified factors altered nuclear morphology without affecting the levels of lamin proteins, which are known prominent regulators of nuclear shape. In contrast, a major group of nuclear shape regulators were modifiers of repressive heterochromatin. Biochemical and molecular analysis uncovered a direct physical interaction of histone H3 with lamin A mediated via combinatorial histone modifications. Furthermore, disease-causing lamin A mutations that result in disruption of nuclear shape inhibited lamin A-histone H3 interactions. Finally, oncogenic histone H3.3 mutants defective for H3K27 methylation resulted in nuclear morphology abnormalities. Altogether, our results represent a systematic exploration of cellular factors involved in determining nuclear morphology and they identify the interaction of lamin A with histone H3 as an important contributor to nuclear morphology in human cells.

cell biology↗

bioRxiv2022

HiFENS: High-throughput FISH detection of endogenous pre-mRNA splicing isoforms

Splicing factors play an essential role in regulation of alternative pre-mRNA splicing. While much progress has been made in delineating the mechanisms of the splicing machinery, the identity of signal transduction pathways and upstream factors that regulate splicing factor activity is less well understood. A major challenge in the discovery of upstream regulatory factors of pre-mRNA splicing is the scarcity of functional genomics screening methods to monitor splicing outcomes of endogenous genes. Here, we have developed HiFENS (High throughput FISH Detection of ENdogenous Splicing Isoforms), a high-throughput imaging assay based on hybridization chain reaction (HCR) and used HiFENS to screen for cellular factors that regulate alternative splicing of endogenous genes. We demonstrate optimized detection with high specificity of endogenous splicing isoforms and multiplexing of probes for accurate detection of splicing outcomes with single cell resolution. As proof-of-principle, we perform an RNAi screen of 702 human kinases and identify potential candidate upstream splicing regulators of the FGFR2 gene. HiFENS should be a useful tool for the unbiased delineation of cellular pathways involved in alternative splicing regulation.

cell biology↗

bioRxiv2022

Structure-function relationships in mitochondrial transcriptional condensates

Phase separation organizes many membraneless structures in cells. The functional consequences of concentrating cellular machinery into biomolecular condensates, however, are largely unclear. One fundamental cellular function that has been linked to condensate formation is transcription. Here, we have reconstituted mitochondrial transcription in condensates from purified components. We find that the core components of the mttranscriptional machinery form multi-phasic, viscoelastic condensates in vitro. Strikingly, the rates of condensate-mediated transcription are substantially lower than equivalent reactions in bulk solution. These condensate-mediated decreases in transcriptional rates are associated with the formation of dynamically arrested vesicular structures that are driven by the production and accumulation of RNA during transcription. Using coarse-grained, equilibrium simulations, we show that the generation of RNA alters the phase behavior and the organization of transcriptional components within condensates and that the in vitro mtcondensates are non-equilibrium structures. Together, our in vitro and in silico approaches shed light on how proteins and (ribo)nucleic acids biophysically self-assemble within mitochondria in vivo. Our results highlight the complex morphologies of transcribing, multicomponent condensates and they illustrate the interdependent structure-function relationships in condensates. Significance StatementMitochondria condense their genome into transcriptionally active mt-nucleoids. These structures fit the definition of biomolecular condensates that form via macromolecular phase separation. We take advantage of the ability to reconstitute mitochondrial transcriptional condensates in vitro from minimal components. We find that the production and accumulation of RNA alters the phase behavior of transcriptional condensates. The altered phase behavior is linked to the formation of arrested, non-equilibrium vesicular structures. Similar changes to phase behavior of proteins and (ribo)nucleic acids can be recapitulated in live mitochondria through knockdown of mt-nucleoid core components. Computer simulations help identify biophysical mechanisms that are needed to maintain the steady-state structures of transcriptional condensates.

biophysics↗