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Targeting TUT1 Depletes Tri-snRNP Pools to Suppress Splicing and Inhibit Pancreatic Cancer Cell Survival.

Pancreatic ductal adenocarcinoma (PDAC) is highly aggressive and lacks effective therapeutic options. Cancer cells frequently become more dependent on splicing factors than normal cells due to increased rates of transcription. Terminal uridylyltransferase 1 (TUT1) is a specific terminal uridylyltransferase for U6 small nuclear RNA (snRNA), which plays a catalytic role in the spliceosome. Here, we found that TUT1 was required for the survival of PDAC cells but not for normal pancreatic cells. In PDAC cells, the uridylylation activity of TUT1 promoted tri-snRNP assembly by facilitating the binding of LSM proteins to U6 snRNA and subsequent tri-snRNP assembly. PDAC cells required higher amounts of tri-snRNP to efficiently splice pre-mRNA with weak splice sites to support the high transcriptional output. Depletion of TUT1 in PDAC cells resulted in inefficient splicing of exons in a group of highly expressed RNAs containing weak splice sites, thereby resulting in the collapse of an mRNA processing circuit and consequently dysregulating splicing required by PDAC cells. Overall, this study unveiled an interesting function of TUT1 in regulating splicing by modulating tri-snRNP levels and demonstrated a distinct mechanism underlying splicing addiction in pancreatic cancer cells.

Sir2 and Fun30 regulate ribosomal DNA replication timing via MCM helicase positioning and nucleosome occupancy.

The association between late replication timing and low transcription rates in eukaryotic heterochromatin is well known, yet the specific mechanisms underlying this link remain uncertain. In Saccharomyces cerevisiae, the histone deacetylase Sir2 is required for both transcriptional silencing and late replication at the repetitive ribosomal DNA (rDNA) arrays. We have previously reported that in the absence of SIR2, a de-repressed RNA PolII repositions MCM replicative helicases from their loading site at the ribosomal origin, where they abut well-positioned, high-occupancy nucleosomes, to an adjacent region with lower nucleosome occupancy. By developing a method that can distinguish activation of closely spaced MCM complexes, here we show that the displaced MCMs at rDNA origins have increased firing propensity compared to the nondisplaced MCMs. Furthermore, we found that both activation of the repositioned MCMs and low occupancy of the adjacent nucleosomes critically depend on the chromatin remodeling activity of FUN30. Our study elucidates the mechanism by which Sir2 delays replication timing, and it demonstrates, for the first time, that activation of a specific replication origin in vivo relies on the nucleosome context shaped by a single chromatin remodeler.

A new vaccination approach for Salmonellosis employing a multi-epitope vaccine based on live microbial cell factory from Lactococcus lactis.

A major health and financial burden in the chicken sector is salmonella infection. It is difficult to create an oral vaccination that can provide strong intestinal mucosal immunity in birds, particularly cross-protection against several Salmonella serotypes. As a result, the poultry industry needs a powerful oral vaccination platform that uses live bacterial vectors to prevent various Salmonella serotypes. The genetically engineered L. lactis was given orally to birds as a vaccine after a multi-epitope vector was created using a reverse vaccinology technique. After the plasmid was digested, the target group produced a 72 kDa protein called multi-epitop. Birds that received the L. lactis/pNZ8121-Multi epitope vaccination showed increased levels of interferon (IFN-γ) and NFkB1α, increased transcription rates of cytokines, and a significant presence of IgY antibodies specific to the multi epitope gene in their serum. Salmonella infection is a severe health and economic burden in the poultry industry, according to spleen sections from the L. lactis/pNZ8121-Multi epitope. Developing an oral vaccine that can provide birds robust intestinal mucosal immunity-specifically, cross-protection against many Salmonella serotypes-is challenging. The results provide a fresh method for creating new immunological candidate multi-epitome genes by using the food-grade, non-pathogenic Lactococcus lactis as a protein cell factory. This method provides a unique technique to assess the long-term sustainability, cost, safety, and usefulness of experimental pharmaceutical products.

Feed-forward loop improves the transient dynamics of an antithetic biological controller.

Integral controller is widely used in industry for its capability of endowing perfect adaptation to disturbances. To harness such capability for precise gene expression regulation, synthetic biologists have endeavoured in building biomolecular (quasi-)integral controllers, such as the antithetic integral controller. Despite demonstrated successes, challenges remain with designing the controller for improved transient dynamics and adaptation. Here, we explore and investigate the design principles of alternative RNA-based biological controllers, by modifying an antithetic integral controller with prevalently found natural feed-forward loops (FFL), to improve its transient dynamics and adaptation performance. With model-based analysis, we demonstrate that while the base antithetic controller shows excellent responsiveness and adaptation to system disturbances, incorporating the type-1 incoherent FFL into the base antithetic controller could attenuate the transient dynamics caused by changes in the stimuli, especially in mitigating the undesired overshoot in the output gene expression. Further analysis on the kinetic parameters reveals similar findings to previous studies that the degradation and transcription rates of the circuit RNA species would dominate in shaping the performance of the controllers.

Association between TLR-2 and TLR-9 gene polymorphisms (rs5743708 and rs5743836) and susceptibility to psoriatic arthritis in Egyptian patients

Psoriasis (PsO) is a chronic immune-mediated disease of the skin. Psoriatic arthritis (PsA) is a prevalent chronic inflammatory disease that is associated with joint destruction and disability. The presence of PsO is the single greatest risk factor for the development of PsA. Toll-like receptors (TLRs) are trans-membrane proteins coded by the toll genes family. They are expressed in different cell types including immune and non-immune cells. Polymorphisms in TLR genes that lead to changes in these receptors or interfere with the transcription rates of their messenger ribonucleic acid (mRNA), may be involved in the chronic inflammatory immune response observed in PsA. This study involved 50 patients with PsA, 50 patients with cutaneous PsO and 50 age and sex matched normal subjects as controls. We aimed to assess TLR-2 (rs5743708) and TLR-9 (rs5743836) gene polymorphisms as potential risk factors for PsA in Egyptian patients with cutaneous PsO. Genotyping and allele frequencies were performed using Real Time polymerase chain reaction (qRT-PCR). Toll-like receptor-2 (TLR-2) rs5743708 and TLR-9 rs5743836 polymorphisms were associated with increased risk of PsO as an autoimmune disease, however they were not related to increase susceptibility to PsA in this cohort study of Egyptian patients.

Camelina circRNA landscape: Implications for gene regulation and fatty acid metabolism.

Circular RNAs (circRNAs) are closed-loop RNAs forming a covalent bond between their 3' and 5' ends, the back splice junction (BSJ), rendering them resistant to exonucleases and thus more stable compared to linear RNAs. Identification of circRNAs and distinction from their cognate linear RNA is only possible by sequencing the BSJ that is unique to the circRNA. CircRNAs are involved in the regulation of their cognate RNAs by increasing transcription rates, RNA stability, and alternative splicing. We have identified circRNAs from C. sativa that are associated with the regulation of germination, light response, and lipid metabolism. We sequenced light-grown and etiolated seedlings after 5 or 7 days post-germination and identified a total of 3447 circRNAs from 2763 genes. Most circRNAs originate from a single homeolog of the three subgenomes from allohexaploid camelina and correlate with higher ratios of alternative splicing of their cognate genes. A network analysis shows the interactions of select miRNA:circRNA:mRNAs for regulation of transcript stabilities where circRNA can act as a competing endogenous RNA. Several key lipid metabolism genes can generate circRNA, and we confirmed the presence of KASII circRNA as a true circRNA. CircRNA in camelina can be a novel target for breeding and engineering efforts.

Decoding the biogenesis of HIV-induced CPSF6 puncta and their fusion with the nuclear speckle.

Viruses rely on host cellular machinery for replication. After entering the nucleus, the HIV genome accumulates in nuclear niches where it undergoes reverse transcription and integrates into neighboring chromatin, promoting high transcription rates and new virus progeny. Despite anti-retroviral treatment, viral genomes can persist in these nuclear niches and reactivate if treatment is interrupted, likely contributing to the formation of viral reservoirs. The post-nuclear entry dynamics of HIV remain unclear, and understanding these steps is critical for revealing how viral reservoirs are established. In this study, we elucidate the formation of HIV-induced CPSF6 puncta and the domains of CPSF6 essential for this process. We also explore the roles of nuclear speckle scaffold factors, SON and SRRM2, in the biogenesis of these puncta. Through genetic manipulation and depletion experiments, we demonstrate the key role of the intrinsically disordered region of SRRM2 in enlarging nuclear speckles in the presence of the HIV capsid. We identify the FG domain of CPSF6 as essential for both puncta formation and binding to the viral core, which serves as the scaffold for CPSF6 puncta. While the low-complexity regions (LCRs) modulate CPSF6 binding to the viral capsid, they do not contribute to puncta formation, nor do the disordered mixed charge domains (MCDs) of CPSF6. These results demonstrate how HIV evolved to hijack host nuclear factors, enabling its persistence in the host. Of note, this study provides new insights into the underlying interactions between host factors and viral components, advancing our understanding of HIV nuclear dynamics and offering potential therapeutic targets for preventing viral persistence.

An autonomous microbial sensor enables long-term detection of TNT explosive in natural soil

Microbes can be engineered to sense target chemicals for environmental and geospatial detection. However, when engineered microbes operate in real-world environments, it remains unclear how competition with natural microbes affect their performance over long time periods. Here, we engineer sensors and memory-storing genetic circuits inside the soil bacterium Bacillus subtilis to sense the TNT explosive and maintain a long-term response, using predictive models to design riboswitch sensors, tune transcription rates, and improve the genetic circuit’s dynamic range. We characterize the autonomous microbial sensor’s ability to detect TNT in a natural soil system, measuring single-cell and population-level behavior over a 28-day period. The autonomous microbial sensor activates its response by 14-fold when exposed to low TNT concentrations and maintains stable activation for over 21 days, exhibiting exponential decay dynamics at the population-level with a half-life of about 5 days. Overall, we show that autonomous microbial sensors can carry out long-term detection of an important chemical in natural soil with competitive growth dynamics serving as additional biocontainment.

Exploring transcription modalities from bimodal, single-cell RNA sequencing data.

There is a growing interest in generating bimodal,single-cell RNA sequencing (RNA-seq) data for studying biological pathways. These data are predominantly utilized in understanding phenotypic trajectories using RNA velocities; however, the shape information encoded in the two-dimensional resolution of such data is not yet exploited. In this paper, we present an elliptical parametrization of two-dimensional RNA-seq data, from which we derived statistics that reveal four different modalities. These modalities can be interpreted as manifestations of the changes in the rates of splicing, transcription or degradation. We performed our analysis on a cell cycle and a colorectal cancer dataset. In both datasets, we found genes that are not picked up by differential gene expression analysis (DGEA), and are consequently unnoticed, yet visibly delineate phenotypes. This indicates that, in addition to DGEA, searching for genes that exhibit the discovered modalities could aid recovering genes that set phenotypes apart. For communities studying biomarkers and cellular phenotyping, the modalities present in bimodal RNA-seq data broaden the search space of genes, and furthermore, allow for incorporating cellular RNA processing into regulatory analyses.

In Silico-Designed G-Quadruplex Targeting Peptide Attenuates VEGF-A Expression, Preventing Angiogenesis in Cancer Cells.

Vascular endothelial growth factor-A (VEGF-A) is a growth factor and pluripotent cytokine that promotes angiogenesis in cancer cells, transitioning to an angiogenic phenotype. The binding of VEGF-A protein to VEGF receptors (VEGFR-1 and VEGFR-2) initiates a cascade of events that stimulates angiogenesis by facilitating the migration and enhancing the permeability of endothelial cells. The proximal promoter of the VEGF gene encompasses a 36-base pair region (from -85 to -50) that can form a stable G-quadruplex (G4) structure in specific conditions. The activity of the VEGF promoter is reliant on this structure. During cancer progression, the VEGF-A G4 succumbs to cellular pressure and fails to maintain a stable structure. This shifts the balance to form a duplex structure, increasing the transcription rate. Earlier research has tried to develop small-molecule ligands to target and stabilise G4, demonstrating the possibility of suppressing VEGF expression. However, they either lack specificity or toxic. Peptides, on the other hand, are significantly less studied as G4 binders. Here, we designed a peptide that successfully binds and stabilises the VEGF-A G4 while reducing its gene expression. This further alters the expression fate of the VEGF-A signalling cascade and blocks angiogenesis in cancer cells. We employed high-resolution nuclear magnetic resonance (NMR) spectroscopy and molecular dynamics simulation to elucidate the chemical details of G4-peptide interaction. In addition, we used qPCR and western blot techniques to investigate the expression pattern of the molecules implicated in the VEGF-A signalling cascade. The study explores the intricate relationship between peptides and quadruplex structures, revealing valuable insights that can improve the design of pharmacophores targeting the dynamic quadruplex structure. The results of our study are encouraging, opening possibilities for advancements in, the characterisation and optimisation of peptides as G-quadruplex ligands in view of their potential therapeutic uses.

Genome-wide quantification of polycistronic transcription in Leishmania major.

Leishmania major is a human-pathogenic, obligate parasite and the etiological agent of the most prevalent, cutaneous form of leishmaniasis, which is an important neglected, tropical disease with ~1.2 million new infections per year. Leishmania, and the whole order Trypanosomatida, are early eukaryotes with highly diverged gene expression and regulation pathways, setting them apart from their mammalian hosts and from most other eukaryotes. Using precision run-on sequence analysis, we performed a genome-wide mapping and density analysis of RNA polymerases in isolated nuclei of the protozoan parasite Leishmania major. We map transcription initiation sites at divergent strand switch regions and head-tail regions within the chromosomes and correlate them with known sites of chromatin modifications. We confirm continuous, polycistronic RNA synthesis in all RNA polymerase II-dependent gene arrays but find small varying RNA polymerase activities in polycistronic transcription units (PTUs), excluding gene-specific transcription regulation, but not PTU-specific variations. Lastly, we find evidence for transcriptional pausing of all three RNA polymerase classes, hinting at a possible mechanism of transcriptional regulation.IMPORTANCELeishmania spp. are pathogens of humans and animals and cause one of the most important neglected tropical diseases. Regulation of gene expression in Leishmania but also in the related Trypanosoma is radically different from all eukaryotic model organisms, dispensing with regulated, gene-specific transcription, and relying instead on highly regulated translation. Our work sheds light on the initiation, elongation, and termination of transcription, maps unidirectional, polycistronic transcription units, provides evidence for transcriptional pausing at or near starting points of RNA synthesis, and quantifies the varying transcription rates of the polycistronic transcription units. Our results will further the understanding of these important pathogens and should provide a valuable resource for researchers in the field of eukaryotic microbiology.

Kv3.3 Expression Enhanced by a Novel Variant in the Kozak Sequence of KCNC3.

Pathogenic variants in KCNC3, which encodes the voltage-gated potassium channel Kv3.3, are associated with spinocerebellar ataxia type 13. SCA13 is a neurodegenerative disease characterized by ataxia, dysarthria and oculomotor dysfunction, often in combination with other signs and symptoms such as cognitive impairment. Known disease-causing variants are localized in the protein coding regions and predominantly in the transmembrane and voltage sensing domains. In a patient with an ataxic movement disorder and progressive cognitive decline, the c.-6C>A variant was detected in the Kozak sequence of KCNC3. The Kozak sequence is responsible for efficient initiation of translation. Functional analysis of the new c.-6C>A variant and the upstream 5'-UTR region of KCNC3 by luciferase assays, quantitative PCR and methylation analysis shows increased protein expression but no effect on transcription rate. Therefore, increased translation initiation of KCNC3 transcripts compared to wild-type Kozak sequence seems to be the cause of the increased expression. Variants in the regulatory elements of disease-causing genes probably play an underestimated role.

Storm: Incorporating transient stochastic dynamics to infer the RNA velocity with metabolic labeling information

The time-resolved scRNA-seq (tscRNA-seq) provides the possibility to infer physically meaningful kinetic parameters, e.g., the transcription, splicing or RNA degradation rate constants with correct magnitudes, and RNA velocities by incorporating temporal information. Previous approaches utilizing the deterministic dynamics and steady-state assumption on gene expression states are insufficient to achieve favorable results for the data involving transient process. We present a dynamical approach, Storm (Stochastic models of RNA metabolic-labeling), to overcome these limitations by solving stochastic differential equations of gene expression dynamics. The derivation reveals that the new mRNA sequencing data obeys different types of cell-specific Poisson distributions when jointly considering both biological and cell-specific technical noise. Storm deals with measured counts data directly and extends the RNA velocity methodology based on metabolic labeling scRNA-seq data to transient stochastic systems. Furthermore, we relax the constant parameter assumption over genes/cells to obtain gene-cell-specific transcription/splicing rates and gene-specific degradation rates, thus revealing time-dependent and cell-state-specific transcriptional regulations. Storm will facilitate the study of the statistical properties of tscRNA-seq data, eventually advancing our understanding of the dynamic transcription regulation during development and disease.

Kidney mRNA-protein expression correlation: what can we learn from the Human Protein Atlas?

The Human Protein Atlas, with more than 10 million immunohistochemical images showing tissue- and cell-specific protein expression levels and subcellular localization information, is widely used in kidney research. The Human Protein Atlas contains comprehensive data on multi-tissue transcript and protein abundance, allowing for comparisons across tissues. However, while visual and intuitive to interpret, immunohistochemistry is limited by its semi-quantitative nature. This can lead to mismatches in protein expression measurements across different platforms. We performed a comparison of the Human Protein Atlas' kidney-specific RNA sequencing and immunohistochemistry data to determine whether the mRNA and protein abundance levels are concordant. Our study shows that there is a discordance between mRNA and protein expression in the kidney based on the Human Protein Atlas data. Using an external validation mass spectrometry dataset, we show that more than 500 proteins undetected by immunohistochemistry are robustly measured by mass spectrometry. The Human Protein Atlas transcriptome data, on the other hand, exhibit similar transcript detection levels as other kidney RNA-seq datasets. Discordance in mRNA-protein expression could be due to both biological and technical reasons, such as transcriptional dynamics, translation rates, protein half-lives, and measurement errors. This is further complicated by the heterogeneity of the kidney tissue itself, which can increase the discordance if the cell populations or tissue compartment samples do not match. As such, shedding light on the mRNA-protein relationship of the kidney-specific Human Protein Atlas data can provide context to our scientific inferences onrenal gene and protein quantification.

CSIG-01. DECREASING EXPRESSION OF TRAF PROTEINS WITH INCREASING MALIGNANCY WITH LOWEST EXPRESSION IN IDH-WILDTYP GLIOBLASTOMA

Abstract The tumor necrosis factor receptor-associated factors (TRAF) are cytoplasmatic adapter proteins that can interact directly with the intracellular domains of cell surface receptors, such as the TNF receptor superfamily. TRAFs are thus involved in cellular processes like proliferation or apoptosis. Seven members are known; all expressed in the cytosol of various cell and tissue types. Increased levels of TRAF4 in GBM tissue are related to cell proliferation and migration. About 25 % of grade I meningioma show mutations in TRAF7, which are associated with a lower risk of malignant transformation. Therefore expression patterns of all seven TRAF members are evaluated in different glioma grades. Following tumor samples were obtained during neurosurgery and shock frozen in liquid nitrogen: peritumoral tissue, astrocytoma and oligodendroglioma (G2, G3) and glioblastoma (primary and recurrent). Transcription rate of TRAF1-7 was measured via qPCR. Correlation of TRAF expression level and patient overall survival was evaluated using TCGA datasets of the TCGA-LGG” and “TCGA-GBM” projects. With the exception of TRAF5, a decreased mRNA level with increasing malignancy could be found for all TRAFs (TRAF1 p=n.s.; TRAF2 p=0.0026; TRAF3 p<0.0001; TRAF4 p=0.0021; TRAF6 p<0.0001; TRAF7 p<0.0001). IDHwt glioblastoma showed significant lower expression level compared to IDHmut glioblstoma in all TRAFs except for TRAF1 (TRAF2 p<0.0001; TRAF3 p=0.0346; TRAF4 p<0.0001; TRAF6 p=0.0447; TRAF7 p=0.0081). TRAF5 again showed a reverse effect (p=0.0065). Correlation of TRAF expression level in low grade glioma and patient survival revealed longer overall survival with low TRAF1,2,4 and TRAF5 expression (p<0.0001, p= 0.0178, p=0.0307, p<0.0001). No difference in survival rate in high grade glioma could be found.

CNSC-03. HIGH EXPRESSION OF PICCOLO AND SYNTAXIN-1A IN PAEDIATRIC MEDULLOBLASTOMA DUE TO BOTH: HIGH NEURONAL FRACTION AND ENDOGENOUS EXPRESSION

Abstract Emerging evidence indicates that the “healthy” nervous system contributes to the pathogenesis of cancer. Tumor cells were found to form ultra-long protrusions with distinct synapses, mainly in the infiltrative zone, resulting in a neuron-to-brain tumor synaptic communication. Located at the presynapse, proteins of the cytomatrix of the active zone have been identified to maintain highly specialized functions in neurons. Further, the proteins were shown to be involved in progression in various tumors, but little is known about their role in brain tumors. This study examined the expression of CAZ proteins in glioma and medulloblastoma. Tumor samples of Astrocytoma, glioblastoma, pilocytic astrocytoma and medulloblastoma were obtained during neurosurgical resection. Transcription rate of following CAZ proteins was measured via qPCR: SNAP25, VAMP1, VAMP2, SYT1, PCLO, BSN, STX1A. To examine amount of neuronal tissue TUBB3 and for glial fraction GFAP were quantified. A medulloblastoma cell line (DAOY) was furthermore analyzed to evaluate origin of high expressed proteins. Medulloblastoma showed high expression of PCLO (pilocytic astrocytoma vs. medulloblastoma p=0.0126) and STX1A (pilocytic astrocytoma vs. medulloblastoma p=0.0006) compared to. Likewise a high transcriptional level of TUBB3 could be seen with significance to pilocytic astrocytoma (2.2 ± 1.4 vs. 0.2 ± 0.17; p = 0.0018), which could indicate a high amount of neuronal tissue in given medulloblastoma samples. To rule out a causal link between high proportion of neuronal tissue and high CAZ expression, a paediatric medulloblastoma cell line was analysed. High expression of PCLO and STX1A in absence of TUBB3 was shown (3.8 and 2.01 vs. 0.059), indicating endogenous expression of CAZ proteins in medulloblastoma.

Predicting synthetic mRNA stability using massively parallel kinetic measurements, biophysical modeling, and machine learning.

mRNA degradation is a central process that affects all gene expression levels, though it remains challenging to predict the stability of a mRNA from its sequence, due to the many coupled interactions that control degradation rate. Here, we carried out massively parallel kinetic decay measurements on over 50,000 bacterial mRNAs, using a learn-by-design approach to develop and validate a predictive sequence-to-function model of mRNA stability. mRNAs were designed to systematically vary translation rates, secondary structures, sequence compositions, G-quadruplexes, i-motifs, and RppH activity, resulting in mRNA half-lives from about 20 seconds to 20 minutes. We combined biophysical models and machine learning to develop steady-state and kinetic decay models of mRNA stability with high accuracy and generalizability, utilizing transcription rate models to identify mRNA isoforms and translation rate models to calculate ribosome protection. Overall, the developed model quantifies the key interactions that collectively control mRNA stability in bacterial operons and predicts how changing mRNA sequence alters mRNA stability, which is important when studying and engineering bacterial genetic systems.

Sir2 and Fun30 regulate ribosomal DNA replication timing via MCM helicase positioning and nucleosome occupancy.

The association between late replication timing and low transcription rates in eukaryotic heterochromatin is well-known, yet the specific mechanisms underlying this link remain uncertain. In Saccharomyces cerevisiae , the histone deacetylase Sir2 is required for both transcriptional silencing and late replication at the repetitive ribosomal DNA arrays (rDNA). We have previously reported that in the absence of SIR2 , a derepressed RNA PolII repositions MCM replicative helicases from their loading site at the ribosomal origin, where they abut well-positioned, high-occupancy nucleosomes, to an adjacent region with lower nucleosome occupancy. By developing a method that can distinguish activation of closely spaced MCM complexes, here we show that the displaced MCMs at rDNA origins have increased firing propensity compared to the nondisplaced MCMs. Furthermore, we found that both, activation of the repositioned MCMs and low occupancy of the adjacent nucleosomes critically depend on the chromatin remodeling activity of FUN30 . Our study elucidates the mechanism by which Sir2 delays replication timing, and it demonstrates, for the first time, that activation of a specific replication origin in vivo relies on the nucleosome context shaped by a single chromatin remodeler.

Chromatin Buffers Torsional Stress During Transcription.

Transcription through chromatin under torsion represents a fundamental problem in biology. Pol II must overcome nucleosome obstacles and, because of the DNA helical structure, must also rotate relative to the DNA, generating torsional stress. However, there is a limited understanding of how Pol II transcribes through nucleosomes while supercoiling DNA. In this work, we developed methods to visualize Pol II rotation of DNA during transcription and determine how torsion slows down the transcription rate. We found that Pol II stalls at ± 9 pN·nm torque, nearly sufficient to melt DNA. The stalling is due to extensive backtracking, and the presence of TFIIS increases the stall torque to + 13 pN·nm, making Pol II a powerful rotary motor. This increased torsional capacity greatly enhances Pol II's ability to transcribe through a nucleosome. Intriguingly, when Pol II encounters a nucleosome, nucleosome passage becomes more efficient on a chromatin substrate than on a single-nucleosome substrate, demonstrating that chromatin efficiently buffers torsional stress via its torsional mechanical properties. Furthermore, topoisomerase II relaxation of torsional stress significantly enhances transcription, allowing Pol II to elongate through multiple nucleosomes. Our results demonstrate that chromatin greatly reduces torsional stress on transcription, revealing a novel role of chromatin beyond the more conventional view of it being just a roadblock to transcription.

MITOCHONDRIAL MEMBRANE POTENTIAL IS A FUNCTIONALLY RELEVANT AXIS OF NON-GENETIC CELLULAR HETEROGENEITY IN GLIOBLASTOMA

Abstract AIMS Glioma stem cells (GSCs) are believed to underpin treatment resistance in glioblastoma by virtue of their self- renewal capabilities and presumed transcriptional plasticity. Understanding how cell state diversity is established and maintained is crucial to make therapeutic gains. We have previously shown mitochondrial membrane potential (MMP) to be a functionally relevant source of extrinsic noise in embryonic and haematopoietic stem cells. This study evaluates cellular MMP as an axis of variability in GSCs. METHOD MMP was measured by flow cytometry in patient-derived GSC lines using cationic dyes that diffuse reversibly across the mitochondrial membrane in a voltage dependent manner. MMP-high and low populations (top and bottom 10-20%) were fractionated and subjected to RNA-sequencing and assays of proliferation and clonogenicity. Correlation between MMP and global transcription rate was investigated through a uridine analogue (5-EU) incorporation assay. The relationship between MMP and quiescence was assessed, with quiescence defined as inducible histone2B-GFP label retention after 10 days. Timecourse data for MMP was obtained through sequential staining. RESULTS RNA sequencing identified differential gene expression and exon utilization according to MMP status. MMP- high cells upregulated cell cycle genes, while MMP-low cells exhibited a diverse signature suggestive of a more differentiated state. A greater proportion of MMP-high cells were in cycle, but MMP-low cells formed more colonies. MMP positively correlated with global transcription rate in both cycling and quiescent GSCs. Time- course experiments revealed dynamic periodicity in cellular MMP. CONCLUSION The MMP axis reveals transcriptional and functional heterogeneity in GSCs and importantly unmasks phenotypic variability within the treatment-refractory quiescent compartment. The positive correlation between MMP and transcription rate, along with splicing differences, suggests a mechanism underlying functional diversity. Given the oscillatory dynamics of MMP, we postulate that stable-low MMP may define a deeply quiescent, therapy-resistant state. Evaluating MMP as a tractable regulator of GSC fate is warranted.