Biology subjects

Kutter, C.

Publications and source records attributed to Kutter, C..

14 recordsLinked to original sources

bioRxiv2026

tTEscanR: A user-friendly integrative R package for quantifying and visualizing translation efficiency from sequencing data in diverse biological systems

Translation elongation relies on accurate codon-anticodon pairing. Here, we present tTEscanR, an R package designed to investigate this translational interface. By quantifying mRNA codon demand alongside tRNA anticodon availability, tTEscanR provides scalable estimates of translation rates directly from standard transcriptomic and chromatin accessibility count matrices, where genomic features are represented as rows and experimental conditions as columns. tTEscanR seamlessly integrates into existing bulk and single-cell pipelines and provides modular functions for quality control, filtering, normalization, statistical analysis, and visualization. A structured data object centralizes workflow outputs and associated metadata. A built-in multilevel visualization module generates customizable, publication-ready graphical outputs to facilitate data interpretation and reproducibility of the complex translational landscape. We demonstrate the utility of tTEscanR across cancer biology, aging, and neurodegeneration datasets, uncovering critical translational regulatory programs overlooked by conventional analyses. tTEscanR is available in an open-source repository as a standalone tool or workflow plug-in.

genomics↗

bioRxiv2026

In vivo evidence for bleb-induced survival signaling in metastatic melanoma

Bleb signaling is a cellular process in which pressure-driven plasma membrane protrusions generate localized micron-scale membrane curvature that recruits cytosolic septin complexes, assembling signaling hubs that promote cell survival1. Our prior work showed that this morphology-encoded signaling pathway is necessary to sustain anchorage-independent survival of BRAF and NRAS mutant melanoma cells in vitro. However, whether bleb-induced signaling occurs in vivo and contributes to cancer progression is unclear. Here, we develop complementary in vivo and ex vivo assays spanning human patient samples and mouse xenograft models to mechanistically interrogate bleb signaling directly in physiologic contexts. We observe that bleb-associated septin hubs are exclusively formed in poorly adherent amoeboid tumor cells at the invasive margin, within malignant effusions, and at distant metastatic sites, while well-adhered cells in the tumor interior show no signs of septin hub formation. Accordingly, pharmacological pathway disruption specifically kills disseminated tumor cells within these low-adhesion microenvironments while having no appreciable effect on adhered cells in the tumor interior, resulting in reduced metastatic burden and delayed disease recurrence in vivo. This work confirms septin-mediated bleb signaling as a previously unrecognized vulnerability of disseminated cancer cells and demonstrates that this morphology-encoded pathway operates in vivo to support disease progression, preserving cancer cell viability under conditions in which cell survival signals from the environment are muted. These findings suggest novel opportunities to target survival signaling in micrometastatic disseminated cancer cells. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=150 SRC="FIGDIR/small/697601v1_ufig1.gif" ALT="Figure 1"> View larger version (61K): org.highwire.dtl.DTLVardef@1770384org.highwire.dtl.DTLVardef@87e567org.highwire.dtl.DTLVardef@1e5d6b2org.highwire.dtl.DTLVardef@724c5f_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2024

Comprehensive analysis of the RBP regulome reveals functional modules and drug candidates in liver cancer

RNA binding proteins (RBPs) are essential components of the transcriptomic regulome. Identifying the RBP regulome in cancer cells is crucial to discovering and understanding carcinogenesis mechanisms and providing new therapeutic targets. Here, we aimed to reveal the regulome of liver cancer upon specific perturbations. To this end, we applied a consensus Gene Regulatory Network (GRN) approach using knockdown data for the liver cancer cell line HepG2. By incorporating multiple GRNs from diverse inference methods, we constructed a highly precise GRN. To validate our results, we comprehensively evaluated the consensus GRN, focusing on characterizing the most relevant aspects of the liver cancer regulome. This included utilizing eCLIP-seq and RAPseq data to verify RBP interactions and binding sites. In addition, we performed an enrichment analysis of network modules and drug repurposing based on the inferred GRN. Taken together, our findings demonstrate the critical roles of RBP regulatory interactions in liver cancer that can be employed to improve treatment strategies.

cancer biology↗

bioRxiv2023

Estrogen receptor activation remodels TEAD1 gene expression to alleviate nonalcoholic fatty liver disease

IntroductionThe occurrence of obesity-related hepatic malignancies differs between sexes, suggesting the involvement of sex hormones. Female sex hormones maintain cell homeostasis through estrogen receptor (ER) signaling and protect from developing nonalcoholic fatty liver disease (NAFLD) in mice and humans. RationaleTo understand recovery from high-fat diet (HFD)-induced liver disease in males upon estrogen treatment, we comprehensively characterized molecular changes in the liver upon selective activation of estrogen receptors (ERs) to identify novel therapeutic targets downstream of estrogen signaling. MethodsTo dissect hepatic ER isoform-driven responses, we integrated liver transcriptomes from female and male HFD mice treated with or without four different estrogen agonists, along with multiomics data, including bulk, single-cell and spatial transcriptomics, chromatin profiling, machine learning models and advanced microscopy. Patient cohorts and primary human hepatocyte spheroids datasets were included. ResultsOnly males developed liver steatosis. We found that selective activation of either ER or ER{beta} reduced HFD-induced hepatic steatosis in male mice. Systemic ER activation restored HFD-induced aberrant gene expression of cellular processes across liver cell types, including hepatocytes. Profiling of marked histones revealed that ER activation modulated promoter and enhancer sites and identified 68 estrogen-sensitive enhancer-gene pairs. Most of these genes were similarly deregulated in human nonalcoholic fatty liver disease (NAFLD) patients, including the transcription factor TEAD1. TEAD1 expression increased in NAFLD patients, and inhibiting TEAD ameliorated steatosis in spheroids by suppressing lipogenic pathways. ConclusionsSystemic activation of ER or ER{beta} modulates molecular pathways in the liver to counteract NAFLD. Our study identified TEAD1 as a key ER-sensitive gene and suggested that its inhibition poses a therapeutic strategy to combat NAFLD without the undesired side effects elicited by estrogen signaling. Clinical research relevanceWe identified drug targets downstream of estrogen signaling, including TEAD1, and demonstrate that TEAD inhibition improves steatosis by suppressing lipogenic pathways. Basic research relevanceThe targeted activation of nuclear ERs recovers high-fat diet-induced molecular and physiological liver phenotypes by remodeling core pathways beyond lipid metabolism. ER-responsive enhancers regulate central metabolic genes of clinical significance in NAFLD patients, highlighting the potential impact of this research on understanding liver cell plasticity. HIGHLIGHTSO_LIsteatosis in livers of high-fat diet (HFD) male mice was effectively reduced by selective activation of estrogen receptors (ER and ER{beta}) with four different agonists. C_LIO_LIER agonist treatments successfully reversed HFD-induced changes in gene regulation and expression, revealing new treatment targets involving previously unconnected molecular pathways. C_LIO_LIestrogen-sensitive enhancers regulated important genes, including TEAD1, emerging as pivotal NAFLD regulators significantly impacting metabolic processes. C_LIO_LIhigh TEAD1 gene expression in NAFLD patients correlated with disease severity, underscoring its clinical significance in disease progression. C_LIO_LIinhibiting TEAD with small molecules alleviated steatosis by suppressing lipogenic pathways, resembling some of the same beneficial effects as estrogen treatment. C_LI

genomics↗

bioRxiv2023

CRISPR-Cas9-mediated genome engineering exaggerates genomic deletion at 10q23.31 including the PTEN gene locus mimicking cancer profiles

The CRISPR-Cas9 system is a powerful tool for studying gene functions and has tremendous potential for disease treatment. However, precise genome editing requires thorough assessments to minimize unintended on- and off-target effects. Here, we report an unexpected deletion of a 287 kb region on Chromosome 10 (10q23.31) in chronic myelogenous leukemia HAP1 cells, which are frequently used in CRISPR screens. The deleted region encodes regulatory genes, including PAPSS2, ATAD1, KLLN, and PTEN. We found that this deletion was not a direct consequence of CRISPR-Cas9 off-targeting but rather occurred frequently by the process of generating CRISPR-Cas9-modifed cells. The deletion was associated with global changes in histone acetylation and gene expression, affecting fundamental cellular processes such as cell cycle and DNA replication. We detected this deletion in cancer patient genomes. As in HAP1 cells, the deletion contributed to similar gene expression patterns among cancer patients despite interindividual differences. Overall, our findings suggest that the unintended deletion of 10q23.31 can confound CRISPR-Cas9 studies, highlights the importance of assessing unintended genomic changes in CRISPR-Cas9-modified cells and may have clinical significance in cancer research. HighlightsO_LICRISPR-Cas9-modified HAP1 cells carry an unexpected large genomic deletion at 10q23.31 encompassing four protein-coding genes frequently expressed across various cell types. C_LIO_LIThe 10q23.31 deletion is accompanied by global changes in histone modification and transcriptomes. C_LIO_LIThe generation of CRISPR-Cas9-modified cells rather than Cas9 activity increases the frequencies of the deletion at 10q23.31. C_LIO_LIThe 10q23.31 deletion identified in HAP1 cells resembles a commonly occurring deletion pattern in cancer patients. C_LI

cancer biology↗

bioRxiv2023

Co-inhibition of topoisomerase 1 and BRD4-mediated pause release selectively kills pancreatic cancer via readthrough transcription

Pancreatic carcinoma is one of the most lethal cancers and the absence of efficient therapeutic strategies results in poor prognosis. Transcriptional dysregulation due to alterations in KRAS and MYC impacts initiation, development, and survival of this tumor type. Using patient-derived xenografts of pancreatic carcinoma driven by KRAS and MYC oncogenic transcription, we show that co-inhibition of Topoisomerase 1 (TOP1) and bromodomain containing protein 4 (BRD4) synergistically induce tumor regression through targeting promoter pause-release, a rate-limiting step in transcription elongation. By comparing the nascent transcriptome with the recruitment of elongation and termination factors along genes, we found that co-inhibition of TOP1 and BRD4, while globally impairing RNA production, disturbs recruitment of proteins involved in termination. Thus, RNA polymerases continue transcribing downstream of genes for hundreds of kilobases leading to readthrough transcription. This pervasive transcription also occurs during replication, perturbing replisome progression and leading to DNA damage. The synergistic effect of TOP1 and BRD4 inhibition is specific for cancer cells leaving normal cells unharmed, highlighting the sensitivity of the tumor to these transcriptional defects. This preclinical study provides a mechanistic understanding of the benefit of combining TOP1 and BRD4 inhibitors to treat pancreatic carcinomas addicted to oncogenic drivers of high transcription and replication. One Sentence SummaryTOP1 and BRD4 inhibitors synergize to selectively kill pancreatic cancer in vivo via readthrough transcription without emergence of drug resistance

cancer biology↗

bioRxiv2021

Large-scale identification of RBP-RNA interactions by RAPseq refines essentials of post-transcriptional gene regulation

Over the past decade, thousands of putative human RNA binding proteins (RBPs) have been identified and increased the demand for specifying RNA binding capacities. Here, we developed RNA affinity purification followed by sequencing (RAPseq) that enables in vitro large-scale profiling of RBP binding to native RNAs. First, by employing RAPseq, we found that vertebrate HURs recognize a conserved RNA binding motif and bind predominantly to introns in zebrafish compared to 3UTRs in human RNAs. Second, our dual RBP assays (co-RAPseq) uncovered cooperative RNA binding of HUR and PTBP1 within an optimal distance of 27 nucleotides. Third, we developed T7-RAPseq to discern m6A-dependent and - independent RNA binding sites of YTHDF1. Fourth, RAPseq of 26 novel non-canonical RBPs revealed specialized moonlighting interactions. Last, five pathological IGF2BP family variants exhibited different RNA binding patterns. Overall, our simple, scalable and versatile method enables to fast-forward RBP-related questions. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/467743v1_ufig1.gif" ALT="Figure 1"> View larger version (106K): org.highwire.dtl.DTLVardef@1f77ce0org.highwire.dtl.DTLVardef@12778fcorg.highwire.dtl.DTLVardef@77ff8corg.highwire.dtl.DTLVardef@2a7f24_HPS_FORMAT_FIGEXP M_FIG C_FIG HIGHLIGHTSO_LIRAPseq reveals in vitro-derived RBP-RNA interactomes C_LIO_LIthe vertebrate-conserved HUR binding motif adapted to species-unique RNA features C_LIO_LIco-RAPseq and T7-RAPseq uncover binding cooperativity and modification dependencies C_LIO_LInon-canonical RBPs have specialized RNA interactomes C_LI

genomics↗

bioRxiv2021

Nanoengineered DNA origami with repurposed TOP1 inhibitors targeting myeloid cells for the mitigation of neuroinflammation

Targeting myeloid cells, especially microglia, for the treatment of neuroinflammatory diseases such as multiple sclerosis (MS), is underappreciated. Here, we screened a library of compounds and identified the topoisomerase 1 (TOP1) inhibitor camptothecin (CPT) as a promising drug candidate for microglial modulation. CPT and its FDA-approved analog topotecan (TPT) inhibited inflammatory responses in microglia and macrophages, and ameliorated neuroinflammation in mice. Transcriptomic analysis of sorted microglia revealed an altered transcriptional phenotype following TPT treatment, with Ikzf1 identified as a potential target. Importantly, TOP1 expression was found elevated in several neuroinflammatory conditions, including human MS brains. To achieve targeted delivery to myeloid cells we designed a nanosystem using DNA origami and loaded TPT into it (TopoGami). TopoGami also significantly suppressed the inflammatory response in microglia and mitigated disease progression in MS-like mice. Our findings suggest that TOP1 inhibition represents a therapeutic strategy for neuroinflammatory diseases, and the proposed nanosystem may foster future research and drug development with a demand to target myeloid cells.

immunology↗

bioRxiv2021

CRISPR/Cas9 deletions induce adverse on-target genomic effects leading to functional DNA in human cells

The CRISPR/Cas9 system is widely used to permanently delete genomic regions by inducing double-strand breaks via dual guide RNAs. However, on-target consequences of Cas9 deletion events have yet to be fully investigated. To characterize Cas9-induced genotypic abnormalities in human cells, we utilized an innovative droplet-based target enrichment approach followed by long-read sequencing and coupled it to a customized de novo sequence assembly. This approach enabled us to dissect the sequence content at kilobase scale within an on-target genomic locus. We here describe extensive genomic disruptions by Cas9, involving a genomic duplication and inversion of the target region as well as integrations of exogenous DNA and interchromosomal DNA fragment rearrangements at the double-strand break sites often at the same time. Although these events altered the genomic composition of the on-target region, we found that the aberrant DNA fragments are still functional, marked by active histones and bound by RNA polymerase III. In HAP1 cells, the integration of the target-derived fragments accelerated cell proliferation in deletion clones. Our findings broaden the consequential spectrum of the Cas9 deletion system, reinforce the necessity of meticulous genomic validations and rationalize extra caution when interpreting results from a deletion event.

molecular biology↗

bioRxiv2021

Analysis at single-cell resolution identifies a stable mammalian tRNA-mRNA interface and increased translation efficiency in neurons

The correlation between codon and anticodon pools influences the efficiency of translation, but whether differences exist in these pools across individual cells is unknown. We determined that codon usage and amino acid demand are highly stable across different cell types using single-cell RNA-sequencing atlases of adult mouse and fetal human. After demonstrating the robustness of ATAC-sequencing for analysis of tRNA gene usage, we quantified anticodon usage and amino acid supply in adult mouse and fetal human single-cell ATAC-seq atlases. We found that tRNA gene usage is overall coordinated across cell types, except in neurons which clustered separately from other cell types. Integration of these datasets revealed a strong and statistically significant correlation between amino acid supply and demand across almost all cell types. Neurons have an enhanced translation efficiency over other cell types, driven by an increased supply of tRNAAla (AGC) anticodons. This results in faster decoding of the Ala-GCC codon, as determined by cell-type specific ribosome profiling, and a reduction of tRNAAla (AGC) anticodon pools may be implicated in neurological pathologies. This study, the first such examination of codon usage, anticodon usage, and translation efficiency at single-cell resolution, identifies conserved features of translation elongation across mammalian cellular diversity and evolution.

genomics↗

bioRxiv2021

Molecular programming in utero modulates hepatic lipid metabolism and adult metabolic risk in obese mother offspring in a sex-specific manner

Male and female offspring of obese mothers are known to differ significantly in their metabolic adaptation and later development of complications. We investigated the sex-dependent responses in obese offspring of mice with maternal obesity, focusing on changes in liver glucose and lipid metabolism. Maternal obesity prior to and during gestation led to hepatic insulin resistance and inflammation in male offspring, while female offspring were protected. These sex differences were explained by more efficient transcriptional and posttranscriptional reprogramming of metabolic pathways to prevent the damaging effects of maternal obesity in females compared to males. These differences were sustained later in life, resulting in a better metabolic balance in female offspring. In conclusion, sex and maternal obesity drive transcriptional and posttranscriptional regulation of major metabolic processes in offspring liver differently, explaining the sexual dimorphism in obesity-associated metabolic risk.

physiology↗

bioRxiv2021

ZFP207 controls pluripotency by multiple post-transcriptional mechanisms

The pluripotent state is not solely governed by the action of the core transcription factors Oct4, Sox2, and Nanog, but also by a series of co-transcriptional and post-transcriptional events, including alternative splicing (AS) and the interaction of RNA-binding proteins (RBPs) with defined subpopulations of RNAs. Zinc Finger Protein 207 (ZFP207) is an essential transcription factor for mammalian embryonic development. Here, we employ multiple functional analyses to characterize the role of ZFP207 in mouse embryonic stem cells (ESCs). We find that ZFP207 plays a pivotal role in ESC maintenance, and silencing of Zfp207 leads to severe neuroectodermal differentiation defects. In striking contrast to human ESCs, ZFP207 does not transcriptionally regulate stem cell and neuronal-related genes but exerts its effects by control AS networks and acting as an RBP. Our study expands the role of ZFP207 to maintain ESC identity, and underscores ZFP207 functional versatility with key roles in neural fate commitment.

cell biology↗

bioRxiv2021

FOXO1 and FOXO3 cooperatively regulate innate lymphoid cell development

The natural killer (NK) and non-cytotoxic innate lymphoid cells (ILC) lineages play vital role in the regulation of the immune system. Yet understanding of mechanisms controlling NK/ILC development remains incomplete. The evolutionary conserved FOXO family of forkhead transcription factors are critical regulators of cellular processes. We found that the loss of FOXO1 and FOXO3 together caused impaired activation of the NK gene expression program and reduced ETS binding already at the common lymphoid progenitor (CLP) level and a block at the ILC progenitor (ILCP) to NK progenitor transition. FOXO controlled NK cell maturation in organ specific manner and their ability to respond to IL-15. At the ILCP level, disruption of the ILC lineage specific gene programs was associated with broad perturbation of the generation of the non-cytotoxic ILC subsets. We concluded that FOXO1 and FOXO3 cooperatively regulate ILC lineage specification at the progenitor level as well as the generation of mature ILCs.

cell biology↗

bioRxiv2020

5'XP sRNA-seq: A simple sequencing method to identify small RNA with and without 5' phosphorylation in a single library from low input samples

Small RNA (sRNA) sequencing has been critical for our understanding of many cellular processes, including gene regulation. Nonetheless, the varying biochemical properties of sRNA, such as 5 nucleotide modifications, makes many sRNA subspecies incompatible with common protocols for sRNA sequencing. Here we describe 5XP-seq that outlines a novel strategy that solves this problem. By tagging 5P sRNA during library preparation, 5XP-seq combines an open approach that includes all types of 5-terminal modifications (5X), with a selective approach for 5-phosphorylated sRNA (5P). We show that 5XP-seq not only enriches phosphorylated miRNA and piRNA but successfully discriminates these sRNA from all other sRNA species. We further demonstrate the importance of this strategy by successful inter-species validation of sRNAs that would have otherwise failed, including human to insect translation of several tRNA (tRFs) and rRNA (rRFs) fragments. By combining 5 insensitive library strategies with 5 sensitive tagging, we have solved an intrinsic bias in modern sRNA sequencing that will help us reveal the true complexity and the evolutionary significance of the sRNA world.

molecular biology↗