Biology subjects

Iskar, M.

Publications and source records attributed to Iskar, M..

11 recordsLinked to original sources

bioRxiv2026

Modular control of CpG islands by chromatin-opening transcription factors specifies transcription start sites

CpG islands (CGIs) constitute the dominant class of promoters in vertebrate genomes, yet the mechanisms underlying their transcription initiation remain unclear. Using deep learning and acute perturbation of twelve transcription factors (TFs), we show that chromatin-opening TFs are the primary determinants of initiation site selection, thereby governing promoter output. The dominant TF defines a nucleosome-free region and specifies where transcription begins by binding closest to the transcription start site. Loss of this TF redirects transcription initiation to an adjacent secondary TF position, partially buffering gene expression. Promoter variant analyses validate that this modular control of initiation is encoded in cis and programmable. This principle extends to initiation at enhancers and TATA-box promoters, where chromatin-opening TFs unmask the TATA box for focused initiation. These findings point to a common sequence grammar for transcription across promoters and enhancers, supporting a minimalist and evolvable architecture of cis-regulatory regions.

molecular biology↗

bioRxiv2025

Nuclear rerouting of paracrine Fgf3 in source cells represses target genes to pattern morphogen responses

Morphogen gradients direct tissue patterning by inducing dose-dependent transcriptional responses, yet ligand-producing cells often respond differently from their neighbors. Using the zebrafish lateral line organogenesis model, we uncover a cell-autonomous role for the paracrine ligand Fgf3. Transcriptomic profiling and quantitative single-molecule imaging identify target genes, including the chemokine scavenger cxcr7b, whose expression decreases both when FGF receptor signaling is inhibited and when Fgf3 is overexpressed. High-resolution live imaging reveals nuclear accumulation of Fgf3 in producing cells, whereas neighbors receive only extracellular ligand, a feature also observed in other embryonic tissues. Mosaic gain- of-function and nanobody-mediated degradation demonstrate that the nuclear pool of Fgf3 autonomously represses specific targets without impairing canonical receptor signaling. Structure-guided comparative assays indicate nuclear targeting as a latent property of several paracrine FGFs. Dual secreted-nuclear functionality of FGF ligands may represent an intrinsic symmetry-breaking mechanism during organogenesis.

developmental biology↗

bioRxiv2024

Pipeline Olympics: continuable benchmarking of computational workflows for DNA methylation sequencing data against an experimental gold-standard

DNA methylation is a widely studied epigenetic mark and a powerful biomarker of cell type, age, environmental exposures, and disease. Whole-genome sequencing following selective conversion of unmethylated cytosines into thymines via bisulfite treatment or enzymatic methods remains the reference method for DNA methylation profiling genome-wide. While numerous software tools facilitate processing of DNA methylation sequencing reads, a comprehensive benchmarking study has been lacking thus far. In this study, we systematically compared complete computational workflows for processing DNA methylation sequencing data using a dedicated benchmarking dataset generated with five genome-wide profiling protocols. As an evaluation reference, we employed highly quantitative locus-specific measurements from our preceding benchmark of targeted DNA methylation assays. Based on this experimental gold-standard assessment and several comprehensive metrics, we identified workflows that consistently demonstrated superior performance and revealed major workflow development trends. To facilitate the sustainability of our benchmark, we implemented an interactive workflow execution and data presentation platform, adaptable to user-defined criteria and seamlessly expandable to future software.

bioinformatics↗

bioRxiv2024

Chromatin-dependent motif syntax defines differentiation trajectories

Transcription factors recognizing short DNA sequences within gene regulatory regions are crucial drivers of cell identity. Despite recent advances, their specificity remains incompletely understood. Here, we address this by contrasting two TFs, NGN2 and MyoD1, which recognize ubiquitous E-box motifs yet instigate distinct cell fates--neurons and muscles, respectively. Following controlled induction in embryonic stem cells, we monitor binding across differentiation trajectories, employing an interpretable machine-learning approach integrating pre-existing DNA accessibility data. This reveals a chromatin-dependent motif syntax, delineating both common and factor-specific binding and predicting genome engagement with high precision. Shared binding sites reside in open chromatin, locally influenced by nucleosomes. In contrast, factor-specific binding in closed chromatin involves NGN2 and MyoD1 acting as pioneer-factors, influenced by multi-motifs, rotational spacing, flanking sequences, and specific interaction partners, accounting for subsequent lineage divergence. Extending our methodology to other models demonstrates how such combination of opportunistic-binding and context-specific chromatin opening underpin transcription factor specificity driving differentiation trajectories.

genomics↗

bioRxiv2023

Systematic assessment of ISWI subunits reveals that NURF creates local accessibility for proper CTCF function

Catalytic activity of the ISWI family of remodelers is critical for nucleosomal organization and DNA binding of transcription factors, including the insulator protein CTCF. To define which subcomplex mediates these diverse functions, we derived a panel of isogenic mouse stem cell lines each lacking one of six ISWI accessory subunits. Individual deletions of subunits of either CERF, RSF, ACF, WICH or NoRC subcomplexes only moderately affect the chromatin landscape, while removal of the NURF-specific subunit BPTF leads to drastic reduction in chromatin accessibility and SNF2H ATPase localization around CTCF sites. While this affects adjacent nucleosome occupancy, it only modestly impacts CTCF binding itself. In the absence of accessibility, the structural function of CTCF is nevertheless impaired resulting in lower occupancy of cohesin and cohesin release factor, and reduced physical insulation at these sites, highlighting the need of NURF-mediated remodeling for open chromatin and proper CTCF function. These results separate local CTCF binding from insulator function in nuclear organization and reveal a specific role for NURF in mediating SNF2H localization and chromatin opening at bound CTCF sites. They designate local accessibility as critical for cohesin positioning and establishment of physical insulation.

molecular biology↗

bioRxiv2022

T cell landscape definition by multi-omics identifies Galectin-9 as novel immunotherapy target in chronic lymphocytic leukemia

Failure of cancer immunotherapy is linked to T cell exhaustion. To decipher the underlying mechanisms, we explored the T cell landscape in blood, bone marrow and lymph node samples of patients with chronic lymphocytic leukemia (CLL), and spleen samples of a CLL mouse model. By single-cell RNA-sequencing, mass cytometry (CyTOF), and multiplex image analysis of tissue microarrays, we identified a disease-specific accumulation of distinct regulatory T cell subsets and T cell exhaustion stages and their trajectories in CLL lymph nodes. Integration of T cell receptor sequencing data revealed a clonal expansion of CD8+ precursor exhausted T cells (TPEX), suggesting their CLL reactivity. Interactome analyses identified the TIM3 ligand Galectin-9 as a novel immunoregulatory molecule in CLL. Blocking of Galectin-9 in CLL-bearing mice slowed down disease development and reduced the number of TIM3-expressing T cells. Galectin-9 expression correlated with shorter survival of patients with CLL, renal cell carcinoma or glioma. Statement of significanceOur findings for the first time define the T cell landscape in CLL lymph nodes and reshape the current understanding of T cell exhaustion in this malignancy. They further introduce Galectin-9 as novel immune checkpoint with a high potential to overcome resistance to PD1 targeting drugs in CLL and beyond.

cancer biology↗

bioRxiv2022

Readout of histone methylation by Trim24 locally restricts chromatin opening by p53

The genomic binding sites of the transcription factor (TF) and tumour suppressor p53 are unusually diverse in regards to their chromatin features, including histone modifications, opening the possibility that chromatin provides context-dependence for p53 regulation. Here, we show that the ability of p53 to open chromatin and activate its target genes is indeed locally restricted by its cofactor Trim24. Trim24 binds to both p53 and unmethylated lysine 4 of histone H3, thereby preferentially locating to those p53 sites that reside in closed chromatin, while it is deterred from accessible chromatin by lysine 4 methylation. The presence of Trim24 increases cell viability upon stress and enables p53 to impact gene expression as a function of the local chromatin state. These findings link histone methylation to p53 function and illustrate how specificity in chromatin can be achieved, not by TF-intrinsic sensitivity to histone modifications, but by employing chromatin-sensitive cofactors which locally modulate TF function.

genomics↗

bioRxiv2022

Proteogenomics refines the molecular classification of chronic lymphocytic leukemia

Cancer heterogeneity at the proteome level may explain differences in therapy response and prognosis beyond the currently established genomic and transcriptomic based diagnostics. The relevance of proteomics for disease classifications remains to be established in clinically heterogeneous cancer entities such as chronic lymphocytic leukemia (CLL). Here, we characterized the proteome and transcriptome in-depth alongside genetic and ex-vivo drug response profiling in a clinically well annotated CLL discovery cohort (n= 68). Unsupervised clustering of the proteome data revealed six subgroups. Five of these proteomic groups were associated with genetic features, while one group was only detectable at the proteome level. This new group was characterized by accelerated disease progression, high spliceosomal protein abundances associated with aberrant splicing, and low B cell receptor signaling protein abundances (ASB-CLL). We developed classifiers to identify ASB-CLL based on its characteristic proteome or splicing signature in two independent cohorts (n= 165, n= 169) and confirmed that ASB-CLL comprises about 20 % of CLL patients. The inferior overall survival observed in ASB-CLL was independent of both TP53- and IGHV mutation status. Our multi-omics analysis refines the classification of CLL and highlights the potential of proteomics to improve cancer patient stratification beyond genetic and transcriptomic profiling. Single sentence summaryWe performed the largest proteogenomic analysis of CLL, linked proteomic profiles to clinical outcomes, and discovered a new poor outcome subgroup (ASB-CLL).

cancer biology↗

bioRxiv2021

A synergistic interaction between HDAC- and PARP inhibitors in childhood tumors with chromothripsis

Chromothripsis is a form of genomic instability characterized by the occurrence of tens to hundreds of clustered DNA double-strand breaks in a one-off catastrophic event. Rearrangements associated with chromothripsis are detectable in numerous tumor entities and linked with poor prognosis in some of these, such as Sonic Hedgehog medulloblastoma, neuroblastoma and osteosarcoma. Hence, there is a need for therapeutic strategies eliminating tumor cells with chromothripsis. Defects in DNA double-strand break repair, and in particular homologous recombination repair, have been linked with chromothripsis. Targeting DNA repair deficiencies by synthetic lethality approaches, we performed a synergy screen using drug libraries (n = 375 compounds, 15 models) combined with either a PARP inhibitor or cisplatin. This revealed a synergistic interaction between the HDAC inhibitor romidepsin and PARP inhibition. Functional assays, transcriptome analyses, and in vivo validation in patient-derived xenograft mouse models confirmed the efficacy of the combinatorial treatment.

cancer biology↗

bioRxiv2018

The landscape of viral associations in human cancers

Potential viral pathogens were systematically investigated in the whole-genome and transcriptome sequencing of 2,656 donors as part of the Pan-Cancer Analysis of Whole Genomes using a consensus approach integrating three independent pathogen detection pipelines. Viruses were detected in 382 genomic and 68 transcriptome data sets. We extensively searched and characterized numerous features of virus-positive cancers integrating various PCAWG datasets. We show the high prevalence of known tumor associated viruses such as EBV, HBV and several HPV types. Our systematic analysis revealed that HPV presence was significantly exclusive with well-known driver mutations in head/neck cancer. A strong association was observed between HPV infection and the APOBEC mutational signatures, suggesting the role of impaired mechanism of antiviral cellular defense as a driving force in the development of cervical, bladder and head neck carcinoma. Viral integration into the host genome was observed for HBV, HPV16, HPV18 and AAV2 and associated with a local increase in copy number variations. The recurrent viral integrations at the TERT promoter were coupled to high telomerase expression uncovering a further mechanism to activate this tumor driving process. High levels of endogenous retrovirus ERV1 expression is linked to worse survival outcome in kidney cancer.

cancer biology↗

bioRxiv2018

Rapid proteotyping reveals cancer biology and drug response determinants in the NCI-60 cells

We describe the rapid and reproducible acquisition of quantitative proteome maps for the NCI-60 cancer cell lines and their use to reveal cancer biology and drug response determinants. Proteome datasets for the 60 cell lines were acquired in duplicate within 30 working days using pressure cycling technology and SWATH mass spectrometry. We consistently quantified 3,171 proteotypic proteins annotated in the SwissProt database across all cell lines, generating a data matrix with 0.1% missing values, allowing analyses of protein complexes and pathway activities across all the cancer cells. Systematic and integrative analysis of the genetic variation, mRNA expression and proteomic data of the NCI-60 cancer cell lines uncovered complementarity between different types of molecular data in the prediction of the response to 240 drugs. We additionally identified novel proteomic drug response determinants for clinically relevant chemotherapeutic and targeted therapies. We anticipate that this study represents a significant advance toward the translational application of proteotypes, which reveal biological insights that are easily missed in the absence of proteomic data.

systems biology↗