Biology subjects

Skok, J.

Publications and source records attributed to Skok, J..

9 recordsLinked to original sources

bioRxiv2026

Cohesin collisions maintain ordered nucleosome architecture at boundaries and promoters

Cohesin is best known for its role in loop extrusion, while nucleosome phasing at regulatory elements is usually attributed to local DNA-bound factors and remodelers. Here we identify a previously unrecognized role for cohesin-mediated extrusion in maintaining local nucleosome architecture at CTCF sites and transcription start sites. Using single-molecule nano-NOMe-seq during SCC1 depletion, cell-cycle progression and Sororin perturbation, we show that CTCF-bound sites contain distinct nucleosome architectures ranging from ordered CTCF-footprinted arrays to footprint-free nucleosomal and inaccessible configurations.. In unperturbed cells, ordered CTCF-footprinted nucleosome arrays were strongest at a boundary-enriched class of CTCF sites without regulatory elements. By contrast, CTCF sites overlapping regulatory elements showed stronger aggregate CTCF ChIP-seq signal despite weaker footprinting and less regular nucleosome phasing, indicating that boundary-like nucleosome architecture is not predicted by CTCF occupancy alone. At TSSs, promoter-proximal CTCF defined a distinct state balance: CTCF-positive promoters were enriched for accessible and footprinted configurations, whereas CTCF-negative promoters showed proportionately fewer footprinted states and were dominated by footprint-free phased arrays. Acute SCC1 depletion disrupted nucleosome organization at CTCF sites without regulatory elements and at promoters with promoter-proximal CTCF, despite retention of aggregate CTCF ChIP-seq signal at CTCF-bound sites. SCC1 depletion also altered nucleosome organization at promoters lacking promoter-proximal CTCF, highlighting that cohesin-dependent nucleosome patterning is not simply a CTCF-barrier phenomenon. Cell-cycle and Sororin analyses further separated extrusion-associated states from Sororin-stabilized post-replicative cohesin, highlighting that nucleosome order depends on effective cohesin-barrier encounters rather than cohesin occupancy alone. Together, these findings establish cohesin collisions as an active local mechanism that patterns nucleosomes at boundaries and promoters.

molecular biology↗

bioRxiv2025

A developmentally regulated long-range enhancer-promoter contact mediates human neural development

SOX2 is a core pluripotency factor in human embryonic stem cells (hESCs), but upon differentiation to the three germ layers, its expression is preserved selectively in neuroectoderm. The mechanisms regulating SOX2 transcription in distinct developmental stages remain incompletely understood. Here, we demonstrate that a distant enhancer 550 kb from the human SOX2 locus is selectively activated in neural stem cells (NSCs) and establishes long-range contact with the SOX2 gene. CRISPR-Cas9 excision of the enhancer has no effect in hESCs but reduces SOX2 transcription in NSCs and impairs neuroectodermal differentiation and forebrain specification in teratomas and cerebral organoids. CRISPR excision of a CTCF recognition motif adjacent to the enhancer does not affect enhancer activation in neuroectoderm but reduces chromatin looping and SOX2 transcription to partially reproduce phenotypes seen with enhancer deletion. Our findings indicate that the development of the human nervous system depends on a developmentally regulated long-range contact between a distant enhancer and the SOX2 locus.

developmental biology↗

bioRxiv2024

A targetable developmental program co-regulates angiogenesis and immune evasion

Ultraviolet (UV)-induced DNA mutations produce genetic drivers of cutaneous melanoma initiation and numerous neoantigens that can trigger anti-tumor immune responses in the host. Consequently, melanoma cells must rapidly evolve to evade immune detection by simultaneously modulating cell-autonomous epigenetic mechanisms and tumor-microenvironment interactions. Angiogenesis has been implicated in this process; although an increase of vasculature initiates the immune response in normal tissue, solid tumors manage to somehow enhance blood flow while preventing immune cell infiltration. By comparing the expression of transcription factors (TFs) across early-stage melanoma, naevi, and other cancer types, we found the homeodomain-containing TF HOXD13 drives a melanoblast-like developmental program, which is upregulated in melanoma and strongly correlated with angiogenesis and immune cell exclusion. Using transcriptomics, 3D chromatin profiling, and in vivo models, we demonstrate that HOXD13 upregulation promotes tumor growth in vivo by concomitantly enhancing angiogenesis and suppressing T-cell infiltration. HOXD13 orchestrates 3D chromatin contacts between distal enhancers and promoters, simultaneously activating VEGFA, SEMA3A, and CD73. VEGFA and SEMA3A remodel the tumor vasculature and CD73 elevates extracellular levels of adenosine, a vasodilator and immune suppressor that binds adenosine receptors (AdR) on endothelial and T cells. In line with these findings, HOXD13-induced growth advantage in vivo was significantly reversed by the concomitant administration of VEGFR and AdR inhibitors. By revealing a dual pro-angiogenic and immunosuppressive HOXD13-CD73/VEGF gene regulatory axis, we identify a subset of patients who might benefit from combinations of AdR and VEGFR inhibitors which are both currently being tested in clinical trials.

cancer biology↗

bioRxiv2022

Cell type-specific prediction of 3D chromatin architecture

The mammalian genome is spatially organized in the nucleus to enable cell type-specific gene expression. Investigating how chromatin organization determines this specificity remains a challenge. Methods for measuring the 3D chromatin organization, such as Hi-C, are costly and bear strong technical limitations, restricting their broad application particularly in high-throughput genetic perturbations. In this study, we present C.Origami, a deep neural network model that performs de novo prediction of cell type-specific chromatin organization. The C.Origami model enables in silico experiments to examine the impact of genetic perturbations on chromatin interactions in cancer genomes and beyond. In addition, we propose an in silico genetic screening framework that enables high-throughput identification of impactful genomic regions on 3D chromatin organization. We demonstrate that cell type-specific in silico genetic perturbation and screening, enabled by C.Origami, can be used to systematically discover novel chromatin regulatory mechanisms in both normal and disease-related biological systems.

genomics↗

bioRxiv2021

SOX2-phosphorylation toggles a bistable differentiation-switch in squamous cell carcinoma

The fate choice between stem cell self-renewal and differentiation is regulated by bistable transcriptional networks, which are balanced in homeostasis and imbalanced in tumors. Yet, how stem cells switch from self-renewal to differentiation remains a conundrum. Here, we discover a molecular mechanism that allows stem cell-like tumor propagating cells (TPCs) in squamous cell carcinomas (SCCs) to switch from a mutually exclusive SOX2-PITX1-TP63 self-renewal circuit to a KLF4 driven differentiation program, dependent on the relative occupancy of a novel Klf4-regulatory enhancer cluster (Klf4EC944) by SOX2 or KLF4, respectively. We find SOX2 occupies this site in TPCs to inhibit Klf4 transcription, but upon phosphorylation SOX2 becomes evicted from Klf4EC944, allowing residual KLF4 to occupy this site instead, boost the expression of KLF4 and its downstream targets, and differentiate self-renewing TPCs into post-mitotic SCC cells. This mechanism allows SOX2 to promote self-renewal and tumor formation, while preserving the differentiation potential in SCC cells. Our data suggest that stochastic cell fate decisions depend on the effective concentration of enzymatically regulated transcription factors. The surprising specificity by which SOX2-phosphorylation governs the bistable Klf4EC944 network-switch in SCCs reveals a conceptual framework for the identification of similar switches in other stem cell and cancer types and their potential development into cell type specific differentiation therapies for diseases in which tissue homeostasis has gone awry.

cancer biology↗

bioRxiv2020

CRISPR-Decryptr reveals cis-regulatory elements from noncoding perturbation screens

Clustered Regularly Interspace Short Palindromic Repeats (CRISPR)-Cas9 genome editing methods provide the tools necessary to examine phenotypic impacts of targeted perturbations in high-throughput screens. While these technologies have the potential to reveal functional elements with direct therapeutic applications, statistical techniques to analyze noncoding screen data remain limited. We present CRISPR-Decryptr, a computational tool for the analysis of CRISPR noncoding screens. Our method leverages experimental design: accounting for multiple conditions, controls, and replicates to infer the regulatory landscape of noncoding genomic regions. We validate our method on a variety of mutagenesis, CRISPR activation, and CRISPR interference screens, extracting new insights from previously published data.

bioinformatics↗

bioRxiv2019

EpiMethylTag simultaneously detects ATAC-seq or ChIP-seq signals with DNA methylation

Activation of regulatory elements is thought to be inversely correlated with DNA methylation levels. However, it is difficult to determine whether DNA methylation is compatible with chromatin accessibility or transcription factor (TF) binding if assays are performed separately. We developed a low input, low sequencing depth method, EpiMethylTag that combines ATAC-seq or ChIP-seq (M-ATAC or M-ChIP) with bisulfite conversion, to simultaneously examine accessibility/TF binding and methylation on the same DNA.

genomics↗

bioRxiv2019

RNA interactions with CTCF are essential for its proper function

The function of the CCCTC-binding factor (CTCF) in the organization of the genome has become an important area of investigation, but the mechanisms of how CTCF dynamically contributes to genome organization is not clear. We previously discovered that CTCF binds to large numbers of endogenous RNAs; promoting its oligomerization. Here we found that inhibition of transcription or interfering with CTCF ability to bind RNA through mutations of two of its 11 zinc fingers that are not involved with CTCF binding to its cognate site in vitro, zinc finger-1 (ZF1) or -10 (ZF10), disrupt CTCF association to chromatin. These mutations alter gene expression profiles as CTCF mutants lose their ability to promote local insulation. Our results highlight the importance of RNA as a structural component of the genome, in part by affecting the association of CTCF with chromatin and likely its interaction with other factors. O_LITranscriptional inhibition disrupts CTCF binding to chromatin C_LIO_LIRNA-binding regions (RBR) in CTCF are found within ZF1 and ZF10 C_LIO_LILocal insulation is markedly decreased in ZF1{triangleup} and ZF10{triangleup} mutant rescues C_LIO_LIGene expression and chromatin organization are disrupted by RBR mutants C_LI

molecular biology↗

bioRxiv2018

The impact of endogenous retroviruses on nuclear organization and gene expression

BackgroundThe organization of chromatin in the nucleus plays an essential role in gene regulation. When considering the mammalian genome it is important to take into account that about half of the DNA is comprised of transposable elements. Given their repetitive nature, reads associated with these elements are generally discarded or randomly distributed among elements of the same type in genome-wide analyses. Thus, it is challenging to identify the activities and properties of individual transposons. As a result, we only have a partial understanding of how transposons contribute to chromatin folding and how they impact gene regulation.\n\nResultsUsing adapted PCR and Capture-based chromosome conformation capture (3C) approaches, collectively called 4Tran, we take advantage of the repetitive nature of transposons to capture interactions from multiple copies of endogenous retrovirus (ERVs) in the human and mouse genomes. With 4Tran-PCR, reads are selectively mapped to unique regions in the genome. This enables the identification of TE interaction profiles for individual ERV families and integration events specific to particular genomes. With this approach we demonstrate that transposons engage in long-range intra-chromosomal interactions guided by the separation of chromosomes into A and B compartments as well as topologically associated domains (TADs). In contrast to 4Tran-PCR, Capture-4Tran can uniquely identify both ends of an interaction that involve retroviral repeat sequences, providing a powerful tool for uncovering the individual TE insertions that interact with, and potentially regulate target genes.\n\nConclusions4Tran provides new insight into the manner in which transposons contribute to chromosome architecture and identifies target genes that transposable elements can potentially control.

genomics↗