Biology subjects

Bartosovic, M.

Publications and source records attributed to Bartosovic, M..

8 recordsLinked to original sources

bioRxiv2026

Single-cell chromatin landscapes visualize epigenetic barriers and reveal lineage-specific Polycomb-mediated repression

Understanding how chromatin state contributes to developmental trajectories remains central to deciphering cell specification and differentiation. Using dual-modality nano-CUT&Tag, we profiled two antagonistic histone modifications--active H3K27ac and repressive H3K27me3--in thousands of single cells from Drosophila embryos across early lineage diversification and terminal differentiation. Joint embedding of both marks enabled robust cell-type classification and revealed increasing epigenetic specificity over developmental time. We ordered cells by developmental age and epigenomic similarity, and defined an epigenetic potential metric that visualizes repressive chromatin barriers as landscapes that predict transcriptional activity. While many genes conform to a classical model in which expression resides in low-potential epigenetic valleys, a substantial subset shows co-occurrence of H3K27ac, H3K27me3, and transcription within the same cell lineage. This indicates that Polycomb-mediated H3K27me3 repression frequently acts within, rather than solely between, lineages. Consistently, tissue-specific E(z) knockdown demonstrates that partial loss of H3K27me3 predominantly de-represses lineage-matched genes rather than inducing fate conversion. Systematic analysis showed that H3K27me3 occurs in multiple distributional modes, ranging from ubiquitous to highly cell-type-specific deposition, co-occuring with accessible but silent gene promoters. These findings demonstrate that cell-type-specific deployment of H3K27ac and H3K27me3 sculpts epigenetic potential landscapes that shape developmental gene expression patterns.

developmental biology↗

bioRxiv2025

G-quadruplex profiling in complex tissues using single-cell CUT&Tag

G-quadruplexes (G4) are non-canonical DNA structures that gained increasing attention for their potential roles in gene regulation, with implications in neurodegenerative diseases and cancer. Despite their biological significance, G4 structures have not been studied systematically across tissues and cell types. In this study, we employ G4 single-cell CUT&Tag (G4 scCUT&Tag) to characterize G4 landscapes in postnatal mouse brain cells, leveraging single-cell analytical approaches commonly used in scRNA-Seq and scATAC-Seq datasets. Using conventional single-cell omics workflows to process and explore our data, we distinguished different cell lineages based on G4 heterogeneity and established that a subset of lineage-specific genes show unique promoter G4s. Multi-omics integration with scRNA-Seq gene expression profiles, using both a covariance-based technique (canonical correlation analysis) and a transfer learning-based approach, enabled a more detailed annotation of cell types. These integrations not only revealed significant correlation of G4 and gene expression signals, but demonstrated that G4 scCUT&Tag enables detailed examination of G4 heterogeneity in complex tissues and supports integrative analysis of G4 profiles with other omics layers, offering new insights into the epigenomic landscapes of the developing brain.

developmental biology↗

bioRxiv2024

Multiplexed spatial mapping of chromatin features, transcriptome, and proteins in tissues

The phenotypic and functional states of a cell are modulated by a complex interactive molecular hierarchy of multiple omics layers, involving the genome, epigenome, transcriptome, proteome, and metabolome. Spatial omics approaches have enabled the capture of information from different molecular layers directly in the tissue context. However, current technologies are limited to map one to two modalities at the same time, providing an incomplete representation of cellular identity. Such data is inadequate to fully understand complex biological systems and their underlying regulatory mechanisms. Here we present spatial-Mux-seq, a multi-modal spatial technology that allows simultaneous profiling of five different modalities, including genome-wide profiles of two histone modifications and open chromatin, whole transcriptome, and a panel of proteins at tissue scale and cellular level in a spatially resolved manner. We applied this technology to generate multi-modal tissue maps in mouse embryos and mouse brains, which discriminated more cell types and states than unimodal data. We investigated the spatiotemporal relationship between histone modifications, chromatin accessibility, gene and protein expression in neuron differentiation revealing the relationship between tissue organization, function, and gene regulatory networks. We were able to identify a radial glia spatial niche and revealed spatially changing gradient of epigenetic signals in this region. Moreover, we revealed previously unappreciated involvement of repressive histone marks in the mouse hippocampus. Collectively, the spatial multi-omics approach heralds a new era for characterizing tissue and cellular heterogeneity that single modality studies alone could not reveal.

genetics↗

bioRxiv2024

Single-nuclei histone modification profiling of the adult human central nervous system unveils epigenetic memory of developmental programs

The adult human central nervous system (CNS) is remarkably complex, with neural cells displaying extensive transcriptional heterogeneity. However, how different layers of epigenetic regulation underpin this heterogeneity is poorly understood. Here, we profile the adult human CNS from distinct regions, for chromatin accessibility at the single-nuclei level. In addition, we simultaneously co-profiled the histone modifications H3K27me3 and H3K27ac at the single nuclei-level, providing their first map in all major human CNS cell types. We unveil primed chromatin signatures at HOX loci in spinal cord-derived human oligodendroglia (OLG) but not microglia. These signatures were reminiscent of developmental OLG but were decoupled from robust gene expression. Moreover, using high-resolution Micro-C, we show that induced pluripotent stem cell (iPS) derived human OLGs exhibit a HOX chromatin architecture compatible with the primed chromatin in adult OLGs, and bears a strong resemblance not only to OLG developmental architecture, but also high-grade pontine gliomas. Thus, adult OLG retain epigenetic memory from developmental states, which might enable them to promptly transcribe Hox genes, in contexts of regeneration, but also make them susceptible to gliomagenesis.

neuroscience↗

bioRxiv2022

Multimodal chromatin profiling using nanobody-based single-cell CUT&Tag

Probing epigenomic marks such as histone modifications at a single cell level in thousands of cells has been recently enabled by technologies such as scCUT&Tag. Here we developed a multimodal and optimized iteration of scCUT&Tag called nano-CT (for nano-CUT&Tag) that allows simultaneous probing of three epigenomic modalities at single-cell resolution, using nanobody-Tn5 fusion proteins. nano-CT is compatible with starting materials as low as 25 000 cells and has significantly higher resolution than scCUT&Tag, with a 16-fold increase in the number of fragments per cells. We used nano-CT to simultaneously profile chromatin accessibility, H3K27ac and H3K27me3 in a complex tissue - juvenile mouse brain. The obtained multimodal dataset allowed for discrimination of more cell types/states that scCUT&Tag, and inference of chromatin velocity between ATAC and H3K27ac in the oligodendrocyte (OL) lineage. In addition, we used nano-CT to deconvolute H3K27me3 repressive states and infer two sequential waves of H3K27me3 repression at distinct gene modules during OL lineage progression. Thus, given its high resolution, versatility, and multimodal features, nano-CT allows unique insights in epigenetic landscapes in different biological systems at single cell level.

molecular biology↗

bioRxiv2022

Human tRNA methyltransferase ALKBH8 targets wider repertoire of tRNAs and non-coding RNAs

Transfer RNAs acquire a large plethora of chemical modifications. Among those, modifications of the anticodon loop play important roles in translational fidelity and tRNA stability. Four human wobble U containing tRNAs obtain 5-methoxycarbonylmethyluridine (mcm5U34) or 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U34), which play a role in decoding. This mark involves a cascade of enzymatic activities. The last step is mediated by Alkylation Repair Homolog 8 (ALKBH8). In this study, we performed a transcriptome-wide analysis of the repertoire of ALKBH8 RNA targets. Using a combination of HITS-CLIP-seq and RIP-seq analyses, we uncover ALKBH8-bound RNAs. It targets an additional wobble U-containing tRNA tRNALys(UUU). More interestingly, the spectrum of bound RNAs includes other types of non-coding RNAs, such as C/D box snoRNAs, 7SK RNA or some miRNAs, respectively.

molecular biology↗

bioRxiv2021

Spatial-ATAC-seq: spatially resolved chromatin accessibility profiling of tissues at genome scale and cellular level

Cellular function in tissue is dependent upon the local environment, requiring new methods for spatial mapping of biomolecules and cells in the tissue context. The emergence of spatial transcriptomics has enabled genome-scale gene expression mapping, but it remains elusive to capture spatial epigenetic information of tissue at cellular level and genome scale. Here we report on spatial-ATAC-seq: spatially resolved chromatin accessibility profiling of tissue section via next-generation sequencing by combining in situ Tn5 transposition chemistry and microfluidic deterministic barcoding. Spatial chromatin accessibility profiling of mouse embryos delineated tissue region-specific epigenetic landscapes and identified gene regulators implicated in the central nerve system development. Mapping the accessible genome in human tonsil tissue with 20m pixel size revealed spatially distinct organization of immune cell types and states in lymphoid follicles and extrafollicular zones. This technology takes spatial biology to a new realm by enabling spatially resolved epigenomics to improve our understanding of cell identity, state, and fate decision in relation to epigenetic underpinnings in development and disease.

genomics↗

bioRxiv2020

Single-cell profiling of histone modifications in the mouse brain

The development of the mouse central nervous system (CNS) involves coordinated execution of transcriptional and epigenetic programs. These programs have been extensively studied through single-cell technologies in a pursuit to characterize the underlying cell heterogeneity. However, histone modifications pose additional layers of both positive and negative regulation that defines cellular identity. Here we show that the Cut&Tag technology can be coupled with a droplet-based single cell library preparation platform to produce high quality chromatin modifications data at a single cell resolution in tens of thousands of cells. We apply single-cell Cut&Tag (scC&T) to probe histone modifications characteristic of active promoters (H3K4me3), active promoters and enhancers (H3K27ac), active gene bodies (H3K36me3) and inactive regions (H3K27me3) and generate scC&T profiles for almost 50,000 cells. scC&T profiles of each of these histone modifications were sufficient to determine cell identity and deconvolute at single cell level regulatory principles such as promoter bivalency, spreading of H3K4me3 and promoter-enhancer connectivity. Moreover, we used scC&T to investigate the single-cell chromatin occupancy of transcription factor Olig2 and the cohesin complex component Rad21. Our results indicate that analysis of histone modifications and transcription factor occupancy at a single cell resolution can provide unique insights of epigenomic landscapes in the CNS. We also provide an online resource that can be used to interactively explore the data at https://castelobranco.shinyapps.io/BrainCutAndTag2020/.

neuroscience↗