Biology subjects

Polasek-Sedlackova, H.

Publications and source records attributed to Polasek-Sedlackova, H..

5 recordsLinked to original sources

bioRxiv2025

Epigenetic reprogramming guides sexual dimorphism during floral development in Silene latifolia

Dioecy, the condition in which male and female individuals exist as separate plants, represents a fascinating and relatively rare reproductive strategy, offering unique opportunities to study the genetic and epigenetic regulation of sexual dimorphism. While sex determining genes underlying dioecy have already been described for several plant species, the role of epigenetic modifications in meristematic cell populations remains poorly understood. In this study we describe the spatio-temporal deposition of three epigenetic markers during early stages of floral development in model dioecious species Silene latifolia. We selected H3K4me1, H3K9me2 and active Ser2 phosphorylated form of RNA Polymerase II (Pol-IIS2ph), to assess levels of chromatin condensation and transcriptional activity of meristematic cells during key developmental stages. Utilizing the novel approach of an AI-assisted nuclei segmentation and high-content imaging we created a single-cell resolution atlas for male and female floral meristems. Our results show a relationship between transcription activity and sex determination during early meristem development. Moreover, our results suggest that H3K9me2 deposition in the developing meristem is linked to sex-specific chromatin reprogramming events, such as pollen mother cell formation during anther maturation. Overall, these results offer new insights into the role of chromatin regulation during floral meristem development and improves our understanding of sexual dimorphism in dioecious species.

plant biology↗

bioRxiv2025

Quantitative AI-based DNA fiber workflow to study replication stress

Replication stress (RS) is a prominent source of genome instability and human diseases. Understanding its molecular mechanism through various quantitative and unbiased methodologies is essential for the advancement of treatment strategies. One of the powerful methods to study DNA replication dynamics and its alterations at the single-molecule resolution is the DNA fiber assay. However, this method relies exclusively on manual image acquisition and analysis, making it time-consuming and susceptible to user bias. Here, we present a quantitative AI-based DNA fiber (qAID) workflow enabling imaging and multiparameter analysis of thousands of DNA fibers within several dozen minutes. Our workflow quantifies key parameters, including DNA fiber frequency, length, and symmetry, while also allowing visual inspection of individual DNA fibers using unbiased image galleries. The robustness of the workflow is demonstrated by comprehensive datasets of biologically relevant experiments performed by three independent laboratories. Overall, qAID workflow provides a fast and effective examination of replication dynamics and its alterations at the single-molecule resolution.

cell biology↗

bioRxiv2025

PAF15-PCNA assembly exhaustion governs lagging strand replication and replisome integrity

Genome replication in eukaryotic cells is surveyed by the S-phase checkpoint, which orchestrates sequential replication origin activation to avoid exhaustion of hitherto poorly defined rate-limiting replisome components. Here, we find that excessive activation of replication origins depletes chromatin-bound PCNA and lagging strand components, thereby limiting additional PCNA loading at new origins when checkpoint control is disrupted. PAF15 (PCNA-associated factor 15) emerges as a dosage-sensitive regulator of PCNA, delineating the dynamic range of global genome duplication and defining distinct roles for PCNA on the leading and lagging strands. Through its high-affinity PIP motif and interaction within the DNA encircling channel of PCNA, PAF15 stabilizes PCNA exclusively on the lagging strand, optimizing and rate-limiting lagging strand processing. On the other hand, misregulation of PAF15--whether by overexpression or mislocalization to the leading strand--impairs replication fork progression and leads to cell death. These defects are mitigated by TIMELESS and CLASPIN, which restrain PAF15-PCNA interactions beyond the lagging strand. E2F4-mediated repression orchestrates PAF15 expression in normal and cancer cells, maintaining its optimal dosage for lagging strand-specific interactions with PCNA. Thus, the S-phase checkpoint functions in concert to restrict origin activation when lagging strand PAF15-PCNA assembly is exhausted, linking a previously concealed strand-specific rate limitation to overall replication dynamics.

biochemistry↗

bioRxiv2024

CRL4DCAF12 regulation of MCMBP ensures optimal licensing of DNA replication

The minichromosome maintenance (MCM2-7) protein complexes are central drivers of genome duplication. Distinct protein pools, parental and nascent MCMs, and their precise equilibrium are essential to sustain error-free DNA replication1. However, the mechanism responsible for generating these pools and maintaining their equilibrium remains largely unexplored. Here, we identified CRL4DCAF12 as a new factor controlling the assembly of nascent MCM complexes. During MCM biogenesis, MCMBP facilitates the assembly and transport of newly synthesized MCM3-7 subcomplexes into the nucleus2,3. Once in the nucleus, the MCM2 subunit must be incorporated into the MCM3-7 subcomplex, while MCMBP needs to be removed. CRL4DCAF12 facilitates the degradation of MCMBP and thereby regulates the assembly of MCM2-7 complexes. The absence of CRL4DCAF12 adversely affects the level of chromatin-bound nascent MCMs, resulting in accelerated replication forks and genome instability. Collectively, our findings uncovered the molecular mechanism underlying nascent MCM production essential to counteract genome instability and tumor formation.

cell biology↗

bioRxiv2024

Development of a cell-permeable Biotin-HaloTag ligand to explore functional differences between protein variants across cellular generations

HaloTag technology represents a versatile tool for studying proteins. Fluorescent HaloTag ligands employed in sequential labeling led to the discovery of distinct protein variants for histones, cohesins, and MCM complexes. Nonetheless, an efficient biochemical approach to separate the distinct protein variants to study their biological functions is missing. Principally being a gap in technology, the HaloTag toolbox lacks affinity ligands displaying good cell permeability and efficient affinity capture. Here, we describe the design, synthesis, and validation of a new cell-permeable Biotin-HaloTag ligand, which allows rapid labeling of Halo-tagged proteins in live cells and their efficient separation using streptavidin pull-down. Our work outlines how to use the herein-developed affinity ligand in sequential labeling to biochemically separate distinct protein variants and study their biological properties. The approach holds immense potential for addressing fundamental questions concerning essential cellular processes, including genome duplication and chromatin maintenance.

cell biology↗