Biology subjects

Roukos, V.

Publications and source records attributed to Roukos, V..

7 recordsLinked to original sources

bioRxiv2026

The nuclear actin cytoskeleton supports DNA double-strand break repair via VCP-mediated extraction of the KU70/80 complex from damaged chromatin

Double-strand breaks (DSBs) are critical lesions in genomic DNA, and their accurate repair is essential for maintaining genome stability. The nuclear actin cytoskeleton has been implicated in homology-directed repair (HDR) of DSBs. However, the underlying mechanism remains poorly understood. Here, we report that Myosin VI (Myo6), an actin-based motor protein, cooperates with F-actin in end resection and DSB mobilization. Our findings reveal that Myo6 directly interacts with both KU70 and the ubiquitin-dependent segregase VCP to facilitate the extraction of the KU70/80 complex from chromatin. This process is supported by F-actin, revealing an interplay between nuclear actin dynamics and the DSB repair machinery. By elucidating the function of Myo6 and its direct interactions with key repair factors, our study provides mechanistic insight into how repair mechanisms rely on nuclear actin to safeguard genome integrity.

cell biology↗

bioRxiv2026

The phosphoproteomic landscape of the DNA damage response

The DNA damage response (DDR) comprises an intricate network of protein-protein interactions and signaling pathways activated by DNA lesions and genomic instability. Central to this response is protein phosphorylation, which orchestrates DNA repair, cell cycle checkpoint activation, and chromatin organization. The response of the human phosphoproteome to different DNA damage-inducing agents and the functional role of regulated phosphorylation sites remains insufficiently characterized. Here, we systematically profiled the cellular phosphoproteome following exposure to eleven DNA damage-inducing agents that humans encounter physiologically or during cancer therapy. We identified a core set of DNA damage responsive phosphorylation sites, along with DDR signatures associated with DNA double strand breaks, replication stress and a pleiotropic response. Regulated phosphorylation sites are enriched within intrinsically disordered regions (IDRs), often forming clusters of nearby modifications that can affect IDR conformations or overlap with short linear interaction motifs. We discover that the RNA damage response predominantly shapes the changes induced by reactive aldehyde formaldehyde, alkylating agent methyl methanesulfonate and oxidative stress. Finally, we demonstrate that the proteasome-associated ubiquitin E3 ligase UBE3A is targeted by ATM and ATR kinases, thus linking proteasome regulation with the DDR.

cell biology↗

bioRxiv2026

RepliCNN: High-resolution inference of the DNA replication program from strand-specific 3' DNA end sequencing

During S phase, the genome is replicated in a tightly regulated spatiotemporal order described as DNA replication timing (RT). Discontinuous lagging-strand synthesis produces Okazaki fragments whose strand-specific distribution reflects replication dynamics. Here, we present RepliCNN, a deep learning framework based on one-dimensional convolutional neural networks to predict RT from Okazaki fragment distributions obtained from strand-specific 3' DNA end sequencing methods such as GLOE-Seq, TrAEL-seq, or OK-Seq. RepliCNN also automatically annotates replication origins, termination zones, replication fork directionality, and origin efficiency genome-wide from a single dataset. Benchmarking on public and in-house human and yeast datasets using leave-one-chromosome-out cross-validation demonstrates high predictive accuracy in both wild-type and perturbation experiments, enabling comprehensive analyses of replication dynamics from strand-specific DNA 3' end sequencing data. HighlightsO_LIRepliCNN enables integrated analysis of replication timing, fork directionality and replication features from strand-specific 3' DNA end sequencing data. C_LIO_LIHigh-resolution replication dynamics can be inferred from a single experiment, bypassing complex multi-fraction labelling approaches. C_LIO_LIThe framework generalizes across experimental protocols, datasets, and species. C_LIO_LIThis enables cost-effective comparative analysis of replication programs across biological conditions. C_LI

bioinformatics↗

bioRxiv2023

High clonal diversity and spatial genetic admixture in early prostate cancer and surrounding normal tissue

Somatic copy number alterations (SCNAs) are pervasive in advanced human cancers, but their prevalence and spatial distribution in early-stage, localized tumors and their surrounding normal tissues are poorly characterized. Here, we performed multi-region, single-cell DNA sequencing to characterize the SCNA landscape across multiple tumor-rich and normal tissue regions ([~]125 mm3 tissue cubes) obtained from prostatectomy performed in two patients with localized prostate cancer. We identified two distinct populations of cells with abnormal karyotypes, one marked by sparse deletions or amplifications ( pseudo-diploid cells) and the second characterized by genome-wide copy number changes reminiscent of monster cells previously described in colorectal cancer. Pseudo-diploid cells formed numerous small-sized subclones ranging from highly spatially localized to broadly spread subclones mainly featuring (sub-)chromosomal arm deletions. In contrast, monster cells harbored whole-chromosome gains and losses and were mostly singular events detected throughout the prostate, including normal tissue regions. Targeted deep sequencing of cancer-associated genes revealed a more confined pattern of mutations overlapping with tumor-rich regions, although we also detected mutations in regions deemed normal based on morphological assessment and bulk RNA-seq. Highly localized pseudo-diploid subclones were confined within tumor-rich regions and typically carried deletions involving chromosome (chr) 6 and 13, resulting in simultaneous loss of multiple tumor-suppressor genes, including FOXO1 and FOXO3 encoding two transcription factors belonging to the Forkhead family previously implicated in prostate carcinogenesis. Tumor-rich regions also contained mutations in genes frequently mutated in prostate cancer, including FOXA1, LRP1B, SPOP, and SPTA1. Our study reveals that SCNAs are widespread in both normal and tumor regions across the prostate gland of patients with localized prostate cancer and suggests that a subset of pseudo-diploid cells harboring chromosomal deletions that result in the loss of specific tumor-suppressor genes drive tumorigenesis in the aging prostate.

cancer biology↗

bioRxiv2020

RNA polymerase II is required for spatial chromatin reorganization following exit from mitosis

Mammalian chromosomes are three-dimensional entities shaped by converging and opposing forces. Mitotic cell division induces drastic chromosome condensation, but following reentry into the G1 cell cycle phase, condensed chromosomes unwind to reestablish interphase organization. Here, we use a cell line allowing auxin-mediated degradation of RNA polymerase II to test its role in this transition. In situ Hi-C showed that RNAPII is required for compartment and loop formation following mitosis. RNAPs often counteract loop extrusion and, in their absence, longer and more prominent loops arise. Evidence from chromatin fractionation, super-resolution imaging and in silico modeling attribute these effects to RNAPII-mediated cohesin loading at active promoters upon reentry into G1. Our findings reconcile the role of RNAPII in gene expression with that in chromatin architecture.

genomics↗

bioRxiv2018

Spatial chromosome folding and active transcription drive DNA fragility and formation of oncogenic MLL translocations

How spatial chromosome organization influences genome integrity is still poorly understood. Here we show that DNA double-strand breaks (DSBs) mediated by topoisomerase 2 (TOP2) activities, are enriched at chromatin loop anchors with high transcriptional activity. Recurrent DSBs occur at CTCF/cohesin bound sites at the bases of chromatin loops and their frequency positively correlates with transcriptional output and directionality. The physiological relevance of this preferential positioning is indicated by the finding that genes recurrently translocating to drive leukemias, are highly transcribed and are enriched at loop anchors. These genes accumulate DSBs at recurrent hot spots that give rise to chromosomal fusions relying on the activity of both TOP2 isoforms and on transcriptional elongation. We propose that transcription and 3D chromosome folding jointly pose a threat to genomic stability, and are key contributors to the occurrence of genome rearrangements that drive cancer.

cell biology↗

bioRxiv2018

Spatially clustered loci with multiple enhancers are frequent targets of HIV-1

HIV-1 recurrently targets active genes that are positioned in the outer shell of the nucleus and integrates in the proximity of the nuclear pore compartment. However, the genomic features of these genes and the relevance of their transcriptional activity for HIV-1 integration have so far remained unclear. Here we show that recurrently targeted genes are delineated with super-enhancer genomic elements and that they cluster in specific spatial compartments of the T cell nucleus. We further show that these gene clusters acquire their location at the nuclear periphery during the activation of T cells. The clustering of these genes along with their transcriptional activity are the major determinants of HIV-1 integration in T cells. Our results show for the first time the relevance of the spatial compartmentalization of the genome for HIV-1 integration, thus further strengthening the role of nuclear architecture in viral infection.

genomics↗